Pan ephb tyrosine kinase forward signaling inhibitors in ephb-associated diseases

Abstract

The present disclosure pertains to a method of treating or preventing a disease in a subject by administering to the subject an inhibitor of an EphB receptor. The present disclosure also pertains to inhibitors of an EphB receptor. The methods and inhibitors may be utilized to treat or prevent various diseases in subjects, such as metabolic disorder, pain, or combinations thereof.

Description

PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049TITLEPan EphB tyrosine kinase forward signaling inhibitors in EphB -associated diseasesCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 684,938, filed on August 20, 2024. The entirety of the aforementioned application is incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under 1R01NS136485-01, awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] A need exists for developing more effective therapeutics for treating or preventing pain and metabolic disorders. Numerous embodiments of the present disclosure aim to address the aforementioned need.SUMMARY

[0004] In some embodiments, the present disclosure pertains to a method of treating or preventing a disease in a subject by administering to the subject an inhibitor of an EphB receptor. Additional embodiments of the present disclosure pertain to inhibitors of an EphB receptor.

[0005] The inhibitors of the present disclosure may include various structures. For instance, in some embodiments, the inhibitors of the present disclosure include, without limitation, one or more of the following structures:PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049derivatives thereof, salts thereof, or combinations thereof.

[0006] The methods and inhibitors of the present disclosure may be utilized to treat or prevent various diseases in subjects. For instance, in some embodiments, the disease includes, without limitation, a metabolic disorder, pain, or combinations thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] A more complete understanding of the subject matter of the present disclosure may be obtained by reference to the following Detailed Description when taken in conjunction with the accompanying Drawings wherein:

[0008] FIG. 1 illustrates a method of treating or preventing a disease in a subject by administering an EphB receptor inhibitor to the subject.

[0009] FIG. 2 illustrates chemical structures of EphA / B tyrosine kinase inhibitors. Targeting EphB tyrosine kinase receptor selectively is challenging due to the high degree of sequence.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0010] FIG. 3 illustrates synthesis of STA-A to STA-F. Reagents: COMF1; (l-Cyano-2-ethoxy- 2-oxoethylidenaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate and DMF; N, N dimethyl formamide.

[0011] FIGS. 4A-4H illustrate the structure-based drug design of the second generation of thienopyridine-based analogs (STA-001 to STA-016). FIG. 4A shows the surface representation for the catalytic binding domain of hEphBl. FIG. 4B shows a design strategy for STA compounds. FIGS. 4C-4D show the surface representation showing CTC to fill in S2 and S3 of hEphBl kinase domain supported with 3D visualization showing dotted hydrogen bonds with T697 and M700. FIGS. 4E-4F show the surface representation showing the potential of STA- 001 to fill in S2 and S3 of hEphBl kinase domain supported with 3D visualization showing dotted hydrogen bonds with E668. FIGS. 4G-4H show the surface representation showing the potential of STA-013 to fill in S1-S3 of hEphBl kinase domain supported with 3D visualization showing dotted hydrogen bonds with M700.

[0012] FIG. 5 illustrates the synthesis of the second generation of thienopyridine-based analogs (STA-001 to STA-016). Reagents: COMU; (l-Cyano-2-ethoxy-2- oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate, DIPEA; N,N-Diisopropylethylamine, DMF; N,N dimethyl formamide; NaH; Sodium hydride, TEA; Triethyl amine.

[0013] FIGS. 6A-6Q illustrate dose-response curves of STA-related analogs against EphBl, EphB2, and EphB4 tyrosine kinase activity using ADP-Glo assay. In vitro protein kinase assay (ADP-Glo Assay) was used, where different concentrations of STA-compounds ranging from 10 pM to 10-3pM were tested for their inhibitory activity in three different kinase domains. IC50 values were calculated based on the residual activity (percent) of the compounds’ ability to inhibit three protein kinases for two independent biological runs.

[0014] FIGS. 7A-7E illustrate that STA-013 selectively inhibits EphBl, EphB2, and EphB4 tyrosine kinases using premier kinome profiling assay. FIG. 7A shows the STA013 structure.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049FIGS. 7B-7D show the IC50 curves of STA013 in EphBl, EphB2, and EphB4 protein kinase activity assays. FIG. 7E shows the kinome profiling tree representing the inhibitory profiles of STA-013 against 140 kinases via premier kinome profiling assay. Residual activity was calculated using two independent biological replicates, represented by mean ± SD.

[0015] FIGS. 8A-8B illustrate that STA-013 inhibits the lipid accumulation after differentiation to adipocytes in the embryonic mouse 3T3-L1 fibroblast. FIG. 8A shows Oil Red O staining for differentiated 3T3-L1 treated with vehicle or STA-013 in a dose-dependent manner, while the nuclei are counterstained blue with Haematoxylin Gill III solution. FIG. 8B shows the quantification of Oil Red O staining sensitivity relative percentage to control (n = 3). Data are mean ± s.e.m.; one-way ANOVA. ***p < 0.001****P < 0.0001.

