Small molecule mimetics of carboxypeptidase e and related compositions, methods, and systems

Abstract

Small molecule mimetics of Carboxypeptidase E (CPE) and related compositions, methods, and systems for providing cytoprotection. Compounds of Formula I, which encompasses specific sub-sets including hydrazone-based (Formula II, III, IV), urea-based (Formula V), and amide-based (Formula VI) scaffolds are configured to bind selectively to the 5-Hydroxytryptamine Receptor 1E (5-HTR1E) to serve as functional mimetics of the neurotrophic protein CPE. Upon binding, the compounds trigger a specific biased signaling pathway that involves the recruitment of beta-arrestin, phosphorylation of ERK, and upregulation of the anti-apoptotic protein BCL-2. The compounds exhibit nanomolar binding affinity and significant cytoprotective activity against oxidative stress in human neurons and peripheral tissues. Also methods for identifying such mimetics using a hybrid virtual screening protocol, synthetic methods comprising a Betti reaction sequence, and therapeutic methods for treating conditions associated with oxidative stress, such as neurodegenerative disorders (e.g., Alzheimer's Disease) and peripheral organ injury, via intranasal or systemic administration.

Description

Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT SMALL MOLECULE MIMETICS OF CARBOXYPEPTIDASE E AND RELATED COMPOSITIONS, METHODS, AND SYSTEMSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is claims priority to US provisional application No.63 / 746,513, entitled “Therapeutics to Prevent Neurodegeneration, Amyloidosis, Tau Hyperphosphorylation, and Cognitive Dysfunction,” filed on January 17, 2025 with docket number CIT 9265-P. the content of which is incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under Grant No. GM 145239 and Grant No. HD000056 awarded by the National Institutes of Health and under Grant No. CBET2311117 awarded by the National Science Foundation. The government has certain rights in the invention.

[0003] This invention was also made with support under Grant No. KK2432-10 awarded by the Korea Research Institute of Chemical Technology, Grant No. CAP23011-200 awarded by the National Research Council of Science and Technology of the Korea and Grant No. RS-2024-00344154 awarded by the National Research Foundation funded by the Ministry of Science and ICT of the Korean Government.FIELD

[0004] The present disclosure relates to cellular signaling pathways activated by carboxypeptidase E (CPE) via the 5-HTR1E receptor and in particular small molecule mimetics of carboxypeptidase E and related compositions, methods and systems.BACKGROUND

[0005] Carboxypeptidase E (CPE), also known as Neurotrophic Factor- 1 (NF-1), is a protein recognized for its role in cellular signaling and neuroprotection. Beyond its enzymatic functions, CPE acts extracellularly to promote cell survival and resilience against various forms of physiological stress, including oxidative stress. Its activity isTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT mediated through specific receptor interactions that trigger protective intracellular pathways.

[0006] Replicating the precise receptor binding and signaling properties of a large protein such as CPE with synthetic compounds presents significant structural and functional hurdles. Accordingly, despite the biochemical and therapeutic potential of CPE, the identification of compounds capable of mimicking its specific biological functions remains challenging.SUMMARY

[0007] Described herein are small molecule compounds configured to mimic the biological activity of Carboxypeptidase E (CPE) and related compositions, methods and system. In particular, the present disclosure provides structural and functional mimetics having a structure configured to bind to the 5-HTR1E receptor to activate cytoprotective signaling pathways, such as the β-arrestin / ERK / BCL-2 pathway.

[0008] According to a first aspect, a small molecule is described of Formula I:R2R5(I)wherein n is 0 or 1; andwherein when n is 0, Cl, C2, C3, C4, and C6 comprise a 5-membered aromatic ringwherein any of Cl, C2, C3, C4, or C6 is optionally substituted with a Nitrogen, Oxygen, or Sulfur heteroatom; andTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT wherein when n is 1, Cl, C2, C3, C4, C5 and C6 comprise a 6-membered aromatic ring wherein any of Cl, C2, C3. C4, C5 or C6 is optionally substituted with a Nitrogen,Oxygen, or Sulfur heteroatom; andwherein Al, A2, and A3 are each independently selected from Carbon, Nitrogen, Sulfur, or Oxygen; andwherein each of Al, A2, and A3 is optionally substituted with hydrogen, alkyl, or oxo(=0) groups; andwherein X comprises a carboxylic acid, ester, amide, sulfonic acid, sulfinic acid, sulfonamide, hydroxamic acid phosphonic acid or nitro group; andwherein Rl, R2, R4, and R5 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen,Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms; andwherein R3 comprises a Sulfur, Nitrogen, Oxygen, or Phosphorus-containing functional group; andand wherein Y is a 4-, 5- or 6-membered ring such that Al and X are separated by at least3 atoms of the ring;or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0009] According to a second aspect, a small molecule of Formula I is described comprising a phenyl scaffold wherein ring Y is a 6-membered aromatic ring (e.g., phenyl) and A1-A3 form a hydrazone linkage. In some of those embodiments, the small molecule comprises a hydrazone scaffold and has Formula IIFormula IIwhereinTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Rl, R2, R4 and R5 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms;R3 comprises an S, N, O or P-containing functional group; andn is 0 or 1whereinwhen n is 0. Cl, C2, C3, C4, and C6 comprise a 5-membered aromatic ring and any of Cl, C2, C3, C4, or C6 is optionally substituted with a Nitrogen, Oxygen, or Sulfur heteroatom; andwhen n is 1. Cl, C2, C3, C4, C5 and C6 comprise a 6-membered aromatic ring andany of Cl, C2, C3, C4, C5 or C6 is optionally substituted with a Nitrogen, Oxygen, or Sulfur heteroatom; andwhereinX comprises an ester, amide or acid group containing at least one E=O bond where E isC, S or P, or wherein X is a nitro group -NO2; andAl is NH, A2 is N and A3 is C.

[0010] According to a third aspect, a small molecule of Formula I is described wherein ring Y is a 6-membered aromatic ring (e.g., phenyl), the linker A1-A3 forms a hydrazone moiety, and wherein R2 is a non-hydrogen / non-alkoxy substituent selected to enhance stability. Accordingly in those embodiments, the small molecule comprises a hydrazone scaffold and has Formula IIITitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Formula IIIwhereinRl, R4, R5 and R6 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms; andR2 is an alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms such that R2 is not H or an alkoxy group;R3 comprises an S, N, O or P-containing functional group; andn is 0 or 1whereinwhen n is 0, Cl, C2, C3, C4, and C6 comprise a 5-membered aromatic ring and any ofCl, C2, C3, C4, or C6 is optionally substituted with a Nitrogen, Oxygen, or Sulfur heteroatom; andwhen n is 1, Cl, C2, C3, C4, C5 and C6 comprise a 6-membered aromatic ring and any of Cl, C2, C3, C4, C5 or C6 is optionally substituted with a Nitrogen, Oxygen, orSulfur heteroatom; andwhereinX comprises an ester, amide or acid group containing at least one E=O bond where E is C,S or P, or wherein X is a nitro group -NO2

[0011] According to a fourth aspect, a small molecule of Formula I is configured to include a specific hydrazone linker regardless of ring type. Accordingly, in the small molecule having the structure of Formula I, the linker Al -A3 can form a hydrazone moiety, while ring Y remains broad (4-, 5-, or 6-membered). In those embodiments the small molecule is thus a hydrazone small molecule having the structure of Formula IV:Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCTFormula IVwherein R6 is selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms; andwherein X, Y, Rl, R2 R3, R4, R5, and n are defined as in Formula I.

[0012] According to a fifth aspect, a small molecule of Formula I is described comprising a urea linker. Accordingly, in those embodiments, a small molecule is described which has Formula VFormula Vwherein X comprises an ester, amide, or acid group containing at least one E=O bond whereE is C, S, or P, or is a nitro group; and wherein Rl, R2, R4, and R5 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms; and wherein R3 comprises a Sulfur, Nitrogen, Oxygen, or Phosphorus-Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT containing functional group (e.g., a sulfonate ester).

[0013] According to a sixth aspect, a small molecule of Formula I is described comprising an amide linker.. Accordingly, in those embodiments, a small molecule is described which has Formula VIZoFormula VIwherein X and Z each independently are a nitro group -NO2or comprises an ester, amide, or acid group containing at least one E=O bond where E is C, S, or P,; and wherein any of the aromatic rings can be optionally substituted with alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with heteroatoms N, O. S, F, Cl, Br and / or I.

[0014] According to a seventh aspect, described herein is a method for identifying small molecule mimetics of Carboxypeptidase E (CPE). The method comprises a virtual screening protocol, preferably a hybrid protocol that integrates ligand-based and structurebased approaches, performed by: (a) defining a pharmacophore model based on the interaction interface between CPE and the 5-HTR1E receptor, wherein the interface is partitioned into distinct binding regions (e.g., SI, S2, and S3) corresponding to specific peptide fragments of the CPE protein; (b) screening a library of small molecule compounds against said pharmacophore model to identify initial hits; and (c) validating said hits by molecular docking into a structural model of the 5-HTR1E receptor binding pocket.

[0015] According to an eighth aspect, described herein is a general method of synthesizing a small molecule mimetic as described herein, encompassing compounds of Formula I, and specifically including compounds of Formula II, Formula III, Formula IV, Formula V and Formula VI. The method broadly comprises the following steps:(i) performing a Betti reaction (or Mannich-type condensation) between a substituted phenol comprising a linker precursor group selected from an aldehyde or a nitroTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT group, a formaldehyde source, and a primary or secondary amine moiety to form an (aminoalkyl)hydroxy-aromatic intermediate;(ii) protecting the amino group of the resulting intermediate (e.g., with a Boc group);(iii) reacting the protected intermediate with a sulfonyl electrophile to install a sulfonate or sulfonamide group;(iv) converting the linker precursor group into a reactive coupling moiety, wherein:a) if the precursor group is an aldehyde and a hydrazone scaffold (e.g., Formula II,III or IV) is desired, maintaining the aldehyde group;(b) if the precursor group is an aldehyde and a urea or amide scaffold (e.g., Formula V or VI) is desired, oxidizing the aldehyde to a carboxylic acid; or(c) if the precursor group is a nitro group and a urea scaffold (e.g., Formula V) is desired, reducing the intermediate (e.g., via hydrogenation with Pd / C) to convert the nitro group into an aromatic amine (aniline);(v) reacting the resulting intermediate with a complementary linker-forming partner to form the bridged pharmacophore, wherein:(a) the aldehyde intermediate is condensed with a hydrazine derivative to yield a hydrazone scaffold (Formula II, III and IV);(b) the carboxylic acid intermediate is coupled with an amine to yield an amide scaffold (Formula VI);(c) the carboxylic acid intermediate is converted to a hydroxamic acid / isocyanate sequence and trapped with an amine to yield a urea scaffold (Formula V); or(d) the aromatic amine (aniline) intermediate is coupled with an isocyanate (e.g., generated from tert-butyl 4-aminobenzoate) to yield a urea scaffold (Formula V): and(vi) optionally deprotecting the amino group and any other protecting groups to yield the final compound.

[0016] According to a ninth aspect, described herein is a pharmaceutical compositionTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT comprising a therapeutically effective amount of at least one small molecule mimetic as described herein (e.g., a compound of Formula I, Formula II, Formula III, FormulaIV, Formula V or Formula VI), or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier, vehicle, or excipient. In some embodiments, the composition comprises a combination of two or more distinct small molecule mimetics. The composition can be formulated for specific routes of administration, such as to intranasal administration (e.g., sprays, drops, in-situ gels) or systemic injection, to facilitate delivery to the central nervous system.

[0017] According to a tenth aspect, described herein is a method and related system for activating the 5-Hydroxytryptamine Receptor 1E (5-HTR1E). The method comprises contacting a cell expressing the 5-HTR1E receptor with at least one small molecule mimetic of the disclosure in an amount sufficient to bind to the receptor and initiate downstream signaling, such as |3-arrestin recruitment, ERK phosphorylation, or BCL-2 upregulation. This method can be performed in vitro, for example in cellular assays (e.g., luciferase reporter assays, dynamic mass redistribution assays) to evaluate receptor kinetics, or in vivo to modulate receptor activity in a biological system.

[0018] The system for activating the 5-Hydroxy tryptamine Receptor IE (5-HTR1E) and detecting said activation. In some embodiments, the system comprises: (a) at least one small molecule mimetic as described herein (e.g., a compound of Formula I or II); and (b) a biological substrate comprising cells expressing the 5-HTR1E receptor. In further embodiments, the system may additionally comprise (c) detection reagents configured to quantify downstream signaling events, such as β-arrestin recruitment or ERK phosphorylation. For example, the system may function as a research tool or assay kit, wherein the detection reagents comprise luciferase reporter substrates, fluorescent biosensors, or antibodies specific for phosphorylated signaling protein

[0019] According to an eleventh aspect, the disclosure provides a method of treating, preventing, or attenuating the progression of a condition associated with 5-HTR1E receptor dysfunction or a condition responsive to 5-HTR1E receptor activation. The method comprises administering to a subject in need thereof a therapeutically effective amount ofTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT a compound of the disclosure (e.g., Formula I). In some embodiments, the condition is a neurodegenerative disorder selected from Alzheimer’s disease, Parkinson’s disease, or tauopathy. In other embodiments, the condition is a peripheral disorder characterized by oxidative stress or mitochondrial dysfunction, such as acute kidney injury (AKI), ischemiareperfusion injury, or cardiomyopathy.

[0020] The system for treating or preventing a condition associated with 5-HTR1E receptor dysfunction or for mitigating oxidative stress-induced cytotoxicity in a subject.The system broadly comprises: (a) a pharmaceutical composition comprising a therapeutically effective amount of one or more small molecule mimetics of the disclosure (e.g., Formula I); and (b) a delivery device configured to administer said composition to a target tissue within the subject.

[0021] According to a twelfth aspect, described herein is a method and system for treating or preventing a neurodegenerative condition or preventing neuronal cell death. The method comprises administering to a subject in need thereof a therapeutically effective amount of one or more small molecule mimetics of the disclosure. In preferred embodiments, the administration is configured to mitigate oxidative stress-induced cytotoxicity. The condition treated may be Alzheimer's Disease, Parkinson's Disease, or other neurodegenerative disorders characterized by amyloidosis, tau hyperphosphorylation, or cognitive dysfunction.

[0022] The system for treating or preventing a neurodegenerative condition or preventing neuronal cell death comprises: (a) a pharmaceutical composition comprising a therapeutically effective amount of one or more small molecule mimetics of the disclosure; and (b) a delivery device configured to administer said composition to a subject. In preferred embodiments, the delivery device is an intranasal delivery device, such as a nasal spray pump, a dropper, or a nebulizer, configured to bypass the blood-brain barrier and deliver the composition directly to the central nervous system. The system may further comprise instructions for a dosage regimen, for example, administering the composition twice daily to mitigate oxidative stress-induced cytotoxicity.

[0023] The small molecules of the disclosure and related compositions methods andTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT systems can be used for promoting cellular survival and resilience against physiological stress, including oxidative stress, as will be understood by a skilled person upon reading of the present disclosure

[0024] The small molecules herein described and related products, compositions, methods, and systems, allow in several embodiments the non-invasive activation of neuroprotective signaling pathways normally regulated by the Carboxypeptidase E protein.

[0025] The small molecules herein described and related products, compositions, methods, and systems, allow in several embodiments the provision of a synthetically accessible therapeutic alternative to large recombinant proteins.

[0026] The small molecules herein described and related products, compositions, methods, and systems, allow in several embodiments for highly potent therapeutic efficacy, characterized by binding affinities superior to that of the native Carboxypeptidase E protein.

[0027] The small molecules herein described and related products, compositions, methods, and systems, allow in several embodiments the treatment of neurodegenerative conditions such as Alzheimer's Disease at least through the selective recruitment of f>-arrestin.

[0028] The small molecules herein described, along with related products, compositions, methods, and systems, provide for the mitigation of oxidative stress-induced cellular damage. While primary embodiments are directed to neuroprotection, the utility of the disclosed compounds extends to both neuronal and non-neuronal tissues. Experimental data demonstrating potent cytoprotection in HEK293 (Human Embryonic Kidney) cells indicates that the mechanism of action — specifically the modulation of BCL-2 and ERK pathways via the 5-HTR1E receptor — is conserved in peripheral organs. Accordingly, in several embodiments, the compounds are configured for protecting peripheral tissues, such as the kidney, heart, and liver, against ischemic, hypoxic, or oxidative injury, independent of their action in the central nervous system.

[0029] The small molecules herein described, and related products, compositions, methods, and systems, allow in several embodiments the selective modulation of the 5-HTR1E receptor. Specifically, the compounds demonstrate selectivity against structurallyTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT related serotonin subtypes, such as 5-HTR1A and 5-HTR1F. Furthermore, the therapeutic application of these molecules is supported by a favorable in vivo safety profile. In murine models, administration of the effective neuroprotective dose resulted in no observable adverse physiological effects or behavioral toxicity, suggesting that the compounds avoid the off-target activation of serotonin receptors associated with cardiotoxicity (e.g., 5-HTR2B) or other systemic adverse events.

[0030] The compounds described herein, and related products compositions, methods, and systems, allow in several embodiments for the non-invasive treatment of degenerative disorders. Unlike large protein therapeutics (e.g., neurotrophic factors), these small molecules exhibit superior physicochemical stability and may be formulated for diverse routes of administration, including intranasal, oral, or systemic delivery. Exemplary applications comprise methods of treating neurodegenerative diseases (e.g., Alzheimer's disease), protecting cells from cytotoxicity, and modulating the 5-HTR1E receptor for therapeutic or research purposes. Consistent with the evolutionary conservation of the 5-HTR1E receptor in peripheral tissues of vertebrate species (e.g., cardiac and hepatic tissues), the disclosed methods further extend to the cytoprotection of non-neuronal organs, such as the heart and liver.

[0031] The small molecules and related products compositions, methods, and systems provide significant clinical and manufacturing advantages over protein-based therapeutics often utilized for neuroprotection. Unlike large protein therapeutics, such as neurotrophic factors, the small molecule mimetics of the present disclosure exhibit robust metabolic stability and batch-to-batch consistency. Furthermore, they are synthetically accessible via scalable chemical processes, ensuring cost efficiency for mass production. Due to their low molecular weight and favorable physicochemical properties, such as lipophilicity, these compounds are amenable to non-invasive delivery routes, including intranasal sprays and oral dosage forms, which are typically ineffective for large biologies. This formulation flexibility significantly enhances patient compliance and clinical usability compared to invasive injection-based therapies.

[0032] In addition to therapeutic applications in neurodegenerative diseases, the utility ofTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT the compounds extends to cytoprotective applications in non-neuronal tissues. Consistent with the evolutionary conservation of the 5-HTR1E receptor in peripheral tissues of vertebrate species, including cardiac and hepatic tissues, the disclosed methods are applicable to the protection of organs such as the heart and liver. Consequently, the disclosure provides a versatile platform for stabilizing mitochondrial function and mitigating oxidative stress across diverse organ systems, distinct from the specific neuroprotective applications in the central nervous system. The compounds may further be utilized in combination therapies with other active agents or as research tools for elucidating 5-HTR1E signaling pathways.

[0033] The small molecules herein described, and related products, compositions, methods, and systems, can be used in connection with applications wherein activation of the 5-HTR1E receptor (or functionally analogous cognate receptors), selective recruitment of -arrestin, phosphorylation of ERK, and / or upregulation of anti-apoptotic proteins such as BCL-2 are desired. Exemplary applications comprise therapeutic methods for the treatment or prevention of conditions associated with serotonergic and mitochondrial dysfunction. These include neurodegenerative disorders (e.g., Alzheimer's Disease, Parkinson's Disease, and TBI), wherein the molecules function to prevent neuronal cell death, amyloidosis, and tau hyperphosphorylation. The applications further extend to neuropsychiatric and vascular disorders (e.g., depression, anxiety, and migraine) and peripheral cytotoxic conditions (e.g., acute kidney injury, myocardial ischemia, and hepatic failure), wherein the molecules provide broad cytoprotection against oxidative stress in both neuronal and non-neuronal tissues. Additionally, applications comprise the use of the small molecules as pharmacological tools or research reagents for investigating biased signaling pathways in vitro or in vivo.

[0034] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features and objects will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF DRAWINGS

[0035] The accompanying drawings, which are incorporated into and constitute a part ofTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT this specification, illustrate one or more embodiments of the present disclosure and, together with the description of example embodiments, explain the principles and implementations of the disclosure.

[0036] Figure 1A: General structure of candidates for R-group screening based on the structure of hit compound, Z124, where R₃ is -SO₂-aryl and R₁, R₂, R₄, R₅, and R₆ are H, alkyl, cycloalkyl, aryl or heterocyclic groups optionally substituted with heteroatoms.

[0037] Figure IB: Top designed candidates through R-group screening based on the structure of hit compound, Z124, in which R₁ is -SO₂-aryl and R₂ is an alkyl or cycloalkyl or heterocyclic group optionally substituted with heteroatoms.

[0038] Figure 2 illustrates the hybrid virtual screening approach utilized to discover novel small molecules exhibiting neuroprotective properties similar to Carboxypeptidase E (CPE). Figure 2 Panel (A) provides a schematic overview of the high-throughput virtual screening workflow. Figure 2 Panel (B) depicts the ligand-based pharmacophore model derived from the CPE hypothesis, illustrating its correlation with the 5-HTR1E binding site shown in Figure 2 Panel (C). Figure 2 Panel (C) further highlights key interactions identified through mutation studies that informed the structure-based virtual screening.Figure 2 Panel (D) displays the 2D chemical structure of the virtual screening hit compound. Z49535124 (Z124). Panel (E) shows the predicted binding model of Z124 complexed with the 5-HTR1E receptor.

[0039] Figure 3 presents experimental data characterizing the biological activity of lead compound Z124. Figure 3 Panel (A) is a bar graph showing relative luciferase activity in HTLA cells expressing HTR1E treated with Z124 compared to CPE. In particular, the bar graph shows the relative luciferase activity normalized against WT-CPE. * p<0.05.Figure 3 Panel (B) shows a line graph displaying the dose-response binding kinetics of Z124 determined via dynamic mass redistribution (DMR) assays. Each concentration represents an n=4. Analysis by Graph Pad prism determined the binding affinity of 27.4+ 2.3 nM. Figure 3 Panel (C) shows a bar graph illustrating the neuroprotective effect of Z124 on cell cytotoxicity in 5-HTR1E stable HEK.293 cells challenged with hydrogenTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT peroxide (H2O2).

[0040] Figure 4: Experimental results of Z124 selected compound from virtual screening.Figure 4 Panel A reports the results Luciferase assay showing comparison of binding affinity of Z 124 and CPE to HTR1E. The Presto-tango reporter system was used to measure the binding affinity between compound Z124 and 5-HTR1E. HTR1E transfected HTLA cells were treated with 50nM of compound Z124 or 50nM CPE for 6 hours and luciferase activity was measured using whole cell extracts from transfected cells. The bar graph shows the relative luciferase activity normalized against WT-CPE. Figure 4 Panel B reports results, 5-HTR1E stable HEK293 cells were treated with 50nM of compound Z124 for 24 hours followed by 500uM H₂O₂ for 4 hours, then the LDH activity was measured using cell medium after centrifugation. The bar graph shows the relative cytotoxicity for Z124 is similar to DMSO treated cells. Values are mean ± SEM.

[0041] Figure 5 illustrates the structural optimization and predicted binding modes of selected lead compounds. In particular Figure 5 Panel A shows the 2D chemical structures of the optimized hydrazone derivatives R9 and R10. Figure 5 Panel B depicts the predicted3D binding mode of compound R9 within the HTR1E receptor binding pocket. Figure 5 Panel C depicts the predicted 3D binding mode of compound R10 within the HTR1E receptor binding pocket.

[0042] Figure 6 reports the structure of the top 10 designed candidates through R- group screening based on the structure of selected compound, Z124.

[0043] Figure 7 depicts the 2D chemical structures of synthesized derivatives DI and D2 derived from R5.

[0044] Figure 8 is a schematic diagram illustrating the general synthetic route employed for the preparation of compounds R9, R10, DI, and D2.

[0045] Figure 9 is a proton nuclear magnetic resonance (1H NMR) spectrum of the intermediate compound 3-((ethylamino)methyl)-4-hydroxybenzaldehyde (R10-01).Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0046] Figure 10 is an infrared (IR) spectrum of the intermediate compound 3-((ethylamino)methyl)-4-hydroxybenzaldehyde (R 10-01 ).

[0047] Figure 11 is a carbon-13 nuclear magnetic resonance (13C NMR) spectrum of the intermediate compound 3-((ethylamino)methyl)-4-hydroxybenzaldehyde (R10-01)).

[0048] Figure 12 is a 1H NMR spectrum of the protected intermediate compound tertbutyl ethyl(5-formyl-2-(tosyloxy)benzyl)carbamate (R 10-02).

[0049] Figure 13 is an infrared (IR) spectrum of the protected intermediate compound tertbutyl ethyl(5-formyl-2-(tosyloxy)benzyl)carbamate (R10-02).

[0050] Figure 14 is a 13C NMR spectrum of the protected intermediate compound tertbutyl ethyl(5-formyl-2-(tosyloxy)benzyl)carbamate (R10-02).

[0051] Figure 15 is a 1H NMR spectrum of the final Compound R10.

[0052] Figure 16 is a 13C NMR spectrum of the final Compound R10.

[0053] Figure 16A is a fluorine- 19 nuclear magnetic resonance (19F NMR) spectrum of the final Compound R10.

[0054] Figure 17 is a 1H NMR spectrum of the intermediate compound 3-((l-(tert-butoxycarbonyl)-2-methylhydrazinyl)methyl)-4-hydroxybenzaldehyde (R9-01 ).

[0055] Figure 18 is a 1H NMR spectrum of the tosylated intermediate compound tertbutyl 2-(5-formyl-2-(tosyloxy)benzyl)-2-methylhydrazine-l -carboxylate (R9-02).

[0056] Figure 19 is an infrared (IR) spectrum of the tosylated intermediate compound tertbutyl 2-(5-formyl-2-(tosyloxy)benzyl)-2-methylhydrazine-l-carboxylate (R9-02).

[0057] Figure 20 is a 13C NMR spectrum of the tosylated intermediate compound tertbutyl 2-(5-formyl-2-(tosyloxy)benzyl)-2-methylhydrazine-l-carboxylate (R9-02).

[0058] Figure 21 is a 1H NMR spectrum of the final Compound R9.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0059] Figure 22 is a 13C NMR spectrum of the final Compound R9.

[0060] Figure 23 is a fluorine- 19 nuclear magnetic resonance (19F NMR) spectrum of the final Compound R9.

[0061] Figure 24 is a 1H NMR spectrum of the intermediate compound 3- ((dimethylamino)methyl)-4-hydroxybenzaldehyde (D 1 -01 ).

[0062] Figure 25 is a 1H NMR spectrum of the tosylated intermediate compound 2- ((dimethylamino)methyl)-4-formylphenyl 4-methylbenzenesulfonate (Dl-02).

[0063] Figure 26 is an infrared (IR) spectrum of the tosylated intermediate compound 2- ((dimethylamino)methyl)-4-formylphenyl 4-methylbenzenesulfonate (Dl-02).

[0064] Figure 27 is a 13C NMR spectrum of the tosylated intermediate compound 2- ((dimethylamino)methyl)-4-formylphenyl 4-methylbenzenesulfonate (Dl-02).

[0065] Figure 28 is a 1H NMR spectrum of the final Compound DI.

[0066] Figure 29 is a 13C NMR spectrum of the final Compound DI.

[0067] Figure 30 is a fluorine- 19 nuclear magnetic resonance (19F NMR) spectrum of the final Compound DI.

[0068] Figure 31 is a 1H NMR spectrum of the intermediate compound 4-hydroxy-3- (piperidin- 1 -ylmethyl)benzaldehyde (D2-01 ).

[0069] Figure 32 is an infrared (IR) spectrum of the intermediate compound 4-hydroxy- 3-(piperidin-l-ylmethyl)benzaldehyde (D2-01).

[0070] Figure 33 is a 13C NMR spectrum of the intermediate compound 4-hydroxy-3- (piperidin- 1 -ylmethyl)benzaldehyde (D2-01 ).

[0071] Figure 34 is 1H NMR spectrum of the tosylated intermediate compound 4-formyl-2-(piperidin- l-ylmethyl)phenyl 4-methylbenzenesulfonate (D2-02).Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0072] Figure 35 is an infrared (IR) spectrum of the tosylated intermediate compound 4-formyl-2-(piperidin- l-ylmethyl)phenyl 4-methylbenzenesulfonate (D2-02).

[0073] Figure 36 is a 13C NMR spectrum of the tosylated intermediate compound 4-formyl-2-(piperidin- l-ylmethyl)phenyl 4-methylbenzenesulfonate (D2-02).

[0074] Figure 37 is a 1H NMR spectrum of the final Compound D2.

[0075] Figure 37A is a 13C NMR spectrum of the final Compound D2.

[0076] Figure 38 is a fluorine- 19 nuclear magnetic resonance (19F NMR) spectrum of the final Compound D2.

[0077] Figure 39 presents biological data characterizing the binding affinity and receptor subtype selectivity of the synthesized compounds. Figure 39 Panel A shows a bar graph displaying the relative luciferase activity, indicative of [3-arrestin recruitment, in HTLA cells expressing 5-HTR1E treated with 50 nM of compounds DI, D2, R9, R10, Z124, or the positive control CPE. Figure 39 Panel B shows a line graph displaying the dosedependent binding kinetics to the target 5-HTR1E receptor determined by Dynamic Mass Redistribution (DMR) assays for Serotonin (5-HT), CPE, R9, and R10. Figure 39 PanelC shows a line graph displaying the binding kinetics of the lead compound R9 against the off-target receptor 5-HTR1A. Figure 39 Panel (D) is a line graph displaying the binding kinetics of the lead compound R9 against the off-target receptor 5-HTR1F, demonstrating the receptor subtype specificity of the compound.

[0078] Figure 40 presents biological data demonstrating the neuroprotective efficacy of the synthesized compounds against oxidative stress. Figure 40 Panel A shows a bar graph showing the percentage of cytotoxicity in HTRlE-expressing HEK293 cells pre-treated with 50 nM of compounds DI, D2, R9, or R10 followed by hydrogen peroxide challenge.Figure 40 Panel B shows a bar graph showing the cytotoxicity levels in wild-type HEK293 cells (lacking the HTR1E receptor) under identical treatment conditions, demonstrating receptor-dependent activity. Figure 40 Panel C shows a bar graph showing the neuroprotection conferred by the compounds in primary human cortical neuronsTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT challenged with H₂O₂.

[0079] Figure 41 presents Western blot analysis demonstrating the activation of neuroprotective signaling pathways by the synthesized compounds. In particular, Figure41 Panel A displays representative Western blot images and the corresponding densitometric quantification of phosphorylated ERK1 / 2 (pERK) relative to total ERK1 / 2 (tERK) in HTR1 E-expressing HEK293 cells treated with compounds R9 or RIO compared to vehicle. Figure 41 Panel B displays representative Western blot images and the corresponding densitometric quantification of the anti-apoptotic protein BCL-2 normalized to the loading control GAPDH under identical treatment conditions.

[0080] Figure 42 illustrates example delivery systems for embodiments provided herein.DETAILED DESCRIPTION

[0081] Described herein are small molecule compounds configured to mimic the biological activity of Carboxypeptidase E (CPE) and related compositions, methods and system.

[0082] The term "small molecule" as used herein refers to a low molecular weight organic compound that may regulate a biological process. Structurally, a small molecule is generally a non-peptidic compound, typically having a molecular weight of less than 900 Daltons, characterized by a specific arrangement of functional groups, such as aromatic rings, hydrogen bond donors, and hydrogen bond acceptors, capable of binding to a biological macromolecule such as a protein, nucleic acid, or receptor. These compounds are chemically distinguishable from biopolymers such as proteins, nucleic acids, or polysaccharides, and are often designed to have physicochemical properties suitable for pharmaceutical administration, such as solubility, stability, and the ability to cross biological barriers including cell membranes or the blood-brain barrier. Examples of small molecules include natural products, metabolites, synthetic organic compounds, pharmaceutical drugs, and chemical probes used in research.

[0083] The term " Carboxypeptidase E" or " CPE" (also known as Neurotrophic Factor-α1 or NF-1α) refers to a polypeptide belonging to the hormone-processing carboxypeptidaseTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT family. Structurally, CPE is defined by a specific amino acid sequence comprising a signal peptide, a pro-region, and a catalytic domain. It exists in various isoforms, including soluble and membrane-bound forms. Functionally, the protein acts as an exopeptidase that cleaves C-terminal basic residues from peptide precursors and also functions as an extracellular signaling molecule or neurotrophic factor independent of its enzymatic activity. Examples include the native human CPE protein, mammalian orthologs such as murine or rat CPE, and biologically active fragments or variants thereof that retain neurotrophic activity.

[0084] The term "mimic" or "mimetic" as used herein refers to an agent or compound that imitates the structure and / or function of a reference molecule. Structurally, a mimic is chemically distinct from the reference molecule but possesses structural features, such as charge distribution, shape, hydrophobicity, or spatial orientation of functional groups, that allow it to interact with the same biological target as the reference molecule. A mimic may be a structural mimic, containing chemical groups that resemble the reference pharmacophore, or a functional mimic, eliciting a similar biological response, such as receptor activation or signal transduction, regardless of structural homology. Examples of mimetics include peptidomimetics designed to mimic peptide chains, bioisosteres, and synthetic agonists that replace endogenous protein ligands.