[0016] FIGS. 9A-9M illustrate that STA-013 modulated the mitochondrial respiration and inhibited fatty acid oxidation. FIG. 9A shows the seahorse XFe24 mitochondria stress test, measured as the OCR by sequential injection of 1 pM oligomycin, 1 pM FCCP, 1 pM rotenone, and 2 pM antimycin A in differentiated 3T3-L1 cells pretreated with 1 pM STA-013 or vehicle (0.1 % DMSO) presented as the parameters of mitochondrial respiration. FIG. 9B shows the basal respiration. FIG. 9C shows the proton leak. FIG. 9D shows the ATP production. FIG. 9E shows the maximal respiration. FIG. 9F shows the spare respiratory capacity. FIG. 9G shows the non- mitochondrial oxygen consumption. FIG. 9H shows the coupling efficiency. FIG. 91 shows the spare respiratory capacity, and n=10 independent biological replicates. FIG. 9J shows the seahorse XFe24 glycolysis stress test in differentiated 3T3-L1 measured as the extracellular acidification rate (ECAR) by sequential injection of 20 mM glucose, 1 pM oligomycin, and 100 mM 2-deoxy-d-glucose (2-DG). FIG. 9K shows quantifying glycolysis and glycolytic capacity in differentiated 3T3-L1 pre-treated with IpM of STA-013 or vehicle-treated cells. FIG. 9L shows the seahorse XFe24 traces of exogenous palmitic acid oxidation measured as the OCR following sequential injection of 200 pM palmitic acid conjugated to BSA or BSA control, 1 pM oligomycin, 1 pM FCCP, 1 pM rotenone, and 2 pM antimycin A. FIG. 9M shows the quantification of exogenous palmitic acid oxidation measured in differentiated 3T3-L1 cellsPCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049 pretreated with 1 pM STA-013 or vehicle (0.1% DMSO) for 30 minutes and n=5 independent biological replicates. Data are mean ± s.e.m.; unpaired two-sided / -test, (ns) non-significant, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0017] FIGS. 10A-10O illustrate STA-013 promoted whole-body weight loss, restored glucose homeostasis, and mitigated insulin resistance in a high-fat diet (HFD)-induced obesity mouse model. FIG. 10A shows a schematic for STA-013 administration to HFD-induced obese mice at 25 mg / kg, i.p. for 6 weeks. FIGS. 10B-10C show that STA-013 significantly reduced whole-body weight for HFD-obese mice compared to vehicle-treated HFD-obese mice. FIGS. 10D-10E show that STA-013 restored glucose homeostasis for HFD-obese mice significantly compared to vehicle-treated HFD-obese mice using the intraperitoneal glucose tolerance test (IPGTT). FIGS. 10F-10G show that STA-013 significantly mitigated insulin resistance for HFD-obese mice compared to vehicle-treated HFD-obese mice using an intraperitoneal insulin tolerance test (1P1TT). FIGS. 10H-10I show that STA-013 reduced fat-mass while preserving the lean muscle mass in HFD-obese mice compared to vehicle-treated HFD-obese mice using NMR (Minispec LF110®, Bruker) n=(8-10). FIGS. 10J-10K show that STA-013 induced intrascapular brown adipose tissue (BAT)-specific thermogenesis through the utilization of a high-resolution FLIR T560® infra-red thermal imaging camera (FLIR® Systems Inc.). FIGS. 10L-10M show that STA-013 decreased total cholesterol and free fatty acids in the plasma for HFD-obese mice significantly compared to vehicle-treated HFD-obese mice (n=5). FIGS. 10N-100 show that STA-013 decreased fat deposition in livers from HFD-obese mice significantly, compared to vehicle-treated HFD-obese mice using oil red staining (n=4). Data are mean ± s.e.m.; unpaired two-sided Z-test. (ns) non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0018] FIGS. 11A-11D illustrate in vivo pharmacokinetics (PK) profiling of STA-013. FIG. 11A shows plasma concentration in C57B6 / J mice (n=3) after IV bolus of STA-013 (25mg / kg). FIG. 11B shows PK Parameters after IV Bolus. FIG. 11C shows plasma concentration in C57B6 / J mice (n=5) after IP administration of STA-013 (25mg / kg). FIG. 11D shows PK Parameters after IP administration. Data are presented as mean ± SD.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0019] FIGS. 12A-12J illustrate metabolic profiling of STA-013-treated DIO mice using an indirect calorimetry (Comprehensive Lab Animal Monitoring System, CLAMS®) in dark and light cycles for three days (n=4 / group). FIGS. 12A-12B show VO2. FIGS. 12C-12D show VCO2. FIGS. 12E-12F show RER. FIGS. 12G-12H show the energy expenditure (EE). FIGS. 12I-12J show the food intake. Data is presented as mean ± SEM; statistical significance was analyzed using an unpaired two-sided t-test. Data in panels (FIGS. 12G-12H) were analyzed by repeated measures linear mixed models using body lean mass and 20% fat mass as a covariate, where (ns) non- significant, **P < 0.01, ***P < 0.001, and ****P<0.0001.

[0020] FIGS. 13A-13H illustrate Western blotting analysis for isolated livers and brown adipose tissue (BAT) from vehicle and STA-013 treated HFD-obese mice (n=5). FIG. 13A shows a schematic for STA-013 administration to HFD-induced obese mice at 25 mg / kg, i.p for 6 weeks. FIGS. 13B-13E show the Western blots and quantification of p-EphB / EphB, p-AKT / AKT, and InsR-p / Actin for isolated BAT from vehicle and STA-013 treated HFD-obese mice. FIGS. 13F- 13H show the Western blots and quantification of p-EphB / EphB, p-AKT / AKT, and InsR-p / Actin for isolated livers from vehicle and STA-013 treated HFD-obese mice. STA-013 showed a significant reduction in the phosphorylation of EphB signaling associated with a significant increase in the p-AKT, indicating activation of insulin signaling, compared to vehicle-treated cohorts. This was associated with an increase in InsR concentration to suggest the reversal effect of STA-013 for insulin receptor degradation to restore glucose homeostasis and insulin sensitivity, compared to vehicle. Data are mean ± s.e.m.; unpaired two-sided t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

[0021] FIGS. 14A-14D illustrate transcriptomic signature for isolated livers from STA-013 and vehicle-treated HFD-obese mice (n=3). FIG. 14A shows the co-expression Venn diagram, which presents the number of genes uniquely expressed with the overlapping regions, showing the number of genes co-expressed within the vehicle and STA-013 treated groups. FIG. 14B shows the gene ontology (GO) enrichment analysis results for isolated livers from the vehicle, and STA- 013 treated HFD-obese mice, where the most significant 30 GO Terms were selected for display.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049FIG. 14C shows the up-regulated GO-enriched pathways involved in glucose and carbohydrate metabolism, insulin regulation, and response to leptin. FIG. 14D shows a heat map showing up regulated gene expression from isolated livers from the vehicle, and STA-013 treated HFD- obese mice (Relative normalized gene expression FPKM) *P < 0.05.