[0085] In embodiments herein described, the small molecules described herein function as both structural and functional mimetics of the CPE protein. Structurally, they are designed to replicate the spatial orientation of key amino acid residues found on the surface of the CPE protein that interface with the 5-HTR1E receptor. Functionally, these small molecules bind to the 5-HTR1E receptor and trigger a specific neuroprotective signaling cascade comprising β-arrestin recruitment, ERK phosphorylation, and BCL-2 upregulation.

[0086] The term "prodrug" refers to a compound that, after administration, is metabolized or otherwise chemically converted within the body to yield a pharmacologically active compound of the disclosure. Prodrugs are frequently utilized to optimize the physicochemical properties of the parent drug, such as solubility, stability, orTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT bioavailability, or to enhance delivery to a specific target tissue (e.g., crossing the bloodbrain barrier). In the context of the present disclosure, prodrugs include, but are not limited to, ester or amide derivatives of the functional groups defined for X (e.g., where X is a carboxylic acid, the prodrug may be an alkyl ester thereof) or R3 (e.g., sulfonate esters), which are hydrolyzed in vivo to release the active acidic or ionic species

[0087] In particular in embodiments herein described, small molecules of the disclosure provide structural and functional mimetics having a structure configured to bind to the 5-Hydroxytryptamine Receptor IE (5-HTR1E) receptor to activate cytoprotective signaling pathways, such as the β-arrestin / ERK / BCL-2 pathway.

[0088] The term "5-Hydroxy tryptamine Receptor IE" or "5-HTR1E" refers to a specific subtype of the G protein-coupled receptor (GPCR) superfamily that binds the endogenous neurotransmitter serotonin. Structurally, the 5-HTR1E receptor is characterized by a core architecture common to Class A GPCRs, comprising seven transmembrane $\alpha$-helices connected by extracellular and intracellular loops, possessing a specific ligandbinding pocket that dictates its selectivity profile.

[0089] Functionally, the 5-HTR1E receptor is distinguished by its coupling to intracellular signaling pathways governing cellular survival and mitochondrial integrity. Unlike subtypes primarily mediating neuro transmission, activation of the 5-HTR1E receptor triggers a cytoprotective cascade — mediated, for example, via Gi / o protein coupling or β-arrestin recruitment — that results in the phosphorylation of extracellular signal-regulated kinase (ERK) and the modulation of BCL-2 family proteins. Through this mechanism, the receptor functions as a physiological sensor and regulator of oxidative stress, mitigating mitochondrial dysfunction and preventing apoptosis in response to ischemic or cytotoxic insults.

[0090] Methods to detect or identify the 5-HTR1E receptor include radioligand binding assays using tritiated serotonin or specific antagonists to determine receptor density and affinity; antibody-based techniques such as Western blotting, immunohistochemistry, or immunofluorescence to visualize protein expression and localization; and nucleic acidTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT amplification methods such as quantitative PCR (qPCR) or RNA sequencing to measure mRNA transcript levels in specific tissues. Examples of the 5-HTR1E receptor include the human receptor encoded by the HTR1E gene, predominantly expressed in the cerebral cortex and hippocampus, as well as mammalian orthologs expressed in peripheral tissues (e.g., cardiac and hepatic tissues) where they contribute to systemic cytoprotection.

[0091] The term "cytoprotective signaling" refers to biological processes and intracellular transduction cascades that preserve cellular viability and function in response to noxious stimuli. Structurally, cytoprotective signaling involves the coordinated activation of specific kinases, transcription factors, and effector proteins that stabilize cellular organelles and inhibit programmed cell death. Methods to detect or identify cytoprotective signaling include cell viability assays such as the MTT or CellTiter-Glo assays to measure metabolic activity; lactate dehydrogenase (LDH) release assays to quantify membrane integrity; flow cytometry or fluorescent microscopy using markers like Annexin V or TUNEL to detect apoptosis; and the use of fluorescent probes such as DCFDA to measure the reduction of reactive oxygen species (ROS). Examples of cytoprotective signaling include the activation of antioxidant response elements (ARE), the PI3K / Akt survival pathway, and the specific β-arrestin-dependent signaling mechanisms described herein.

[0092] The term "pathway" refers to a series of interactions among molecules in a cell that leads to a certain product or a change in a cell state. Structurally, a pathway is defined by a specific sequence of biochemical reactions, such as phosphorylation, cleavage, or translocation, transmitting a signal from a receptor to an intracellular target. Methods to detect or identify a pathway involve the use of specific chemical inhibitors or small interfering RNA (siRNA) to block specific steps and observe the downstream consequences; phosphoproteomic analysis to map kinase activity; and reporter gene assays configured to light up upon activation of a specific transcription factor associated with the pathway. Examples of pathways include metabolic pathways and signal transduction pathways such as the MAPK / ERK pathway.

[0093] The term "arrestin" refers to a family of adapter and scaffolding proteins that regulate the signaling and trafficking of G protein-coupled receptors. Structurally, arrestinsTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT are bilobed proteins capable of binding to phosphorylated residues on the intracellular tail of activated GPCRs. Methods to detect or identify arrestin activity or recruitment include Bioluminescence Resonance Energy Transfer (BRET) or Fluorescence Resonance Energy Transfer (FRET) assays (e.g., the Tango assay) which measure the physical proximity of arrestin to a specific receptor; co-immunoprecipitation assays to isolate receptor-arrestin complexes; and confocal microscopy to visualize the translocation of Green Fluorescent Protein (GFP)-tagged arrestin from the cytoplasm to the cell membrane. Examples include visual arrestin, β-arrestin 1, and β-arrestin 2.

[0094] The term " ERK" or " Extracellular Signal-Regulated Kinase" refers to a specific subfamily of protein-serine / threonine kinases. Structurally, ERK proteins are characterized by a kinase domain containing a T-E-Y activation motif that requires dual phosphorylation for enzymatic activity. Methods to detect or identify ERK include the use of phosphospecific antibodies in Western blotting or ELISA to distinguish between the activated (phosphorylated) form and the total protein pool; kinase activity assays using specific substrates; and immunofluorescence to track the nuclear translocation of the kinase upon activation. Examples include the isoforms ERK1 (MAPK3) and ERK2 (MAPK1).

[0095] The term " BCL-2" or " B-cell lymphoma 2" refers to a regulatory protein that inhibits cell death (apoptosis). Structurally, BCL-2 is an integral membrane protein typically localized to the mitochondrial membrane, characterized by Bcl-2 homology domains. Methods to detect or identify BCL-2 include Western blotting to quantify protein expression levels; quantitative PCR to measure gene expression; and flow cytometry using intracellular staining to assess the abundance of BCL-2 relative to pro-apoptotic proteins like BAX. Examples include the human BCL-2 protein and its functional analogs that promote cellular longevity.

[0096] The term " HTR1E related pathway" in the context of the present disclosure refers to the specific signal transduction axis activated by the interaction of the small molecule mimetics with the 5-HTR1E receptor. Structurally, this pathway is defined by the specific sequence of β-arrestin recruitment, ERK phosphorylation, and BCL-2 upregulation.Methods to detect or identify this related pathway involve a combinational approach, suchTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT as demonstrating that the neuroprotective effect of a compound is abolished by the specific knockdown of β-arrestin or the 5-HTR1E receptor, or by the co-administration of MEK / ERK inhibitors, thereby confirming the causal link between receptor binding and BCL-2 expression. This related pathway is distinct from canonical G-protein signaling and represents the biased signaling mechanism utilized by the small molecular of the disclosure.

[0097] In one aspect, the present disclosure provides small molecule mimetics designed to modulate the 5-HTR1E receptor, in which a specific spatial arrangement capable of bridging the SI binding pocket (comprising residue Asp86) and the S2 binding pocket (comprising residue Argl64) of the 5-HTR1E receptor. Structural modeling indicates that these residues are separated by a span of approximately 13 Å. Accordingly, small molecules of the present disclosure are configured as molecular spacers that present terminal moieties at this distance to facilitate simultaneous interaction with both pockets.As used herein in the context of this spatial span, 'approximately 13 Å' encompasses a range of 13 Å ± 3 Å (i.e., from 10 Å to 16 Å), sufficient to bridge the gap between the Asp86 and Arg164 residues of the receptor.

[0098] Accordingly, small molecules of the present disclosure are configured as molecular spacers that present terminal moieties at this distance and orientation to facilitate simultaneous interaction of the functional moieties and related functional groups with both pockets.

[0099] The terms “present” and “presented” as used herein with reference to a compound or functional group indicates attachment performed to maintain the chemical reactivity of the compound or functional group as attached. The term “attach” or “attached” as used herein, refers to connecting or uniting by a bond, link, force or tie in order to keep two or more components together, which encompasses either direct or indirect attachment where, for example, a first molecule is directly bound to a second molecule or material, or one or more intermediate molecules are disposed between the first molecule and the second molecule or material.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0100] In particular, in some embodiments HTR1E binders herein described are defined generally by the structure of Formula I:R2R5Formula Iin whichn is an integer selected from 0 or 1;when n is 0, the ring atoms Cl, C2, C3, C4, and C6 form a 5-membered aromatic ring, wherein any of said carbon atoms is optionally substituted or replaced with a nitrogen, oxygen, or sulfur heteroatom:when n is 1, the ring atoms Cl, C2, C3, C4, C5, and C6 form a 6-membered aromatic ring, wherein any of said carbon atoms is optionally substituted or replaced with a nitrogen, oxygen, or sulfur heteroatom;Al, A2, and A3 represent the linker region and are each independently selected from a Carbon, Nitrogen, Sulfur, or Oxygen atom optionally substituted with hydrogen, alkyl, or oxo (=0) groups;Y represents a 4-, 5- or 6-membered ring system. In distinct contrast to the Right-Hand Core (C1-C6), Ring Y is not required to be aromatic and may comprise saturated, partially saturated, or aromatic rings, and may optionally comprise heteroatoms;X is a functional group comprising a carboxylic acid, ester, amide, sulfonic acid, sulfinic acid, sulfonamide, hydroxamic acid, phosphonic acid or nitro group;Ri, R2, R4, and Rs are each independently selected from hydrogen, alkyl, cycloalkyl,Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms; andR3 comprises a Sulfur, Nitrogen, Oxygen, or Phosphorus-containing functional group.

[0101] In small molecules of Formula I, the moiety X is spatially positioned relative to the linker to meet the configuration established by structural modeling. Specifically, the ring Y is configured such that atom Al and the group X are separated by at least 3 atoms of the ring Y. This geometric constraint ensures the molecule maintains the necessary linear extension, calculated to be approximately 13 Å, to bridge the gap between the Asp86 and Arg 164 residues of the receptor. This requirement for linear extension guides the selection of substitution patterns on the ring Y. For example, in embodiments where Y is a 6-membered ring, such as a phenyl, cyclohexyl, or piperidine ring, this constraint generally favors a 1,4-substitution pattern, also known as para-substitution. This arrangement places the linker atom Al and the headgroup X at maximal distance from one another on the ring, optimizing the reach of the molecule. Conversely, in embodiments where Y is a 5-membered ring, such as furan, pyrrolidine, or cyclopentane, or a 4-membered ring, such as cyclobutane, the constraint generally favors a 1,3-substitution pattern. While the bond angles differ from 6-membered systems, a 1,3-arrangement on these smaller rings projects the substituents away from each other, thereby contributing to the necessary spatial span.

[0102] In some embodiments of the small molecule of Formula I, the ring Y acts as a structural scaffold that holds the polar moiety X in position, and the present disclosure contemplates broad variations of this scaffold to modulate physicochemical properties without disrupting the binding length. In embodiments where Y is an aromatic ring, the molecule adopts a rigid, planar conformation which may reduce the entropic cost of binding. Alternatively, in embodiments where Y is a saturated or partially saturated ring, such as cyclohexane or piperidine, the ring may adopt non-planar conformations, including chair or boat forms. These variations allow the vector of group X to be fine-tuned to adjust the angle of approach to the Arg 164 residue. Furthermore, the use of saturated rings increases the fraction of sp3-hybridized carbons (Fsp3) in the molecule, a property often associated with improved clinical safety and metabolic stability. The ring Y may furtherTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT include heteroatoms to adjust local polarity or introduce additional hydrogen-bond acceptors to interact with the corridor between the SI and S2 pockets.

[0103] The ring defined by Y may vary in size and heteroatom content, and may be saturated or unsaturated. In embodiments where Y is a six-membered ring, this ring may be entirely carbon-based (e.g., cyclohexyl or phenyl), wherein each carbon atom of the ring are additionally bound to hydrogen. Alternatively, the ring may comprise one or more nitrogen atoms, forming a pyridine, quinoline, or isoquinoline (one nitrogen); a pyridazine, pyrimidine, pyrazine, cinnoline, quinazoline, quinozaline, or phthalazine (two nitrogens);or a 1,2,3-triazine. 1,2, 4- triazine, or 1,3.5-triazine (three nitrogens), or saturated variants.The six-membered ring may also contain oxygen or sulfur, forming a pyran or thiopyran or saturated variants such as a cyclic ether or cyclic thioether. Any of these rings may be substituted with alkyl, cycloalkyl, aryl, or heterocyclic groups.

[0104] The ring Y is selected from a 4-, 5-, or 6-membered cyclic scaffold. This explicitly includes 4-membered rings such as cyclobutane, azetidine, and oxetane. While 6-membered rings (e.g., phenyl, piperidine) provide a linear vector, 4-membered rings utilize a diagonal substitution pattern (1,3-substitution) to satisfy the spatial separation required between the linker and the group X.

[0105] In embodiments where Y is a five-membered ring, this ring may be entirely carbon-based (e.g., cyclopentyl). This ring may contain a single nitrogen atom (pyrrole), two nitrogen atoms (pyrazole or imidazole), three nitrogen atoms (1,2,3-triazole or 1,2,4-triazole), or four nitrogen atoms (tetrazole) or saturated variants. The five-membered ring may also comprise mixed heteroatoms, such as nitrogen and oxygen (isoxazole, oxazole, 1,2,3-oxadiazole, 1,3,4-oxadiazole, 1,2,4-oxadiazole, or 1,2,5-oxadiazole) or nitrogen and sulfur (isothiazole, thiazole, 1,2,3-thiadiazole, 1,3,4-thiadiazole, 1,2,4-thiadiazole, or 1,2,5-thiadiazole) or saturated variants. Additionally, the ring may contain a single oxygen atom (furan) or a single sulfur atom (thiophene) or saturated variants such as a cyclic ether or cyclic thioether. Any of these rings may be substituted with alkyl, cycloalkyl, aryl, or heterocyclic groups.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0106] In embodiments where Y is a four-membered ring, this ring may be entirely carbon-based (e.g., cyclobutyl). This ring may contain a single nitrogen atom (azetidine) or two nitrogen atoms (diazetidine), or unsaturated variants. This ring may contain a single oxygen atom (oxetane) or a single sulfur atom (thietane), or unsaturated variants. Any of these rings may be substituted with alkyl, cycloalkyl, aryl, or heterocyclic groups.

[0107] In small molecule of Formula I head group X functions as a polar anchor, likely interacting with arginine residues within the receptor. In some embodiments of the small molecule of Formula I, the moiety X serves as the anchor in the S2 pocket and is designed to engage the basic residue Arg 164. While a carboxylic acid represents a canonical interaction via an ionic salt bridge, the present disclosure encompasses logical bioisosteric replacements such as sulfonic acids, sulfinic acids, sulfonamides, hydroxamic acids, phosphonic acids, nitro groups and tetrazoles, that maintain this binding mode while improving permeability or stability. For instance, X can be selected from tetrazoles, sulfonamides, acyl sulfonamides or nitro groups. These groups mimic the polarity and planar geometry of a carboxylate but often exhibit better membrane permeability and longer biological half-lives. Additionally, variations of X, such as hydroxamic acids or phosphonic acids, allow for the modulation of the pKa of the functional group. Adjusting the pKa is expected to result in the moiety being ionized at the specific pH of the binding pocket, thereby maximizing the electrostatic attraction to Arg 164.

[0108] Furthermore, in embodiments of Formula I, X can be modified to form a prodrug, such as an ester, amide, or anhydride, or may exist as a pharmaceutically acceptable salt formed with a cation selected from alkali metals, alkaline earth metals, transition metals, or organic ammonium or phosphonium cations.

[0109] In embodiments of Formula I, the aromatic core defined by atoms Cl through C6is not limited to phenyl rings. The core may explicitly comprise a 5- or 6-membered heteroaryl ring containing 1 to 3 heteroatoms selected from Nitrogen, Oxygen, and Sulfur (e.g., pyridine, pyrimidine, thiophene, thiazole). In such embodiments, the numbering Cl-C6 refers to the ring positions, wherein one or more carbon atoms are replaced by said heteroatoms.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0110] The linker substituent R3 is an important determinant of receptor affinity, mapped to the SI binding region. In many embodiments, R3 comprises an S, N, O, or P-containing functional group capable of electrostatic or hydrogen-bonding interactions. Preferred embodiments feature a sulfonate ester, such as a p-toluenesulfonyloxy group, but the scope includes sulfonamides, sulfoxides, sulfones, thioethers, phosphate esters, and phosphite esters. Additionally, R3 may comprise a heteroaromatic group containing sulfur, nitrogen, oxygen, or phosphorus configured to mimic the electrostatics of the sulfonate moiety.

[0111] The substituent R3 is an important determinant of receptor affinity, mapped to theSI binding region. In many embodiments. R3 comprises an S. N. O, or P-containing functional group capable of electrostatic or hydrogen-bonding interactions. Preferred embodiments feature a sulfonate ester, such as a p-toluenesulfonyloxy group, but the scope includes sulfonamides, sulfoxides, sulfones, thioethers, phosphate esters, and phosphite esters. Additionally, R3 may comprise a heteroaromatic group containing sulfur, nitrogen, oxygen, or phosphorus configured to mimic the electrostatics of the sulfonate moiety.

[0112] Furthermore, while the R3 substituent in the lead compounds is typically exemplified as a sulfonate ester (e.g., p-toluenesulfonyloxy) designed to interact with aspartate residues in the receptor's SI site, the scaffold of Formula I is not limited to this single functionality. The disclosure contemplates bioisosteres at the R3 position that preserve the essential electrostatic interaction, including sulfonamides, sulfones, sulfoxides, and thioethers, as well as phosphate or nitrate esters.

[0113] The substituent R2 represents a primary site for structural diversification, mapping to the SI binding region. While R2 may comprise broadly diverse functional groups including alkyl, alkoxy, ester, or amide groups, particular focus is placed on amine-containing functionalities. In specific embodiments, R2 comprises an aminoalkyl group, which may be linear, branched, or cyclic, such as a piperidine, pyrrolidine, or dimethylamine motif. These amino groups may be protonated to form positively charged species, forming salts with pharmaceutically acceptable anions such as chloride, bromide, sulfonate, acetate, citrate, or fumarate. The remaining substituents Rl, R4, and R5, are independently variable and may be selected from hydrogen, alkyl, aryl, heteroaryl, orTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT halides. In certain structural variations, adjacent substituents (e.g., R1 and R2) may be joined to form fused five- or six-membered rings, thereby restricting the conformational flexibility of the molecule to favor the bioactive pose.

[0114] These structures were rationally designed and selected based on synthetic feasibility and in silico binding free energy. For example, specific embodiments described herein, such as those designated R9 and RIO, incorporate hydrazino or ethylamino motifs that were accessible via specific synthetic routes, such as the Betti reaction or Mannich-type condensations. Although initial optimization prioritized these specific amine structures for their rapid synthesis and stability, the disclosure broadly encompasses analogous structures identified in the virtual screen, including acyclic and cyclic amine variants (e.g., the DI and D2 analogues) which target the same hydrophobic and electrostatic features of the receptor's S 1 site.

[0115] An important feature of the amine-containing R2 substituents of Formula I is their ability to exist in protonated forms under physiological conditions. Consequently, the present disclosure explicitly includes pharmaceutically acceptable salts of the compounds of Formula I. The protonated amine functionality may form stable salts with a wide variety of counterions. Beyond simple halides like chloride or bromide, the disclosure contemplates the use of organic anions often used in pharmaceutical development to modulate solubility or bioavailability, including but not limited to citrate, succinate, maleate, fumarate, pamoate, tartrate, acetate, and trifluoroacetate. This capacity for salt formation allows the physicochemical profile of the neuroprotective agent to be tuned for specific formulation requirements, such as intranasal delivery.

[0116] In some embodiments, small molecules of Formula I, can encompasses variations at the R2 position that modulate the electronic environment of the Right-Hand Core without abolishing receptor binding. In certain embodiments, R2 is selected from an alkoxy group, including but not limited to a methoxy, ethoxy, or isopropoxy group. Experimental observations indicate that while the introduction of an alkoxy group at this position (e.g., a methoxy group) may result in reduced potency relative to amine-substituted analogues, such compounds retain measurable pharmacological activity at the HTR1E receptor.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Accordingly, embodiments wherein R2 is an oxygen-linked alkyl group are explicitly included within the scope of Formula I to capture the full spectrum of biologically active mimetics.

[0117] In some embodiments of the small molecules of Formula I, the substituents Rl, R4, and R5 are each independently selected to comprise an alcohol, amine, imine, aldehyde, ketone, carboxylic acid, ester, ether, amide, alkene, alkyne, thioether, epoxy, hydrazine, aziridine, alkyl, fluoroalkyl, chloroalkyl, haloalkyl, aryl, fluoroaryl, chloroaryl or haloaryl functional group, or a combination thereof. Similarly, the substituent R2 may comprise an alcohol, amine, imine, aldehyde, ketone, carboxylic acid, ester, ether, amide, alkene, alkyne, thioether, epoxy, hydrazine, aziridine, alkyl, fluoroalkyl, chloroalkyl, haloalkyl, aryl, fluoroaryl, chloroaryl or haloaryl functional group, or a combination thereof. Unlike Formula III. Formula I broadly encompasses embodiments wherein R2 is an alkoxy group, such as a methoxy group.

[0118] With respect to the substituent R3, which maps to the SI binding pocket, embodiments of Formula I include compounds wherein R3 comprises a sulfonate ester or an alkyl or aryl-substituted sulfonate ester. In alternative embodiments. R3 comprises a sulfonamide, a sulfoxide, a sulfone, or a thioether. The disclosure further contemplates embodiments wherein R3 comprises a carboxylic ester, carboxylic amide, or carboxylic anhydride; a nitrate ester or a nitrite ester; or a phosphate ester or a phosphite ester.Additionally, R3 may comprise a heteroaromatic group containing one of S, N, O, or P.

[0119] Structural rigidity may be introduced into the scaffold through ring fusion. In some embodiments, Rl and R2, or R4 and R5 are joined to form a five or six-membered ring by additional covalent bonds. Furthermore, the spacer atoms Al, A2 and A3 may be joined by additional covalent bonds to form a five or six-membered ring, thereby constraining the linker conformation.

[0120] The aromatic core defined by atoms Cl through C6 may vary in size and heteroatom content. In embodiments where n=l, the core is a six-membered ring. This ring may be entirely carbon-based (e.g., phenyl). Alternatively, the ring may comprise one orTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT more nitrogen atoms, forming a pyridine, quinoline, or isoquinoline (one nitrogen); a pyridazine. pyrimidine, pyrazine, cinnoline. quinazoline. quinozaline, or phthalazine (two nitrogens); or a 1,2,3-triazine, 1,2,4-triazine, or 1,3,5-triazine (three nitrogens). The sixmembered ring may also contain oxygen or sulfur, forming a pyran or thiopyran. Any of these rings may be substituted with alkyl, cycloalkyl, aryl, or heterocyclic groups.

[0121] In embodiments where n=0, the aromatic core is a five-membered ring. This ring may contain a single nitrogen atom (pyrrole), two nitrogen atoms (pyrazole or imidazole), three nitrogen atoms (1,2, 3 -triazole or 1,2,4-triazole), or four nitrogen atoms (tetrazole).The five-membered ring may also comprise mixed heteroatoms, such as nitrogen and oxygen (isoxazole, oxazole, 1,2,3-oxadiazole, 1,3,4-oxadiazole, 1,2,4-oxadiazole, or 1,2,5-oxadiazole) or nitrogen and sulfur (isothiazole, thiazole, 1,2,3-thiadiazole, 1,3,4-thiadiazole, 1,2,4-thiadiazole, or 1,2,5-thiadiazole). Additionally, the ring may contain a single oxygen atom (furan) or a single sulfur atom (thiophene).

[0122] The polar head group X is broadly defined to include carboxylic acids, sulfonic acids, sulfinic acids, sulfonamides, hydroxamic acids, phosphonic acids and nitro groups.X may also exist as an ester or amide derivative of these groups. In many embodiments, X is deprotonated to form a salt, wherein the counterion is an alkali metal cation (Li+, Na+, K+, Rb+, Cs+), an alkaline earth metal cation (Mg2+, Ca2+, Sr2+, Ba2+), a transition metal cation (Zn2+, Cu2+. Ag+), or an organic cation such as ammonium (NH4+) or alkylammonium (NR4+). Similarly, any basic functional group within Rl, R2, R4, R5, orR6 may be protonated to form a salt with an anion selected from halides (chloride, bromide, iodide), sulfonates, carbonates, bicarbonates, nitrates, sulfates, phosphates, or carboxylates (acetate, citrate, fumarate, maleate, tartrate, succinate, pamoate, trifluoroacetate). Accordingly, in some embodiments the genus of Formula I encompasses any scaffold wherein the combination of the Linker, the geometry of Ring Y, and the electronic character of X cooperatively satisfy the spatial requirements established by the structural modeling as will be understood by a skilled person upon reading of the present disclosure.

[0123] In embodiments of Formula I the geometric requirements for receptor recognitionTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT (encompassing various linker types including amides and hydrazones), and the specific moieties can be selected in view of the metabolic stability of the resulting chemical structure, and / or ability to bind the specific SI and S2 pockets of the HTR1E receptor.

[0124] In accordance with the present disclosure, while preferred embodiments of Formula I utilize a hydrazone linkage (e.g., wherein Al and A2 are nitrogen and A3 is carbon), the present disclosure broadly contemplates any three-atom linker configuration capable of maintaining the requisite spatial separation between the aromatic domains.Suitable alternative linkers include amides, as subset of, hydrazines, hydrazides, urea or cyclic linkers wherein the atoms A1-A3 form part of a fused ring system. The disclosure includes variations where Al, A2, and A3 are each Carbon; where Al and A2 are Nitrogen and A3 is Carbon; where Al is Oxygen and A2 / A3 are Carbon; or where Al is Sulfur and A2 / A3 are Carbon. In certain cases any of Al, A2 and A3 may optionally substituted with hydrogen, alkyl, or oxo (=0) groups, for example embodying carbonyl (C-O). sulfonyl (S=O), and nitro (N=O) groups, or alkylated forms of the atoms Al, A2 and A3.

[0125] Formula I constitutes a generic structural platform encompassing the specific variations defined herein as Formulas II, III, IV, V, and VI as will be understood by a skilled person upon reading of the present disclosure Accordingly, any structural feature, substituent definition, or functional limitation described in the context of Formulas II- VI is expressly contemplated as a specific embodiment within the broader scope of FormulaI. The specific sub-genera described below (Formulas II-VI) represent preferred selections of the variables n, Y, X, A1-A3, and R1-R5 defined in Formula I.

[0126] In some embodiments, the CPE mimetics small molecules of Formula I comprise a phenyl scaffold in which Ring Y is a 6-membered aromatic ring (e.g., phenyl)

[0127] In particular in a specific embodiment of, the small molecule of Formula I comprises a phenyl scaffold wherein ring Y is a 6-membered aromatic ring and the linker A1-A3 forms a hydrazone linkage. Accordingly, provided herein is a sub-set of small molecules of Formula I the structure of Formula IITitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCTR5Formula IIwhereinRi, R2, R4, and R5 are each independently selected from H, alkyl, cycloalkyl, aryl or heterocyclic groups optionally substituted with heteroatoms N, O. S, F, Cl, Br and / or I;R3 comprises an S, N, O or P-containing functional group; andn is 0 or 1whereinwhen n = 0, Cl, C2, C3, C4 and C6 comprise a 5-membered aromatic ring where any of Cl, C2, C3, C4 and C6 is optionally substituted with a N, O or S heteroatom or heteroatoms; andwhen n = 1, Cl, C2, C3, C4, C5 and C6 comprise a 6-membered aromatic ring where any of Cl, C2, C3, C4, C5 and C6 is optionally substituted with a N, O or S heteroatom or heteroatoms; andwhereinX comprises an ester, amide or acid group containing at least one E=O bond where E isC, S or P, or wherein X is a nitro group -NO2; and

[0128] Al is NH, A2 is N and A3 is C. The small molecules of the Formula II generally comprise a tripartite structure consisting of a central hydrazone spacer connecting a substituted aromatic ring scaffold to a ring structure functionalized with a polar head group moiety.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0129] In embodiments of the present disclosure wherein the small molecule of FormulaI have the structure of Formula II the linker region connecting the aromatic rings is a hydrazone where Al and A2 are Nitrogen and A3 is Carbon.

[0130] In preferred embodiments of Formula II the substituents X, Rl, R2, R3, R4 andR5 have the variation specified for Formula I.

[0131] In a specific set of preferred embodiments, the small molecule of Formula II combines the following features: X is a carboxylic acid (COOH); n=l; the aromatic core(C1-C6) is a phenyl ring; and the linker is a hydrazone (A1=NH, A2=N, A3=C). In this specific scaffold, Rl, R4, and R5 are preferably hydrogen. Further, in this specific scaffold, R3 preferably comprises a toluenesulfonate ester (-OS(O2)-C& H4CH3).

[0132] Within the specific preferred scaffold defined immediately above (X=COOH, Phenyl-Hydrazone, R3=Tosylate), the substituent R2 may be selected from a variety of specific amine-containing groups, including: cyclo-[CHCH2CH2NH-];CH2CHN=C(CH3)NH2; CH2NHCH2SH; CH2C(H)(NH)2OH; CH2-cyclo-[NHCH2CH2-]; CH2C(CCH)NHCH3; CH2NHCH2-cyclo-[CH2CH20-]; CH2NHCH2F;CH2N(CH3)NH2; CH2NHCH2CH3; CH2N(CH3)2; and CH2-cyclo-[NCH2CH2CH2CH2CH2-], When R2 comprises one of these amino groups, it may be protonated to form a salt with a pharmaceutically acceptable anion such as chloride, bromide, iodide, benzenesulfonate, esylate, camsylate, mesylate, acetate, citrate, fumarate, maleate, tartrate, succinate, pamoate, or trifluoroacetate.

[0133] Alternatively, within the same specific preferred scaffold (X=COOH, Phenyl-Hydrazone, R3=Tosylate), the substituent R2 may be a methoxy group (OCH3). This specific embodiment corresponds to the hit compound Z124 identified in the initial screen.

[0134] While preferred embodiments utilize an amine at the R2 position, the scope of Formula II explicitly includes compounds wherein R2 is an alkoxy group (e.g., methoxy, ethoxy). As demonstrated by Compound Z124, alkoxy-substituted analogues retain significant binding affinity and neuroprotective efficacy, serving as functional mimetics of the CPE pharmacophore. While experimental data support the conclusion that basic amineTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT side chains can exhibit enhanced potency, analogues wherein R2 is an alkoxy group — such as a methoxy, ethoxy, or isopropoxy group — retain measurable binding affinity for the HTR1E receptor. Accordingly, in specific embodiments wherein X is a carboxylic acid andR3 is a toluenesulfonate ester, R2 is selected from a C1-C4 alkoxy group, particularly a methoxy group.

[0135] In some embodiments, hydrazone small molecules of the disclosure are hydrazones of Formula IIIFormula IIIwhereinRi, R4, R5. and Re are each independently selected from H, alkyl, cycloalkyl, aryl or heterocyclic groups optionally substituted with heteroatoms N, O, S, F, Cl, Br and / orI;R2 is an alkyl, cycloalkyl, aryl or heterocyclic group optionally substituted with heteroatoms N, O, S, F, Cl, Br and / or I such that R2 is not H or an alkoxy group;R3 comprises an S, N, O or P-containing functional group; andn is 0 or 1whereinwhen n = 0, Cl, C2, C3, C4 and C6 comprise a 5-membered aromatic ring where any of Cl, C2, C3, C4 and C6 is optionally substituted with a N, O or S heteroatom or heteroatoms; andTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT when n = 1, Cl, C2, C3, C4, C5 and C6 comprise a 6-membered aromatic ring where any of Cl, C2, C3. C4, C5 and C6 is optionally substituted with a N. O or S heteroatom or heteroatoms; andwhereinX comprises an ester, amide or acid group containing at least one E=O bond where E isC, S or P, or wherein X is a nitro group -NO2.

[0136] In a further subset of Formula I (and Formula II), the substituent R2 is selected to enhance metabolic stability or receptor fit. Accordingly, provided herein is a specific embodiment of Formula I having the structure of Formula III. These compounds are chemically distinct from the hydrazone of Formula II by virtue of specific structural modifications at the R2 position. While the general hydrazone scaffold and the polar head group X share commonalities with the broader genus of Formula II, the compounds of Formula III are defined by the proviso that the substituent R2 is selected from alkyl, cycloalkyl, aryl, or heterocyclic groups, excluding hydrogen or simple alkoxy moieties.This structural distinction is based on secondary R-group screening and optimization efforts targeting the SI binding pocket of the 5-HTR1E receptor. The inventors have determined that specific functionalization at this position, particularly with amine-containing alkyl groups, provides novel chemical entities that maintain or enhance the cytoprotective activity observed in the parent scaffold while offering improved physicochemical properties or synthetic accessibility.

[0137] In preferred embodiments of Formula III the substituents X, Rl, R3, R4 and R5 have the variation specified for Formula II.

[0138] In preferred embodiments, R2 comprises an alcohol, amine, imine, aldehyde, ketone, carboxylic acid, ester, amide, alkene, alkyne, thioether, epoxy, hydrazine, aziridine, alkyl, fluoroalkyl, chloroalkyl, haloalkyl, aryl, fluoroaryl, chloroaryl or haloaryl functional group or combination of groups.