[0022] FIGS. 15 A- 151 provide experimental results indicating that STA-013 represents a novel therapeutic strategy for diabetic cardiomyopathy. FIG. 15A provides the STA-013 structure. FIG. 15B provides IC50 curves of STA-013 in EphBl, EphB2, and EphB4 protein kinase activity assays. FIG. 15C provides representative echocardiography M-mode images for diabetic cardiomyopathy vehicle and STA-013 treated mice groups. FIG. 15D provides heart weight / body weight ratio. FIG. 15E provides a percentage of ejection fraction. FIG. 15F provides a percentage of fractional shortening. FIG. 15G provides representative images of Masson's trichrome staining of heart sections showing fibrotic tissue in diabetic cardiomyopathy vehicle groups compared to STA-013 treated group. FIG. 15H provides a percentage of collagen deposition positive tissue. FIG. 151 provides a schematic overview of the effect of STA013 as an EphB tyrosine kinase inhibitor.DETAILED DESCRIPTION

[0023] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0024] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, citedPCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049 in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0025] Pain and metabolic disorders present numerous public health concerns. However, current methods and therapeutics for treating pain and metabolic disorders have numerous limitations.

[0026] For instance, although opioids are commonly used for chronic pain treatment, they are only partially efficacious for short-term pain management. Moreover, the response to the long-term use of opioids is widely variable. Despite the magnitude of the pain epidemic and the opioid crisis, there has been little progress in the development of non-opioid alternative therapies.

[0027] For instance, peripheral neuropathic pain (PNP), which is defined by neuralgia and painful polyneuropathy, is a highly prevalent type of pain that results in significant morbidity and disability. PNP is an associated manifestation for a series of different diseases, such as autoimmune diseases, diabetes, different types of cancers, neurofibromatosis, viral infections and toxins exposure.

[0028] Current therapies for treatment of PNP include over-the-counter (OTC) drugs, such as ibuprofen and acetaminophen, in addition to prescription pain medications including opioids, anticonvulsants and antidepressants. However, these therapies are seldom effective. For instance, opioids, which as outlined above are ineffective for treatment of chronic pain, require dose escalation, which further contributes to the ongoing opioid crisis.

[0029] Similarly, obesity is a chronic disease with a fast-growing prevalence of up to 42.4%, which has presented a significant financial burden to the U.S. healthcare system. Additionally, current estimates highlight the global epidemic status of obesity at more than about 57%. The significant consequences of obesity are associated with the progression of various chronic disorders such as hyperglycemia, type 2 diabetes (T2D), dyslipidemia, cardiovascular disease, and heart failure.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049Obesity is also associated with chronic pain, and peripheral neuropathy is a common sequelae of T2DM.

[0030] Obesity usually occurs due to higher caloric intake than are burned by exercise or normal daily activities. This in turn leads to an increased body mass index and abnormal or massive fat accumulation.

[0031] Obesity is also associated with leptin resistance, which in turn leads to fat accumulation in non-adipose tissues like the pancreas and skeletal muscle. Leptin resistance also leads to increased fatty acid oxidation, free fatty acid production, and induction of peripheral insulin resistance that in turn leads to lipotoxicity, diminished glucose oxidation and the development of T2D.

[0032] Obesity and T2D are independent risk factors for cardiovascular disease and heart failure that can lead to an inability to respond to normal changes in fuel availability. The inability of the heart to appropriately utilize glucose, heavy reliance on fatty acids for energy production, and oxidative stress lead to cardiometabolic disorders and myocardial infarction.

[0033] The current non-surgical therapeutic interventions for treatment of obesity include: (1) dietary caloric restrictions; (2) administration of Orlistat that can inhibit pancreatic lipases to prevent absorption of ingested fats up to 32% to be excreted in the feces leading to steatorrhea; (3) administration of a combination of Naltrexone (opioid-based antagonist) and bupropion (antidepressant) to suppress appetite and hunger centers in the hypothalamus with severe adverse effects; (4) administration of centrally acting serotonin agonists and combinations of centrally acting appetite suppressing and antiepileptic drugs; and (5) glucagon-like peptide-1 (GLP-1) receptor agonists.

[0034] Recently, GLP-1 receptor agonists, featuring Liraglutide, are believed to be the new blockbusters targeting obesity. Such GLP-1 receptor agonists show significant potential to induce dose-dependent weight loss with patients living with T2D. However, there are different reports about the adverse effects associated with Liraglutide (Saxenda®) administration, such as chronic intestinal pseudo-obstruction (CIP) and loss of lean mass.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0035] As such, an urgent need exists for developing more effective therapeutics for treating or preventing pain and metabolic disorders. For instance, there is an urgent need for developing novel non-opioid, and non-addicting therapies that are effective in chronic pain management. Similarly, there is a need to develop non-surgical obesity treatments with novel therapeutic strategies while tackling integrated molecular targets contributing to the progression of obesity, concurrent pain in obesity and T2DM, and associated metabolic disorders. Numerous embodiments of the present disclosure aim to address the aforementioned needs.

[0036] In some embodiments illustrated in FIG. 1, the present disclosure pertains to a method of treating or preventing a disease in a subject by administering to the subject an inhibitor of an EphB receptor (steps 10 and 12). Additional embodiments of the present disclosure pertain to inhibitors of an EphB receptor. In some embodiments, the inhibitors of the present disclosure may be suitable for use in treating or preventing various diseases in various subjects. As set forth in more detail herein, the methods and inhibitors of the present disclosure can have numerous embodiments.

[0037] Treatment or prevention of diseases

[0038] The methods and inhibitors of the present disclosure may be utilized to treat or prevent various diseases in subjects. For instance, in some embodiments, the disease includes, without limitation, a metabolic disorder, pain, or combinations thereof.

[0039] In some embodiments, the disease to be treated or prevented includes a metabolic disorder. In some embodiments, the metabolic disorder includes, without limitation, obesity, insulin resistance, metabolic syndrome, hyperglycemia, diabetes, type 2 diabetes (T2D), dyslipidemia, diabetic cardiomyopathy, or combinations thereof. In some embodiments, the metabolic disorder includes obesity.