[0139] In preferred embodiments of Formula III. the R2 substituent comprises a nitrogen-containing functional group, such as a substituted or unsubstituted aminoalkylTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT moiety.

[0140] In preferred embodiments of the small molecules of Formula III, the substituentR6 may vary as for Rl, R2 R4 and R5 for Formula I above.

[0141] The present disclosure also contemplates embodiments of Formula III wherein, the hydrazone N-H bond may be replaced by a covalent bond joining to R6 to form a five or six-membered ring.

[0142] In a specific preferred set of embodiments, the small molecule of Formula III is defined by a combination of features wherein X is a carboxylic acid (COOH); n=l; the aromatic core (C1-C6) is a phenyl ring wherein C1-C6 are carbon atoms; In this preferred scaffold, Rl. R4, R5, and R6 are typically hydrogen, and R3 comprises a toluenesulfonate ester.

[0143] In some of these preferred embodiments of small molecules Formula III, whereinX is a carboxylic acid and R3 is a toluenesulfonate ester, the substituent R2 can be selected from the group consisting of: cyclo- [CHCH2CH2NH-]; CH2CHN=C(CH3)NH2;CH2NHCH2SH; CH2C(H)(NH)2OH; CH2-cyclo-[NHCH2CH2-]; CH2C(CCH)NHCH3;CH2NHCH2-cyclo-[CH2CH2O-]; CH2NHCH2F; CH2N(CH3)NH2; CH2NHCH2CH3;CH2N(CH3)2; and CH2-cyclo-[NCH2CH2CH2CH2CH2-]. In any of these specific embodiments, the amino group on R2 may be protonated such that the substituent carries a positive charge. The counterion for this salt may be selected from chloride, bromide, iodide, benzenesulfonate, esylate, camsylate, mesylate, acetate, citrate, fumarate, maleate, tartrate, succinate, pamoate, trifluoroacetate, nitrate, sulfate, or phosphate.

[0144] In some embodiments, the configuration of CPE mimics of Formula I can comprise the chemical fixation of the linker region, defined as A1-A2-A3 in the general formula, into a specific hydrazone motif selected to prevent the instability observed for amide-based analogues.

[0145] Accordingly, in some embodiments, the generic variables of Formula I can specifically defined so that the linker atoms A1-A2-A3 are selected to form a -C=N-N-Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT hydrazone bridge. This specific conjugation provides the requisite planarity to maintain the linear extension of approximately 13 Angstroms while offering superior stability in physiological fluids compared to amide isosteres. Additionally, in some of those embodiments, a substituent R6 is introduced at the terminal nitrogen of the hydrazone, corresponding to the variable R1 or A3 substitution in Formula I, and is selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups. The Ring Y and Headgroup X retain the definitions provided for Formula I, preserving the interaction with the Arg 164 residue.

[0146] In some embodiments, the configuration of CPE mimics of Formula I comprises the chemical fixation of the linker region into a specific hydrazone motif, selected to provide enhanced metabolic stability while maintaining the required pharmacophoric geometry. Accordingly, provided herein is a hydrazone-specific sub-genus of Formula I having the structure of Formula IV:R2Formula IV

[0147] In Formula IV, the linker region defined by A1-A2-A3 in Formula I is specifically defined as a hydrazone motif. This conjugated system provides conformational rigidity, maintaining the planarity required to span the receptor pockets, while exhibiting superior resistance to enzymatic degradation compared to amides and other bioisosteres.

[0148] In certain embodiments of Formula IV, the substitution pattern is selected to further enhance stability and patentable distinction. Specifically, wherein n is 1, the substituent R2 is selected from an alkyl, cycloalkyl, aryl, or heterocyclic group optionallyTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT substituted with heteroatoms, with the proviso that R2 is not hydrogen or an alkoxy group.Without wishing to be bound by theory, the introduction of steric bulk or lipophilicity at the R2 position may serve to shield the hydrazone linkage from metabolic attack or provide additional hydrophobic contacts within the receptor binding cleft.

[0149] Further, R6 in Formula IV is selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms. The flexibility of Ring Y (being aromatic or non-aromatic) allows for the optimization of physicochemical properties, such as fraction of sp3 carbons (Fsp3). to improve solubility and blood-brain barrier permeability.

[0150] Accordingly, while Formula I broadly permits various substituents at the C2 orR2 position, in the specific embodiment particularly wherein n is 1, R2 is selected from an alkyl, cycloalkyl, aryl, or heterocyclic group, with the specific proviso that R2 is not hydrogen or an alkoxy group. This exclusion is based on structure-activity relationship data indicating that sterically demanding or lipophilic groups at the R2 position, as opposed to the smaller proton or electron-donating alkoxy groups, are preferred for maximizing the interaction of this specific hydrazone scaffold with the hydrophobic sub-pockets of the receptor.

[0151] The present disclosure further recognizes that the hydrazone linkage defined in Formula IV introduces specific stereochemical considerations pivotal to receptor binding.The carbon-nitrogen double bond of the hydrazone moiety may exist in either an E-configuration or a Z-configuration. In preferred embodiments, the compound of FormulaIV is isolated or synthesized predominantly in the configuration that maximizes the linear extension of the molecule, typically the E-configuration, to ensure the optimal bridging distance between the SI and S2 pockets. However, the scope of Formula IV explicitly encompasses both individual stereoisomers and mixtures thereof, as well as tautomeric forms wherein the double bond may shift in solution.

[0152] In some embodiments of small molecule of Formula IV, in which the R6 attached to the hydrazone nitrogen, this position serves as a distinct vector for modulating theTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT pharmacokinetic profile of the mimetic. In certain embodiments, R6 is a small alkyl group, such as a methyl, ethyl, or isopropyl group, selected to cap the nitrogen and reduce polarity, thereby facilitating blood-brain barrier penetration. In alternative embodiments, R6 is a functionalized alkyl chain comprising a solubilizing moiety, such as a morpholino-ethyl, dimethylamino-ethyl, or hydroxy-ethyl group. These polar variations at R6 allow for the adjustment of aqueous solubility without disrupting the core interactions of the hydrazone bridge or the R2 substituent.

[0153] In some embodiments of small molecule of Formula IV in which R2 is excluded from being hydrogen or an alkoxy group, the present disclosure contemplates specific hydrophobic moieties that satisfy this requirement. In particular embodiments, R2 is a Cl-C6 alkyl group, including but not limited to methyl, ethyl, isopropyl, tert-butyl, or cyclopropyl. In other embodiments. R2 is a halogenated alkyl group, such as a trifluoromethyl, difluoromethyl or chloromethyl group, which provides both the requisite steric bulk to shield the linker and increased metabolic stability against oxidative defluorination. Furthermore, R2 may be selected from an aryl or heteroaryl group, such as a phenyl or pyridyl ring, which can engage in pi-stacking interactions with aromatic residues within the receptor binding site.

[0154] in some embodiments of small molecule of Formula IV the structure encompasses variations in the Left-Hand Ring Y that synergize with the stability of the hydrazone linker. While the hydrazone bridge provides a semi-rigid tether, the selection of Ring Y as a saturated heterocycle, such as a piperidine or piperazine, introduces a defined degree of flexibility at the distal end of the molecule. This combination — a rigid linker coupled with a flexible headgroup scaffold — allows the polar moiety X to scan the local environment of the Arg 164 residue for the optimal electrostatic pairing. Conversely, embodiments wherein Ring Y is an aromatic ring, such as a phenyl or thiophene, create a fully conjugated system extending from the Right-Hand Core through the hydrazone linker to the headgroup, resulting in a highly planar molecule optimized for intercalation into narrow receptor clefts.

[0155] In alternative embodiments of Formula I, the linker atoms A1-A3 are selected toTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT form a urea motif to provide a hydrolytically stable bridge for the pharmacophore domains.Thus, the present disclosure provides a urea-based sub-genus of Formula I having the structure of F ormula V:.R2R5Formula Vwherein X comprises an ester, amide, or acid group containing at least one E-0 bond whereE is C, S, or P, or is a nitro group; and wherein Rl, R2, R4, and R5 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms; and wherein R3 comprises a Sulfur, Nitrogen, Oxygen, or Phosphorus-containing functional group (e.g., a sulfonate ester).

[0156] . In those embodiments where the small molecule of Formula I have the structure of Formula V, the urea linkage is an alternative to the hydrazone and amide linkers described herein, the present disclosure defines a class of mimetics utilizing a urea linkage to bridge the pharmacophore domains. Accordingly, in some embodiments, the small molecule of Formula I takes the form of a small molecule having Formula V:

[0157] The distinguishing feature of Formula V is the linker region connecting the Left-Hand Ring Y to the Right-Hand Aromatic Core. In Formula V, this linker is defined as a urea motif comprising a carbonyl group flanked by two nitrogen atoms. Unlike the hydrazone linker utilized in Formula II, which contains a carbon-nitrogen double bond, the urea linker provides a distinct physicochemical profile characterized by enhanced hydrolytic stability and a unique hydrogen-bond donor and acceptor array. While preferredTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT embodiments utilize a primary urea motif wherein the nitrogen atoms are unsubstituted, in alternative embodiments, one or both of the linker nitrogens may be substituted with a Cl-C4 alkyl group or a C3-C6 cycloalkyl group. This N-alkylation allows for the modulation of the molecule's lipophilicity and blood-brain barrier permeability by removing a hydrogen bond donor.

[0158] Regarding the specific substituents for the urea scaffold of Formula V, while theR-groups generally follow the broad definitions of the Master Genus of Formula I, specific combinations are preferred to maximize stability and potency. In preferred embodiments, R3 is a sulfonate ester, such as a p-toluenesulfonyloxy group, or a metabolically stable bioisostere such as a sulfonamide or sulfone. Consistent with the structure-activity relationship of the lead compounds, R2 is preferably a nitrogen-containing motif, such as an aminomethyl, dimethylamine, or piperidine group, positioned to interact with the S 1 sub-pocket of the receptor. Furthermore, in Formula V, the Right-Hand Core is preferably a phenyl ring wherein n is 1.

[0159] In other specific embodiments of Formula V, X is a tert-butyl or methyl ester, R3 is a sulfonate ester, such as a p-toluenesulfonyloxy group, or a metabolically stable bioisostere such as a sulfonamide or sulfone. Consistent with the structure-activity relationship of the lead compounds, R2 is preferably a nitrogen-containing motif, such as an aminomethyl, dimethylamine, or piperidine group, positioned to interact with the S 1 sub-pocket of the receptor. Furthermore, the Right-Hand Core is preferably a phenyl ring wherein n is 1.

[0160] A specific functionality of the urea scaffold of Formula V is its resistance to hydrolysis. Unlike hydrazones, which may equilibrate or hydrolyze in highly acidic environments, the urea linkage is chemically inert under physiological pH ranges. This characteristic makes Formula V a preferred scaffold for embodiments where high metabolic stability is required, such as oral formulations where the drug must survive the gastric environment before systemic absorption.

[0161] in some embodiments, in alternative to the stable hydrazone species of FormulasTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT I, II III and IV, the present disclosure defines a distinct class of mimetics representing the direct structural realization of the primary pharmacophore defined by Formula I, utilizing a carbonyl-based linkage, such as an amide or ester to bridge the S 1 and S2 binding pockets.

[0162] Accordingly, in some embodiments, in the compound of Formula I the linker region defined by variables Al, A2, and A3 is selected to form a carbonyl-containing bridge. This structural arrangement provides a high degree of complementarity to the receptor active site. Specifically, the carbonyl oxygen serves as a hydrogen-bond acceptor, while the amide nitrogen, if present, serves as a hydrogen-bond donor, structurally mimicking the peptide backbone of endogenous ligands or peptide substrates.

[0163] Therefore, in those embodiments, the generic variables of Formula I are defined such that the linker region comprises a motif selected from an amide, a reverse-amide, or an ester linkage, thereby fixing the spatial relationship between the Right-Hand Core and the Left-Hand Ring Y. The Ring Y and Headgroup X retain the definitions provided for Formula I, ensuring the requisite linear extension of approximately 13 Angstroms to the Argl64 residue is maintained. Furthermore, the variables n, Rl, R2, and R3 are selected from the broad definitions of Formula I to maximize receptor affinity.

[0164] In further alternative embodiments of Formula I, the linker atoms Al -A3 are selected to form an amide motif. Thus, provided is an amide-based sub-genus of FormulaI having the structure of Formula VIOFormula VIwherein X and Z each separately comprises an ester, amide, or acid group containing at least one E=O bond where E is C, S, or P, or may be a nitro group; and wherein any of the aromatic rings can be optionally substituted with alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with heteroatoms N, O, S, F, Cl, Br and / or I.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0165] The substituents X and Z function as polar interaction heads designed to engage specific residues within the receptor binding pocket. In various embodiments, X and Z are independently selected from a carboxylic acid, a sulfonic acid, a sulfinic acid, a sulfonamide, a hydroxamic acid, phosphonic acid or a nitro group. The disclosure also encompasses prodrug forms or derivatives wherein X and / or Z comprises an ester or an amide of a carboxylic acid, sulfonic acid, sulfinic acid, sulfonamide, hydroxamic acid, or phosphonic acid.

[0166] In many pharmaceutical applications, it is desirable to utilize these compounds in their salt forms. Accordingly, in some embodiments, X and / or Z is deprotonated to form a salt. The counterion for such salts may be selected from an alkali metal cation such as lithium, sodium, potassium, rubidium, or cesium; an alkaline earth metal cation such as magnesium, calcium, strontium, or barium; a transition metal cation such as zinc, copper, or silver; or a transition metal oxocation such as the vanadyl ion. Alternatively, the counterion may be an organic cation, such as an ammonium cation, a phosphonium cation, an alkylammonium cation, or an alkylphosphonium cation. The counterion may carry a charge of +1, +2, +3, or +4.

[0167] In a specific preferred embodiment corresponding to a high- scoring hit identified in the virtual screen, the compound of Formula VI is defined wherein X is a carboxylic acid (-COOH) and Z is a primary amide (-CONH2). This specific configuration is hypothesized to span the SI and S3 binding regions of the 5-HTR1E receptor, providing a distinct binding mode compared to the hydrazone-based mimetics of Formulas II and III.Such a spacer will still successfully present the terminal functionalized rings at the precise distance and orientation to bridge the SI (Asp86) and S2 (Argl64) binding pockets of the5-HTR1E receptor (approx. 13A span).

[0168] In another specific preferred embodiment the compound of Formula VI is defined wherein X is a methyl or tert-butyl ester and Z is a primary amide (-CONH2).

[0169] In embodiments of the disclosure the small molecule of Formula VI encompassed both directional isomers of the amide linkage. In some embodiments, the linker region Al-Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT A2-A3 of Formula VI can be configured as a forward amide, wherein the carbonyl group is proximal to the Right-Hand Core, or as a reverse amide, wherein the carbonyl group is proximal to the Left-Hand Ring Y. The inversion of the amide bond is expected to alter the vector of the hydrogen-bond donor and acceptor sites within the linker tunnel.Consequently, in those embodiments the reverse amide configuration is expected to allow the mimetic to engage specific hydrogen-bonding residues in the receptor channel that can be inaccessible to the forward amide, potentially altering the selectivity profile of the compound for the HTR 1 E receptor over distinct serotonin receptor subtypes.

[0170] In some embodiments, the amide nitrogen within the linker of Formula VI provides a specific site for chemical modification to modulate physical properties. While the primary embodiment comprises a secondary amide (-NH-), in some embodiments Formula VI can further includes tertiary amides wherein the linker nitrogen is substituted with a lower alkyl group, such as a methyl or ethyl group. This N-alkylation serves two functions. First, it eliminates a hydrogen bond donor, which can reduce the energy penalty for desolvation and improve the lipophilicity of the molecule, thereby enhancing bloodbrain barrier permeability. Second, N-alkylation introduces steric constraints that can bias the conformational equilibrium of the amide bond towards a cis- or trans-geometry, allowing for the pre-organization of the molecule into the bioactive conformation.

[0171] While biological assays suggest that the linker region of Formula VI can be more susceptible to enzymatic hydrolysis than the hydrazones of Formula IV, the compounds of Formula Viexhibit potent intrinsic binding affinity due to the favorable electrostatic interactions of the carbonyl group as will be understand by a skilled person upon reading of the present disclosure. Consequently, small molecules of Formula VI can be used for example in embodiment for use in in vitro pharmacological tools, competitive binding assays, or as prodrug scaffolds wherein the active pharmacophore is released or modified in situ as will be understood by a skilled person upon reading of the present disclosure.

[0172] In some embodiments, while the susceptibility of the amide linker in Formula VIto enzymatic hydrolysis distinguishes it from the stable hydrazones of Formula IV, this characteristic is leveraged in specific application as a "soft drug" design strategy. In suchTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT embodiments, the compound of Formula VI can be formulated for local administration, such as intranasal or intrathecal delivery, to effect immediate therapeutic modulation of CNS receptors. Upon diffusion into the systemic circulation, the metabolically labile linker facilitates rapid degradation into inactive metabolites, thereby minimizing systemic exposure and reducing the risk of peripheral side effects. Accordingly, in some embodiments, Formula VI can be a preferred embodiment for therapeutic applications requiring a high therapeutic index with short duration of action or strictly localized effects.

[0173] The small molecules of the disclosure have been identified using a specialized method for identifying small molecule mimetics of Carboxypeptidase E (CPE), comprising a rational virtual screening protocol.

[0174] As used herein, the term 'virtual screening protocol' refers to a computational method used in drag discovery to search libraries of small molecules to identify those structures which are most likely to bind to a drag target, typically a protein receptor or enzyme. The protocol generally involves the in silica assessment of large chemical libraries using computer algorithms to select a subset of 'hit' compounds for subsequent experimental validation. Virtual screening protocols generally comprise two main approaches: Ligand-Based Virtual Screening (LBVS), which relies on the properties of known active ligands (or peptide fragments) to identify new compounds with similar pharmacophoric features (e.g., shape, electrostatics); and Structure-Based Virtual Screening (SBVS), which involves docking candidate molecules into a 3D structural model of the target protein's binding site to predict binding modes and affinity. In the context of the present disclosure, a 'hybrid' virtual screening protocol refers to a method that sequentially integrates both ligand-based and structure-based approaches to filter and prioritize compounds.

[0175] In broad embodiments, the method comprises the steps of: (a) defining a pharmacophore model based on the interaction interface between CPE and the 5-HTR1E receptor, wherein the interface is partitioned into distinct binding regions (designated herein as SI, S2, and S3) corresponding to specific peptide fragments of the CPE protein;(b) screening a library of small molecule compounds against said pharmacophore model to identify initial hits; and (c) validating and prioritizing said hits by molecular docking intoTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT a structural model of the 5-HTR1E receptor binding pocket.

[0176] In embodiments of the method identifying small molecule CPE mimetics herein described, step (a) the pharmacophore definition and partitioning is directed to perform the replacement of the complex regulatory functions of a large endogenous protein (approx.50-53 kDa) with a small molecule. Because a small molecule cannot replicate the full, extensive surface area of the native protein-receptor interface, the method involves a rational decomposition of the binding site.

[0177] In particular, in embodiments herein described of the method identifying small molecule CPE mimetics, the pharmacophore definition and partitioning comprises dividing the CPE interaction region into three functional "hot spots" or sites:• Site SI: Corresponding to a specific electrostatic interaction region (e.g., involving Aspartate residues).• Site S2: Corresponding to a hydrophobic or steric fit region, that also comprises specific electrostatic interactions (e.g., involving Arginine residues). This site is targeted by the polar head group (X) of the small molecule mimetics, such as the carboxylic acid moiety of Formula IV, which is configured to engage the basic Arg 164 residue via an ionic salt bridge or hydrogen bond.• Site S3: Corresponding to an auxiliary binding region utilized by non-hydrazone scaffolds.

[0178] The hypothesis for partitioning the binding interface into these distinct sites is based on CPE mutational studies identifying residues critical for binding, specifically mutants Ml (K302A), Mil (K302A+D306A+D75A), and M15 (D259A+E260A+W319A). Specifically, the maximum distance between key residues within each site was constrained to within approximately 11 A to ensure feasibility for small-molecule recognition. The pharmacophore models were generated using shapematching software, for example OpenEye Scientific Software’s ROCS and EON packages.

[0179] By using specific peptide fragments of the native CPE protein as 3D templates, pharmacophore models are generated that define the essential spatial arrangement of hydrogen bond donors, acceptors, and hydrophobic features required to mimic the protein'sTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT " pharmacophore " at these specific sites..

[0180] In embodiments of the method identifying small molecule CPE mimetics herein described, step (b): Hybrid Screening (LBVS) is directed to screen available libraries of compounds to select compounds having chemical features allowing mimicking the CPE peptide fragment. In particular once the pharmacophore models for SI, S2, and / or S3 are defined, the method proceeds to screen large commercially available chemical libraries.This Ligand-Based Virtual Screening (LBVS) step filters millions of compounds to identify those that match the 3D pharmacophore query. This step effectively prioritizes compounds that possess the correct chemical features to mimic the CPE peptide fragments, regardless of their underlying chemical scaffold.

[0181] Regarding the specific libraries utilized in step (b), a chemical library comprising approximately 6.2 million compounds was assembled from commercial suppliers, including ChemBridge (~1.4 million compounds), ChemDiv (-1.8 million compounds), and Enamine (-3.0 million compounds). This Ligand-Based Virtual Screening (LBVS) step filters millions of compounds to identify those that match the 3D pharmacophore query.This step effectively prioritizes compounds that possess the correct chemical features to mimic the CPE peptide fragments, regardless of their underlying chemical scaffold. In some embodiments virtual screening can be performed separately for each of the SI, S2, and S3 binding sites.

[0182] In embodiments of the method identifying small molecule CPE mimetics herein described, Step (c): Structural Validation (SB VS) is directed to validate the hits identified in the ligand-based phase using Structure-Based Virtual Screening (SBVS). In this step, the selected small molecules are docked into a structural model of the 5-HTR1E receptor.This step verifies that the pharmacophore-matched compounds can physically fit within the receptor's orthosteric or allosteric binding pockets without steric clash and calculates a predicted binding energy. This hierarchical workflow — moving from protein-template pharmacophores to receptor-docking validation — ensures that the final candidates are not just chemical look-alikes of the peptide, but actual binders of the receptor.

[0183] In embodiments of step (c) of the method herein described, the selected smallTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT molecules are docked into a structural model of the 5-HTR1E receptor. In some embodiments, a Cryo-EM structure of the human 5-HTR1E receptor (e.g., PDB ID: 7E33) was utilized as the structural template wherein missing regions (e.g., residues 159-171 and213-281) were generated using homology modeling to complete the receptor structure.Initial ligand docking poses were generated using docking software such as Schrodinger Glide (e.g., in SP mode), followed by molecular dynamics (MD) simulations performed using software such as Schrodinger Desmond. Ligand binding free energies were subsequently calculated using the Molecular Mechanics-Generalized Born Surface Area(MM-GBSA) method to prioritize candidate compounds. This step verifies that the pharmacophore-matched compounds can physically fit within the receptor's orthosteric or allosteric binding pockets without steric clash and calculates a predicted binding energy.

[0184] In some embodiments of the method identifying small molecule CPE mimetics herein described, this algorithm allows for the identification of small molecules that do not merely bind the receptor but functionalize it to trigger specific biased signaling pathways (e.g., $\beta$-arrestin recruitment). By focusing on the SI, S2, and S3 sub-regions, the method successfully identifies high-affinity binders (such as the hydrazones of Formula II / III and the amides of Formula VI) that mimic the neuroprotective activity of the native protein while offering superior physicochemical properties, such as blood-brain barrier permeability and synthetic accessibility.

[0185] During visual inspection of the virtual screening hits, compounds were evaluated for their ability to form appropriate three-dimensional interactions with key pharmacophoric residues. Compounds were explicitly excluded if they exhibited unstable or implausible conformations, or if they contained scaffolds known to be promiscuous (PAINS), functional groups associated with known toxicity, or chemically reactive motifs.Further filtering excluded structures with poor synthetic feasibility, excessive numbers of chiral centers, high molecular weight, or unfavorable planarity likely to complicate synthesis or optimization. This visual inspection ensured the selection of chemically tractable and biologically relevant candidates beyond automated scoring alone.

[0186] In preferred embodiments, the virtual screening protocol is a hybrid virtual screening protocol. This protocol uniquely integrates Ligand-Based Virtual ScreeningTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT (LBVS) and Structure-Based Virtual Screening (SBVS) to translate the macromolecular interactions of a large protein (CPE) into the chemical space of synthetically accessible small molecules.

[0187] It will be understood by those skilled in the art that while the hybrid protocol described above is preferred for its ability to filter vast chemical space (via LBVS) before expending computational resources on detailed docking (via SBVS), the general virtual screening protocol of the disclosure is not limited to this sequential combination. In alternative "non-hybrid" embodiments, the method may comprise utilizing Structure-Based Virtual Screening (SBVS) alone, wherein the library is screened directly against the5-HTR1E structural model without prior pharmacophore filtering. Conversely, in other embodiments, the method may comprise utilizing Ligand-Based Virtual Screening (LBVS) alone, identifying hits based solely on their pharmacophoric similarity to the CPE peptide fragments (SI, S2, S3) without subsequent docking validation. The hybrid protocol is distinct from these general approaches in that it requires the output of the ligand-based screen to serve as the input for the structure-based validation, thereby creating a hierarchical filter that maximizes hit enrichment.In those preferred embodiments in which the virtual screening protocol is a hybrid protocol, the method sequentially utilizes ligand-based and structure-based techniques to maximize hit rate and biological relevance. In such embodiments, step (b) comprises performing a Ligand-Based Virtual Screening (LBVS) to filter the library for compounds matching the3D shape and electrostatic features of the CPE peptide fragments (e.g., using shapematching software such as ROCS and electrostatic comparison software such as EON).Subsequently, step (c) comprises performing a Structure-Based Virtual Screening (SBVS) wherein the hits identified in step (b) are docked into a Cryo-EM structure of the 5-HTR1E receptor (e.g., PDB ID: 7E33), wherein missing regions (e.g., residues 159-171 and 213— 281) were generated using homology modeling, followed by molecular dynamics (MD) simulations and calculation of binding free energies (e.g., via MM-GBSA) to confirm physical fit and affinity. The hybrid protocol may further comprise a selection step comprising visually inspecting the docked compounds to exclude candidates exhibiting unstable conformations, promiscuous scaffolds, toxicophores, chemically reactive motifs,Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT or poor synthetic feasibility.

[0188] In some embodiments, described herein is a method for synthesizing a small molecule mimetic, encompassing the broad genus of Formula I, and specifically including compounds of Formula II, Formula III, Formula IV, Formula V and Formula VI.

[0189] According to the present disclosure, the the small molecule mimetic as described herein, encompassing compounds of Formula I, and specifically including compounds of, Formula II, Formula III, Formula IV, Formula V and Formula VI can be manufactured with methods and corresponding systems comprising the following steps:(i) performing a Betti reaction (or Mannich-type condensation) between a substituted phenol comprising a linker precursor group selected from an aldehyde or a nitro group, a formaldehyde source, and a primary or secondary amine moiety to form an (aminoalkyl)hydroxy-aromatic intermediate;(ii) protecting the amino group of the resulting intermediate (e.g., with a Boc group);(iii) reacting the protected intermediate with a sulfonyl electrophile to install a sulfonate or sulfonamide group;(iv) converting the linker precursor group into a reactive coupling moiety, wherein:(a) if the precursor group is an aldehyde, optionally oxidizing the intermediate to convert the aldehyde into a carboxylic acid, or (b) if the precursor group is a nitro group, reducing the intermediate (e.g., via hydrogenation with Pd / C) to convert the nitro group into an aromatic amine (aniline);(v) reacting the resulting intermediate with a complementary linker-forming partner to form the bridged pharmacophore, wherein:(a) the aldehyde intermediate is condensed with a hydrazine derivative to yield a hydrazone scaffold (Formula II, III and IV);(b) the carboxylic acid intermediate is coupled with an amine to yield an amide scaffold (Formula VI);(c) the carboxylic acid intermediate is converted to a hydroxamic acid / isocyanateTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT sequence and trapped with an amine to yield a urea scaffold (Formula V); or(d) the aromatic amine (aniline) intermediate is coupled with an isocyanate (e.g., generated from tert-butyl 4-aminobenzoate) to yield a urea scaffold (Formula V);and(vi) optionally deprotecting the amino group and any other protecting groups to yield the final compound.

[0190] Accordingly in a first set of embodiments of the synthesis method of small molecules herein described, the method comprises a sequence of reactions beginning with a Betti reaction or Mannich-type condensation of a substituted aromatic aldehyde and an amine moiety comprising at least one alkyl group to form an (aminoalkyl)hydroxy-aromatic intermediate. This intermediate is subsequently subjected to a protection step wherein the amino group is protected. The protected (aminoalkyl)hydroxy-aromatic intermediate is then reacted with a sulfonyl electrophile to install a sulfonate group, yielding a sulfonated aldehyde intermediate. This sulfonated aldehyde is then reacted with a linker-forming partner to form the bridged pharmacophore. In one route, this aldehyde is condensed with a hydrazine to form a hydrazone scaffold (as in Formula II, III and IV).Alternatively, this aldehyde is first oxidized to a carboxylic acid and then coupled with an amine or acid derivative to form an amide scaffold (as in Formula VI ). In yet another route, for aromatic aldehydes, the aldehyde is oxidized to a carboxylic acid, coupled with hydroxylamine to form a hydroxamic acid, followed by activation with sulfonyl chloride and mild base to give the isocyanate which is then trapped with a suitable amine to give a urea scaffold (as in Formula V). Finally, the protecting group is removed in a deprotection step to yield the target small molecule mimetic.

[0191] In an alternative and preferred embodiment for the synthesis of urea-linked scaffolds (e.g., Formula V), the method proceeds via a nitro-aromatic intermediate to utilize orthogonal protecting group strategies. This route specifically avoids base-mediated saponification steps that may degrade the sulfonate ester pharmacophore.

[0192] In particular in embodiments comprising a urea linkage according to Formula VTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT may be prepared by performing a Betti or Mannich reaction with an aromatic nitrophenol a formaldehyde source, and a primary or secondary amine moiety to form an (aminoalkyl)nitro-aromatic intermediate, followed by protection of the amino group and subsequent reaction of this protected intermediate with a sulfonyl electrophile to install a sulfonate or sulfonamide group as above. Reduction of the protected nitro-intermediate is then carried out to convert the nitro group into an aromatic amine using a reducing a reagent such as Pd-C / H2. Subsequent reaction of the reacting the resulting sulfonated aromatic amine with an isocyanate allows the formation of the urea- bridged pharmacophore, for which deprotecting the amino group will yield the desired final compound.

[0193] In some embodiments, the method thus comprises: (i) performing a Betti reaction(or Mannich-type condensation) between p-nitrophenol, a formaldehyde source, and a primary or secondary amine moiety to form an (aminoalkyl)nitro-phenol intermediate: (ii) protecting the amino group (e.g., with a Boc group) and reacting the phenolic hydroxyl with a sulfonyl electrophile to install the sulfonate group (e.g., O-tosylation); (iii) reducing the nitro group of the protected intermediate to an aniline (aromatic amine) using a reducing agent, such as hydrogen gas with a palladium on carbon (Pd / C) catalyst; (iv) reacting the resulting aniline intermediate with an isocyanate to form the urea linkage; and(v) performing a global deprotection to yield the final compound.

[0194] In specific embodiments of this route, the isocyanate electrophile is generated in situ or ex-situ by treating tert-butyl 4-aminobenzoate with triphosgene(bis (trichloromethyl) carbonate). This selection of the tert-butyl ester is critical; unlike methyl or ethyl esters which require basic hydrolysis, the tert-butyl ester is cleaved under acidic conditions. Accordingly, step (v) comprises a global deprotection using trifluoroacetic acid (TFA), which simultaneously removes the Boc protecting group from the scaffold amine and hydrolyzes the tert-butyl ester to the carboxylic acid, thereby preserving the integrity of the acid-stable / base-labile O-tosyl group

[0195] In some embodiments of the method, the starting aromatic aldehyde is 4-hydroxybenzaldehyde. In other embodiments, particularly where the mimetic comprises a5-membered core (where n is 0). the starting material is a hydroxylated 5 -memberedTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT heterocyclic aldehyde, such as a hydroxy- thiophene carboxaldehyde or hydroxy-furan carboxaldehyde. The aromatic aldehyde may be substituted with an alkyl, cycloalkyl, aryl, or heterocyclic group, which may itself be optionally substituted with heteroatoms such as nitrogen, oxygen, sulfur, or fluorine. Regarding the amine component utilized in the initial Betti reaction, consistent with the mechanistic requirements of the condensation, the amine is selected from a primary amine or a secondary amine. Various embodiments employ amines such as dimethylamine, methylamine, piperidine, morpholine, or substituted anilines. The disclosure further encompasses embodiments where the amine is substituted with an alkyl, cycloalkyl, aryl, or heterocyclic group, optionally substituted with heteroatoms.

[0196] With respect to the protection step, in some embodiments, the protecting group selected is a tert-butyloxycarbonyl (BOC) group. In other embodiments, the protecting group is a benzyloxycarbonyl (CBZ) group. The choice of protecting group dictates the specific conditions of the final deprotection step. For instance, in embodiments where the protecting group is BOC, the deprotection step generally comprises the addition of an acid, such as trifluoroacetic acid. Conversely, in embodiments where the protecting group is CBZ, the deprotection step generally comprises hydrogenation using hydrogen gas and a suitable catalyst, such as palladium on carbon (Pd / C).

[0197] The sulfonylation step involves the reaction of the protected intermediate with a sulfonyl electrophile. In some embodiments, this electrophile is an alkyl sulfonyl halide or an aryl sulfonyl halide. The electrophile may be substituted with an alkyl, cycloalkyl, aryl, or heterocyclic group, optionally containing heteroatoms. In a specific preferred embodiment, the sulfonyl electrophile is toluenesulfonyl chloride, thereby installing a p-toluenesulfonate ester at the R3 position of the scaffold.

[0198] The linker-forming step utilizes a reagent complementary to the aldehyde to form the requisite bridge. In embodiments directed to Formula II, III and IV. the step comprises a condensation with a hydrazine derivative to form a hydrazone linker. In some such embodiments, the hydrazine is substituted with an alkyl, cycloalkyl, aryl, or heterocyclic group, optionally substituted with heteroatoms. In specific embodimentsTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT corresponding to the synthesis of the lead compounds described herein, the hydrazine is 4-hydrazinobenzoic acid. Alternatively, in embodiments directed to Formula VI, the aldehyde may be oxidized to a carboxylic acid and coupled with an amine, or converted to an amine and coupled with a carboxylic acid, to generate the amide linker. In other alternative embodiments directed to Formula V, the aldehyde may be oxidized to a carboxylic acid, converted to a hydroxamic acid via coupling with hydroxylamine (e.g. using an activating agent such as EDC, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and triphenylphosphine), converting the hydroxylamine to a isocyanate by reacting with sulfonyl chloride and mild base (e.g., potassium carbonate) followed by trapping this isocyanate by reaction with an amine. In other alternative embodiments directed to Formula V, the starting material is a nitrophenol such as 4-nitrophenol, and the isocyanate is formed from tert-butyl 4-aminobenzoate by treatment with triphosgene [1].

[0199] In some embodiments, a method for the synthesis of the small molecule mimetics described herein, specifically utilizing a Betti reaction or Mannich-type condensation to assemble the core scaffold. With respect to the reactants employed in this condensation step, it is critical to specify that the amine component is selected from a primary amine or a secondary amine, or alternatively, a primary or secondary aniline. While previous general descriptions may have referenced amines broadly, the present method specifically excludes the use of tertiary amines as the initiating reactant for this step. This distinction is based on the mechanistic requirement of the reaction; tertiary amines lack the necessary proton to allow for the condensation onto the aldehyde component to form the requisite iminium intermediate, which is the electrophilic species intercepted by the phenol. Accordingly, the nitrogen-containing substituent R9 or RIO in the resulting scaffold is derived explicitly from a primary or secondary precursor.

[0200] Regarding the reaction conditions for this condensation step, the method necessitates the application of thermal energy to facilitate the formation of the carboncarbon bond. The reaction is typically conducted in a polar solvent system capable of solubilizing the reactants and intermediates. Suitable solvents for this step include, but are not limited to, ethanol, methanol, dimethylformamide (DMF), dimethyl sulfoxideTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT (DMSO), and tetrahydrofuran (THF). The selection of aprotic polar solvent such as ethanol is particularly advantageous for stabilizing the transition state of the iminium formation.

[0201] Following the assembly of the core amine scaffold, the method involves a sulfonylation step, for example, the introduction of a tosyl group, to generate the functionalized intermediate. This reaction is typically performed in a non-nucleophilic organic solvent, such as dichloromethane (DCM), although other aprotic solvents are tolerated. While the sulfonylation proceeds efficiently at ambient temperature (room temperature), the method is robust and tolerates heating if required to drive the reaction to completion or solubilize less soluble substrates. In specific embodiments, to activate the sulfonyl chloride reagent and accelerate the reaction kinetics, a nucleophilic catalyst such as 4-dimethylaminopyridine (DMAP) is included in the reaction mixture.

[0202] In embodiments involving the formation of the hydrazone linker (e.g., for compounds of Formula II, III or IV), the synthetic method may further comprise a deprotection step, particularly when a Boc-protected hydrazine intermediate is employed.The present disclosure provides two distinct protocols for this transformation. In a first "one-pot" protocol, the hydrazone formation is conducted in the presence of an acid, such as trifluoroacetic acid (TFA), acting as a co- solvent. This allows for the simultaneous deprotection of the Boc group and formation of the hydrazone linkage. In a second, sequential protocol, the acid (TFA) is added only after the hydrazone formation is complete. This sequential addition is preferred in the synthesis of specific analogues, such as those designated R9, where immediate exposure to strong acid in the presence of unreacted starting materials may lead to competitive side reactions or degradation.

[0203] Furthermore, while the primary method is described for the synthesis of monomeric species using mono-amines, the present disclosure theoretically contemplates the extension of this method to diamine precursors. The use of a diamine in the Betti reaction context provides a potential route to dimeric HTR1E binders. However, the practitioner will appreciate that such a modification introduces challenges in stoichiometry control, as the bifunctional nature of the amine significantly increases the propensity for multiple condensation events, potentially leading to the formation of oligomeric orTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT polymeric byproducts rather than the discrete dimeric species.

[0204] In some embodiments, the present disclosure provides compositions comprising at least one small molecule mimetic as described herein, encompassing the broad genus of Formula I, and specifically including compounds of Formula I, Formula II, FormulaIII, Formula IV, Formula V and / or Formula VI. In broad embodiments, these compositions are not limited solely to pharmaceutical applications but encompass any formulation wherein the small molecule is combined with a suitable vehicle, carrier, solvent, or excipient to facilitate its use in a specific context. Depending on the intended application, the composition may be formulated as a pharmaceutical product for therapeutic intervention in vivo, or as a chemical reagent or biological probe for research assays in vitro.

[0205] The term "vehicle" as used herein generally indicates a diluent, solvent, adjuvant, excipient, or carrier with which the small molecule mimetic is combined or administered.The selection of the vehicle is dictated by the intended use of the composition. In embodiments directed to research or laboratory applications (non-pharmaceutical compositions), the vehicle may comprise a laboratory-grade solvent or buffer suitable for maintaining the stability and solubility of the compound in an experimental setting. Unlike pharmaceutical formulations which require physiological compatibility, these research compositions are optimized for chemical stability and long-term storage. Experimental observations indicate that the compounds of the present disclosure exhibit limited solubility in pure aqueous media but are readily soluble in organic solvents. Accordingly, exemplary vehicles for these non-therapeutic embodiments include organic solvents such as dimethyl sulfoxide (DMSO), ethanol, or methanol. In one embodiment, the composition comprises a concentrated solution of the compound of Formula I, II, IV, V or VI dissolved in DMSO. To maintain the integrity of the compound, particularly the hydrazone linkage which may be sensitive to hydrolysis over extended periods, the composition is preferably stored at sub-zero temperatures, for example at or below -20 degrees Celsius. In these contexts, the composition serves as a research tool, useful for modulating 5-HTR1E receptor activity in isolated cells (e.g., investigating signaling kinetics such as beta-arrestinTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT recruitment) or serving as a positive control in competitive binding assays.

[0206] In embodiments wherein the composition is formulated for therapeutic use, the small molecule mimetic is combined with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition. The term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce severe allergic or untoward reactions when administered to a subject.Exemplary pharmaceutically acceptable vehicles comprise sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water, saline solutions, aqueous dextrose, and glycerol solutions are preferably employed as vehicles, particularly for injectable solutions. The composition may further contain minor amounts of wetting or emulsifying agents. pH buffering agents, or stabilizers to enhance the shelf-life and efficacy of the active agent.

[0207] An important advantage of the small molecule mimetics described herein is their ability to be administered via non-invasive routes that facilitate delivery to the central nervous system. Accordingly, in preferred pharmaceutical embodiments, the composition is formulated for intranasal administration. Intranasal delivery offers a direct pathway to the brain by bypassing the blood-brain barrier via the olfactory and trigeminal nerve pathways, thereby reducing systemic side effects and avoiding first-pass metabolism.Formulations for intranasal administration may take the form of drops, sprays, powders, or aerosols.

[0208] In a specific preferred embodiment, the intranasal formulation is designed as an in-situ gel system. An in-situ gel is a formulation that exists as a low-viscosity liquid at room temperature or during storage, allowing for easy administration as a spray or drop, but undergoes a phase transition to a semi-solid or viscous gel upon contact with the nasal mucosa. This sol-to-gel transition may be triggered by physiological factors such as temperature (thermosensitive), pH (pH-sensitive), or the presence of ions (ion- sensitive) in the nasal cavity. The formation of a gel in situ increases the residence time of the formulation in the nasal cavity, prevents rapid mucociliary clearance, and enhances theTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT permeation of the small molecule mimetic across the nasal epithelium into the CNS. Such in-situ gel formulations typically comprise a thermosensitive polymer or mucoadhesive gelling agent. Suitable polymers for these systems include poloxamers, specifically Poloxamer 407 (Pluronic F127) or Poloxamer 188, as well as chitosan, alginates, gellan gum, carbomers, or hydroxypropyl methylcellulose (HPMC). The composition may further comprise permeation enhancers, such as cyclodextrins or bile salts, fusidic acid derivatives, and phosphatidylcholines to further facilitate transport across the biological membrane.

[0209] Regarding the dosage regimens encompassed by the present disclosure, it is acknowledged that the precise therapeutically effective amount for human subjects will depend on multiple factors, including the specific condition being treated, the route of administration, and the age, weight, and metabolic status of the patient. While preclinical studies utilizing murine models have demonstrated therapeutic efficacy at a dosage of approximately 25 mg / kg, the translation of this dose to human subjects requires adjustment based on pharmacokinetic data and species- specific metabolic rates. Accordingly, the present disclosure defines the dosage broadly and without limitation. In general embodiments, the effective amount may range from about 0.01 mg / kg to about 100 mg / kg of body weight per day. However, given the lack of established human pharmacokinetic and toxicity profiles at this stage, the disclosure contemplates that the attending physician will titrate the dosage to achieve the desired receptor occupancy and therapeutic effect while minimizing adverse events.

[0210] Alternatively, the composition can be formulated for systemic delivery, including intravenous, intramuscular, or subcutaneous injection. For these embodiments, the small molecules may be formulated in sterile aqueous solutions which may contain buffers, antioxidants, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient. These systemic formulations are particularly useful for applications where peripheral cytoprotection is desired, or where the small molecule mimetic is used to treat conditions affecting organs other than the central nervous system.

[0211] In view of the expected application of the compounds herein described to metabolic disorders and peripheral conditions, the present disclosure further contemplatesTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT formulations adapted for oral administration. While oral delivery provides convenience for chronic administration, the inventors recognize that the chemical linkers utilized in Formula VI (amides), Formula V (ureas) and particularly Formula II, III and IV (hydrazones) may be susceptible to acid-catalyzed hydrolysis in the gastric environment.Accordingly, in preferred oral embodiments, the compound is formulated as a tablet or capsule comprising an enteric coating. This coating is selected from pH-sensitive polymers, such as cellulose acetate phthalate or methacrylic acid copolymers (e.g., Eudragit), which remain intact in the acidic stomach (pH ~1.5) but dissolve in the neutral environment of the small intestine. This delayed-release strategy ensures that the intact pharmacophore is delivered to the site of absorption, thereby maximizing systemic bioavailability for the treatment of peripheral organ dysfunction or metabolic regulation.

[0212] Furthermore, for embodiments directed to the treatment of peripheral neuropathic pain or localized inflammatory conditions, the disclosure provides topical or transdermal formulations. These preparations may take the form of creams, ointments, gels, or transdermal patches. In such embodiments, the small molecule mimetic is dispersed in a lipophilic vehicle or matrix capable of penetrating the stratum corneum to access peripheral nerve endings (e.g., nociceptors) without necessarily achieving high systemic blood levels.Transdermal patches may further comprise permeation enhancers, such as dimethyl sulfoxide (DMSO) or propylene glycol, and a rate-controlling membrane to provide continuous, controlled delivery of the active agent over a prolonged period, thereby improving patient compliance in chronic pain management.

[0213] Additionally, to enhance delivery specifically to neural cells and across the bloodbrain barrier (BBB) beyond the intranasal route, the present disclosure encompasses colloidal carrier systems. In these embodiments, the compound of Formula I is encapsulated within liposomes, solid lipid nanoparticles (SLNs), or polymeric nanoparticles. These nanocarriers serve a dual function: protecting the labile linker region from enzymatic degradation in the bloodstream and facilitating transport across the BBB via receptor-mediated transcytosis. In highly specific embodiments, the surface of the nanocarrier is modified with targeting ligands, such as transferrin, insulin, orTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT apolipoprotein E (ApoE) peptides, which specifically bind to receptors on the brain capillary endothelial cells, thereby acting as a " Trojan horse" to deliver the cargo directly into the central nervous system parenchyma.

[0214] In some embodiments, a method and a related system are described for activating the 5-Hydroxytryptamine Receptor 1E (5-HTR1E). In a broad embodiment, the method comprises contacting a cell expressing the 5-HTR1E receptor with at least one small molecule mimetic of the disclosure (e.g., Formula I, including I, II, III, IV, V or VI) in an amount sufficient to bind to the receptor and initiate downstream signaling. Unlike nonspecific serotonin agonists, the small molecules described herein are configured to selectively trigger specific cytoprotective pathways, including the recruitment of betaarrestin, the phosphorylation of Extracellular Signal-Regulated Kinase (ERK), and the upregulation of the anti-apoptotic protein BCL-2. This method is versatile and may be performed in vitro, for example in high-throughput cellular assays to evaluate receptor kinetics and ligand efficacy, or in vivo / ex vivo to modulate receptor activity in a biological system for research or physiological study.

[0215]

[0101] In some in vitro embodiments, the activation of the receptor is quantified using a luciferase reporter assay, such as the Presto-Tango system. In this embodiment, the method involves transfecting a suitable cell line, such as HTLA cells (human transactivator cells), with a plasmid encoding the 5-HTR1E receptor gene (e.g., the Tango-5-HTR1E plasmid). Following a suitable incubation period to allow for protein expression, the cells are reseeded and subsequently contacted with the small molecule mimetic. In exemplary protocols described herein, the cells are treated with the compound at a concentration of approximately 50 nM for a duration of about 4 hours. The activation of the receptor is then detected by lysing the cells and adding a luciferase substrate, wherein the resulting luminescence — measured by a plate reader — correlates directly with the degree of [3-arrestin recruitment and receptor activation.

[0216]

[0102] In other in vitro embodiments, the method utilizes Dynamic Mass Redistribution (DMR) to determine binding affinity and receptor kinetics in a label-free manner. This approach typically employs a biosensor system, such as the Corning Epic BTTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT system. In this embodiment, cells stably transfected with the 5-HTR1E receptor (e.g., HEK293 cells) are seeded onto optical microplates and incubated to form a monolayer.The method proceeds by establishing a baseline measurement of the cellular response, followed by the addition of the small molecule mimetic. The binding of the mimetic to the receptor triggers a mass redistribution within the cell, which causes a measurable shift in the index of refraction of the biosensor, reported as a picometer wavelength shift. By monitoring this shift over time — for example, comparing the steady state to the shift observed 20 minutes post-addition — the specific binding characteristics and dissociation constants of the compound can be calculated.

[0217] In embodiments directed to in vivo activation within a biological system (e.g., a laboratory animal model), the "contacting" step comprises administering the small molecule mimetic to the subject via a route capable of accessing the central nervous system.In preferred embodiments, the manner of contacting is intranasal administration, achieved using a delivery device such as a nasal spray pump, nebulizer, or insufflator calibrated to target the olfactory epithelium. Alternatively, contacting may be achieved via systemic routes (e.g., intravenous or intraperitoneal injection) utilizing formulations capable of crossing the blood-brain barrier. Regarding the dosage for such in vivo activation, while the compounds demonstrate high affinity (approximately 15-fold higher than the native protein), a broad effective range is contemplated to ensure robust receptor occupancy.Accordingly, the mimetic is administered in a dosage range of from about 10 mg / kg to about 100 mg / kg of body weight, typically administered once or twice daily.

[0218] The disclosure further provides a system or kit for activating the 5-HTR IE receptor and detecting said activation. While the individual biological components or detection methodologies utilized herein may be known in the art, the present disclosure provides a novel assembly wherein these standard research tools are combined with the specific small molecule mimetics of Formula I (including Formulas II, III,, IV, V and VI) to enable the precise, non-peptide modulation of the receptor. Accordingly, in some embodiments, the system comprises a multicomponent assembly including: (a) at least one small molecule mimetic as described herein; and (b) a biological substrate comprising cellsTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT expressing the 5-HTR1E receptor or genetic material suitable for generating such cells.

[0219] In specific embodiments, the system comprises the specific vectors or genetic materials required to establish the activation assay. For instance, in embodiments utilizing the [3-arrestin recruitment assay, the system includes a plasmid vector encoding a fusion protein of the 5-HTR1E receptor and a reporter component (e.g., a Tango-type plasmid encoding a GPCR-TEV protease fusion), along with transfection reagents suitable for introducing said vector into a host cell line. By packaging the novel mimetic with the specific genetic hardware required to visualize its activity, the system functions as a comprehensive research tool, allowing researchers to screen for downstream signaling bias or to utilize the mimetic as a high-affinity positive control in competitive binding experiments against unknown libraries.

[0220] In further embodiments, the system additionally comprises detection reagents specifically configured to quantify the downstream signaling events initiated by the small molecule mimetic. For example, when the system is configured as a screening kit, the detection reagents may comprise luciferase reporter substrates (e.g., luciferin), fluorescent biosensors, or antibodies specific for phosphorylated signaling proteins such as p-ERK.The inclusion of the disclosed high-affinity mimetics (e.g., the hydrazone species of Formula II, III and IV) provides a standardized reference agonist that allows for the calibration of these detection reagents, ensuring reproducible quantification of receptor kinetics across different experimental runs.

[0221] Additionally, the scope of the system extends to physical platforms used in label-free assays. In embodiments utilizing Dynamic Mass Redistribution (DMR), the system comprises the small molecule mimetic in combination with a biosensor-embedded microplate, such as an optical microplate compatible with the Coming Epic system. In this embodiment, the mimetic is provided in a solubilized form or as a lyophilized powder optimized for reconstitution and addition to the biosensor plate. This combination transforms the generic biosensor hardware into a specific diagnostic tool for profiling the HTR1E receptor's response to non-peptide ligands, thereby facilitating the identification of novel neuroprotective pathways.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0222] In some embodiments, the present disclosure provides a method and a related therapeutic system for treating or preventing a condition or preventing cell death in an individual in need thereof.

[0223] The term "treatment" or "treating" is used herein to refer to the medical management of a patient with the intent to cure, ameliorate, stabilize, or reduce the severity of a disease, pathological condition, or disorder. In the specific context of the present disclosure, treatment encompasses the administration of the small molecule mimetics described herein to a subject who has already been diagnosed with or is suspected of having a neurodegenerative condition. This includes the administration of the compound in an amount sufficient to alleviate specific symptoms, such as cognitive dysfunction, or to reverse underlying pathological processes, such as the mitigation of oxidative stress-induced cytotoxicity or the reduction of neuroinflammation. Treatment also includes the stalling or slowing of the progression of neurodegeneration, for example, by preventing further neuronal cell death in a subject suffering from traumatic brain injury or Alzheimer's Disease.

[0224] The term "prevention" or "preventing" refers reducing the likelihood of disease onset, delaying the onset of symptoms, or diminishing the severity of the disease should it occur. In the context of the disclosure, prevention specifically refers to the protection of neuronal integrity prior to an insult or in early-stage disease states. For example, the administration of the small molecule mimetics may be performed to prevent neuronal cell death in subjects predisposed to neurodegenerative disorders due to genetic markers, such as amyloidosis or tau hyperphosphorylation susceptibility, or environmental risk factors, or to protect neurons against secondary injury mechanisms following an acute event such as mild traumatic brain injury.

[0225] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to a vertebrate animal, preferably a mammal. In preferred embodiments, the individual is a primate, including human beings and non-human primates such as monkeys, apes, and lemurs. The term also encompasses commercially relevant mammals such as farm animals, sport animals, and pets, as well as laboratory animals such as mice, rats, andTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT guinea pigs used in the screening and validation of the compounds described herein. In the context of the methods of treatment described, the individual is typically a human subject in need of neuroprotection or modulation of the 5-HTR1E receptor.

[0226] The term "condition" or "disorder" refers to any state of health that deviates from the normal structural or functional state of the organism. As used herein, the term specifically refers to neurodegenerative conditions characterized by the progressive loss of structure or function of neurons, including the death of neurons. Exemplary conditions treated or prevented by the methods of the disclosure include Alzheimer's Disease, Parkinson's Disease, and other disorders characterized by pathological features such as amyloidosis, tau hyperphosphorylation, or cognitive dysfunction. The term further encompasses acute conditions involving neuronal damage, such as traumatic brain injury, mild traumatic brain injury, and conditions associated with oxidative stress-induced cytotoxicity in both the central nervous system and peripheral tissues.

[0227] The wording “Oxidative Stress-Associated Condition" refers to any physiological or pathological state characterized by an imbalance between the systemic manifestation of reactive oxygen species (ROS) and a biological system's ability to detoxify the reactive intermediates or to repair the resulting damage. This term encompasses neurodegenerative disorders (e.g., Alzheimer’s disease, Parkinson’s disease, TBI) and peripheral ischemic or cytotoxic disorders (e.g., acute kidney injury, myocardial infarction, hepatic ischemia, and chemotherapy-induced cytotoxicity).

[0228] The therapeutic operability of the disclosed disclosure is defined by the capacity of the small molecule mimetics to functionally engage the 5-HTR1E receptor or its evolutionary cognates to elicit a cytoprotective signaling response. Specifically, administration of the compound is effective to trigger a specific intracellular cascade — characterized by Gi / o protein coupling and / or [3-arrestin recruitment — that results in the phosphorylation of extracellular signal-regulated kinase (ERK) and the upregulation or stabilization of BCL-2 family proteins. It is this conserved signaling mechanism that governs the utility of the disclosure across diverse species and tissue types.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0229] Accordingly, in embodiments where a specific subject (e.g., a murine model) or tissue type lacks a direct genetic ortholog of the human 5-HTR1E receptor, the term "5-HTR1E modulation" encompasses the activation of functionally analogous cognate receptors (e.g., 5-HTR1D or related serotonin receptor subtypes) that preserve the aforementioned ERK / BCL-2 cytoprotective pathway. The validity of this mechanism is evidenced by the robust neuroprotection observed in murine in vivo studies despite the pseudogene status of Htr1e in rodents.

[0230] By virtue of this specific receptor operability, the compounds are defined as therapeutic agents for Conditions Associated with Serotonergic and Mitochondrial Dysfunction. These conditions can categorized into: (i) Neurodegenerative Disorders: including Alzheimer’s disease. Parkinson’s disease, and Traumatic Brain Injury (TBI), wherein the operability manifests as the prevention of neuronal apoptosis; (ii) Neuropsychiatric and Vascular Disorders: including depression, anxiety, and migraine, wherein the operability manifests as the mitigation of oxidative stress and preservation of cellular integrity via the activation of cytoprotective signaling pathways; and (iii) Peripheral Cytotoxic Disorders: including Acute Kidney Injury (AKI), myocardial ischemia, and hepatic failure, wherein the operability manifests as the mitigation of oxidative stress in epithelial or endothelial cells via the conserved ERK / BCL-2 axis.

[0231] In embodiments herein described, the method generally comprises administering to the subject a therapeutically effective amount of one or more small molecule mimetics of the disclosure, preferably those of Formula II, Formula III, and Formula IV. The administration is configured to deliver the small molecule to the central nervous system, where it binds to the 5-HTR1E receptor to initiate a cytoprotective signaling cascade. In preferred embodiments, this signaling mitigates oxidative stress-induced cytotoxicity, reverses neurodegeneration, and improves cognitive function by recruiting P-arrestin, phosphorylating ERK, and upregulating the anti-apoptotic protein BCL-2.

[0232] The compounds, compositions, and systems described herein are useful in the treatment and prevention of a broad spectrum of disorders associated with cellular stress and apoptosis. The therapeutic utility of the present disclosure is grounded in the discoveryTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT that activation of the 5-HTR1E receptor triggers a robust cytoprotective signaling cascade — specifically involving the recruitment of P-arrestin, the phosphorylation of ERK, and the stabilization of mitochondrial function via BCL-2 upregulation.Consequently, the methods described herein are not limited to a single organ system but are applicable to any tissue wherein the 5-HTR1E receptor is expressed and where oxidative stress or excitotoxicity is a driver of pathology.

[0233] Accordingly, the present disclosure provides methods for treating conditions characterized by "mitochondrial compromise" or "oxidative cytotoxicity." In the central nervous system, this manifests as neurodegeneration, where the method functions to prevent neuronal loss against toxic insults such as amyloid-beta or hyperphosphorylated tau. In peripheral tissues, such as the kidney or heart, this same mechanism functions to protect epithelial or endothelial cells against acute ischemic insults (e.g., during surgery or infarction) or chronic oxidative damage. Thus, the administration of the small molecule mimetics of Formula I constitutes a generalized cytoprotective therapy, adaptable via specific routes of administration (e.g., intranasal for CNS, systemic for peripheral) to target the site of injury.

[0234] In some embodiments, the conditions treatable by the method include a broad spectrum of neurodegenerative disorders. In specific embodiments, the condition is Alzheimer's Disease or Parkinson's Disease, where the upregulation of BCL-2 via the mimetic's activity counteracts the pathological effects of amyloidosis or tau hyperphosphorylation. The method is also applicable to the treatment of traumatic brain injury (TBI), including mild traumatic brain injury (mTBI). In such acute applications, the administration of the small molecule mimetic serves to reduce neuroinflammation and prevent the secondary neuronal loss associated with oxidative stress following the injury.Furthermore, the cytoprotective properties of the compounds against oxidative stress extend the utility of the method to other conditions characterized by cellular degeneration, potentially including ischemic stroke, retinal degeneration, or other hypoxic injuries.

[0235] A preferred mode of administration for the method is intranasal delivery. This route is particularly advantageous for the disclosed small molecules as it allows for directTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT transport to the brain via the olfactory and trigeminal nerve pathways, thereby bypassing the blood-brain barrier and minimizing systemic exposure. In an exemplary therapeutic regimen, the small molecule mimetic is administered via nasal instillation, drops, or spray.

[0236] To demonstrate the efficacy and establish the parameters of this administration route, specific in vivo protocols have been developed which may guide the clinical application of the method. In an exemplary evaluation method utilized herein, the small molecule mimetic is administered to a subject by nasal instillation at a dosage of approximately 10-100 mg / kg. The administration is performed twice daily, with doses spaced approximately 8 hours apart to maintain therapeutic plasma or CNS levels. To ensure maximal absorption and minimize tracheal suctioning, the subject is preferably held in a supine position, for example at an angle of 70 to 90 degrees, during administration.The formulation is delivered in small aliquots, alternating between nostrils to allow for sufficient inhalation and absorption by the nasal mucosa. This regimen, when maintained for a period before and after an injury or disease onset (e.g., one week prior to and three weeks post-injury), results in significant neuroprotection as evidenced by reduced silver staining (indicating less neurodegeneration) and reduced immunostaining for inflammatory markers in brain sections.

[0237] Regarding the route of administration, the method is characterized by significant versatility. While preclinical efficacy has been primarily established via intranasal administration to demonstrate direct central nervous system effects, the scope of the disclosure is not limited thereto. The term "non-invasive activation" as used herein broadly encompasses systemic routes of administration. Accordingly, in certain embodiments, the compound is administered orally, for example in the form of a tablet, capsule, or liquid suspension. In other embodiments, the compound is administered parenterally, including intravenous (IV) injection or infusion, which ensures rapid systemic distribution and bioavailability. These systemic routes are particularly suitable for chronic therapeutic regimens where patient compliance and ease of administration are paramount.

[0238] Notwithstanding the utility of systemic delivery, the method further comprises embodiments utilizing intranasal administration as a preferred mode for targeting the brain.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT This route leverages the olfactory and trigeminal nerve pathways to bypass the blood-brain barrier, delivering the active pharmacophore directly to the cerebrospinal fluid and brain parenchyma. This approach minimizes systemic exposure and potential peripheral side effects while maximizing the concentration of the drug at the target 5-HTR1E receptors in the hippocampus and cortex.

[0239] Furthermore, for specific clinical presentations — such as advanced stage neurodegeneration or cases where the blood-brain barrier is intact and impermeable to systemic agents — the method explicitly contemplates invasive, direct- to-CNS delivery. In such embodiments, the contacting step comprises introducing the compound directly into the cerebrospinal fluid. This may be achieved via an intrathecal injection or, in more acute interventions, via an intracerebroventricular (ICV) route. In this specific embodiment, a cannula or catheter is surgically implanted through the cranium into the lateral ventricles of the brain, allowing for the direct infusion of the small molecule mimetic into the ventricular system. This route guarantees immediate receptor engagement and is particularly valuable for "rescue" therapies or for high-concentration local delivery that would be toxic if administered systemically.

[0240] The methods described herein are particularly directed to the treatment of " Tauopathies," a class of neurodegenerative disorders characterized by the pathological aggregation of tau protein. Specifically, the administration of the disclosed HTR1E mimetics initiates a signaling cascade that prevents the formation of neurotoxic tau tangles and protects neurons from ApoE4-mediated toxicity. Accordingly, the disease to be treated is selected from the group consisting of Alzheimer’s Disease (AD), Frontotemporal Dementia (FTD), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), and chronic traumatic encephalopathy (CTE). By activating the HTR1E receptor, the method restores the neuroprotective axis lost during the progression of these conditions, preserving cognitive function and neuronal viability.

[0241] In another aspect, the present disclosure extends beyond therapeutic intervention for established disease states to encompass prophylactic administration. In such embodiments, the compounds of Formula I. and specifically Formula II, III, IV, V orTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT VI, are administered to a subject identified as being at risk for developing a neurodegenerative disorder or cognitive deficit, prior to the onset of overt clinical symptoms. This "at-risk" population may be identified through genetic screening (e.g., carriers of APOE-e4 alleles), biomarker analysis, or family history. In this context, the administration of the HTR1E mimetic serves to delay the onset of the disease, slow the progression of pre-symptomatic or post-symptomatic pathology, or enhance cognitive reserve, thereby providing a neuroprotective benefit that preserves neuronal function before significant degeneration occurs.

[0242] In addition to monotherapy, the present disclosure contemplates the use of the described small molecule mimetics in combination therapy with other active pharmaceutical agents. In certain embodiments, the compound of Formula II, III, IV, V or Formula VI can be administered simultaneously, sequentially, or separately with a second therapeutic agent. The selection of the second agent is guided by the specific condition being treated. For example, in the context of Alzheimer’s disease or related dementias, the HTR1E binder may be combined with an acetylcholinesterase inhibitor (such as donepezil, rivastigmine, or galantamine) or an NMDA receptor antagonist (such as memantine). Without wishing to be bound by theory, it is believed that the concurrent activation of the HTR1E receptor and the modulation of cholinergic or glutamatergic pathways may yield additive or synergistic effects on cognitive function.

[0243] In addition to neurodegenerative disorders, the small molecules of the disclosure and methods are applicable to other indications involving cellular stress modulation. While distinct from the orthosteric modulation of other serotonin subtypes, the compounds of Formula I may be utilized in the treatment of neuropsychiatric conditions, including depression, anxiety disorders, and stress-related pathology. Furthermore, given the expression of the 5-HTR1E receptor in peripheral vascular tissues, the compounds are provided as alternative therapeutic agents for the treatment of migraine and tension headaches via the stabilization of vascular endothelial function. While the primary mechanism comprises cytoprotection, the modulation of 5-HTR1E to reduce oxidative cytotoxicity or its crosstalk with related signaling pathways supports utility in these broaderTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT neurological contexts.

[0244] Furthermore, the utility of the disclosed compounds extends to the treatment of conditions beyond classical neurodegeneration. Given the distribution of 5-HT receptors, the compounds of the present disclosure may find application in the treatment of a broader spectrum of neurological and non-neurological disorders. These comprise neurological conditions such as depression and anxiety, where preservation of neuronal function via mitochondrial stabilization is beneficial; metabolic disorders, where peripheral cytoprotection plays a regulatory role; and pain management, specifically in neuropathic pain conditions where oxidative damage to peripheral nerve is implicated.

[0245] In some embodiments the small molecules of the disclosure can be used in connection with methods of treating or preventing peripheral ischemia and organ injury In those additional embodiments, the disclosure provides a method of treating or preventing tissue damage in a peripheral organ, specifically damage resulting from ischemiareperfusion injury or oxidative stress. As supported by the robust cytoprotective effect observed in renal lineage cells (HEK293), the small molecule mimetics of Formula I are useful in the treatment of conditions such as Acute Kidney Injury (AKI), Myocardial Infarction, and oxidative damage associated with chronic renal failure.

[0246] In these peripheral embodiments, the method of contacting differs from the neuroprotective regimen. The compound is preferably administered via a systemic route (e.g., intravenous infusion or oral administration) to maximize bioavailability in the target organ, rather than the intranasal route used for CN S targeting. For example, in the treatment of Acute Kidney Injury, the compound may be administered intravenously to a subject prior to or during a high-risk surgical procedure (e.g., cardiac surgery) to prophylactically mitigate oxidative stress in the renal tubules.

[0247] In a further aspect, the specific binding affinity of the compounds described herein renders them valuable as research or diagnostic tools. In such embodiments, the compound of Formula I can be modified to incorporate a detectable label, such as a radioactive isotope (e.g.. Tritium, Carbon- 14, or Fluorine- 18), a fluorophore, or a heavy metal chelate.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT These labeled analogues function as imaging ligands for Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), allowing for the in vivo mapping of HTR1E receptor distribution in the human brain. Clinically, such diagnostic tools are useful for quantifying receptor occupancy during drug development or for stratifying patients based on receptor density prior to initiating therapy. In a research setting, the labeled compounds serve as standard probes in competitive binding assays to screen novel candidates or to elucidate downstream signaling pathways associated with HTR1E activation.