[0040] In some embodiments, the disease to be treated or prevented includes pain. In some embodiments, the pain includes, without limitation, chronic pain, acute pain, neuropathic pain, peripheral neuropathic pain (PNP), chemotherapy-induced neuropathic pain, or combinations thereof. In some embodiments, the pain includes PNP.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0041] Subjects

[0042] The methods and inhibitors of the present disclosure may be utilized to treat or prevent diseases in various subjects. For instance, in some embodiments, the subject is a human being. In some embodiments, the subject is a non-human mammal. In some embodiments, the non-human mammal includes, without limitation, a horse, a rabbit, a mouse, a rat, a pig, a sheep, a cow, a dog, or a cat. In some embodiments, the non-human mammal is a domestic animal, such as a dog or a cat.

[0043] In some embodiments, the subject is suffering from a disease. In some embodiments, the subject is vulnerable to a disease. In some embodiments, the subject is a metabolically unhealthy and obese subject.

[0044] Administration of inhibitors

[0045] The methods of the present disclosure may administer the inhibitors of the present disclosure to subjects in various manners. For instance, in some embodiments, the administration occurs by a method that includes, without limitation, intravenous administration, subcutaneous administration, transdermal administration, topical administration, intraarterial administration, intrathecal administration, intracranial administration, intraperitoneal administration, intraspinal administration, intranasal administration, intraocular administration, oral administration, intratumor administration, or combinations thereof. In some embodiments, the administration occurs by intraperitoneal administration.

[0046] Targeting of EphB receptors

[0047] The inhibitors of the present disclosure may target various EphB receptors. For instance, in some embodiments, the targeted EphB receptor includes, without limitation, EphBl, EphB2, EphB4, EphB5, or combinations thereof. In some embodiments, the EphB receptor includes, without limitation, EphBl, EphB2, EphB4, or combinations thereof. In some embodiments, the EphB receptor includes an EphB 1 receptor.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0048] Inhibitors

[0049] The inhibitors of the present disclosure may include various structures. For instance, in some embodiments, the inhibitors of the present disclosure include, without limitation, one or more of the following structures:derivatives thereof, salts thereof, or combinations thereof.

[0050] In some embodiments, the salt forms of the compounds of the present disclosure include, without limitation, chloride, sulfate, citrate, maleate, or combinations thereof. In some embodiments, each of Ri, R2, R3, R4 and R5 in the aforementioned structures independently includes, without limitation, aromatic groups, cyclic groups, heterocyclic groups, polycyclic groups, polyaromatic groups, phenyl groups, pyridyl groups, 2-pyridyl groups, 3-pyridyl groups, 4-pyridyl groups, morpholine groups, piperidine groups, pyrrolidine groups, oxazolidine groups, thiazolidine groups, n-methyl piperazine, methanamine groups, (lH-Indol-4-yl) methanamine,PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049(lH-Indol-5-yl) methanamine, (lH-Indol-6-yl) methanamine, derivatives thereof, salts thereof, or combinations thereof.

[0051] In some embodiments, the inhibitors of the present disclosure include the following structure:

[0052] In some embodiments, Rs in the aforementioned structure includes, without limitation, phenyl groups, 2-pyridyl groups, 3-pyridyl groups, 4-pyridyl groups, morpholine groups, piperidine groups, n-methyl piperazine, derivatives thereof, salts thereof, or combinations thereof.

[0053] In some embodiments, the inhibitors of the present disclosure include the following structure:

[0054] In some embodiments, the inhibitors of the present disclosure include the following structure:PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0055] In some embodiments, the inhibitors of the present disclosure include, without limitation, one or more of the following structures:PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049derivatives thereof, salts thereof, or combinations thereof.

[0056] In some embodiments, the inhibitors of the present disclosure include the following structure:

[0057] In some embodiments, the inhibitors of the present disclosure include the following structure:PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0058] In some embodiments, the inhibitors of the present disclosure include, without limitation, one or more of the following structures:PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049(STA-021) (STA-022) derivatives thereof, salts thereof, or combinations thereof.

[0059] In some embodiments, the inhibitors of the present disclosure include the following structure:

[0060] In some embodiments, the inhibitors of the present disclosure include, without limitation, one or more of the following structures:PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049(HBA-023) derivatives thereof, salts thereof, or combinations thereof.

[0061] In some embodiments, the inhibitors of the present disclosure include the following structure:

[0062] In some embodiments, the inhibitors of the present disclosure include, without limitation, one or more of the following structures;(HBA-026)PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049(HBA-028)(HBA-029)(HBA-030)PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049(HBA-031)(HBA-032)(HBA-033)(HBA-034)PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049(HBA-035) derivatives thereof, salts thereof, or combinations thereof.

[0063] In some embodiments, the inhibitors of the present disclosure are in a pharmaceutically acceptable composition. In some embodiments, the pharmaceutically acceptable composition includes one or more physiologically acceptable carriers or excipients. In some embodiments, the pharmaceutically acceptable composition can also include formulation materials for modifying, maintaining, or preserving various conditions, including pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, and / or adsorption or penetration of the compounds. Suitable formulation materials include, without limitation, amino acids (e.g., glycine), antimicrobials, antioxidants (e.g., ascorbic acid), buffers (e.g., Tris-HCl), bulking agents (e.g., mannitol and glycine), chelating agents (e.g., EDTA), complexing agents (e.g., hydroxypropyl-beta-cyclodextrin), or combinations thereof.

[0064] Additional Embodiments

[0065] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way.

[0066] Example 1. Discovery of Pan-EphB Tyrosine Kinase Inhibitor for Metabolic Syndrome Sparing EphB3 Signaling in micePCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0067] The global prevalence of metabolic syndrome has created one of the most pressing public health dilemmas and significant financial burden to the healthcare system. Despite the surge of glucagon-like peptide- 1 agonists, recent studies showed that 40% of body weight loss is due to lean mass loss, raising the concern about induction of musculoskeletal arthritis. Therefore, there is an urgent need to develop novel therapeutic strategies to tackle the progression of metabolic disorders with minimal adverse effects.