[0248] The disclosure further describes systems, and articles of manufacture configured for the administration of the small molecules of the disclosure. In a general embodiment, the system comprises: (a) a pharmaceutical composition comprising a therapeutically effective amount of a small molecule mimetic of Formula I (including Formulas II, III, IV, V and VI); and (b) a delivery means for administering said composition to a subject.The nature of the delivery means is selected based on the specific condition — neurological or peripheral — to be treated.

[0249] In some embodiments the system can further comprises instructions for use, detailing the method of administering the composition for the treatment or prevention of the indicated condition. For example, the instructions may direct the user to administer the composition twice daily to mitigate oxidative stress-induced cytotoxicity. Regarding the dosage regimen, the instructions reflect the specific potency of the disclosed compounds.As exemplified by lead compounds R9 and RIO, which exhibit dissociation constants () in the low nanomolar range (approximately 15 -fold higher affinity than the native protein), the system is configured to deliver a therapeutically effective dose that may be significantly lower than that predicted by standard allometric scaling of murine data. While preclinical models will utilize dosages of 10-100mg / kg or initially 25 mg / kg, the superior affinity of these mimetics allows for efficacy at lower human equivalent doses, which are to be definitively established via pharmacokinetic profiling but are encompassed within the broad instructions of the kit.

[0250] With respect to the timing of administration, the systems described herein areTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT applicable to both therapeutic and prophylactic paradigms as well as non-therapeutic applications according to methods herein described. For example, the instructions can guide administration to "at-risk" subjects prior to the onset of overt clinical symptoms (prophylactic use) or to subjects following a clinical diagnosis (therapeutic use).Furthermore, the instructions can detail specific administration techniques to enhance efficacy. For example, mimicking the supine positioning used in preclinical studies, the instructions or the device design itself (e.g., via a specific nozzle angle) may facilitate gravity-assisted transport of the compound along the nasal-olfactory pathway to the cribriform plate.

[0251] In some embodiments directed to the treatment of neurodegenerative disorders (e.g., Alzheimer’s Disease, TBI), the system comprises the pharmaceutical composition in a formulation optimized for intranasal or intracerebral delivery.

[0252] In particular in some embodiment, the system comprises a container holding the composition and a nasal spray device, atomizer, or nebulizer. The device is specifically calibrated to atomize the composition into droplets of a size (e.g., < 10 microns) that facilitates deposition on the olfactory epithelium, thereby enabling direct transport to the central nervous system via the olfactory nerve fibers.

[0253] In acute embodiments, the system comprises a sterile, preservative-free formulation of the compound in a pre-filled syringe or vial, paired with an intrathecal catheter or intracerebroventricular (ICV) cannula for direct infusion into the cerebrospinal fluid.

[0254] For embodiments directed to the treatment of peripheral organ damage (e.g., Acute Kidney Injury, Myocardial Infarction), the system comprises the pharmaceutical composition in a formulation optimized for systemic distribution.

[0255] In embodiments, where the system is directed to intravenous administration of the small molecules of the disclosure, the system can comprise a concentrate of the compound (e.g., a lyophilized powder of a hydrazone salt of Formula II, III or IV) in a sealed vial, packaged together with a compatible diluent (e.g., saline or dextrose solution) and an IVTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT infusion set. This system is configured for rapid administration in clinical settings, such as during cardiac surgery or post-ischemic resuscitation.

[0256] In embodiments where the system is directed to oral administration of the small molecule of the disclosure. Alternatively, the system may comprise an oral dosage form (e.g., tablets or capsules) packaged in a blister pack or bottle, combined with instructions for a prophylactic dosing regimen (e.g., for chronic renal protection).

[0257] In some embodiments, the small molecules herein described can be comprise in a delivery device, possibly comprised within a system of the disclosure, the configuration of the delivery device can be selected based on the specific anatomical location of the condition to be treated.

[0258] In embodiments directed to neurodegenerative conditions (e.g., Alzheimer’s disease) or preventing neuronal cell death, the delivery device is preferably an intranasal delivery device, such as a nasal spray pump, dropper, or nebulizer. This device is specifically configured to atomize or dispense the composition for deposition on the olfactory epithelium, thereby bypassing the blood- brain barrier to deliver the active agent directly to the central nervous system.

[0259] In embodiments directed to peripheral tissue damage (e.g., acute kidney injury, myocardial ischemia), the delivery device comprises a systemic administration tool, such as a pre-filled syringe, an intravenous infusion bag, or an oral dispenser. These devices are configured to deliver the composition into the systemic circulation to ensure rapid bioavailability in organs such as the kidney, heart, or liver

[0260] In some embodiments, therapeutic system herein described for treating or preventing a condition. In broad embodiments, the system comprises: (a) a pharmaceutical composition comprising a therapeutically effective amount of one or more small molecule mimetics of Formula I (including compounds of Formula II, Formula III, Formula IV, Formula V and Formula VI); and (b) a delivery device configured to administer said composition to a subject. Consistent with the preferred method of treatment described herein, the delivery device can preferably be an intranasal delivery device for conditionTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT where the route of administration is known to be effected. Suitable devices include a nasal spray pump, a dropper, a nebulizer, or an atomizer. In preferred embodiments, the device is specifically designed to atomize the composition into particles of a size optimal for deposition in the olfactory region of the nasal cavity, thereby maximizing transport to the central nervous system via the olfactory nerve pathway.

[0261] In some embodiments, the system can comprise one or more additional active pharmaceutical agents in combination with the compounds of the disclosure. This second agent may be selected from standard-of-care treatments such as acetylcholinesterase inhibitors (e.g., donepezil) or NMDA receptor antagonists (e.g., memantine). These components may be formulated together in a single fixed-dose unit or packaged separately within the kit for simultaneous or sequential administration. This combination of a potent, synthetically accessible small molecule mimetic with a targeted delivery system and potential combination therapies represents a novel therapeutic approach for addressing the unmet medical need in Alzheimer's Disease and related neurodegenerative disorders.

[0262] In further embodiments, the system includes a diagnostic component to stratify patients who will benefit most from 5-HTR1E activation. The system may comprise the therapeutic compound in combination with a reagent for detecting a specific biomarker (e.g., a kit for genotyping APOE alleles or measuring serum biomarkers of kidney injury like NGAL). This "theranostic" system ensures the targeted administration of the compound to subjects exhibiting the specific molecular pathology (e.g., 5-HTR1E downregulation or high oxidative stress) that the disclosure is designed to correct.

[0263] In some embodiments the system can comprise a therapeutic system, for treating or preventing a neurodegenerative condition. In some embodiments, the system comprises:(a) a pharmaceutical composition comprising a therapeutically effective amount of one or more small molecule mimetics as described herein; and (b) a delivery device configured to administer said composition to a subject. Consistent with the preferred method of treatment, the delivery device is preferably an intranasal delivery device. Suitable devices include a nasal spray pump, a dropper, a nebulizer, or an atomizer, specifically designed to atomize the composition into particles of a size optimal for deposition in the olfactoryTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT region of the nasal cavity.

[0264] Recognizing the multifactorial nature of ischemic and degenerative diseases, the system may further comprise a second therapeutic agent. For CNS systems, this can include an acetylcholinesterase inhibitor (e.g., donepezil) or an anti-amyloid antibody. For peripheral systems, this may include a standard-of-care agent such as a diuretic, an antihypertensive, or an anti-inflammatory drug. These agents can be formulated as a fixed-dose combination with the mimetic of Formula I or provided as separate units within the same packaging.

[0265] In some embodiments, the system takes the form of a kit of parts or article of manufacture comprising the pharmaceutical composition described herein. The kit generally comprises a container holding a therapeutically effective amount of the compound, or a pharmaceutically acceptable salt thereof. The container may be a bottle, vial, blister pack, or pre-filled syringe, adapted to maintain the stability of the formulation.This is particularly critical for embodiments comprising the hydrazone- linked species (Formula II, III or IV), where the container and formulation are selected to prevent hydrolytic degradation during storage. In specific embodiments designed for immediate use, the kit comprises the delivery device (such as the spray pump or dropper) pre-filled with a precise unit dose of the mimetic to ensure accurate dosing and facilitate patient compliance.

[0266] Additional components can include labeled polynucleotides, labeled primer such as barcoded with an adapter sequence for next generation sequencing, labels, microfluidic chip, reference standards, and additional components identifiable by a skilled person upon reading of the present disclosure.

[0267] The terms “label” and “labeled molecule” as used herein refer to a molecule capable of detection, including but not limited to radioactive isotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes, enzymes substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, nanoparticles, metal sols, ligands (such as biotin, avidin, streptavidin or haptens) and the like. The term “fluorophore” refers to a substanceTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT or a portion thereof which is capable of exhibiting fluorescence in a detectable image. As a consequence, the wording “labeling signal” as used herein indicates the signal emitted from the label that allows detection of the label, including but not limited to radioactivity, fluorescence, chemoluminescence, production of a compound in outcome of an enzymatic reaction and the like.

[0268] In embodiments herein described, the components of the kit can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here disclosed. The kit will normally contain the compositions in separate containers.Instructions, for example written or audio instructions, on paper or electronic support such as tapes, CD-ROMs, flash drives, or by indication of a Uniform Resource Locator (URL), which contains a pdf copy of the instructions for carrying out the assay, will usually be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (i.e. wash buffers and the like).

[0269] Further details concerning the identification of the suitable carrier agent or auxiliary agent of the compositions, and generally manufacturing and packaging of the kit, can be identified by the person skilled in the art upon reading of the present disclosure.

[0270] In general, further details concerning the small molecule mimetics described herein, which are configured for selectively activating the 5-HTR1E receptor, and related products, compositions, methods, and systems for treating neurodegenerative disorders or screening for receptor modulators, including generally the synthesis, formulation, and packaging of the compounds, compositions, devices, and kits, can be identified by the person skilled in the art upon reading of the present disclosure.EXAMPLES

[0271] The small molecule CPE mimetics of the disclosure and related compositions, methods, and systems are further described in the following examples. A skilled person will be able to identify additional small molecule mimetics which are configured for activating the 5-Hydroxytryptamine Receptor 1E (5-HTR1E) and related products, compositions, methods, and systems for treating neurodegenerative conditions in view ofTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT the content of the present disclosure. The following specific examples are given to illustrate the practice of the disclosure, but are not to be considered as limiting the disclosure in any way.

[0272] In particular, exemplary hydrazone and urea-based compounds which are configured for recruiting arrestin, phosphorylating ERK, and upregulating BCL-2 are described in connection with specific experimental tests and procedures, including hybrid virtual screening protocols, synthetic pathways, and in vitro and in vivo bioassays. A skilled person will be able to understand and identify the modifications required to adapt the results illustrated in the exemplary embodiments of this section to additional embodiments of small molecule mimetics which are configured for mitigating oxidative stress-induced cytotoxicity and related products, compositions, methods, and systems for preventing neuronal cell death within the present disclosure.

[0273] The following materials and methods were used for all compounds and their precursors exemplified herein.

[0274] Materials Compound Z124 was purchased from Enamine LLC. (Monmouth Junction, NJ.) Compounds R9, R10, Dland D2 were synthesized as described herein. All compounds were solubilized in DMSO (Sigma Aldrich, St. Louis, MO). Recombinant carboxypeptidase E was custom produced by GenScript (Piscataway. NJ).

[0275] Ligand-based virtual screening Ligand-based virtual screening was conducted using 3D shape- and electrostatic potential-based searching with OpenEye Software. The queries were derived from CPE fragments interacting with HTR1E at three specific sites: amino acids 302-306 of CPE (Q1), associated with M1; amino acids 259-275 of CPE (Q2), associated with M11; and amino acids 260-319 of CPE (Q3), associated with M15. These three peptide fragments were utilized as query molecules to efficiently screen potentially active compounds through shape-based comparison using ROCS (ROCS v.3.6.1.3.OpenEye, Cadence Molecular Sciences, Santa Fe, NM).

[0276] A chemical library comprising 6.2 million compounds, obtained from ChemBridge, ChemDiv, and Enamine suppliers, was employed for the virtual screening.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Low-energy conformers were generated using OMEGA2 (OMEGA v.5.0.0.3. OpenEye, Cadence Molecular Sciences, Santa Fe, NM) with up to 100 conformers for each compound. The best 30,000 hits were selected based on the ROCS_TanimotoCombo score according to their shape similarity compared to the query molecule. For compassion of electrostatic properties, the EON (EON v.2.4.2.3. OpenEye, Cadence Molecular Sciences, Santa Fe, NM) was conducted to re-rank the top 30,000 ROCS hits in the order EON_ET_combo score as the sum of shape and electrostatics similarity. On the basis of this ranking, we selected the top 3000 after visual inspection.

[0277] Structure-based virtual screening Structure-based virtual screening (SBVS) was performed by docking the top 3000 hits from each query identified through LBVS onto the HTR1E at three binding sites: SI, S2, and S3, corresponding to the CPE peptide fragment queries: QI, Q2 and Q3. Molecular docking for the three binding sites was conducted using Schrodinger Suite 2023-1 (Schrodinger LLC, New York, USA, 2023). Protein preparation was revised using Protein Preparation Wizard in Maestro v13.5. The receptor grid box was generated with a 25 Å x 25 Å x 25 Å centered on the residue of the binding cavity. The 3000 compounds were prepared using LigPrep with the OPLS_2005 force field.Compound docking was performed using Glide v 9.8 with the standard precision (SP) method. Based on the docking score with visual inspection, 340 compounds were subsequently selected for further in silico analysis.

[0278] Machine learning prediction model To consider the target-specific chemical profile, machine learning algorithms were employed to calculate physicochemical properties, hydrogen bond acceptors (HBA), hydrogen bond donors (HBD), molecular weight (MW), polar surface area (PSA), and octanol-water partition coefficient (LogP), in accordance with Lipinski’s Rule of Five [2], using ChemAxon (https: / / chemaxon.com / ).Also, a central nervous system multiparameter optimization (CNS MPO) score was calculated using a model generated in ChemAxon. In addition, Absorption, solubility and blood-brain barrier (BBB) penetration were predicted using Pipeline Pilot (https: / / www.3ds.com / products / biovia / pipeline-pilot). These predictive models were applied to the 340 compounds identified through the LBVS-SBVS hybrid method.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0279] MD simulation For the above 340 compounds, we performed MD simulations with an explicit solvent model using Desmond v.7.3 with the OPLS_2005 force filed (Desmond Molecular Dynamics System; D. E. Shaw Research: New York, NY, 2023). The OPM (Orientations of Proteins in Membranes) [3] database was used for the spatial arrangement of the protein structures in the lipid bilayers. We used the human HTR1E structure with Gi protein from Cryo-EM(PDB ID 7E33) [4], We used the initial complex structure from the docking study of the 340 compounds bound to HTR1E. We selected the POPC (1-palmytoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) bilayer model for the membrane, with the membrane position aligned according to the structure obtained from the OPM database in the System Builder of Desmond. The thickness of the membrane model in the Cryo-EM for HTR1E was set to 32.2. We selected TIP3P [5] water as the solvent model, placing it in an orthorhombic box with dimensions of 10 nm x 10 nm x 10 nm. The overall complex structure was neutralized by adding Cl“ counterions. Then, the NaCl salt concentration was adjusted to 0.15 mol / L. Then, short 10 ns MD simulations were carried out with periodic boundary conditions using the NPT ensemble at body temperature (310 K) and pressure (1.01325 bar) for relaxation before simulation.

[0280] Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) calculation The final equilibrium state for the 340 protein-ligand docking poses was rescored with the MM-GBSA approach, as implemented in the Prime MM-GBSA module in Schrodinger Suite 2023-1 (Schrodinger LLC. New York, USA, 2023). We used the OPLS_2005 force field, the VSGB solvation model, and the default Prime parameters for the MM-GBSA calculations. The ligand binding free energy was calculated from 20 frames based on the last 10 ns of the MD trajectory. Finally, the top 36 compounds were selected through visual inspection combined with a machine learning prediction model score.

[0281] In silico R-group design. To improve the binding affinity of Z49535124 (Z124) we used R-group replacement by replacing its substituents using Spark. The 3D structure of Z 124 bound to HTR1E, obtained from the 100 ns MD simulation, was used as the starting molecule with the methoxy binding position targeted for replacement. A ChEMBL common fragment database consisting of 325,467 fragments was utilized for this R-groupTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT replacement. Each of these first-pass fragments was fitted into the original molecule, and fields and field points were calculated for the resulting whole molecules. The resulting molecules were minimized using the XED force field [6], and the top 500 fragments were selected based on their 50% shape and 50% electrostatic field similarity to the starting molecule. 86 designed compounds were selected using an integrated machine learning model to predict physicochemical properties, CNS MPO score, bioavailability, absorption, solubility, and BBB penetration for further in silico study. Constrained docking was performed for these 86 designed compounds using the Schrodinger Glide software by fixing the core structure aligned with Z124. Finally, the top 10 designed compounds were selected based on visual inspection and expected synthetic feasibility.

[0282] Cell Culture HEK293 cells were obtained from ATCC (Manassas, VA). HTLA cells (an HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a 0-arrestin2-TEV fusion gene) and Human HTR1E HEK293 Stable cells were obtained fromDr. Bryan Roth’s laboratory, at UNC, North Carolina. These cells were cultured in DMEM lx media supplemented with 10% FBS and 1% antibiotics. Primary human neurons were purchased from ScienCell (ScienCell, Carlsbad, CA) and cultured in neuron media containing neuron growth factors and antibiotics as indicated by the vendor (ScienCell, Carlsbad, CA).

[0283] Luciferase assay The Prest-Tango luciferase reporter system was used to test the binding affinity of the various compounds to HTR1E. Briefly, 4ug of Tango-HTRIE plasmid was transfected into 1x106HTLA cells using Lipofectamine 2000 (ThermoFisher Scientific) as protocol instructed; 24 h after transfection, 1x104of transfected HTLA cells were reseeded into 96-well plates overnight followed by treatment with each compound for 4 hours. Then the cells were lysed for luciferase assay using a luciferase system (Promega) according to the manual’s instructions. Luminescence was measured using a Synergy HTX plate reader (Biotek, Winooski, VT).

[0284] Dynamic mass redistribution (DMR) assays with Epic BT for Kd determination Whole cell DMR assays were performed using the Corning Epic BT system as previously described [7], [8] One day prior to performing DMR assay, cells were seededTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT onto Corning 384 well Epic BT microplates (Cat # 5040 Corning, MA USA) at a density of 5000 cells / 40 mL / well for HEK293 cells stably transfected with HTR1E. After seeding in DMEM media with 10% FBS, the Epic microplates were incubated for 30 min at room temperature and then transferred to a humidified incubator (37 °C, 5% CO2) for 20-24 hrs.Prior to initiating the DMR assay, cells were washed with assay buffer (Hank’s balanced salt solution with 20 mM HEPES pH=7.4) and automatically aligned on the Epic BT instrument. Mass changes within a cell also cause index of refraction change, resulting in a picometer wavelength shift. This is measured and reported as a dynamic mass redistribution (DMR) change. The DMR was monitored at room temperature for 60 minutes of continuous data acquisition every 3 seconds whole plate read time. With the acquisition paused, the various concentrations of DMSO- containing vehicle, serotonin, compounds Z124, R9, R10 or carboxypeptidase E (CPE) were added to the cells. Data acquisition was re-initiated and continuous acquisition (every 3 seconds) was completed for up to 30 min. The DMR shift change from steady state without ligand was compared to the DMR shift at 20 min post compound addition by Epic Analyzer software for determination of binding characteristics. There were four replicate curves for each compound or vehicle treatment. These data were analyzed using GraphPad Prism 10.1.2 and dose response analysis was completed for each treatment group to determine the binding affinities. These treatments were completed 3 times.

[0285] Lactate dehydrogenase (LDH) cytotoxicity assay The LDH assay was used to measure cytotoxicity with the CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, Cat#G1780) as instructed. Briefly, 3x104HTR1E stable HEK293 cells were seeded into 96-well plates. The next day, the regular DMEM media was removed and 50 nM of human WT-CPE or each compound (dissolved in DMSO) was prepared in serum free media. 200 μl of each treatment was added to the cells and incubated for 2 hours.Cells were then challenged with 500μM H2O2(prepared in serum free media) for 4 hours.50 μl of the debris-free medium was then collected for the LDH assay. A Biotek microplate reader (Winooski, VT) was used to read absorbance at 490 nm, and relative cytotoxicity was calculated against the DMSO-treated cells.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0286] Western blot The cells were first lysed for Western Blots using RIPA Lysis buffer (cat. No. 89901, Thermo Scientific, MA, USA) containing a protease and phosphate inhibitor cocktail (Thermo Scientific, MA, USA). The samples were then quantified using the Bradford assay (Bio-Rad, USA) and separated on 10% NU-PAGE Bis-Tris gels (Invitrogen, MA, USA). The gels were transferred onto nitrocellulose membranes and probed with the appropriate primary and secondary antibodies. The protein bands were then visualized with the help of the Odyssey Infrared Imaging system and the accompanying imaging software (LLCOR Inc, Lincoln, NE). The bands were quantified using the ImageJ densitometry tool (NIH, Bethesda). The data was normalized to the specific housekeeping proteins and the fold change was calculated from independent trials in Excel.

[0287] ERK activation assay The previous day, 3x105HTR1E stable HEK293 cells were seeded into 12-well plates with DMEM media (supplemented with 10% FBS and antibiotic) and incubated at 37°C. The next day, human WT-CPE and compound treatments were prepared in serum-free media and replaced with regular DMEM media. 1mL of each treatment was added to the appropriate wells and incubated for 5 minutes. The treatments were removed, and the cells were lysed with RIPA Buffer. The lysates were then used to run the ERK phosphorylation assay via Western Blotting. Changes in the level of phosphorylated ERK (pERK) were assessed using the pERKl / 2 (T202 / Y204) rabbit antibody (Cat# 9101L, Cell Signaling, Danvers, MA) and the total ERK (tERK) level changes were assessed using the tERKl / 2 mouse antibody (Cat# 4696S, Cell Signaling); followed by appropriate secondary anti-mouse (Cat#926-68022) and anti-rabbit (Cat#926-32213) antibodies (LI-COR, Lincoln, NE). The fluorescence of the anti-rabbit and antimouse secondary antibodies appears at 800 nm and 680 nm, respectively. The Odyssey Infrared Imaging system was used to visualize the bands, and densitometric analysis using ImageJ (NIH, Bethesda) was done to quantify the bands. pERK levels were normalized to tERK levels, and fold change was calculated from several independent trials.

[0288] BCL2 Western Blot The previous day, 3x105HTR1E stable HEK293 cells were seeded into 12-well plates with DMEM media (supplemented with 10% FBS andTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT antibiotic) and incubated at 37°C. The next day, human WT-CPE and compound treatments were prepared in serum-free media and replaced with regular DMEM media. 1mL of the treatments were added to the appropriate wells and incubated for 6 hours at 37°C. After incubation with the treatments, 0.5mL of 200μM H2O2(prepared in serum-free media) was added into each well and incubated overnight. The next day, the lysate was collected, and Western blotting was carried out on the samples. BCL2 rabbit primary antibody (Cat#3498S, Cell Signaling Technology) and actin (Cat#5441, MilliporeSigma, Burington, MA) were used, followed by the appropriate anti-mouse (Cat#926-68022) and anti-rabbit (Cat#926-32213) secondary antibodies (LI-COR, Lincoln, NE). The blots were visualized using the Odyssey Infrared Imaging System and densitometry was done using ImageJ (NIH, Bethesda). BCL2 protein levels were normalized to actin and fold change was calculated from several independent trials.

[0289] Statistical Analysis The data is representative of triplicates for each experiment and at least 3 separate trials (N). The data were analyzed using a 2-tail Student’s t-test; with statistical significance set at p-value < 0.05.Example 1: Overview of the approach for the identification of CPE mimetic small molecules

[0290] Multiple lines of experimental evidence have shown that carboxypeptidase E (CPE), also known as Neurotrophic factor- 1 (NF-al), possesses neuroprotective activity acting extracellularly, independent of its intracellular enzymatic function. [9],

[0010] , Moreover, studies have shown that gene therapy using Adeno-associated virus (AAV)-NF-otl / CPE injected bilaterally into the hippocampus of Alzheimer Disease mouse models:3xTg-AD pre- symptomatically and 5xFAD post-symptomatically. prevented and reversed neurodegeneration, amyloidosis, tau hyperphosphorylation, and cognitive dysfunction in these animals, respectively. [11, 12] It has also been reported that injection of two agomirs into the hippocampus of 9-month-old APP / PS 1 AD mice that up-regulated CPE expression mitigated AD pathology in these mice.

[0013] . These approaches are invasive, and our goal is to design small molecule (CPE mimics) that can be delivered intranasally to treat neurodegenerative diseases such as Alzheimer Disease.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0291] Recent studies demonstrated complementary experimental and computational findings regarding the interaction between NF-al / CPE and the 5-HTR1E receptor, a GPCR expressed in humans and primates but absent in mice.

[0014] Experiments and molecular dynamics (MD) simulations revealed that a 200-amino acid segment of NF-al / CPE (residues 150-350) mediates binding to 5-HTR1E, specifically targeting the extracellular loops.

[0015] 1.5 μs of MD simulations were conducted to predict the binding site of CPE on the extracellular loops of 5-HTR1E, identifying six polar interactions crucial for this binding. Additionally, computational phosphorylation of serine (Ser) and threonine (Thr) residues in the intracellular loops (ICL) of NF-al / CPE predicted P-arrestin binding, indicating activation of P-arrestin by CPE. MD calculations further demonstrated that phosphorylated Ser and Thr residues in the C-terminal tail and ICL3 facilitate the formation of salt bridges and hydrogen bonds between p-arrestinl and ICL2 / ICL3, resulting in p-arrestinl activation. (11) Further experiments confirmed that CPE interaction with 5-HTR1E triggers a signal transduction mechanism through P-arrestin / ERK signaling, promoting cell survival and protecting human neurons against oxidative and neuroexcitotoxic stress. [15, 16]

[0292] Subsequent validation (12) confirmed the binding site of NF-al / CPE to 5-HTR1E, as previously predicted by Caltech. [15, 16] Experimental measurements were performed of binding and activation using site-directed mutations of the residues in NF-al / CPE identified as key to the interaction. (

[0017]

[0018] ) These experimental findings were further corroborated by additional MD simulations. The combined studies confirmed the accuracy of the predicted binding site and identified specific amino acids critical for the interaction with 5-HTR1E, which are essential for the biological activity of NF-al / CPE. (12).Example 2: Structural Configuration and Pharmacophore Segmentation of exemplary CPE mimetic small molecules

[0293] This example describes the structural genus of the neuroprotective small molecule compounds illustrated in Figure 1A and the pharmacophore segmentation illustrated in Figure IB.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0294] Figure 1A depicts the general structure of exemplary identified hydrazone compounds, designated herein as Formula II. The compound comprises a tripartite structure consisting of a benzoic acid moiety, a hydrazone linker, and a substituted aryl moiety. As shown in the figure, the left-hand portion of the molecule consists of a 4-carboxyphenyl group, which provides a carboxylic acid functionality. In preferred embodiments, this carboxylic acid group is configured to act as a polar interaction site essential for binding to the HTR1E receptor, specifically forming hydrogen bonds with the Arg 164 residue.

[0295] The central portion of the molecule depicted in Figure 1A is a hydrazone linker connecting the benzoic acid moiety to the aryl moiety. This linker is defined by the structure -NH-N=C(R6)-, wherein R6 is selected from hydrogen, alkyl, or other suitable substituents. In the specific embodiments described herein, such as compounds Z124, R9, and RIO, R6 is hydrogen. This linker provides the necessary spacing and geometric orientation to span the CPE binding domain of the receptor.

[0296] The right-hand portion of Formula II in Figure 1A is a substituted phenyl ring comprising substituents Rl, R2, R3, R4, and R5. Based on the structure-activity relationship studies described herein, R3 represents a sulfonate ester group, defined generally as -O-S(O)2-R’, where R’ is an alkyl or aryl group. In preferred embodiments, such as the lead compounds R9 and R10, R3 is a p-toluenesulfonyloxy group. This sulfonate group is spatially oriented to form critical hydrogen bonding interactions with the Asp86 residue of the HTR1E receptor. The substituent R2 represents the primary site of R-group optimization in this study. R2 is selected from hydrogen, alkyl, cycloalkyl, or heterocyclic groups, optionally substituted with heteroatoms including nitrogen, oxygen, sulfur, or fluorine. In the optimized compounds R9 and R10, R2 comprises an aminomethyl or alkylamino functionality, such as a substituted amine, which is configured to provide additional stabilizing interactions within the receptor binding pocket. The remaining substituents Rl, R4, and R5 are independently selected from hydrogen, alkyl, or alkoxy groups.

[0297] Figure IB illustrates the pharmacophore segmentation of the top designedTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT candidates derived from the structure of Formula II. The molecular structure is divided into two functional domains to facilitate the hybrid virtual screening approach employed in the disclosure. The first domain, highlighted with dark gray shading in Figure IB, encompasses the benzoic acid moiety and the hydrazone linker. This region constitutes the phenylhydrazone scaffold, which serves as the structural core ensuring the correct positioning of the functional groups relative to the receptor residues. The second domain, corresponding to the region labeled Q2 (or Site S2) in Figure IB highlighted with, encompasses the substituted aryl moiety. This region serves as the variable pharmacophore head, containing the specific functional groups R1 through R5 designed to interact with specific residues, including Asp86 and Met85, within the SI binding site of the HTR1E receptor. This structural configuration allows the identified small molecules to mimic the binding interactions of the 200-amino acid segment of the native Neurotrophic factor-al / CPE protein, thereby activating the neuroprotective ERK-BCL2 signaling pathway.Example 3: Identification and Virtual Screening of Small Molecules

[0298] Using a method for in silico virtual screening, small molecules were identified capable of protecting human neurons against oxidative or neuroexcitotoxic stress via [3-arrestin / ERK signaling pathways. The method, integrates machine learning algorithms for central nervous system multiparameter optimization (CNS MPO) and blood-brain barrier (BBB) penetration prediction.Virtual Screening Strategy

[0299] To identify novel small-molecule agonists capable of protecting human neurons from oxidative or neurocytotoxic stress via the [3-arrestin / ERK signaling pathway, similar to the neuroprotective effects of CPE protein, a hybrid virtual screening approach was employed combining ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS). This approach focused on the pharmacophore for CPE binding to HTR1E that we identified earlier, as described in Yang et al 2024

[0017] .

[0300] Previous site-directed mutagenesis experiments of this pharmacophore identified three CPE mutants (Ml: K302A; MH: K302A+D306A+D75A; and M15:Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT D259A+E260A+W319A) that showed decreased binding affinity to the HTR1E compared to the wild-type. In particular, CPE Ml (K302A) decreased binding to 64.7%; CPE Mil (K302A+D306A+D275A) decreased binding to 66.8%; and CPE M15 (D259A+E260A+W319A) decreased binding to 64.5%

[0301] Furthermore, these mutants showed dramatically reduced cell-protective activity suggesting that these mutated residues play a critical role in receptor binding and functional activity. In addition, our in-silico MD simulations predicted the binding affinities of these CPE mutants to HTR1E that correlated well with the experimentally determined relative luciferase binding activities. These findings indicate that the identified mutations directly decrease receptor binding. Based on these observations, we propose that the Ml, M15, and M16 mutation sites define key pharmacophores essential for CPE binding to HTR1E.

[0302] Due to the large size of the CPE binding site on the HTR1E, which makes it challenging to find a small molecule to recognize the whole pharmacophore, it was hypothesized that the binding interface of the CPE / HTR1E complex could be portioned into three regions: SI, associated with CPE Ml mutant binding; S2, associated with CPE Ml 1 mutant binding; and S3, associated with CPE M15 mutant binding. This classification was based on distance (< H A) and supported by our mutation studies identifying key interaction residues (Ml: K302A; Mil: K302A+D306A+D75A, and M15:D259A+E260A+W319A)

[0020] Figure 2 Panels A to D) reporting the result of this hybrid virtual screening approach to discover novel small molecules with the neuroprotective properties of CPE.

[0303] Initially, LB VS was conducted using a shape- and field-based pharmacophore model derived from CPE fragments involved in the binding interface with HTR1E at three sites: i) amino acids 302-306 of CPE (Queryl), associated with Ml; ii) amino acids 259-275 of CPE (Query2), associated with Mil; and iii) amino acids 260-319 of CPE (Query3), associated with M15, utilizing the OpenEye

[0019] ,

[0020] software.

[0304] A chemical library was prepared comprising 6.2 million compounds from suppliers ChemBridge, ChemDiv, and Enamine for a 3D similarity search, utilizing theTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT OMEGA software to generate up to 100 low-energy conformers for each molecule.

[0305] Two computational strategies were employed for virtual screening of this large chemical database. First, a 3D shape similarity search was conducted using peptide fragments corresponding to amino acids 302-306 (QI), 259-275 (Q2), and 260-319 (Q3) of CPE as queries, performed using the ROCS software. The top 30,000 hits for each query were selected based on the ROCS_TanimotoCombo score. Second, a 3D electrostatic property similarity search was carried out on these 30,000 selected compounds using the EON software. After analyzing the positive and negative field contour maps and ranking based on EON_ET Combo scores, the top 3,000 hits for each query were selected.

[0306] SBVS was then performed by docking of these top 3,000 hits identified through LBVS onto the HTR1E at the three binding sites, SI, S2 and S3, corresponding to the CPE peptide fragments. Additionally, to consider the target-specific chemical profile for neurodegenerative diseases localized in the brain, machine learning algorithms were integrated to evaluate physicochemical properties, make blood-brain barrier (BBB) penetration predictions

[0021] and perform central nervous system multiparameter optimization (CNS MPO) scores

[0022] .

[0307] A total of 340 compounds were selected using the LBVS-SBVS hybrid method integrated with the machine learning prediction model. Subsequently, a 10 ns MD simulation starting with the initial docking poses and MM-GBSA

[0023]

[0024] calculations were performed to predict the binding free energy of these 340 compounds.

[0308] The top 36 selected compounds, along with their predicted binding free energies and associated binding site hypotheses, are listed in Table 1 below.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Table 1 Top 36 selected compounds through in silico virtual screening with the prec icted binding free energyNo Catalog ID Structure Pred. Hypothesis Hypothesis (Supplier) JG (associated (associated (kcal / 5-HTR1E CPE mol) binding mutant)site)1 Z1455157029 -91.96 SI Ml (Enamine) 0 (Sl_Topi)H2N 0o0oro2 Z49535124 -90.53 S2 Mil (=Z124) ZI (S2_(Enamine) Topi)oJ / / / II (\ / )O o W ——- o ' —1?Z424802890 -70.70 S2 Mil (Enamine)4 Z45720381 -67.82 S2 Mil (Enamine)HHO 05 48046376 -67.23 SI Ml (ChemB ridge)° \= / 1 OHTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Table 1 Top 36 selected compounds through in silico virtual screening with the prec icted binding free energyNo Catalog ID Structure Pred. Hypothesis Hypothesis (Supplier) AG (associated (associated (kcal / 5-HTR1E CPE mol) binding mutant)site)6 Z4910299808 -66.17 S2 Mil (Enamine)[ I o II N N 7J QJ °^0Ht)H7 Z90650485 -65.58 SI Ml (Enamine)I H °008 Z1991830032 -65.27 S2 Mil (Enamine)H H" YrNY~Y^A JHJJJjj ON9 5251-2277 -63.54 S2 Mil (ChemDiv)10 Z224297726 -62.57 S2 Mil (Enamine) 0o / 0HYrHFj(>FFTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Table 1 Top 36 selected compounds through in silico virtual screening with the prec icted binding free energyNo Catalog ID Structure Pred. Hypothesis Hypothesis (Supplier) AG (associated (associated (kcal / 5-HTR1E CPE mol) binding mutant)site)11 Z241357748 -61.63 SI Ml (Enamine)12 45114089 -60.96 SI Ml (ChemBridge) %ZIC4 2h / Z=EO'N1IT13 66094420 -60.42 SI Ml (ChemBridge)_ _ N--f).\NHI2 / \ _ / / 1 H / / ‘Cl NiyNNH214 98974061 -59.17 SI Ml (ChemBridge) NH2N^N Ojl J KN N H1 I 115 K821 -0241 -58.78 S3 Ml 5 (ChemDiv) (S3_ZJN'Y'S HN—^ Y 0 Topi)T \0v HS wTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Table 1 Top 36 selected compounds through in silico virtual screening with the prec icted binding free energyNo Catalog ID Structure Pred. Hypothesis Hypothesis (Supplier) JG (associated (associated (kcal / 5-HTR1E CPE mol) binding mutant)site)16 58801484 -57.10 SI Ml (ChemB ridge)b1 p XOH p= Os / $17 Z4910300022 -56.12 SI Ml (Enamine)\o=o18 Z4067889144 -55.94 SI Ml (Enamine)19 Z4410323848 -55.84 S2 Mil (Enamine)"o o PO20 71818277649 -55.72 SI Ml (Enamine) 0°^oTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Table 1 Top 36 selected compounds through in silico virtual screening with the prec icted binding free energyNo Catalog ID Structure Pred. Hypothesis Hypothesis (Supplier) JG (associated (associated (kcal / 5-HTR1E CPE mol) binding mutant)site)21 31723167 -55.60 S2 Mil (ChemB ridge)Oboz uuo IZp Avr °°22 Z227476540 -55.31 SI Ml (Enamine) N ^^ oozW 0 $\ / O _ zQxz 7o<=°X ft o.. 0X o23 80495772 -54.40 SI Ml (ChemBndge)24 7111521 -54.37 S2 Mil (ChemB ridge)25 8007-4156 -54.16 S3 Ml 5(ChemDiv)Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Table 1 Top 36 selected compounds through in silico virtual screening with the prec icted binding free energyNo Catalog ID Structure Pred. Hypothesis Hypothesis (Supplier) AG (associated (associated (kcal / 5-HTR1E CPE mol) binding mutant)site)26 7961552 -54.07 S2 Mil (ChemB ridge)H00H27 Z82201198 -52.81 S2 Mil (Enamine) 0HO-T. X^^O, )uHHO—28 Z55386846 -52.48 S3 M15 (Enamine) Q N^NH0 HII i %, Br0Y lT if 'o L A29 58115007 -52.30 SI Ml (ChemB ridge)Pl i n"NH2T T n<NYNH230 9343576 -52.20 S2 Mil (ChemB ridge)0 0II J 1 1 H 1H2N^OTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Table 1 Top 36 selected compounds through in silico virtual screening with the prec icted binding free energyNo Catalog ID Structure Pred. Hypothesis Hypothesis (Supplier) JG (associated (associated (kcal / 5-HTR1E CPE mol) binding mutant)site)31 42322114 -51.85 S2 Mil (ChemB ridge)ex6, / Ss^N N- ' QCcnO,32 C447-0314 ° -51.54 SI Ml (ChemDiv)I33 9237118 -51.36 SI Ml (ChemBndge)34 Z5096374150 -51.02 S3 M15 (Enamine) x0Y / / / TV y. - N CNH 0r [ u 1] n |l 1 N H35 Y043-0256 -50.92 S2 Mil (ChemDiv) / y / v T — W. / SSV / / N^AKNIZ<^ n OHW o-v^ H nTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Table 1 Top 36 selected compounds through in silico virtual screening with the prec icted binding free energyNo Catalog ID Structure Pred. Hypothesis Hypothesis (Supplier) JG (associated (associated (kcal / 5-HTR1E CPE mol) binding mutant)site)36 7979809 -50.38 SI Ml (ChemB ridge)

[0309] The specific compounds reported in Table 1 were identified as the highest-scoring candidates for each of the three hypothesized pharmacophore binding regions (SI, S2, and S3). Compound 1 (Z1455157029) was identified as the top-ranked candidate (" Sl_Topl") for the SI binding site, whic yh corresponds to the Ml mutant interaction region.XJIdentification of Lead Compound Z124

[0310] Experimental assays of the top candidates identified the compound Z49535124 (referred to herein as Z124) as a lead structure. Z124 was predicted to bind to the S2 site of the receptor.

[0311] In particular, the top three small molecules (#1, 2, and 15 in Table l)were selected through virtual screening.

[0312] An in-vitro assays was earned out for the top 3 predicted compounds, comprising a Luciferase Assay, dynamic mass redistribution (DMR) assays and a cell cytotoxicity in5-HTR1E stable HEK293 cells challenged with hydrogen peroxide.

[0313] The results of the in vitro assay reported in Figure 3 showed experimentally that only the virtual screening hit Z49535124 (denoted as Z124 hereafter) had good binding activity to HTR1E while also showing protective activity against oxidative stress comparable to CPE. However, Z124 required a concentration higher than the 50 nM CPE determined to be optimally active in mouse and rat neurons [9].Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0314] In particular the luciferase assay reported in Figure 3 panel A and Figure 4 PanelA show a comparison of binding affinity of Z124 and CPE to HTR1E. Compound Z124 effectively binds to HTR1E in HTLA cells. The Presto-tango reporter system was used to measure the binding affinity between compound Z124 and 5-HTR1E. HTR1E transfected HTLA cells were treated with 100 nM of compound Z124 for 6 hours and luciferase activity was measured using whole cell extracts from transfected cells.

[0315] According Figure 3 panel A shows that 100 nM Z124 bound to HEK.293 cells expressing HTR1E in the Presto-tango luciferase reporter system

[0015] is comparable to 50 nM CPE as will be understood by a skilled person.

[0316] The result of the DMR assays reported in Figure 3 Panel B, show that compound Z124 binds to the 5HTR1E receptor on HEK 293 cells. In particular, in order to obtain these results, HEK 293 cells expressing the 5HTR1E receptor in DMEM 10% FBS containing media were plated onto Epic BT 384 well plates and incubated overnight at 37°C and 5% CO2. After 18-24h, the media was removed and replaced with HBSS / Hepes buffer pH 7.4. The plate was mounted onto the Epic BT imager and DMR was determined for 60 minutes, to reach a steady state. The plates were then treated with Compound Z124 from 0.1-300 nM and DMR shift was determined for an additional 30 minutes. The DMR shift post drug treatment was evaluated as a function of Z 124 concentration. The results summarized Fig.3 Panel B indicates that Z124 binds to HTR1E with a Kd 27.4 + 2.3 nM, similar to that for CPE, as shown in Figure 39 Panel B described in Example 10 below as will be understood by a skilled person.

[0317] Th results of the cell cytotoxicity assay reported in Figure 3 Panel C Z124 at 100 nM protects HEK293 cells from H2O2 induced cytotoxicity. 5-HTR1E stable HEK293 cells were treated with 100 nM of Z124 for 24 hours followed by 500uM H₂O₂ for 4 hours, then the LDH activity was measured using cell medium after centrifugation. Z 124 at 100 nM showed protective activity against oxidative stress in HEK293 / HTR1E cells, but not at 50 nM (Fig.3 Panel Cand Figure 4 Panel B)

[0318] Accordingly, the calculations obtained in result of these assays suggest that Z124Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT binds to the S2 site on the HTR1E, which corresponds to the Ml 1 (K302A+D306A+D75A) mutant of CPE. The predicted binding model result from a 100 ns MD simulation, indicates that the acid group of Z 124 forms hydrogen bonds with Arg 164, while the hydrazinylidene group of Z124 forms a hydrogen bond with Lys89. Additionally, the sulfonyl group forms a hydrogen bond with Asp86, and the benzyl group engages in hydrophobic interactions with Met85. However, we observed that the methoxy group on Z124 did not interact with Asp86. Therefore, we proposed that replacement of the methoxy group with a suitable substituent capable of interacting with Asp86 would enhance the binding affinity and hence protective activity. To explore this, we performed hit-to-lead optimization using in silicoR-group design described in Example 2.Example 4 In silico R-group design for hit-to-lead optimization

[0319] R-group design was conducted for lead optimization based on the lead compound of Z124 (Z49535123, S2_Topl) to identify 86 candidate compounds predicted to exhibit comparable or superior efficacy to Z124. This process utilized cheminformatics, machine learning, and molecular modeling techniques incorporating visual inspection.

[0320] In particular, to investigate and enhance the potency of Z 124 R-group searching was conducted using a fragment library database consisting of 325,467 fragments, utilizing Spark in the Cresset

[0025] method. Pharmacophore filtering was applied to identify substituents capable of interacting with Asp86 using the initial binding pose obtained from a 100 ns MD simulation of Z124 bound to HTR1E as the starting structure for R-group replacement. The structure of groups R9 and R10 and related binding model are illustrated in Figure 5 Panels A and B respectively.

[0321] In particular Figure 5 details the results of the R-group optimization process herein described. The specific chemical structures for the two lead candidates, R9 and R10, were selected based on their high predicted affinity and favorable physicochemical properties(see Figure 5 Panel A). Molecular modeling visualizations of these compounds docked into the S2 binding site of the 5-HTR1E receptor were obtained (Figure 5 Panels B and C). The models highlight the key molecular interactions, specifically showing how the engineered amine-containing R-groups of R9 and R10 extend into the sub-pocket to formTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT stabilizing salt bridge or hydrogen bond interactions with the key aspartic acid residue (Asp86), thereby validating the rational design strategy used to enhance binding affinity over the parent compound Z124. Accordingly the predicted binding poses of R9 and RIO indicate that the replaced functional R-group can interact with Asp86 as expected.

[0322] The top 500 fragments were selected initially. Subsequently, 86 designed compounds were chosen through our constrained docking integrated machine learning model for prediction of physicochemical properties, BBB penetration, CNS MPO score, bioavailability, and solubility

[0026] . The structures of the selected candidates aare reported in Figure 6.

[0323] In particular, Figure 6 highlights the top designed compounds predicted to have the highest potential efficacy, including lead compounds R9 and R10. Additionally, derivatives DI and D2 (derived from compound R5) were synthesized, the structures of which are shown in Figure 7. These compounds Z124, R9, R10, R05-D1, andR05-D2 (see Figure 3) in cell line assays all show β-arrestin protection against H₂O₂ induced oxidative stress-induced cytotoxicity comparable to CPE making them good candidates to prevent and reverse neurodegeneration, amyloidosis, tau hyperphosphorylation, and cognitive dysfunction.

[0324] The molecules reported in Figure 6 are also good candidates to prevent and reverse neurodegeneration, amyloidosis, tau hyperphosphorylation, and cognitive dysfunction.Accordingly, these compounds and variations on these compounds in accordance with the present disclosure are comprise.

[0325] This measured data shows that R9 and R10 are very credible candidates for neuroprotection through binding to serotonin 1E and subsequent β-arrestin-ERK. activation. Exemplary compounds having groups R9 and R10 and the compounds shown in Figure 1A and IB and with similar structure are thus encompassed in the CPE mimetic small molecules of the present disclosure.

[0326] Based on these results and the predicted interaction energy with Asp86, the top 10 candidates following visual inspection were proposed for synthesis and experimentalTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT testing the structures of which are shown in Figure 6.

[0327] Figure 7 illustrates the chemical structures of two additional hydrazone derivatives, designated DI and D2. These compounds are structural analogs of the designed candidate R5 and were synthesized to evaluate the impact of modifying the amine substituent at the R2 position. As shown in the figure, DI features a dimethylamine group, while D2 features a piperidine ring. Both compounds retain the core 4-((4-hydroxybenzylidene)hydrazino)benzoic acid scaffold common to Formula II but serve as simplified model compounds for validating the synthetic pathway and establishing structure-activity relationships (SAR).Example 5 Synthesis and purity of compounds R9, R10, D1 and D2.

[0328] Among the compounds identified, R9 and R10 were synthesized, along with D1 and D2 which are derivatives of R5 in which the substituents were modified from an aziridine to dimethylamine and piperidine, respectively, to enhance synthetic feasibility.

[0329] A synthetic sequence toward the desired compounds was developed starting from inexpensive, commercially available p-hydroxybenzaldehyde. A modified Betti reaction was performed to install the aminomethyl moiety, at which point a Boc protection was performed on the intermediate involved in the synthesis of R10. The resulting compounds were then tosylated at the phenol oxygen. Finally, concomitant hydrazone formation and Boc deprotection, if necessary, was performed to reveal the compounds R9, R10, DI, andD2 that were isolated as their trifluoroacetate salt.

[0330] A schematic of the procedure and details of the methods of synthesis and NMR analysis (see Figure 8). For R9, R10 and DI, >95% m / m purity was confirmed by NMR analysis.

[0331] In particular, Figure 8 provides a step-by-step reaction scheme for the chemical synthesis of the claimed hydrazone compounds starting from 4-hydroxybenzaldehyde. The scheme depicts the sequence of: (i) a modified Betti reaction (or Mannich-type condensation) to introduce the amine substituent at the ortho-position; (ii) protection of theTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT amine (e.g., using Boc-anhydride) where necessary; (iii) activation of the phenolic hydroxyl group via tosylation; and (iv) condensation with 4-hydrazinobenzoic acid followed by deprotection to yield the final hydrazone products. This general pathway was adapted as described in the Examples to produce the specific lead compounds R9 and RIO, as well as derivatives DI and D2.

[0332] Figures 9 through 28 present the spectroscopic data generated to validate the identity, chemical structure, and purity of the synthesized compounds described in the Examples. Specifically, Figures 9 through 14 correspond to the synthetic pathway for Compound RIO. These spectra confirm the sequential introduction of the ethylaminomethyl group, the protection of the amine and phenol functionalities, and the final formation of the hydrazone linkage in Compound RIO.

[0333] Figures 15 through 20 correspond to the synthetic pathway for Compound R9, confirming the structure of the boc-protected hydrazine intermediate and the final methylated hydrazone product.

[0334] Figures 21 through 28 correspond to the derivative compounds DI and D2, confirming the structure of the dimethylamine and piperidine analogs, respectively. The spectra display chemical shifts in parts per million relative to the solvent standard, providing structural confirmation of the claimed compositions.

[0335] Figures 29 through 38 present the analytical data used to confirm the molecular identity and purity of the synthesized compounds and the lead reference compound described in the Examples. Specifically, Figures 29, 31, 33, 25, and 37 are mass spectra showing the mass-to-charge ratio (m / z) corresponding to the molecular ions of the respective compounds, verifying their correct molecular weights. Figures 30, 32, 34, 36, and 38 are HPLC traces demonstrating the homogeneity of the samples, confirming that the compounds R9, R10, DI, D2, and Z124 were isolated with greater than 95% purity prior to biological testing. No identifiable impurities were observed. For D2,NMR analysis, corresponding to 94.1% m / m purity of compound was obtained.

[0336] The specific methods of these compounds are described in the followingTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Examples 4 to 7.Example 6; Method of synthesis of the compounds R9, and NMR purity analysis.

[0337] Preparation of Intermediate R9-01 In a 20 mL scintillation vial, a solution of 37% aq. formaldehyde (0.6 mL, 2 equiv) in EtOH (0.5 M) was prepared. To this mixture were added 1-Boc-1-methylhydrazine (585 mg, 2 equiv) and p-hydroxybenzaldehyde (244 mg, 2 mmol) iteratively. The reaction was sealed with a pressure relief cap and the reaction was heated to 90 °C. After 15 h, the reaction was cooled to ambient temperature, at which point the solvent was removed by rotary evaporation and the residue was purified by silica gel chromatography (30% EtOAc / Hexanes) to afford the benzylic hydrazine intermediateR9-01 as an impure mixture in (280 mg, 50% yield).

[0338] Analysis of this crude material by 1H NMR (Figure 17) confirmed the formation of the product by displaying a diagnostic aldehyde singlet at 9.82 ppm, aromatic doublets at 7.74 and 7.02 ppm, and the characteristic tert-butyl singlet at 1.50 ppm.Ntert-butyl 2-(5-formyl-2-hydroxybenzyl)-2-methylhydrazine-1-carboxylate (R9-01)

[0339] Protection and Activation (Intermediate R9-02) This material was telescoped through the next step of the sequence. In particular, a solution of this hydrazine (1 mmol) was prepared in CH₂Cl₂ (0.25 M) under N₂. DIPEA (0.26 mL, 1.5 equiv) was added to this solution at 0 °C. followed by addition of TsCl (210 mg, 1.1 equiv) in one portion. The reaction was warmed to ambient temperature and allowed to stir for 18 h. Upon completion of the reaction by TLC, the reaction was quenched with H2O, and extractions were performed with CH₂Cl₂. The combined organic layers were dried with Na₂SO₄, concentrated, and purified by silica gel chromatography (20 to 50% EtOAc / Hexanes) to deliver R9-02 (307 mg, 71% yield).Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0340] The structure of this intermediate was confirmed by spectroscopic analysis presented in Figure 18 (1H NMR) and Figure 20 (13C NMR). The 1H NMR spectrum revealed the characteristic singlet for the tosyl methyl group at 2.48 ppm and the N-methyl group at 2.64 ppm, alongside the aldehyde proton at 9.98 ppm. High-resolution mass spectrometry (HRMS) further confirmed the molecular formula with a found mass of 434.1504.OTstert-butyl 2-(5-formyl-2-(tosyloxy)benzyl)-2-methylhydrazine-1-carboxylate (R9-02)

[0341] Synthesis of Final Compound R9. Lastly, the tosylated phenol (0.3 mmol) was dissolved in a 10:1:1 mixture of CH₂Cl₂: THF: MeOH followed by addition of 4-hydrazinobenzoic acid (66 mg, 1.5 equiv). The mixture was allowed to stir at ambient temperature for 15 h, and upon determination by LCMS that starting material was converted to Boc-protected R9, TFA (0.5 mL, 22 equiv) was added and the reaction was monitored further by LCMS. Upon quantitative deprotection of the Boc group, the mixture was concentrated and the residue purified by reverse phase chromatography using a C18 column [0 to 100% MeCN / (0.1% TFA / H₂O)]. Fractions containing pure product were concentrated by rotary evaporation to remove acetonitrile, and the water was removed by lyophilization. Compound R9 was isolated as a yellow powder (30 mg, 17% yield).

[0342] The identity and purity of the final compound R9 were rigorously established as >95% m / m by 1H NMR analysis, as shown in Figure 21. The spectrum confirmed the structure with the hydrazone proton appearing as a singlet at 7.82 ppm and the N-methyl singlet shifting to 2.67 ppm, with no identifiable impurities observed. 13C NMR analysis (Figure 22) displayed the expected 23 unique carbon signals, including the carboxyl and hydrazone carbons. 19F NMR confirmed the presence of the trifluoroacetate counterion with a signal at -77.0 ppm (Figure 23). Additionally, High-resolution mass spectrometryTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT (HRMS) confirmed the protonated molecular ion with an observed m / z of 469.1546OTs(E)- 1 -(5-((2-(4-carboxyphenyl)hydrazineylidene)methyl)-2-(tosyloxy)benzyl)- 1 - methylhydrazin-l-ium trifluoroacetate (R9)

[0343] >95% m / m purity was confirmed by 'H NMR analysis. No identifiable impurities were observed.Example 7: Method of synthesis of the compounds RIO, and NMR purity analysis.

[0344] Preparation of Intermediate R10-01 In a 20 mL scintillation vial, a solution of paraformaldehyde (120 mg, 4 equiv) in EtOH (5 mL, 0.2 M) was prepared. To this mixture were added EtNH₂ (0.1 mL 70% aq. soln, 1.2 equiv)) and p-hydroxybenzaldehyde (122 mg, 1 mmol) iteratively. The reaction was sealed with a pressure relief cap and the reaction was heated to 90 °C. After 1.5 h, the reaction was cooled to ambient temperature, at which point the solvent was removed by rotary evaporation and the residue was purified by silica gel chromatography (0 to 100% EtOAc / Hexanes) to afford intermediate R10-01 (84 mg, 47% yield).

[0345] The structure of this aminomethyl intermediate was confirmed by 1H NMR analysis (Figure 9), which displayed the diagnostic aldehyde singlet at 9.83 ppm, aromatic signals consistent with 1,2,4-substitution ( 7.65, 7.54, 6.87 ppm), and the ethyl group signals (quartet at 2.79 ppm, triplet at 1.18 ppm). 13C NMR (Figure 11) and HRMS (found m / z 179.0938) further corroborated the identity of the compound R10-01.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT OH3-((ethylamino)methyl)-4-hydroxybenzaldehyde (R10-01)

[0346] Protection and Activation (Intermediate R10-02) The amine was then dissolved in THF (0.5 M) under N2, and Boc₂O (131 mg, 1.2 equiv) was added followed by DMAP(4 mg, 0.03 equiv). The reaction was allowed to proceed for 18 h, and it was quenched with sat. aq. NaHCO₃. Following multiple extractions with EtOAc, the combined organic layers were dried with Na2SO4 and concentrated in vacuo to remove excess Boc₂O. The crude product was carried forward without additional purification.

[0347] A solution of the protected amine was prepared in CH₂Cl₂ (0.3 M) under N₂.Triethylamine (0.11 mL, 1.5 equiv) and DMAP (12.2 mg, 0.2 equiv) were added to this solution, which was then cooled to 0 °C. TsCl (114 mg, 1.2 equiv) was added as a solution in CH₂Cl₂ (0.8 M). The reaction was warmed to ambient temperature and allowed to stir for 24 h. Upon completion of the reaction by TLC, the reaction was diluted with CH₂Cl₂, washed with brine, and concentrated. The resultant residue was purified by silica gel chromatography (20% EtOAc / Hexanes) to deliver productive R10-02 (97 mg, 44% yield over two steps).

[0348] The 1H NMR spectrum (Figure 12) confirmed the installation of the tosyl group (singlet at 2.45 ppm) and the Boc protecting group (multiplet 1.52-1.23 ppm), alongside the aldehyde peak at 9.95 ppm. 13C NMR (Figure 14) and HRMS analysis (found m / z 456.1455 for [M+Na]⁺) were consistent with the expected structure.OTsTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT 2-(((tert-butoxycarbonyl)(ethyl)amino)methyl)-4-formylphenyl 4- methylbenzenesulfonate (R 10-02)

[0349] Synthesis of Final Compound R10- The tosylated phenol (0.1 mmol) was dissolved in a mixture of CH₂Cl₂ (0.1 M) and TFA (1 M), followed by addition of 4-hydrazinobenzoic acid (15.2 mg, 1 equiv). The mixture was allowed to stir at ambient temperature for 16 h, and upon determination that starting material was consumed by LCMS analysis, the mixture was concentrated and the residue purified by reverse phase chromatography using a C18 column [0 to 100% MeCN / (0.1% TFA / H₂O)]. One fraction containing pure product was concentrated by rotary evaporation to remove acetonitrile, and the water was removed by lyophilization. Compound R10 was isolated as a yellow powder(36 mg, 21% yield).

[0350] The identity and purity of the final compound RIO were confirmed as >95% m / m by 1H NMR analysis, as presented in Figure 15. The spectrum showed characteristic signals including the hydrazone proton ( 7.90 ppm), the tosyl methyl group ( 2.50 ppm), and the ethylamine side chain (quartet at 3.16 ppm, triplet at 1.35 ppm). 13C NMR analysis (Figure 16) provided further structural confirmation, showing 24 carbon signals consistent with the molecular formula. 19F NMR confirmed the presence of the trifluoroacetate salt with a signal at -76.9 ppm (Figure 16A). High-resolution mass spectrometry (HRMS) confirmed the protonated molecular ion with an observed mass of 468.1591 (calc.468.1588).(E)-N-(5-((2-(4-carboxyphenyl)hydrazineylidene)methyl)-2-(tosyloxy)benzyl)ethanaminium trifluoroacetate (R10)Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0351] >95% m / m purity was confirmed by 'H NMR analysis. No identifiable impurities were observed.Example 8: Method of synthesis of the D1 and D2 and NMR purity analysis.

[0352] In a 20 mL scintillation vial, a solution of paraformaldehyde (120 mg, 4 equiv) in EtOH (0.5 M) was prepared. To this mixture were added dimethylamine (0.17 mL 40% aq. soln., 1.3 equiv) and p-hydroxybenzaldehyde (122 mg, 1 mmol) iteratively. The reaction was sealed with a pressure relief cap and heated to 90 °C. After 24 h, the reaction was cooled to ambient temperature, solvent was removed by rotary evaporation, and the residue was purified by silica gel chromatography (50 to 100% EtOAc / Hexanes then 10% MeOH / EtOAc) to afford the benzylic tertiary amine intermediate D1-01 (3-((dimethylamino)methyl)-4-hydroxybenzaldehyde) (114 mg, 64% yield). The structure was confirmed by 1H NMR (Figure 24), which displayed the benzylic methylene singlet at 3.75 ppm and the dimethylamino singlet at 2.38 ppm, alongside the expected aromatic protons. Characterization data was in agreement with the literature.o3-((dimethylamino)methyl)-4-hydroxybenzaldehyde (D1-01)

[0353] Protection and Activation (Intermediate D1-02) A solution of the amine D1-01 was prepared in (0.3 M) under N2. DIPEA (1.5 equiv) was added, followed by addition of TsCl (1.2 equiv) in one portion. The reaction was warmed to ambient temperature and allowed to stir for 18 h. Upon completion by TLC, the reaction was quenched with H2O, extracted with CH₂Cl₂, dried over Na₂SO₄, and purified by silica gel chromatography (1% Et3N / 10% MeOH / 89% CH2CI2) to deliver the tosylated product D1-02 (17% yield). The1H NMR spectrum (Figure 25) confirmed the structure, showing the tosyl methyl singlet at 2.47 ppm, the dimethylamino singlet at 2.18 ppm, and the aldehyde proton at 9.98 ppm.13C NMR (Figure 27) and HRMS (found m / z 334.1111) further validated the identity ofTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT the intermediate.2-((dimethylamino)methyl)-4-formylphenyl 4-methylbenzenesulfonate (D 1-02)

[0354] Synthesis of Final Compound D1

[0003] Lastly, the tosylated phenol D1-02 was dissolved in a mixture of CH₂Cl₂ (0.1 M) and TFA (0.4 M), followed by addition of 4-hydrazinobenzoic acid (1 equiv). The mixture was stirred at ambient temperature for 3 h.Upon consumption of starting material (LCMS), the mixture was concentrated and purified by reverse phase chromatography using a C18 column [0 to 100% MeCN / (0.1% TFA / )].Fractions containing pure product were lyophilized to isolate Compound D1 ((E)-1-(5-((2-(4-carboxyphenyl)hydrazineylidene)methyl)-2-(tosyloxy)phenyl)-N,N-dimethylmethanaminium) as a yellow powder (29% yield).OTs(E)-1-(5-((2-(4-carboxyphenyl)hydrazineylidene)methyl)-2-(tosyloxy)phenyl)-N,N-dimethylmethanaminium (D1)

[0355] The purity of DI was confirmed to be >95% m / m by 1H NMR analysis (Figure 28), with no identifiable impurities observed. The spectrum displayed the hydrazone proton( 7.89 ppm), the tosyl group ( 2.49 ppm), and the dimethylamine signal ( 2.88 ppm). 13C NMR (Figure 29) and HRMS (found m / z 467.1506) were consistent with the assigned structure. 19F NMR confirmed the presence of the trifluoroacetate salt with a signal at -77.0 ppm (Figure 30)Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Example 9: Method of synthesis of D2 (Piperidine Analog)and NMR purity analysis.

[0356] Following the general procedure described in Example 6, paraformaldehyde (120 mg, 4 equiv) and p-hydroxybenzaldehyde (122 mg, 1 mmol) were reacted with piperidine (0.13 mL, 1.3 equiv) in EtOH at 90 °C for 24 h. Purification by silica gel chromatography(1% / 10% MeOH / 89% CH₂Cl₂) afforded the intermediate D2-01 (4-hydroxy-3-(piperidin-l-ylmethyl)benzaldehyde) (160 mg, 73% yield). 1H NMR analysis (Figure 31) confirmed the structure, showing the benzylic singlet at 3.76 ppm and the piperidine ring protons as a multiplet (3.31-1.54 ppm). 13C NMR (Figure 33) and HRMS analysis (found m / z 219.1244) confirmed the molecular weight.4-hydroxy-3-(piperidin- 1 -ylmethyl)benzaldehyde (D2-01)

[0357] Protection and Activation (Intermediate D2-02) Intermediate D2-01 was reacted with TsCl (1.2 equiv) and DIPEA (1.5 equiv) in CH₂Cl₂ (0.3 M) under N₂.Purification by silica gel chromatography (0 to 100% EtOAc / Hexanes) yielded the tosylated product D2-02 (84% yield). The 1H NMR spectrum (Figure 34) displayed the characteristic signals for the tosyl group (singlet, 2.47 ppm) and the aldehyde (singlet, 9.98 ppm), along with the piperidine ring protons. 13C NMR (Figure 36) and HRMS (found m / z 373.1334) confirmed the structure.4-formyl-2-(piperidin-l-ylmethyl)phenyl 4- methylbenzenesulf onate (D2-02)

[0358] Synthesis of Final Compound D2 The tosylated phenol D2-02 was condensedIllTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT with 4-hydrazinobenzoic acid (1 equiv) in CH₂Cl₂ (0.1 M) and TFA (0.4 M) as described for DI. Purification by reverse phase chromatography and lyophilization yielded Compound D2 ((E)- l-(5-((2-(4-carboxyphenyl)hydrazineylidene)methyl)-2-(tosyloxy)benzyl)piperidin-l-ium) as a yellow powder (54% yield).

[0359] Characterization of D21H NMR analysis (Figure 37) confirmed the structure of D2. A minor impurity of 4-hydrazinobenzoic acid was present in a 0.15:1 molar ratio, corresponding to a purity of 94.1% m / m. The spectrum clearly showed the product signals, including the hydrazone proton and the piperidine ring resonances. 13C NMR (Figure 37A) and HRMS (found m / z 507.1819) further confirmed the identity of the compound.19F NMR confirmed the presence of the trifluoroacetate salt with a signal at -77.0 ppm (Figure 38)(£')-l-(5-((2-(4-carboxyphenyl)hydrazineylidene)methyl)-2-(tosyloxy)benzyl)piperidin-l- ium (D2)

[0360] DI: >95% m / m purity was confirmed by ’H NMR analysis. No identifiable impurities were observed. D2: 4-hydrazinobenzoic acid impurity was present in 0.15: 1 mol ratio by NMR analysis, corresponding to 94.1% m / m purity of compound.Example 10. Compounds R9, R10, DI and D2 bind to HTR1E in HEK293-HTLA cells

[0361] Compounds R9, R10, DI and D2 were tested for binding to HTR1E using two different methods. Presto-Tango p-Arrestin Recruitment Assay and Dynamic Mass Redistribution (DMR) Binding and Specificity Assays

[0362] Presto-Tango P-Arrestin Recruitment Assay To evaluate the ability of theTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT compounds to recruit [3-arrestin, a proximal measure of HTR1E activation, the Presto-Tango luciferase reporter system was employed. HTLA cells (HEK.293 cells stably expressing a tTA-dependent luciferase reporter and a -arrestin2-TEV fusion protein) were cultured in DMEM supplemented with 10% dialyzed FBS and antibiotics. The cells were seeded in 6-well plates and transfected with the 5-HTRlE-Tango plasmid using a standard calcium phosphate transfection protocol. Twenty-four hours post-transfection, cells were plated into poly-L-lysine coated 96-well white-bottom plates at a density of 40.000 cells / well. After an additional 24 hours, the culture medium was replaced with serum-free titration medium. The cells were then treated with the test compounds (DI, D2, R9, R10, Z124) or the positive control (CPE) at a final concentration of 50 nM and incubated for 18 hours at 37 °C / 5%. Following incubation, the medium was removed, and Bright-Glo™ luciferase reagent (Promega) diluted in cell culture medium was added. Luminescence was measured using a microplate reader. Data were normalized to vehicle-treated controls and expressed as fold-change in relative light units (RLU). The results are reported in (Figure39, Panel A).