[0068] EphB singaling had been validated in the progression of metabolic syndrome. Applicant leveraged the resolved X-ray crystal structure of the hEphBl kinase domain to introduce thienopyridine-based analogs (termed STA analogs) that showed potential pan-inhibitory profiles for EphBl, EphB2, and EphB4 with no inhibition for EphB3 tyrosine kinase singaling pathways. STA-013 inhibited EphBl, EphB2, and EphB4 tyrosine kinases selectively, as validated by a premier kinase profiling assay against 140 protein kinases, with no inhibitory profile against EphB3 tyrosine kinase. Systemic injections of STA-013 resulted in weight loss with significant reduction of fat mass and preservation of lean mass. This was associated with significant improvement in glucose homeostasis, mitigation of insulin resistance, and inhibition of fatty liver in high-fat diet-induced obese mice. This was allied with modulation of respiratory exchange rates during the dark cycle and brown fat thermogenesis, with no change in food intake. Additionally, STA-013 administration resulted in inhibiting the EphB phosphorylated signal, coupled with increased p-AKT / AKT signaling, to suggest insulin signaling activation. STA-013 showed an elevated signal for InsR-p, which reversed the effect of insulin on InsR degradation, allowing for the restoration of glucose homeostasis and mitigating insulin resistance.

[0069] Example 1,1. Introduction

[0070] Metabolic syndrome (MS) is associated with a cluster of cardiometabolic pathological events including obesity, insulin resistance, type-2 diabetes, cardiovascular diseases, dyslipidaemia, and non-alcoholic steatohepatitis (NASH). Additionally, obesity-induced insulin resistance stimulates pancreatic [3-islcts to secrete more insulin to restore glucose homeostasis, leading to hyperinsulinemia. Insulin signaling is essential for glucose homeostasis, wherePCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049 circulating insulin levels decline in diet-induced and genetically obese mice models when treated with diazoxide or streptozotocin.

[0071] The current non-surgical therapeutic interventions are limited to (1) dietary caloric restrictions, (2) administration of Orlistat that can inhibit pancreatic lipases to prevent absorption of ingested fats up to 32% to be excreted in the faeces leading to steatorrhea, (3) administration of a combination of Naltrexone (opioid-based antagonist) and Bupropion (antidepressant) to suppress appetite and hunger centers in the hypothalamus with severe adverse effects, (4) administration of centrally acting serotonin agonists and combinations of centrally acting appetite suppressing and antiepileptic drug, and (5) agonists of glucagon-like peptide- 1 (GLP-l). Although glucagon-like peptide- 1 (GLP-l) agonists continue their surge in popularity, recent studies showed a significant decrease in lean muscle mass up to 40% and induction of chronic intestinal pseudo-obstruction, increased risk of gastrointestinal disorders, arthritic disorders, interstitial nephritis, and drug- induced pancreatitis. Therefore, there is an urgent need for additional targeted therapies and novel therapeutic strategies for obesity and associated complications with lower risk factors and adverse effects. The large family of Eph (erythropoietin-producing hepatocellular carcinoma) receptor tyrosine kinases is validated in the progression of multiple disorders, including Alzheimer’s disease, neuropathic pain, opioid use disorders, malignancies, fibrotic diseases, and viral infections. EphB l / ephrin-B2 signaling is involved in the progression and development of obesity- associated colorectal tumors. EphBl was initially identified by genome- wide association studies (GWAS) for obesity-related loci in mice and humans. EphB2 receptor tyrosine kinase showed promotion for hepatic fibrosis. Also, Knockdown of Ephb2 in hepatocytes ameliorated inflammation and fibrosis in NASH mouse model. Additionally, genetic and pharmacological modulation of EphB4 improved insulin resistance and glucose intolerance in obese mice. Hepatic overexpression of EphB4 decreased the expression levels of insulin receptor (InsR) and increased hepatic and systemic insulin resistance in vivo, while EphB4 liver- specific knockout mice (EphB4 LKO) improved insulin resistance and glucose intolerance with no effect on the whole-body weight. This validates that EphB4 is critical for insulin-induced liver InsR degradation and the role of the EphB4 tyrosine kinase signaling pathway in insulin resistance and hyperglycemia.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049Collectively, this can suggest that targeting EphB singaling can offer plausible therapeutic strategy to alleviate metabolic syndrome including insulin resistance and liver fibrosis. The EphB receptors have highly conserved intracellular tyrosine kinase catalytic domain sharing homology up to 87%.

[0072] Targeting EphB tyrosine kinase receptor selectively is challenging due to the high degree of sequence structural conservation among EphBl, EphB2, and EphB4 as well as EphA4 tyrosine kinases in subdomain V of the catalytic binding domain. The literature described different multikinase inhibitors including Eph A / B receptors (FIG. 2). For instance, 3-[4-amino-3-(3-chloro-4- fluorophenyl) thieno [3,2-c] pyridin-7-yl] -benzene sulfonamide-based analogs showed EphB4, VEGFR2, and Tie-2 inhibitory profile. Additionally, 3,5-bis substituted anilinopyrimidines-based analogs showed EphB4 inhibitory profiles without comprehensive kinome profiling assays. Similarly, other small molecules showed multi-kinase targeting not limited to EphB tyrosine kinase signaling, such as ALW-II-41-27, Tesevatinib (XL-7647), and NVP-BHG712. Interestingly, ALW-11-49-7 is claimed to be selective EphB2; however, it was co-crystallized with EphA2 (PDB ID: 3dqz) and EphA7 (PDB ID: 3dko). UniPR1447 was reported to target Eph- ephrin interactions, especially EphA and EphB subfamilies. Further, an irreversible and specific inhibitor of the EphB3 kinase domain that covalently binds to the Cys717 residue, which is not present in the other Eph receptors, has been reported.

[0073] Recently, a study recruited DNA-encoded chemistry technology to introduce potent pan- ephrin receptor kinase inhibitors while targeting other kinases such as; SRC, FGR, YES1, and BLK. Therefore, the objectives were tailored to design and synthesize potent and selective pan- EphB tyrosine kinase inhibitors without affecting EphB3 signaling and assess whether the inhibitors can mitigate the progression of complications associated with metabolic syndrome.