[0363] Dynamic Mass Redistribution (DMR) Binding and Specificity Assays Label-free binding kinetics and receptor specificity were measured using a Corning Epic® BT system. To determine the selectivity profile of the lead compounds, three distinct cell lines were utilized: HEK293 cells stably expressing the target 5-HTR1E receptor (for Figure 39 Panel B), HEK.293 cells stably expressing the 5-HTR1A receptor (for Figure 39 Panel C), and HEK293 cells stably expressing the 5-HTR1F receptor (for Panel D). Cells were seeded into fibronectin-coated 384-well Epic® microplates at a density of 10,000 cells / well and cultured for 24 hours. Prior to the assay, cells were washed and incubated in assay buffer (HBSS with 20 mM HEPES, pH 7.4) for 2 hours to establish a baseline.

[0364] The sensor plate was placed in the Epic® reader for equilibration (30 minutes).Baseline wavelengths were recorded, after which the test compounds (Serotonin, CPE, R9, R10) were added across a concentration range (e.g., 0.1 nM to 10 M) using an automated liquid handler. The response, defined as the shift in reflected wavelength (pm) resulting from mass redistribution upon ligand binding, was monitored in real-time for 60 minutes.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Dissociation constants were calculated by fitting the dose-response curves using non-linear regression analysis. The results are reported in (Figure 39, Panels B, C, and D). In particular, specificity was confirmed by the lack of a dose-dependent wavelength shift in Figure 39, Panels C and D compared to the robust response observed in Figure 39, PanelB.

[0365] Accordingly, the Presto-tango reporter system

[0015] was used to measure the binding affinity for the four compounds to HTR1E. HTR1E transfected HTLA cells were treated with 50 nM of each compound for 6 hours and luciferase activity was measured using whole cell extracts from transfected cells. All four compounds, R9, RIO, DI and D2 bound strongly to HTLA-HTR1E cells comparably to CPE, with R9 showing the highest binding (see results of Figure 39 panel A).

[0366] The binding affinity of compound R9 to HTR1E was compared with the two other5-HT family members, HTR1F and HTR1A. While R9 showed a high binding affinity to HTR1E in a dose dependent manner (see Figure 39 Panel B)„ such a pattern was not observed in the other two 5-HT members, HTR1F and HTR1A, indicating a high binding selectivity of R9 for HTR1E (see results of Figure 39 Panel C and Panel D). The binding affinities of serotonin, (5HT), CPE, R9 and R10 was then determined using the dynamic mass redistribution (DMR) assays with Epic BT method. Figure 39 Panel B shows the binding curves, and the calculated binding affinities (Kd) are reported in Table 2. The Kd of R9 and R10 shows binding to HTR1E with much higher affinity than CPE. The Kd for CPE binding to HTR1E using this method is comparable to our previous determination of 13.8 nM using radioactive CPE as ligand [15J.

[0367] Compounds R9, R10, DI and D2 are thus shown protect against H₂O₂-induced cytotoxicity in HEK293 / HTR IE cells and human neurons.

[0368] The efficacy of compounds R9, R10, DI and D2 at 50 nM concentration to protect cells against H₂O₂-induced cytotoxicity was also tested in HEK293-HTR1E cells and in human primary neurons.

[0369] In particular, to validate the neuroprotective potential and receptor specificity ofTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT the compounds, three distinct cell models were utilized, specifically HTRlE-stable HEK293 cells, wild-type HEK293 cells, and primary human cortical neurons. The HTRlE-stable HEK293 cells were human embryonic kidney cells stably transfected with the 5-HTR1E receptor and cultured in DMEM supplemented with 10% fetal bovine serum and selection antibiotics. Wild-type HEK293 cells, which lack the HTR1E receptor, were cultured in DMEM with 10% fetal bovine serum to serve as a negative control for receptor specificity. Cryopreserved primary human cortical neurons were thawed, plated in poly-L-lysine / laminin-coated plates, and maintained in Neurobasal medium supplemented with B27 and GlutaMAX to support neuronal health and phenotype.

[0370] Cytotoxicity was quantified by measuring the release of lactate dehydrogenase (LDH), a stable cytosolic enzyme, into the culture medium upon damage to the plasma membrane. Cells were seeded in 96-well plates at a density of 15,000 cells per well for the HEK293 models or 20,000 cells per well for the primary neurons. After 24 hours of attachment, the culture medium was replaced with serum-free medium containing the test compounds DI, D2, R9, or R10, or a vehicle control of DMSO, at a final concentration of50 nM. The cells were incubated with the compounds for 24 hours to prime the signaling pathways. Following this pre-treatment, oxidative stress was induced by adding hydrogen peroxide to the wells. A concentration of 500 micromolar hydrogen peroxide was used for the HEK293 cells, while a concentration of 300 micromolar was used for the more sensitive primary neurons. The cells were incubated under these stress conditions for 4 hours.

[0371] At the end of the stress incubation, 50 microliters of the cell culture supernatant was carefully harvested from each well and transferred to a new 96-well enzymatic assay plate. An equal volume of CytoTox 96 Reagent was added to each well, and the plates were protected from light and incubated for 30 minutes at room temperature to allow the enzymatic conversion of the tetrazolium salt into a red formazan product. The reaction was terminated by adding 50 microliters of Stop Solution, and the optical density was measured at 490 nm using a microplate reader. Cytotoxicity was calculated as a percentage relative to spontaneous LDH release controls from untreated cells and maximum LDH release controls from lysed cells. The percentage of cytotoxicity was determined by subtractingTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT the spontaneous optical density from the experimental optical density, dividing the result by the difference between the maximum and spontaneous optical densities, and multiplying by 100. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post-hoc test to compare treatment groups against the vehicle-treated stress-induced control.

[0372] The results are shown in Figure 40. In particular Figure 40 Panel A shows that DI, R9, R10 protected HEK-HTR1E cells from cytotoxicity significantly. R9 and R10 both showed similar efficacy as CPE. The specificity of the protective effect via interaction with HTR1E was demonstrated for R9 and R10 in Figure 40 Panel B, showing no protection by these two compounds against H₂O₂ cytotoxicity in HEK293 cells that were not stably transfected with HTR1E.

[0373] The compounds were ten tested for protection against oxidative stress in human primary cortical neurons. Figure 40 Panel C shows that all four compounds DI, D2, R9 and R10 protected human neurons from H₂O₂ -induced cytotoxicity with R10 showing the best activity comparable to CPE.

[0374] Therefore the results of these experiments showed that all four compounds exhibited efficacy comparable to NF-al / CPE. Notably, R9 demonstrated superior performance compared to CPE, with R10 showing efficacy close to R9. The binding constants reported in Table 2.Table 2. Measured binding constants for Serotonin IE binding. The Kd ofSerotonin is similar to values reported in literatureCompound Kd (nM)Serotonin 1.5R9 1.2R10 1.9Z124 28CPE 20.4Example 11: Compounds R9, R10, DI and D2 activate ERK signaling and increase BCL-2 expression to mediate protection against oxidative stress-induced cytotoxicity

[0375] Previously, it was shown that CPE, subsequent to binding to HTR1E activates theTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT ERK signaling pathway to up-regulate BCL-2 expression to mediate neuroprotection

[0015] ,

[0376] To elucidate the molecular mechanism underlying the observed neuroprotective effects, Western blot analysis was performed to monitor the activation of the extracellular signal-regulated kinase (ERK) pathway and the upregulation of the anti-apoptotic protein BCL-2. HTRlE-stable HEK293 cells were seeded in 6-well plates at a density of 500,000 cells per well and cultured for 24 hours. The cells were then serum-starved for 4 hours prior to treatment to minimize basal signaling noise. Cells were treated with the test compoundsR9 or R10, the positive control CPE, or a vehicle control at a concentration of 50 nM. For ERK activation analysis, cells were incubated for 30 minutes, whereas for BCL-2 expression analysis, cells were incubated for 24 hours.

[0377] Following treatment, the cells were washed with ice-cold phosphate-buffered saline and lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with a protease and phosphatase inhibitor cocktail. The lysates were centrifuged at 14,000 revolutions per minute for 15 minutes at 4 degrees Celsius to clarify the supernatant. Total protein concentration was determined using a bicinchoninic acid (BCA) protein assay.Equal amounts of protein, typically 20 micrograms, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes.

[0378] The membranes were blocked with 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 for 1 hour at room temperature to prevent non-specific binding. The membranes were then incubated overnight at 4 degrees Celsius with specific primary antibodies targeting phosphorylated-ERKl / 2, total ERK1 / 2, BCL-2, or GAPDH as a loading control. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) substrate and imaged using a digital chemiluminescence imaging system. Band intensities were quantified using densitometry analysis software, and the relative expression levels were calculated as the ratio of the target protein to the respective loading control.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0379] The results are reported in Figure 41. In particular Figure 41 Panel A shows that all four compounds activated ERK signaling significantly with R9 and RIO having the highest activation, comparable to CPE. Likewise, BCL-2 protein showed the highest increase of >2-fold with R9 and RIO treatment under H₂O₂ induced oxidative stress in HEK293-HTR1E cells (Figure 41 Panel B). This increase of BCL-2 levels was greater than CPE (~1.5 fold) relative to DMSO treated cells. Thus, all the 4 compounds appear to be able to increase ERK phosphorylation to activate ERK signaling, while R9 and RIO show the best efficacy in up-regulating BCL-2 expression to mediate cytotoxic protection, superior to CPE.

[0380] Interestingly, we have shown that these compounds also protected against mouse primary neurons from oxidative stress-induced cytotoxicity via activation of the Erk-Bcl2 pathway (our unpublished data). Future studies will focus on in vivo studies delivering the compounds R9 and R10 intranasally in Alzheimer’s disease mouse models to determine if it will have an effect on reversing AD pathology and memory loss. We recognize the very long route to bringing such drugs to clinical trials and even if our compounds show in vivo activity in mouse models, it may fail in clinical trials due to incompatibility of the models.

[0381] Accordingly, the present examples reports designed, synthesized, and identified 4 small molecules, R9 and R10, as well as DI and D2 which are derivatives of R5, that significantly protect human HEK293 cells and human neurons against H₂O₂-induced oxidative stress. Affinity binding studies reveal that R9 has the best binding affinity with R10 following close, in the 1-2 nanomolar range compared to CPE (Kd 26.5nM). R9 and R10 have the best binding affinity and neuroprotective activity superior or comparable to CPE. These compounds activated ERK signaling. The data also showed that R9 and R10 up-regulated mitochondrial pro-survival protein BCL-2 expression as a mechanism to protect cells against oxidative stress, similar to CPE. Thus, R9 and R10 are potentially excellent candidates to be developed as drugs to treat Alzheimer’s and other neurodegenerative diseases. Nevertheless, there are some limitations to the study that have to be addressed in the future. These include potential toxicity of these compounds in vivo, and the possibility of off target activity, as well as brain bioavailability.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Example 12: CPE mimetics for treatment of Neurodegenerative diseases

[0382] Neurodegenerative diseases such as Alzheimer Disease and Parkinson’s Disease have become prevalent globally in part due to an increased aging population. Hallmarks of these diseases are characterized by degeneration of the nervous system, including apoptosis of nerve cells, activation of microglial cells, deposition of amyloid protein aggregates and tau tangles in the brain, resulting in neuroinflammation and cognitive dysfunction.

[0027] Neuronal cell death is often caused by environmental and oxidative stress and neurotoxicity. There is an unmet need for therapeutics that will treat neurodegeneration.Understanding the mechanisms that can protect against neuronal cell death will facilitate the development of therapeutics that can mitigate these challenges. Indeed, many growth and trophic factors, such as transforming growth factors (TGFs), insulin-like growth factor (IGFs). epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and brain-derived neurotrophic factor (BDNF), are known to play a role in neuroprotection. [28, 29],

[0030]

[0383] Neurodegenerative diseases are widespread among the aging populations globally.In the United States alone, there were 4.7 to 6 million cases of neurodegenerative diseases between 2016 and 2017, which has only continued to rise since then

[0031] . Among the various neurodegenerative diseases, Alzheimer’s disease (AD) and Parkinson’s disease have been especially prevalent. Over 6 million Americans are living with AD in 2022. AD and other neurodegenerative diseases have impacted and continue to impact the health of millions of individuals, costing the U. S. economy billions of dollars each year, with an estimated $100 billion spent on AD alone [32JThus, there is an urgent need for development of therapeutics to treat neurodegenerative diseases.

[0384] AD is characterized by memory loss, neurodegeneration, deposition of amyloid protein plaques and tau tangles in the brain, and activation of microglial cells resulting in neuroinflammation

[0027]

[0033] . Environmental and oxidative stress and neurotoxicity are some of the primary causes of neuronal cell death in AD

[0034] , Deciphering the mechanisms that can prevent and mitigate against neuronal cell death will help in drug discovery for treatment of neurodegenerative diseases. To date, more than 100 agents have beenTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT evaluated in clinical trials, targeting tau, ^-amyloid, neurotransmitter receptors, as well as Apolipoprotein E

[0035] ,

[0036] . Other treatment strategies explored include administering of various growth and trophic factors known to play a role in neuroprotection, such as transforming growth factors (TGFs). insulin-like growth factor (IGFs). epidermal growth factor (EGF), nerve growth factor (NGF), fibroblast growth factor 2 (FGF2), and brain-derived neurotrophic factor (BDNF)

[0037]

[0038]

[0039] . However, these agents have met with little success.

[0385] Recently, various types of experimental evidence have shown that Neurotrophic factor-1 (NF-al), also known as carboxypeptidase E (CPE), possesses strong neuroprotective activity. NF-al / CPE acts extracellularly, independent of its intracellular enzymatic function, and is more effective than BDNF in promoting neuronal survival

[0010] ,[9]. Indeed, studies have shown that gene therapy using Adeno-associated virus (AAV)-NF-al / CPE injected bilaterally into the hippocampus of Alzheimer Disease mouse models:3xTg-AD pre-symptomatically and 5xFAD post-symptomatically, prevented and reversed neurodegeneration, amyloidosis, tau hyperphosphorylation, neuroinflammation, and memory loss in these mice, respectively

[0011] ,

[0040] . In another study, injection of two agomirs into the hippocampus of 9-month-old APP / PS1 AD mice up-regulated CPE expression and reversed AD pathology in these mice

[0041] , NF-al / CPE acts in a multifactorial manner, regulating protein expression in many metabolic pathways including up-regulating pro-survival protein, Bcl2, and down-regulating amyloid precursor protein (APP), Cardl4 (a proinflammatory protein), and Plin4 (a mitophagy inhibitor) to mitigate AD pathology

[0011] ,. Recently, a quantitative proteomic study revealed many differentially expressed proteins comparing with and without AAV-NF-al / CPE treatment in 3xTg-AD mice

[0042] , These included Western blot validated, NF-al / CPE targeted upregulation of autophagy proteins Beclinl and LC3, and down-regulation of Trim28 known to cause dysfunction of microtubule dynamics which occurs with AD progression

[0043] ,

[0044] ,

[0386] The goal of the experimental study described in the Example section is to design small molecules (CPE mimics) that can be delivered intranasally or injected systemicallyTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT to treat neurodegenerative diseases, such as AD. To this end, the experimentals reported in the previous Examples have demonstrated that NF-al / CPE interacts with serotonin IE receptor (HTR1E), a G protein-coupled receptor (GPCR) expressed in humans and primates, but not in mice, which then activates expression of BCL-2 via the -arrestin-ERK signaling pathway

[0015] ,. Complementary experimental and computational molecular dynamics (MD) studies regarding the interaction between NF-al / CPE and the HTR1E receptor revealed that a 200-amino acid segment of NF-al / CPE (residues 150-350) mediates binding to HTR1E, specifically targeting the extracellular loops

[0015] ,

[0017] . The MD simulations predicted the binding site of CPE on the extracellular loops of HTR1E, identifying six polar interactions crucial for this binding. MD calculations further demonstrated that phosphorylated Ser and Thr residues in the C-terminal tail and ICL3 facilitate formation of salt bridges and hydrogen bonds between p-arrestinl and ICL2 / ICL3, resulting in P-arrestinl activation

[0015] ,

[0017] . Site-directed mutagenesis / binding experiments on CPE confirmed the residues involved in CPE interaction with HTR1E that triggers a signal transduction mechanism through p-arrestin / ERK / BCL2 signaling, to promote cell survival and protect human neurons against oxidative and neuroexcitotoxic stress

[0015] ,

[0017] . These studies set the stage for in silico virtual screening to identify small molecules as new drug candidates that bind to the same site as CPE to mimic CPE’s action in neuroprotection.

[0387] In the experiments reported in this section, in silico virtual screening procedure was applied to ~ 6.2 million molecules, based on the pharmacophore validated by our earlier theoretical and experimental studies

[0015] ,

[0017] to discover novel small molecules with similar binding and ERK-BCL2 activation properties as CPE. Among the three top predicted molecules, one molecule (Z124) demonstrated experimental efficacy comparable to NF-al / CPE in biological activity but required a higher concentration than CPE. Next we carried out R-group design optimization based on the hit compound Z124 to identify candidate compounds predicted to exhibit superior efficacy to Z124. Of the top 10 predicted drug candidates, we were able to synthesize two compounds, R9 and R10, as well as two derivatives DI and D2 of R5 in amounts (25 to 40 mg) sufficient for experimental testing. Indeed, we found experimentally that all four bind to HTR1E stably transfected inTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT HEK293 cells, while showing the ability to protect HEK293 cells and human primary neurons against cytotoxicity induced by oxidative stress. Thus, we investigated the ability of these four compounds to activate ERK signaling and increase BCL2 expression under oxidative stress. Our findings reveal all four new small molecules mimic CPE in terms of binding and neuroprotection. Of these R9 and RIO exhibited the best activation of BCL2 expression and protection of human neurons and HEK293 cells against oxidative stress-induced cytotoxicity, with performance comparable to CPE.Example 13: Synthesis of Diaryl Urea Mimetic via Lossen Rearrangement (Prophetic)

[0388] To a solution of the carboxylic acid intermediate (prepared by oxidation of the corresponding Betti aldehyde as described herein) in dichloromethane (DCM) is added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 1.5 eq) and hydroxylamine hydrochloride (1.2 eq). The reaction mixture is stirred at room temperature for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The organic layer is washed with brine, dried over anhydrous sodium sulfate, and concentrated to yield the hydroxamic acid intermediate.

[0389] The crude hydroxamic acid is redissolved in anhydrous tetrahydrofuran (THF). To this solution is added methanesulfonyl chloride (MsCl, 1.1 eq) and triethylamine (3.0 eq) at 0°C. The mixture is allowed to warm to room temperature and stirred for 1 hour to effect the Lossen rearrangement, generating the isocyanate intermediate in situ. Immediately thereafter, tert-butyl 4-aminobenzoate (1.2 eq) and a catalytic amount of 4-dimethylaminopyridine (DMAP) are added. The reaction is stirred for 4 to 12 hours at room temperature. The reaction is quenched by the addition of water and extracted with ethyl acetate. The combined organic layers are washed with IN HC1 and saturated sodium bicarbonate, dried over sodium sulfate, filtered, and concentrated under reduced pressure.The residue is purified by silica gel column chromatography (eluting with a gradient of hexane / ethyl acetate) to afford the title urea- linked compound of Formula V. Mass spectrometry and NMR spectroscopy are performed to confirm the presence of the urea linkage and the integrity of the sulfonate ester.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0390] Accordingly the process can be performed according to the following stepsStep 1: Hydroxamic Acid Formation. To a solution of the carboxylic acid intermediate (prepared by oxidation of the corresponding Betti aldehyde as described herein) in dichloromethane (DCM) is added l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 1.5 eq) and hydroxylamine hydrochloride (1.2 eq). The reaction mixture is stirred at room temperature for 4 hours. The mixture is diluted with water and extracted with ethyl acetate. The organic layer is washed with brine, dried over anhydrous sodium sulfate, and concentrated to yield the hydroxamic acid intermediate.

[0391] Step 2: Lessen Rearrangement and Urea Coupling. The crude hydroxamic acid is redissolved in anhydrous tetrahydrofuran (THF). To this solution is added methanesulfonyl chloride (MsCl, 1.1 eq) and triethylamine (3.0 eq) at 0°C. The mixture is allowed to warm to room temperature and stirred for 1 hour to effect the Lessen rearrangement, generating the isocyanate intermediate in situ. Immediately thereafter, tert-butyl 4- aminobenzoate (1.2 eq) and a catalytic amount of 4-dimethylaminopyridine (DMAP) are added. The reaction is stirred for 4 to 12 hours at room temperature. The reaction is quenched by the addition of water and extracted with ethyl acetate. The combined organic layers are washed withIN HC1 and saturated sodium bicarbonate, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (eluting with a gradient of hexane / ethyl acetate) to afford the protected urea-linked intermediate.

[0392] Step 3: Global Deprotection. To a solution of the protected urea intermediate in DCM is added trifluoroacetic acid (TFA) (e.g., 20% v / v). The mixture is stirred at room temperature until the tert-butyl ester is hydrolyzed and any N-Boc protecting groups are removed (e.g., I -4 hours). The solvent is removed under reduced pressure, and the residue is purified by preparative HPLC to yield the final compound of Formula V. Conditions are selected to ensure the O-tosyl group remains intact during the acidic deprotection. Mass spectrometry and NMR spectroscopy are performed to confirm the presence of the urea linkage and the integrity of the sulfonate ester.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Example 14: Synthesis of Amide-Linked Mimetic (Prophetic)

[0393] Step 1: Pinnick Oxidation. To a solution of the sulfonated aldehyde intermediate(1.0 eq) in a mixture of tert-butanol and water (3:1 v / v) is added 2-methyl-2-butene (10 eq) and sodium dihydrogen phosphate (2.0 eq). The mixture is cooled to 0°C, and sodium chlorite (, 2.0 eq) is added portion-wise. The reaction is stirred at room temperature for 3 hours or until thin-layer chromatography (TLC) indicates complete consumption of the aldehyde. The mixture is diluted with water and extracted with ethyl acetate. The organic layer is washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo to yield the carboxylic acid intermediate, which is used in the subsequent step without further purification.Step 2: Amide Coupling. The crude carboxylic acid (1.0 eq) is dissolved in anhydrous N, N-dimethylformamide (DMF). To this solution is added N, N-diisopropylethylamine (DIPEA, 3.0 eq) and the mixture is stirred for 5 minutes, followed by the addition of HATU(l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, 1.2 eq). After 15 minutes additional stirring, methyl 4-aminobenzoate (1.1 eq) is added and the reaction mixture is stirred at room temperature for 12 to 18 hours. The mixture is then diluted with ethyl acetate and washed successively with IN HC1, saturated aqueous sodium bicarbonate, and brine. The organic phase is dried over anhydrous sodium sulfate, filtered, and concentrated. The crude residue is purified by flash column chromatography on silica gel to afford the title amide-linked compound of Formula VLExample 15; Systems and Devices for administration of small molecules

[0394] Figure 42 illustrates example systems and devices configured for the administration of small molecule therapeutics to a target individual (4200). These delivery mechanisms range from common, self-administered units to complex clinical hardware, selected based on the drug's biochemical properties, the required speed of onset, and patient convenience.

[0395] Inhalers (4205) are handheld devices designed to deliver medication directly to the lungs. Common types include Metered-Dose Inhalers (MDIs), the ubiquitous "puffers" thatTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT use a chemical propellant to push out a specific burst of aerosolized medicine. Another common type is the Dry Powder Inhaler (DPI) which is breath-actuated and requires the patient to breathe in sharply to pull a fine powder formulation deep into the lungs.Furthermore, Soft Mist Inhalers (SMIs) utilize mechanical energy rather than propellants to create a slow-moving mist that some patients find easier to inhale.

[0396] Nasal sprays and atomizers (4210) deliver medication to the highly vascularized nasal mucosa, allowing small molecules to be absorbed rapidly into the bloodstream or even travel directly to the brain via the olfactory nerve. These devices often take the form of multi-dose pumps, such as standard squeeze bottles used for daily allergy medications.Alternatively, they may be unit-dose atomizers designed to deliver a potent, precise dose quickly in emergency situations.

[0397] Unlike handheld inhalers, nebulizers (4215) are larger machines that convert liquid medication into a continuous, fine mist that the patient inhales through a mask or mouthpiece over a period of several minutes. They are particularly useful for small children, the elderly, or patients in severe respiratory distress who cannot use handheld devices effectively. Jet nebulizers use compressed air to create mist and require a compressor unit, whereas ultrasonic or vibrating mesh nebulizers use high-frequency vibrations to aerosolize the liquid.

[0398] A common form of drug delivery is oral solid dosage via pills or capsules (4220), where small molecules are formulated to be swallowed, survive stomach acid, and be absorbed in the intestines. This category includes standard compressed tablets and capsules, which consist of a gelatin shell holding powder or liquid. More sophisticated options include Osmotic-Controlled Release Oral Delivery Systems (OROS), which use a semi-permeable membrane and osmotic pressure to push medication out through a laser-drilled hole at a precise, constant rate.

[0399] Liquid formulations, categorized as drops or liquids (4225), are essential for patients unable to swallow pills or for localized delivery to specific organs. These include oral suspensions and solutions supplied with calibrated dosing tools.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0400] Delivery via injections or intravenous infusion (4230) involves using a needle to bypass skin and mucosal barriers, delivering the drug directly into tissues or the bloodstream for immediate bioavailability. This can be achieved through standard syringes and needles used for manual intramuscular or subcutaneous injections. For selfadministration, autoinjectors offer spring-loaded, pre-filled convenience. In clinical settings, intravenous (IV) infusion pumps are used to deliver fluids and medications into a vein at precise, programmable rates over extended periods.

[0401] Transdermal patches (4235) are adhesive devices containing medication that is slowly absorbed through the skin and into the bloodstream over hours or days, avoiding first-pass liver metabolism and providing steady drug levels. Some, known as matrix patches, have the drug embedded directly into the adhesive layer. Others are reservoir patches, where the drug is held in a liquid reservoir behind a rate-controlling membrane placed against the skin.

[0402] Various other means (4240) exist that utilize advanced bioengineering solutions to overcome specific delivery hurdles. This encompasses gel suspensions, used either for topical applications like diclofenac sodium gel or as injectable "depot" gels that solidify under the skin for long-term release. It also includes nanoparticle carriers, where small molecules are encapsulated within tiny lipid or polymer spheres to protect the drug, improve absorption, or target specific cells. Additionally, subdermal drug delivery systems can involve implanting small rods or pellets beneath the skin to release medication slowly over months or years.

[0403] In summary, described herein are are small molecule mimetics of Carboxypeptidase E (CPE) and related compositions, methods, and systems for providing cytoprotection. The disclosure provides compounds of Formula I, which encompasses specific sub-sets including hydrazone-based (Formula II, III, IV), urea-based (Formula V), and amide-based (Formula VI) scaffolds, are configured to bind selectively to the 5-Hydroxytryptamine Receptor IE (5-HTR1E) to serve as functional mimetics of the neurotrophic protein CPE. Upon binding, the compounds trigger a specific biased signaling pathway that involves the recruitment of beta-arrestin, phosphorylation of ERK, andTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT upregulation of the anti-apoptotic protein BCL-2. The compounds exhibit nanomolar binding affinity and significant cytoprotective activity against oxidative stress in human neurons and peripheral tissues. Also described are methods for identifying such mimetics using a hybrid virtual screening protocol, synthetic methods comprising a Betti reaction sequence, and therapeutic methods for treating conditions associated with oxidative stress, such as neurodegenerative disorders (e.g., Alzheimer's Disease) and peripheral organ injury, via intranasal or systemic administration.

[0404] The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the small molecules compounds, materials, compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.

[0405] The entire disclosure of each document cited (including patents, patent applications, journal articles including related supplemental and / or supporting information sections, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference.All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. However, if any inconsistency arises between a cited reference and the present disclosure, the present disclosure takes precedence.

[0406] The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the disclosure has been specifically disclosed by preferred embodiments, exemplary embodiments and optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art. and that such modifications and variations are considered to beTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT within the scope of this disclosure as defined by the appended claims.

[0407] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. The term "plurality" includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0408] The term "alkyl" as used herein refers to a linear, branched, or cyclic saturated hydrocarbon group typically although not necessarily containing 1 to about 15 carbon atoms, or 1 to about 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups such as cyclopentyl, cyclohexyl and the like. Generally, although again not necessarily, alkyl groups herein contain 1 to about 15 carbon atoms. The term "cycloalkyl" intends a cyclic alkyl group, typically having 4 to 8, or 5 to 7, carbon atoms. The term "substituted alkyl" refers to alkyl substituted with one or more substituent groups, and the terms "heteroatom-containing alkyl" and "heteroalkyl" refer to alkyl in which at least one carbon atom is replaced with a heteroatom. If not otherwise indicated, the terms "alkyl" and "lower alkyl" include linear, branched, cyclic, unsubstituted, substituted, and / or heteroatom-containing alkyl and lower alkyl, respectively.

[0409] The term "heteroatom-containing" as in a "heteroatom-containing alky group" refers to an alkyl group in which one or more carbon atoms is replaced with an atom other than carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon, typically nitrogen, oxygen or sulfur. Similarly, the term "heteroalkyl" refers to an alkyl substituent that is heteroatom-containing, the term "heterocyclic" refers to a cyclic substituent that is heteroatom-containing, the terms "heteroaryl" and "heteroaromatic" respectively refer to "aryl" and "aromatic" substituents that are heteroatom-containing, and the like. It should be noted that a "heterocyclic" group or compound may or may not be aromatic, and further that "heterocycles" may be monocyclic, bicyclic, or polycyclic as described above withTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT respect to the term "aryl." Examples of heteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted alkyl, N-alkylated amino alkyl, and the like. Examples of heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl, quinolinyl, indolyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl, tetrazolyl, etc., and examples of heteroatomcontaining alicyclic groups are pyrrolidine, morpholino, piperazino, piperidino, and additional substituents identifiable by a skilled person.

[0410] The term "alkoxy" as used herein intends an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy" group may be represented as -O-alkyl where alkyl is as defined above. A "lower alkoxy" group intends an alkoxy group containing 1 to 6 carbon atoms. Analogously, "alkenyloxy" and "lower alkenyloxy" respectively refer to an alkenyl and lower alkenyl group bound through a single, terminal ether linkage, and "alkynyloxy" and "lower alkynyloxy" respectively refer to an alkynyl and lower alkynyl group bound through a single, terminal ether linkage.

[0411] The term "aryl" as used herein, and unless otherwise specified, refers to an aromatic substituent containing a single aromatic ring or multiple aromatic rings that are fused together, directly linked, or indirectly linked (such that the different aromatic rings are bound to a common group such as a methylene or ethylene moiety). Aryl groups can contain 5 to 24 carbon atoms, or aryl groups contain 5 to 14 carbon atoms. Exemplary aryl groups contain one aromatic ring or two fused or linked aromatic rings, e.g., phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, benzophenone, and the like." Substituted aryl" refers to an aryl moiety substituted with one or more substituent groups, and the terms "heteroatom-containing aryl" and "heteroaryl" refer to aryl substituents in which at least one carbon atom is replaced with a heteroatom, as will be described in further detail infra.

[0412] The terms "cyclic", "cyclo-", and "ring" refer to alicyclic or aromatic groups that may or may not be substituted and / or heteroatom containing, and that may be monocyclic, bicyclic, or polycyclic. The term "alicyclic" is used in the conventional sense to refer to an aliphatic cyclic moiety, as opposed to an aromatic cyclic moiety, and may be monocyclic, bicyclic or polycyclic.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT

[0413] The term “isomers” as used refers to heterocyclic aromatic groups that have the same core molecular but may differ in atomic connectivity and / or location of unsaturation and is meant to include all possible structural variants. For example, as shown below, “pyrrole isomers” refers to all possible substituted variants of IH-pyrrole and 2H-pyrrole;“indole isomers” refers to all possible substituted variants of 3H-indole, IH-indole and 2H-isoindole, and so on:gH-pytroie 1 h'-0ytro:e 1 Wndote 2H-is®ndote

[0414] Likewise, as shown below, “triazole isomers” refers to all possible substituted variants of 1,2,4-triazole and 1,2,3-triazole; “oxadiazole isomers” refers to all possible substituted variants of 1,2,5-oxadiazole and 1,2,3-oxadiazole, and so on: / / ft" t-i "H H "o"'1,2,4-tnazoie t,2,3-triazote 1,2,3-Qxattoote

[0415] The terms "halo", "halogen", and "halide" are used in the conventional sense to refer to a chloro, bromo, fluoro or iodo substituent or ligand.

[0416] The term alkylene as used herein refers to an alkanediyl group which is a divalent saturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or acyclic structure. Exemplary alkylene includes propane- 1,2-diyl group (-CH(CH3)CH2-) or propane- 1,3-diyl group (-CH2CH2CH2-).

[0417] The term alkenylene refers to an alkenediyl group which is a divalent unsaturated aliphatic group, with two carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon double bond. Exemplary alkylene includes 2-butene-1.4-diyl group (-CH2CH=CHCH2-).

[0418] The term alkynylene refers to an alkynediyl group which is a divalent unsaturatedTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT aliphatic group, with two carbon atoms as points of attachment, a linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon triple bond. Exemplary alkylene includes 2-butyne-l,4-diyl group (-CH2C≡CCH2-).

[0419] The term "substituted" as in "substituted alkyl," "substituted aryl," and the like, is meant that in the alkyl, aryl, or other moiety, at least one hydrogen atom bound to a carbon(or other) atom is replaced with one or more non-hydrogen substituents.

[0420] Examples of such substituents include, without limitation: functional groups such as halo, hydroxyl, sulfhydryl, C1-C24 alkoxy, C2-C24 alkenyloxy, C2-C24 alkynyloxy, C5-C24 aryloxy, C6-C24 aralkyloxy, C6-C24 alkaryloxy, acyl (including C2-C24 alkylcarbonyl (-CO-alkyl) and C6-C24 arylcarbonyl (-CO-aryl)), acyloxy (-O-acyl, including C2-C24 alkylcarbonyloxy (-O-CO-alkyl) and C6-C24 arylcarbonyloxy (-O-CO-aryl)), C2-C24 alkoxycarbonyl (-(CO)-O-alkyl), C6-C24 aryloxycarbonyl (-(CO)-O-aryl), halocarbonyl (-CO)-X where X is halo), C2-C24 alkylcarbonato (-O-(CO)-O-alkyl), C6-C24 arylcarbonato (-O-(CO)-O-aryl), carboxy (-COOH), carboxylate ( COO-), carbamoyl(-(CO)-NH2), mono-(Cl-C24 alkyl)-substituted carbamoyl (-(CO)-NH(C1-C24 alkyl)), di-(Cl-C24 alkyl)-substituted carbamoyl (-(CO)-N(C1-C24 alkyl)2), mono-(C5-C24 aryl)-substituted carbamoyl (-(CO)-NH-aryl), di-(C5-C24 aryl)-substituted carbamoyl (-(CO)-N(C5-C24 aryl)2), di-N-(Cl-C24 alkyl), N-(C5-C24 aryl)-substituted carbamoyl, thiocarbamoyl (-(CS)-NH2), mono-(Cl-C24 alkyl) -substituted thiocarbamoyl (-(CO)-NH(C1-C24 alkyl)), di-(Cl-C24 alkyl)-substituted thiocarbamoyl (-(CO)-N(C1-C24 alkyl)2), mono-(C5-C24 aryl)-substituted thiocarbamoyl (-(CO)-NH-aryl), di-(C5-C24 aryl)-substituted thiocarbamoyl (-(CO)-N(C5-C24 aryl)2), di-N-(Cl-C24 alkyl), N-(C5-C24 aryl)-substituted thiocarbamoyl, carbamide (-NH-(CO)-NH2), cyano(-C=N), cyanato(-O-C=N), thiocyanate (-S-C=N), formyl (-(CO)-H), thioformyl ( (CS)-H), amino (-NH2), mono-(Cl-C24 alkyl)-substituted amino, di-(Cl-C24 alkyl)-substituted amino, mono-(C5-C24 aryl)-substituted amino, di-(C5-C24 aryl)-substituted amino, C2-C24 alkylamido (-NH-(CO)-alkyl), C6-C24 arylamido (-NH-(CO)-aryl), imino (-CR=NH where R = hydrogen, C1-C24 alkyl, C5-C24 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.), C2-C20 alkylimino ( CR=N(alkyl), where R = hydrogen, C1-C24 alkyl, C5-C24 aryl, C6-C24Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT alkaryl, C6-C24 aralkyl, etc.), arylimino (-CR=N(aryl), where R = hydrogen, C1-C20 alkyl, C5-C24 aryl, C6-C24 alkaryl, C6-C24 aralkyl, etc.), nitro (-NO2), nitroso (-NO), sulfo (-SO2-OH), sulfonato (-SO2-O-), C1-C24 alkylsulfanyl (-S-alkyl; also termed "alkylthio"), C5-C24 arylsulfanyl (-S-aryl; also termed "arylthio"), C1-C24 alkylsulfinyl (-(SO)-alkyl), C5-C24 arylsulfinyl (-(SO)-aryl), C1-C24 alkylsulfonyl (-SO2-alkyl), C5-C24 arylsulfonyl (-SO2-aryl), boryl (-BH2), borono (-B(OH)2), boronato (-B(OR)2 whereR is alkyl or other hydrocarbyl), phosphono (-P(0)(0H)2), phosphonato (-P(O)(O“)2), phosphinato (-P(O)(O-)), phospho (-PO2), phosphino (-PH2), silyl (-SiR3 wherein R is hydrogen or hydrocarbyl), and silyloxy (-O-silyl); and the hydrocarbyl moieties C1-C24 alkyl (e.g. C1-C12 alkyl and C1-C6 alkyl), C2-C24 alkenyl (e.g. C2-C12 alkenyl and C2-C6 alkenyl), C2-C24 alkynyl (e.g. C2-C12 alkynyl and C2-C6 alkynyl), C5-C24 aryl (e.g.C5-C14 aryl), C6-C24 alkaryl (e.g. C6-C16 alkaryl), and C6-C24 aralkyl (e.g. C6-C16 aralkyl).

[0421] The term "acyl" refers to substituents having the formula -(CO)-alkyl, -(CO)-aryl, or -(CO)-aralkyl, and the term "acyloxy" refers to substituents having the formula -O(CO)-alkyl, -O(CO)-aryl, or -O(CO)-aralkyl, wherein "alkyl," "aryl, and "aralkyl" are as defined above.

[0422] The term "alkaryl" refers to an aryl group with an alkyl substituent, and the term "aralkyl" refers to an alkyl group with an aryl substituent, wherein "aryl" and "alkyl" are as defined above. In some embodiments, alkaryl and aralkyl groups contain 6 to 24 carbon atoms, and particularly alkaryl and aralkyl groups contain 6 to 16 carbon atoms. Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl, p-cyclohexylphenyl,2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl, 3-ethyl-cyclopenta-l,4-diene, and the like.Examples of aralkyl groups include, without limitation, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl, 4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like. The terms "alkaryloxy" and "aralkyloxy" refer to substituents of the formula -OR wherein R is alkaryl or aralkyl, respectively, as just defined.

[0423] The term “Periodic Table” refers to the version of IUPAC Periodic Table of theTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Elements dated November 28, 2016

[0045] .

[0424] When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible sub-combinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified can be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein can be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations, which is not specifically disclosed herein.

[0425] " Optional" or "optionally" means that the subsequently described circumstance may or may not occur, so that the description includes instances where the circumstance occurs and instances where it does not according to the guidance provided in the present disclosure. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and, thus, the description includes structures wherein a non-hydrogen substituent is present and structures wherein a nonhydrogen substituent is not present. It will be appreciated that the phrase "optionally substituted" is used interchangeably with the phrase "substituted or unsubstituted." Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned can be identified in view of the desired features of the compoundTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT in view of the present disclosure, and in view of the features that result in the formation of stable or chemically feasible compounds. The term "stable", as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0426] A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the disclosure and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the devices, device components, methods steps set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.

[0427] In summary, in several embodiments, described herein are organosilicon compound, related complex that allow performance of fluorocarbon compound or olefin-based reactions and in particular polymerization of olefins to produce polyolefin polymers, and related methods and systems are described.

[0428] In particular, it will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.References1. Ellaby, R. J., et al., Supramolecular sefl-associating amphiphiles as aqueous pollutant scavengers.Organic & Biomolecular Chemistry, 2022. 20(38): p. 7587-7592.2. Lipinski, C. A., Lead-and drug-like compounds: the rule-offive revolution. Drug discovery today: Technologies, 2004. 1(4): p. 337-341.3. Lomize, M. A., et al., 0PM: orientations of proteins in membranes database. Bioinformatics,2006. 22(5): p. 623-625.4. Xu, P., et al., Structural insights into the lipid and ligand regulation of serotonin receptors. Nature,2021. 592(7854): p. 469-473.5. Jorgensen, W. L., et al., Comparison of simple potential function for simulating liquid water. The Journal of chemical physics, 1983. 79(2): p. 926-935.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Vinter, J. G., Extended electron distributions applied to the molecular mechanics of some intermolecular interactions. Journal of computer-aided molecular design, 1994. 8(6): p. 653-668.Kebig, A., et al., An optical dynamic mass redistribution assay reveals biased signaling of dnalsteric GPCR activators. Journal of Receptors and Signal Transduction, 2009. 29(3-4): p. 140- 145.Schroder, R., et al., Deconvolution of complex G protein— coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nature biotechnology, 2010. 28(9): p. 943-949.Xiao, L., X. Yang, and Y. P. Loh, Neurotrophic, gene regulation, and cognitive functions of carboxypeptidase E-neurotrophic factor-a! aud its variants. Frontiers in Neuroscience, 2019.13: p. 243.Xiao, L., et al., Neurotrophic factor-a!, a novel tropin is critical for the prevention of stress-induced hippocampal CA3 cell death and cognitive dysfunction in mice: comparison to BDNF.Translational Psychiatry, 2021. 11(1): p. 24.Xiao, L., et al., Hippocampal delivey of neurotrophic factor-a 1 / carbo ypeptidase E gene prevents neurodegeneration, amyloidosis, memoy loss in Alyheimer’s Disease male mice. Molecular Psychiatry, 2023. 28(8): p. 3332-3342.Xiao, L., et al., Hippocampal delivery of neurotrophic factor-alpha1 / carboxypeptidase E gene prevents neurodegeneration, amyloidosis, memory loss in Alzheimer's Disease male mice. Mol Psychiatry, 2023. 28(8): p. 3332-3342.Jiang, D., et al., Agomirs upregulating carboxypeptidase E expression rescue hippocampal neurogenesis and memoy deficits in Alyheimer’s disease. Transl Neurodegener, 2024. 13(1): p.24.Bai, F., et al., Molecular cloning and pharmacological characterisation of the guinea pig 5-HT1E receptor. Eur J Pharmacol, 2004. 484(2-3): p. 127-39.Sharma, V. K., et al., Novel interaction between neurotrophic factor-a1 / carboxypeptidase E and serotonin receptor, 5-HTR1E, protects human neurons against oxidative / nenroexcitotoxic stress via farrestin / ERK. signaling. Cellular and Molecular Life Sciences, 2022. 79(1): p. 24.Sharma, V. K., et al., Novel interaction between neurotrophic factor-alpha! / carboxypeptidase E and serotonin receptor, 5-HTR E, protects human neurons against oxidative / neuroexcitotoxic stress ria beta-arrestin / ERK signaling. Cell Mol Life Sci, 2021. 79(1): p. 24.Yang, X., et al., Experimental Validation of the Neurotrophic Factor-a1 Binding Site on the Serotonin Receptor 1E (HTR1E) Reponsible for fl-Arrestin Activation and Subsequent Neuroprotection. ACS omega, 2024. 9(39): p. 40749-40758.Yang, X., et al., Predicted molecules followed by experimental validation for protecting human neurons from oxidative stress— induced ytotoxicity. Proceedings of the National Academy of Sciences, 2025. 122(45): p. e2505359122.Hawkins, P. C., et al., Conformer generation with OME. GA: algorithm and validation using high quality structures from the Protein Databank and Cambridge Structural Database. Journal of chemical information and modeling, 2010. 50(4): p. 572-584.Hawkins, P. C., A. G. Skillman, and A. Nicholls, Comparison of shape-matching and docking as virtual screening tools. Journal of medicinal chemistry, 2007. 50(1): p. 74-82.Egan, W. J., K. M. Merz, and J J. Baldwin, Prediction of drug absorption using multivariate statistics. Journal of medicinal chemistry, 2000. 43(21): p. 3867-3877.Wager, T. T., et al., Central nervous system multiparameter optimisation desirability: application in drug discovery. ACS chemical neuroscience, 2016. 7(6): p. 767-775.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Bowers, K. J., ct al. Scalable algorithms for molecular dynamics simulations on commodity clusters.in Proceedings of the 2006 ACM / IEEE Conference on Supercomputing. 2006.Li, J., et al., The VSGB 2.0 model: a next generation energy model for high resolution protein structure modeling. Proteins: Structure, Function, and Bioinformatics, 2011. 79(10): p.2794-2812.Cheeseright, T., et al., Molecular field extrema as descriptors of biological activity: definition and validation. Journal of chemical information and modeling, 2006. 46(2): p. 665-676.Cheng, A. and K. M. Merz, Prediction of aqueous solubility ofi a diverse set of compounds using quantitative structure— properyt relationships. Journal of medicinal chemistry, 2003. 46(17):p. 3572-3580.Zhang, H., et al., Interaction between Aft and tau in the pathogenesis of Altheimer’s disease.International journal of biological sciences, 2021. 17(9): p. 2181.Zhou, X. E., et al., Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein- Coupled Recptors. Cell, 2017. 170(3): p. 457-469 el3.Lyons, R. M. and H. L. Moses, Transforminggrowth factors and the regulation of cell proliferation.EurJ Biochem, 1990. 187(3): p. 467-73.Radin, D. P. and P. Patel, BDNF: An Oncogene or Turn or Suppressor? Anticancer Res, 2017.37(8): p. 3983-3990.Thorpe, K. E., A. I. Levey, and J. Thomas, US burden of neurodegenerative disease.Partnership to Fight Chronic Disease (PFCD), 2021: p. 1-13.2023 Altheimer's disease facts and figures. Alzheimers Dement., 2023(Apr;19(4):l): p.1598-1695.Muzio, L., A. Viotti, and G. Martino, Microglia in neuroinfilammation and neurodegeneration: from understanding to therapy. Frontiers in neuroscience, 2021. 15: p. 742065.Brown, R. C., A. H. Lockwood, and B. R. Sonawane, Neurodegenerative diseases: an overview of environmental risk factors. Environmental health perspectives, 2005. 113(9): p. 1250.Cummings, J., et al., Altheimer's disease drug development pipeline: 2024. Alzheimer's & Dementia: Translational Research & Clinical Interventions, 2024. 10(2): p. el2465.Huang, L.-K., et al., Clinical trials of new drugs for Altheimer disease: a 2020—2023 update.Journal of Biomedical Science, 2023. 30(1): p. 83.Skaper, S. D., Neurotrophic factors: an overview. Neurotrophic factors: methods and protocols, 2017: p. 1-17.Lennon, M. J., et al., Genetic therapies for Altheimer’s disease: a seeping review. Journal of Alzheimer’s Disease, 2021. 84(2): p. 491-504.Tuszynski, M., A clinical trial of AAU2-BDNF gene therapy in early Algheimer’s disease and mild cognitive impairment. 2022.Fan, F.-C., et al., Neurotrophic factor-al / carboxypeptdase E controls progression and reversal of Altheimer's disease pathogenesis in mice. Theranostics, 2025. 15(6): p. 2279.Jiang, D., et al., Agomirs upregulating carboxypeptidase E expression rescue hippocampal neurogenesis and memory deficits in Alzheimer's disease. Translational Neurodegeneration, 2024. 13(1): p. 24.Xiao, L., et al., Neurotrophic Factor-a1 / carboxypptidase E regulates critical protein networks to rescue neurodegeneration, defective synaptogenesis and impaired autophagy in Altheimer’ s Disease mice. Translational Neurodegeneration 14, 59 2025.Chang, J., et al., TRIM28 functions as a negative regulator of aggresome formation. Autophagy, 2021. 17(12): p. 4231-4248.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT Shields, S., ct al., Oxidative stress promotes axonal atrophy through alterations in microtubules and EB1 function. Aging and disease, 2024. 16(6): p. 3706.IUPAC. Periodic Table. 2016; Available from: iupac.org / wp-content / uploads / 2015 / 07 / IUP AC_Periodic_Table-28Novl6.pdf.

Claims

Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT CLAIMS1. A small molecule of Formula I:Formula Iwherein n is 0 or 1; andwherein when n is 0, Cl, C2, C3, C4, and C6 comprise a 5-membered aromatic ring wherein any of Cl, C2, C3, C4, or C6 is optionally substituted with a Nitrogen, Oxygen, or Sulfur heteroatom;and wherein when n is 1, Cl, C2, C3, C4, C5 and C6 comprise a 6-membered aromatic ring wherein any of Cl, C2, C3, C4, C5 or C6 is optionally substituted with a Nitrogen, Oxygen, or Sulfur heteroatom;and wherein Al, A2, and A3 are each independently selected from Carbon, Nitrogen, Sulfur, or Oxygen; andwherein each of Al, A2, and A3 is optionally substituted with hydrogen, alkyl, or oxo (=0) groups; andwherein X comprises a carboxylic acid, ester, amide, sulfonic acid, sulfinic acid, sulfonamide, hydroxamic acid, phosphonic acid or nitro group; andTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT wherein Rl, R2, R4. and R5 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms;and wherein R3 comprises a Sulfur, Nitrogen, Oxygen, or Phosphorus-containing functional group; andand wherein Y is a 4-, 5- or 6-membered ring such that Al and X are separated by at least 3 atoms of the ring;or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

2. The small molecule according to claim 1, wherein Ring Y is a 4-membered ring selected from cyclobutane, azetidine, or oxetane.

3. The small molecule according to claim 1, wherein Ring Y is a 5-membered ring selected from cyclopentane, tetrahydrofuran, pyrrolidine, pyrrole, furan, or thiophene.

4. The small molecule according to claim 1, wherein Ring Y is a 6-membered ring selected from cyclohexane, piperidine, tetrahydropyran or phenyl.

5. The small molecule according to any one of claims 1 to 4, wherein Al, A2, and A3form a hydrazone linker, and optionally wherein the double bond of the hydrazone moiety exists in the E-configuration.

6. The small molecule according to any one of claims 1 to 5, wherein R3 comprises ap-toluenesulfonyloxy group.

7. The small molecule according to any one of claims 1 to 3, wherein R2 is a non- hydrogen / non-alkoxy substituent selected to enhance stability.

8. The small molecule according to any one of claims 1 to 4, wherein R2 is selectedfrom the group consisting of: cyclo-[CHCH2CH2NH-]; CH2CHN=C(CH3)NH2;CH2NHCH2SH; CH2C(H)(NH)2OH; CH2-cyclo-[NHCH2CH2-];Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT CH2C(CCH)NHCH3; CH2NHCH2-cyclo-[CH2CH2O-]; CH2NHCH2F;CH2N(CH3)NH2; CH2NHCH2CH3; CH2N(CH3)2; and CH2-cyclo- [NCH2CH2CH2CH2CH2-].

9. The small molecule according to any one of claims 1 to 8, wherein ring Y is a 6- membered aromatic ring, the linker A1-A3 forms a hydrazone moiety, and whereinR2 is a non-hydrogen / non-alkoxy substituent selected to enhance stability.

10. The small molecule according to any one of claims 1 to 9, having the structure of Formula I, wherein the small molecule comprises a phenyl scaffold wherein ringY is a 6-membered aromatic ring and A1-A3 form a hydrazone linkage.

11. The small molecule according to any one of claims 1 to 10, having Formula II:Formula IIwherein Rl, R2, R4 and R5 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms;R3 comprises an S, N, O or P-containing functional group;and n is 0 or 1;wherein when n is 0. Cl, C2, C3, C4. and C6 comprise a 5-membered aromatic ring and any of Cl, C2, C3, C4, or C6 is optionally substituted with a Nitrogen, Oxygen, or Sulfur heteroatom;Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT and wherein when n is 1, Cl, C2, C3, C4, C5 and C6 comprise a 6-membered aromatic ring and any of Cl, C2, C3, C4, C5 or C6 is optionally substituted with a Nitrogen, Oxygen, or Sulfur heteroatom;and wherein X comprises an ester, amide or acid group containing at least one E=O bond where E = C, S or P, or X is a nitro group -NO2;and A1 is NH, A2 is N and A3 is C.

12. The small molecule according to claim 11, comprising a hydrazone scaffold and having Formula III:Formula IIIwherein Rl, R4, R5 and R6 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms;and R2 is an alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms such that R2 is not H or an alkoxy group;R3 comprises an S, N, O or P-containing functional group; andn is 0 or 1;Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT wherein when n is 0, Cl, C2, C3, C4, and C6 comprise a 5-membered aromatic ring and any of Cl, C2, C3, C4, or C6 is optionally substituted with a Nitrogen, Oxygen, or Sulfur heteroatom;and wherein when n is 1, Cl, C2, C3, C4, C5 and C6 comprise a 6-membered aromatic ring and any of Cl, C2, C3, C4, C5 or C6 is optionally substituted with a Nitrogen, Oxygen, or Sulfur heteroatom;and wherein X comprises an ester, amide or acid group containing at least one E=O bond where E = C, S or P, or X is a nitro group -NO2.

13. The small molecule according to any of claims 1 to 9, having the structure of Formula IV:Formula IVwherein R6 is selected from hydrogen, alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with Nitrogen, Oxygen, Sulfur, Fluorine, Chlorine, Bromine and / or Iodine heteroatoms:and wherein X, Y, Rl, R2, R3, R4, R5, and n are defined as in Formula I.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT 14. The small molecule according to any one of claims 1 to 9, wherein the Linker defined by Al, A2, and A3 comprises a urea linkage (-NH-C(=O)-NH-), and wherein the small molecule has the structure of Formula V:R2R5Formula Vwherein:X is selected from a carboxylic acid, ester, amide, sulfonic acid, sulfinic acid, sulfonamide, hydroxamic acid, phosphonic acid, tetrazole, or nitro group; andn, Rl, R2, R3, R4, and R5 are as defined in claim 1.

15. The small molecule according to claim 14, wherein: n is 1; and C1-C6 form a phenyl ring.

16. The small molecule according to claim 14 or 15, wherein X is selected from a carboxylic acid (-COOH), a methyl ester (-COOMe), a tert-butyl ester (-COOtBu), or a primary amide (-C0NH2).

17. The small molecule according to any one of claims 14 to 16, wherein R3 comprises a sulfonate ester of formula -OS O2R’. wherein R' is an aryl group (e.g.. p-tolyl) or an alkyl group (e.g., methyl).

18. The small molecule according to any one of claims 14 to 17, wherein R2 is selected from: Hydrogen; an alkoxy group or a nitrogen-containing functional group selected from an aminomethyl group, a mono- or di-alkylamino group, or a cyclic amine.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT 19. The small molecule according to any one of claims 1 to 9, comprising an amide linker.

20. The small molecule according to claim 19, having Formula VI:HNFormula VIwherein X and Z each independently are a nitro group -NO2or comprises an ester, amide, or acid group containing at least one E=O bond where E is C, S, or P,;and wherein any of the aromatic rings can be optionally substituted with alkyl, cycloalkyl, aryl, or heterocyclic groups optionally substituted with heteroatoms N, O, S, F, Cl. Br and / or I.

21. The small molecule according to any one of claims 1 to 20, wherein X is a carboxylic acid.

22. The small molecule according to claim 1, selected from the group consisting of:(E)-4-(2-(3-methoxy-4-(tosyloxy)benzylidene)hydrazinyl)benzoic acid (Compound Z124);(E)-l-(5-((2-(4-carboxyphenyl)hydrazineylidene)methyl)-2-(tosyloxy)benzyl)-l- methylhydrazin-l-ium (Compound R9);(E)-N-(5-((2-(4-carboxyphenyl)hydrazineylidene)methyl)-2- (tosyloxy)benzyl)ethanaminium (Compound RIO);(E)-l-(5-((2-(4-carboxyphenyl)hydrazineylidene)methyl)-2-(tosyloxy)phenyl)- N, N-dimethylmethanaminium (Compound DI); andTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT (E)- 1 - (5 - ((2- (4-carboxyphenyl)hydrazineylidene)methyl)-2- (tosyloxy)benzyl)piperidin-l-ium (Compound D2):or a pharmaceutically acceptable salt thereof.

23. The small molecule according to any one of claims 1 to 22, wherein the small molecule is configured to exhibit robust metabolic stability and batch-to-batch consistency.

24. The small molecule according to any one of claims 1 to 23, wherein the small molecule is configured to modulate the 5-HTR1E receptor or functionally analogous cognate receptors.

25. A method for identifying small molecule mimetics of Carboxypeptidase E (CPE),the method comprising a virtual screening protocol performed by:(a) defining a pharmacophore model based on the interaction interface between CPE and the 5-HTR1E receptor, wherein the interface is partitioned into distinct binding regions corresponding to specific peptide fragments of the CPE protein;(b) screening a library of small molecule compounds against said pharmacophore model to identify initial hits; and(c) validating said hits by molecular docking into a structural model of the 5- HTR1E receptor binding pocket.

26. The method according to claim 25, wherein the interface is partitioned into distinct binding regions SI, S2, and S3.

27. The method according to claim 25 or 26, wherein the virtual screening protocol isa hybrid protocol that integrates ligand-based and structure-based approaches.

28. A method of synthesizing a small molecule mimetic according to any one of claims1 to 24, the method comprising:Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT (i) performing a Betti reaction or Mannich-type condensation between a substituted aromatic aldehyde or phenol comprising a linker precursor group, a formaldehyde source, and a primary or secondary amine to form an (aminoalkyl)hydroxy-aromatic intermediate;(ii) protecting the amino group of said intermediate;(iii) reacting the protected intermediate with a sulfonyl electrophile to installa sulfonate group (R3) at the phenolic position;(iv) converting the linker precursor group into a reactive coupling moiety;(v) reacting the resulting intermediate with a complementary linkerforming partner to form the bridged pharmacophore; and(vi) optionally deprotecting the amino group to yield the final compound.

29. The method according to claim 28, wherein the linker precursor group is an aldehyde, and wherein:step (iv) comprises maintaining the aldehyde group; andstep (v) comprises condensing said aldehyde with a hydrazine derivative to yield a hydrazone scaffold (Formula I, III, or IV).30.. The method according to claim 28, wherein:step (i) comprises reacting 4-hydroxybenzaldehyde, paraformaldehyde, and an amine selected from the group consisting of ethylamine, dimethylamine, and piperidine;step (iii) comprises reacting the intermediate with p-toluenesulfonyl chloride (TsCl) to install a tosyl group; andstep (v) comprises condensing the intermediate with 4-hydrazinobenzoic acid.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT 31. The method according to claim 28, wherein the linker precursor group is an aldehyde, and wherein:step (iv) comprises oxidizing the aldehyde to a carboxylic acid; andstep (v) comprises coupling said carboxylic acid with an amine to yield an amide scaffold (Formula VI).

32. The method according to claim 28, wherein the linker precursor group is a nitro group (-NO2), and wherein:step (i) utilizes 4-nitrophenol as the substituted phenol:step (iv) comprises reducing the nitro group to an aniline (aromatic amine); andstep (v) comprises reacting said aniline with an isocyanate to yield a urea scaffold (Formula V).

33. The method according to claim 28, wherein the isocyanate in step (v) is generatedin situ or ex situ by reacting tert-butyl 4-aminobenzoate with triphosgene.

34. The method according to claim 33, wherein step (vi) comprises a global deprotection using acid (e.g., trifluoroacetic acid) to simultaneously remove the amino-protecting group and hydrolyze the tert-butyl ester, while preserving the sulfonate group (R3).

35. The method according to claim 28, wherein the linker precursor group is a carboxylic acid (generated via oxidation of an aldehyde), and wherein step (v) comprises:converting the carboxylic acid to a hydroxamic acid;activating the hydroxamic acid to trigger a Lossen rearrangement to generate an isocyanate intermediate; andTitle: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT trapping said isocyanate with an amine to yield a urea scaffold (Formula V).

36. A pharmaceutical composition comprising a therapeutically effective amount of at least one small molecule mimetic as described in any one of claims 1 to 24, or a pharmaceutically acceptable salt, solvate, or prodrug thereof, and a pharmaceutically acceptable carrier, vehicle, or excipient.

37. The pharmaceutical composition according to claim 36, comprising a combinationof two or more distinct small molecule mimetics.

38. The pharmaceutical composition according to claim 36 or 37, wherein the composition is formulated for oral administration and comprises an enteric coating.

39. The pharmaceutical composition according to claim 36 or 37, formulated for intranasal administration or systemic injection to facilitate delivery to the central nervous system.

40. The pharmaceutical composition according to claims 36 or 37, formulated as an intranasal spray, drop, or in-situ gel.

41. The pharmaceutical composition according to claim 36 to 37, formulated as an in- situ gel comprising a thermosensitive or mucoadhesive polymer.

42. The pharmaceutical composition according to any one of claims 36 to 41, whereinthe composition is encapsulated within a colloidal carrier system selected from liposomes, solid lipid nanoparticles, or polymeric nanoparticles.

43. A method for activating the 5-Hydroxytryptamine Receptor 1E (5-HTR1E), comprisingcontacting a cell expressing the 5-HTR1E receptor with at least one small molecule mimetic according to any one of claims 1 to 24 in an effective amount to bind to the receptor and initiate downstream signaling.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT 44. The method according to claim 31, wherein the downstream signaling comprises beta-arrestin recruitment, ERK phosphorylation, or BCL-2 upregulation.

45. The method according to claim 43 or 44, performed in vitro in cellular assays to evaluate receptor kinetics.

46. The method according to claim 43 or 44, performed in vivo to modulate receptor activity in a biological system.

47. The method according to any one of claims 43 to 46, wherein said activating comprises selective recruitment of beta-arrestin.

48. The method according to any one of claims 43 to 47, wherein said activating comprises upregulation of anti-apoptotic proteins such as BCL-2.

49. A system for activating the 5-Hydroxytryptamine Receptor 1E (5-HTR1E) and detecting said activation, comprising:(a) at least one small molecule mimetic according to any one of claims 1 to 24; and(b) a biological substrate comprising cells expressing the 5-HTR1E receptor.

50. The system according to claim 49, further comprising (c) detection reagents configured to quantify downstream signaling events, such as beta-arrestin recruitment or ERK phosphorylation.

51. The system according to claim 50, wherein the detection reagents comprise luciferase reporter substrates, fluorescent biosensors, or antibodies specific for phosphorylated signaling proteins.

52. A method of treating, preventing, or attenuating the progression of a condition associated with 5-HTR1E receptor dysfunction or a condition responsive to 5- HTR1E receptor activation, comprising administering to a subject in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 24.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT 53. The method according to claim 52, wherein the condition is a neurodegenerative disorder selected from Alzheimer’s disease. Parkinson’s disease, or tauopathy.

54. The method according to claim 52, wherein the condition is a peripheral disorder characterized by oxidative stress or mitochondrial dysfunction, such as acute kidney injury (AKI), ischemia-reperfusion injury, or cardiomyopathy.

55. A system for treating or preventing a condition associated with 5-HTR IE receptor dysfunction or for mitigating oxidative stress-induced cytotoxicity in a subject, comprising:(a) a pharmaceutical composition comprising a therapeutically effective amount of one or more small molecule mimetics according to any one of claims 1 to 24; and(b) a delivery device configured to administer said composition to a target tissue within the subject.

56. The method according to claim 52, wherein the condition is Acute Kidney Injury (AKI) and the compound is administered intravenously.

57. The method according to claim 52, wherein the condition is Myocardial Infarctionand the compound is administered systemically.

58. A method for treating or preventing a neurodegenerative condition or preventing neuronal cell death, comprising administering to a subject in need thereof a therapeutically effective amount of one or more small molecule mimetics according to any one of claims 1 to 24.

59. The method according to claim 58. wherein the administration is performed to mitigate oxidative stress-induced cytotoxicity.

60. The method according to claim 58 or 59, wherein the condition treated is Alzheimer's Disease or Parkinson's Disease.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT 61. A system for treating or preventing a neurodegenerative condition or preventing neuronal cell death, comprising:(a) a pharmaceutical composition comprising a therapeutically effective amount of one or more small molecule mimetics according to any one of claims 1 to 24; and(b) a delivery device configured to administer said composition to a subject.

62. The system according to claim 61, wherein the delivery device is an intranasal delivery device configured to bypass the blood-brain barrier and deliver the composition directly to the central nervous system.

63. The system according to claim 62, wherein the intranasal delivery device is a nasal spray pump, a dropper, or a nebulizer.

64. The system according to claim 63, wherein the delivery device is a systemic administration tool selected from a pre-filled syringe, an intravenous infusion bag, or an oral dispenser.

65. The system according to any one of claims 61 to 64, further comprising instructionsfor a dosage regimen to mitigate oxidative stress-induced cytotoxicity.

66. The small molecule according to any one of claims 1 to 24, for use in providing cytoprotection against oxidative stress or neuroexcitotoxicity in both neuronal and non-neuronal tissues.

67. The small molecule according to any one of claims 1 to 24, for use in the treatmentof neuropsychiatric and vascular disorders, including depression, anxiety, and migraine wherein said treatment comprises mitigating oxidative stress and / or mitochondrial dysfunction.

68. The small molecule according to any one of claims 1 to 24, for use in the treatmentof peripheral cytotoxic conditions, including acute kidney injury, myocardial ischemia, and hepatic failure.Title: " Small Molecule Mimetics Of Carboxypeptidase E..."Inventors: William A. GODDARD et al.Docket No.: P3321-PCT 69. The small molecule for use according to any one of claims 66 to 68, wherein the small molecule is configured and administered for the non-invasive activation of neuroprotective signaling pathways regulated by the Carboxypeptidase E protein.

70. The small molecule according to any one of claims 66 to 68, wherein the small molecule is configured and administered to provide a synthetically accessible therapeutic alternative to large recombinant proteins.

71. A kit comprising:(a) a pharmaceutical composition according to any one of claims 36 to 42; and(b) instructions for administering said composition to an individual to treat a condition associated with serotonergic and mitochondrial dysfunction.

72. The method according to claim 52, wherein the subject lacks a direct genetic ortholog of the human 5-HTR1E receptor, and wherein the compound activates a functionally analogous cognate receptor to elicit a cytoprotective effect.

73. A compound according to any one of claims 1 to 24 for use as a pharmacological tool or research reagent for investigating biased signaling pathways in vitro or in vivo.

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