[0074] Example 1,2, Structure -based drug design of the first generation of thienopyridine-based analogs (STA-A to STA-F)

[0075] Applicant recruited thienopyridine ring inspired by 3-[4-amino-3-(3-chloro-4- fluorophenyl) thieno [3,2-c] pyridin-7-yl] -benzene sulfonamide-based analogs, as shown in FIG.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-0492. Applicant probed different heteroaryl groups, not related to tetracycline, to be called as STA analogs.

[0076] Example 1,3. Synthesis of STA- A to STA-F

[0077] Initially, Applicant synthesized the STA-A to STA-F analogs while probing different heteroaryl functional groups, starting with thieno[2,3-b]pyridine-2-carboxylic acid to undergo amide coupling with different substituted amino heteroaryl based compounds in the presence of 1- Cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethylamino-morpholino-carbenium hexafluorophosphate (COMU), A,A-Diisopropylethylamine (DIPEA), and dimethylformamide at 0°C to room temperature for 24 h (FIG. 3).

[0078] Example 1,4, Testing STA-A to STA-F analogs against EphB tyrosine kinases

[0079] Next, Applicant tested STA-A to STA-F analogs against EphB 1-4 using ADP-Glo kinase chemiluminescent assay for screening the pan-activity for the synthesized analogs against EphBl, EphB2, EphB3, and EphB4. Based on the design, the initial synthesized analogs (STA-A to STA- F) showed unacceptable inhibitory profiles at IC50 > 100 pM (Table 1).Table 1. IC50 profiling of STA-A to STA-F analogs against EphBl, EphB2, EphB3, and EphB4 tyrosine kinase activity using ADP-Glo assay.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0080] Example 1,5. Structure-based drug design of the second generation of thienopyridine-based analogs (STA-001 to STA-016)

[0081] The first generation of STA-related analogs did not show any activities against EphBl-4 tyrosine kinases. Therefore, Applicant leveraged the resolved X-ray crystal structure of the hEphB 1 kinase domain bound to CTC (PDB ID: 6UMW), where CTC showed potential occupancy for hinge binding domain (S2) and DFG gatekeeper motif (S3) sub-pockets oriented towards E668 and F763 (FIGS. 4A-4D).

[0082] This prompted Applicant to extend the second generation of thienopyridine-based analogs at one carbon distance coupled with chain extension. Applicant pursued molecular simulations and a rational-based drug design approach against the hEphB 1 catalytic binding domain using the Open Eye molecular modeling package. The whole library of thienopyridine-based analogs underwent energy minimization using MMFF94 force field, followed by generating multiconformers using OMEGA application. The entire energy minimized library was docked to the prepared receptors of interest (PDB ID: 6UMW) using FRED application to generate a physical property (AG) reflecting the predicted energy profile of ligand-receptor complex. STA-001 showed hydrophobic -hydrophobic interactions with comparable occupancy for the catalytic domain, compared to CTC, while filling the hinge binding domain (S2) and DFG gatekeeper motif (S3) sub-pockets oriented towards E668 with hydrogen bonding (FIGS. 4E-4F). However, Applicant extended the side chain for STA-013 to show hydrophobic-hydrophobic interactions with better occupancy for the catalytic domain and hydrogen bonding with M700, compared to CTC, while fdling filling SI, hinge, and DFG sub-pockets (FIGS. 4G-4H). Surprisingly, this also resulted in shifting the binding mode to SI sub-pocket.

[0083] Example 1,6. Synthesis of Pan-EphB tyrosine kinase inhibitors

[0084] Applicant synthesized the first generation of STA-related analogs adopting chain extension strategy, starting with thieno[2,3-b] pyridine-2-carboxylic acid to undergo amide coupling with (lH-Indol-4-yl) or (lH-Indol-5-yl) methanamine in the presence of (COMF1), N,N-PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049Diisopropylethylamine (DIPEA), and dimethylformamide (DMF) at 0°C to room temperature for 24 h. Next, Applicant optimized the synthetic route to yield the second generation of synthesized analogs via a hydrophobic extension to fill in the SI, hinge region, and DFG sub-pockets of the EphB catalytic binding domain. Applicant validated the synthetic route via two steps: (1) reacting thieno[2,3-b] pyridine-2-carboxylic acid along with (lH-Indol-4-yl) or (lH-Indol-5-yl) methanamine, (2) followed by coupling along with different functional moieties, such as; phenyl; 2-pyridyl; 3-pyridyl; 4-pyridyl; morpholine; piperidine, and n-methyl piperazine as acid chloride(s) in the presence of sodium hydride in basic environment under inert conditions in considerable yields (FIG. 5). Collectively, Applicant synthesized 16 STA-related analogs for further screening as pan-EphB tyrosine kinase inhibitors.

[0085] Example 1,7, Pan-EphB tyrosine kinase profiling of STA-related analogs

[0086] Next, Applicant tested STA-related analogs against EphB 1-4 using ADP-Glo kinase chemiluminescent assay for screening the pan-activity for the synthesized analogs against EphBl, EphB2, EphB3, and EphB4. STA-related analogs showed promising inhibitory profiles against EphB l , EphB2, and EphB4 tyrosine kinases (Table 2 and (FIGS. 6A-6Q). Additionally, the CLogP (lipophilicity profile) and CNSMPO (CNS multi-parameter optimization) scoring profiles of the analogs (Table 2) established their potential in treating pain.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049Table 2. IC50 profiling, ClogP (lipophilicity profile), and CNS MPO Scores (CNS multi-parameter optimization) of the second generation of thicnopyridinc-bascd analogs (STA-001 to STA-22) against EphBl, EphB2, EphB3, and EphB4 tyrosine kinase activity using ADP-Glo assay. Residual activity was calculated using two independent biological replicates, represented by mean ± SEM.

[0087] The structural activity relationship (SAR) analysis showed that substitution of (IH-Indol- yl) methanamine had better potency in the following order: (lH-Indol-4-yl) methanamine (STA- 009, EphBl (IC50 = 11.19+0.60 pM), EphB2 (IC50 = 11.46+1.35 pM), and EphB4 (IC50 = 12.48+0.23 pM)) > (lH-Indol-5-yl) methanamine (STA-001, EphBl (IC50 > 100 pM), EphB2 (IC50 > 100 pM), and EphB4 (IC50 > 100 pM). Eventually, this was sustained along with the nature of substituents, where the phenyl substituent improved the potency against EphBl, EphB2, and EphB4 tyrosine kinase activities Next, Applicant showed the selectivity of STA-013 (concentration, 5 pM) to inhibit EphBl (54%), EphB2 (42%), EphB4 (52%), SGK1 (47%), and PKBb (43%) without affecting the rest of 140 kinases via kinome profiling assay (FIG. 7E).PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0088] Example 1,8. In vitro phenotypic screening of STA-013 in the embryonic mouse 3T3-L1 fibroblast.

[0089] To evaluate the impact of STA-013 on adipocyte differentiation, embryonic mouse 3T3- L1 fibroblasts were treated with varying concentrations of STA-013 and assessed using Oil Red O staining (Sigma-Aldrich, 01391). STA-013 significantly inhibited lipid accumulation in a dosedependent manner when compared to vehicle-treated controls (FIGS. 8A-B).

[0090] Mitochondrial function was assessed using the Seahorse XF Cell Mito Stress Test. The pretreatment with STA-013 resulted in a significant reduction in basal respiration, proton leak, ATP production, and non-mitochondrial oxygen consumption. Conversely, maximal respiration and spare respiratory capacity were increased in STA-013-treated cells relative to controls (FIGS. 9A- I). These changes may reflect a diminished energetic demand or a metabolic shift away from oxidative phosphorylation under basal conditions. Glycolytic function was evaluated using the Seahorse XF Glycolysis Stress Test. Differentiated 3T3-L1 cells pre-treated with 1 pM STA-013 exhibited a significant increase in glycolysis and glycolytic capacity compared to vehicle-treated cells, indicating a shift toward glycolytic metabolism (FIGS. 9J-K). Additionally, to assess fatty acid oxidation, oxygen consumption rate (OCR) was measured following palmitic acid administration using the Seahorse XF palmitate oxidation test. STA-013 pre-treatment (1 pM) significantly reduced palmitate oxidation in differentiated 3T3-L1 cells compared to controls (FIGS. 9L-M).

[0091] Example 1,9. STA-013 promoted weight loss and improved glucose clearance and insulin sensitivity in obese mice induced by a high-fat diet (HFD)

[0092] Next, Applicant evaluated the therapeutic potential of STA-013 as a potent and selective pan-EphB tyrosine kinase inhibitor to mitigate metabolic syndrome. C57BL / 6J mice (6-8 weeks old) were fed a high-fat diet (HFD) for 10 weeks to generate an HFD-obese mouse model. This in vivo model was established to induce obesity, glucose intolerance, hepatic steatosis, and dyslipidaemia as well as investigation of therapeutics preclinically. STA-013 was administeredPCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049 intraperitoneally at three dose levels (12.5, 25 and 50 mg / kg / day, i.p.) in the HFD-induced obesity C57BL / 6J mouse model (FIG. 10A). HFD-obese mice treated with 12.5 mg / kg of STA-013 did not show any significant reduction in body weight over the time of administration. However, STA- 013-treated groups (25 mg / kg / day (i.p.) showed a significant reduction in whole -body weight (FIGS. 10B-C), improved glucose clearance in the IP glucose tolerance test (IPGTT) (FIGS. 10D- E), and insulin sensitivity in the IP insulin tolerance test (IPITT) (FIGS. 10F-G), compared to the vehicle-treated group. Interestingly, STA-013-treated HFD-obese mice showed a significant reduction in the fat mass while preserving the lean muscle mass (FIGS. 10H-I). Interestingly, STA-013-treated HFD obese mice induced interscapular brown adipose tissue (BAT)-specific thcrmogcncsis (FIGS. 10J-K). This was associated with a significant decrease in total cholesterol and free fatty acid in the plasma (FIGS. 10L-M). In addition, Applicant harvested the livers of vehicle and STA-013 HFD-obese mice to be sliced and stained using Oil Red O staining to show a significant reduction in the deposition of fats for STA-013 treated livers, compared to vehicle cohorts (FIGS. 10N-O). Regarding the 50 mg / kg-dose level, STA-013-treated HFD-obese mice also showed significant reduction in whole-body weight associated with reduction in the fat mass (%) as well as increase in the lean mass (%), improved glucose clearance, and restored insulin sensitivity. This was supplemented by conducting in vivo pharmacokinetics studies for intravenous (IV) and IP administration of 25 mg / kg of STA-013. After IV bolus injection, STA-013 showed a more than 10-fold decline in plasma concentration from initial values of 1404 ± 368 ng / mL at 5 min to 115 ±14 ng / mL after 4 h. The terminal half-life at the low concentrations measured up to 24h was estimated at 16.10 ± 4.80 h (FIGS. 11A-B). After IP administration, systemic absorption was rapid with a Cmax of 1126 ± 328 ng / mL occurring at 21 min (FIGS. 11C-D). The concentration-time course after the peak was similar as seen after IV bolus injection and plasma concentrations after 12 h (9.20 ± 4.20 ng / mL) had declined to less than 1% of the peak concentrations. Noncompartmental analysis of the plasma AUC indicated that after a single IV or IP administration only about 10.5% or 4.2%, respectively, of the dose remained in the body after the terminal sampling time. Bioavailability (F) after IP administration was 0.75 as calculated from the dose and the total AUC values after either route of administration.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0093] Example 1.10. In vivo monitoring for vehicle and STA-013-treated DIO mice in energy expenditure chambers

[0094] Importantly, Applicant assessed if the STA013-induced weight loss is due to reduced food intake and / or elevated metabolic rates in DIO mice using metabolic cages (Comprehensive Lab Animal Monitoring System, CLAMS®), compared to vehicle-treated DIO mice for six days. STA- 013-treated HFD mice significantly increased oxygen (VO2) consumption and carbon dioxide (VCO2) production in the light cycle, compared to vehicle-treated DIO mice (FIGS. 12A-D). This was associated with decreased profile in the respiratory exchange rates (RER) during the dark cycle, compared to vehicle-treated DIO mice (FIGS. 12E-F). There was significant decrease in the total energy expenditure (EE) for STA-013 treated HFD mice, compared to the vehicle treated mice in both dark and light cycles (FIGS. 12G-H). This was associated with no change in the food intake to suggest that STA-013-induced weight loss is due to elevated metabolic rates (FIGS. 121- J).

[0095] Example LI L STA-013 improved insulin signaling in isolated brown adipose tissues and livers of HFD-induced obese mice

[0096] To investigate the role of EphB phosphorylation signaling and insulin signaling, liver and brown adipose tissue (BAT) were harvested after 10 weeks of STA-013 administration for immunoblotting analyses. Applicant found that STA-013 at 25 mg / kg inhibited phosphorylation of EphB tyrosine kinase forward signaling in both the liver and BAT. Importantly, this was associated with a significant increase in the p-AKT / AKT signaling, which suggests the activation of insulin signaling in BAT and liver of STA-013-treated HFD obese mice. STA-013-treated mice showed a significant increase in InsR-0 protein levels compared to vehicle in BAT and liver lysates (FIGS. 13A-13H). This supports the ability of STA-013 to reverse the effect of insulin on InsR degradation and enhance insulin signaling.

[0097] Example 1,12. Hepatic transcriptomic signature of STA-013- treated HFD-induced obese micePCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0098] Applicant performed the RNA-sequencing (RNA-seq) analysis of isolated livers from STA-013 and vehicle-treated HFD-induced obese mice to understand the differentially expressed genes and associated regulated pathways. RNA-seq analysis demonstrated that differentially expressed genes were modulated in the STA-013-treated livers, compared with vehicle-treated livers (FIGS. 14A-B). Gene ontology (GO) analysis for STA-013 showed enrichment of upregulated genes, including several carbohydrate / glucose metabolism pathways, insulin regulation, and response to leptin (FIG. 14C). Interestingly, the RNA-Seq showed that STA-013 upregulated Irs2, Lepr, Oxoctl, Stat3, and Pfkm as well as down-regulation of Mgll, Fads3, Fasn, CD36, Acatl, Acaalb, Aldop, and mToR (FIG. 14D). This was further validated by KEGG pathway mapping, which showed the enrichment analysis for several molecular pathways, including fatty acid elongation, fatty acid degradation, and biosynthesis of unsaturated fatty acids, PPAR signaling pathways, Glutathione metabolism, and MAPK signaling pathways.

[0099] Example 1.13. IC50 profiling and CNS-MPO scores for HBA-013 to HBA-035PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049Table 3. IC50 profiling and CNS MPO Scores of HBA-013 to HBA-035.

[0100] Table 3 shows the binding affinity profiles for HBA-related analogs against EphB 1 and EphB2 using an ADP-Glo assay. The results report IC50 + / - SEM (n=2) supported with CNS MPO scoring.

[0101] Example 1,14. Discussion

[0102] Previous reports showed that EphB 1 / 2 signaling plays a role in the pathogenesis of different metabolic diseases, including hepatic fibrosis, lipidosis, and insulin resistance. For example, EphB2‘ / _knock-out mice have attenuated liver fibrosis and inflammation induced by carbon tetrachloride (CCI4) or diet. A recent single-nucleus transcriptomics and epigenomics study in all major hepatocytes in mice and humans during the progression of non-alcoholic steatohepatitis (NASH) and liver fibrosis elucidated the liver- specific metabolic adaptation regulated by EphB2 tyrosine kinase signaling. EphB4 has been shown to play a role in the modulation of insulin-induced InsR degradation with implications for glucose homeostasis and insulin resistance because EphB4- O and the introduction of NVP-BHG712 (VEGFR, EphA, and EphB kinase inhibitor) improved for insulin resistance and glucose intolerance in db / db micePCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049 without affecting body weight. EphB receptors have highly conserved intracellular tyrosine kinase catalytic domain- sharing homology up to 87%, except the EphB3 catalytic binding domain due to CYS717.

[0103] Applicant previously identified the targeting of the EphB tyrosine kinase catalytic binding domain. These findings support the notion of developing pan-EphB tyrosine kinase inhibitors to restore glucose homeostasis and mitigate insulin resistance. However, the anti-obesity and antidiabetic properties of EphB tyrosine kinase inhibitors remain unexplored. Applicant designed and synthesized new analogs that target EphBl, EphBl, and EphB4 tyrosine kinases with no impact on EphB3 catalytic binding domain, designated as pan-EphB tyrosine kinase inhibitors.

[0104] Applicant applied a structure-based drug design approach to develop newly synthesized analogues, not related to tetracycline scaffold. Applicant identified three catalytic sub-pockets SI, hinge region, and DFG to tailor thienopyridine-based scaffolds called STA-related analogs. Applicant optimized a two-step synthetic route to develop the first round of STA-analogs (STA A-F). However, their potencies against EphB 1-4 tyrosine kinases were higher than 100 pM. Later, Applicant developed 16 STA-related analogs with respectable yields. Next, Applicant evaluated the potential of STA-related analogs to inhibit EphBl, EphB2, and EphB4 tyrosine kinase.

[0105] This can be explained due to unexpected shifting within the binding mode to occupy S 1 , hinge, and DFG sub-pockets. Applicant elucidated SAR for STA-related analogs and showed that (lH-Indol-4-yl) based analogs had promising potencies. Also, this revealed the potential of STA- 013 ((lH-Indol-4-yl) methanamine with phenyl substitution) to inhibit EphBl, EphB2, and EphB4 tyrosine kinases. Pharmacological mapping of selective Eph kinase inhibitors is challenging due to the shared homology structures with subdomain V among SRC, MEK1, ERK2, PKA, LKB1, PINK1, EphA2, EphA4, EphBl, EphB2, and EphB4. Next, Applicant addressed the selectivity of STA-013 towards EphBl, EphB2, and EphB4 tyrosine kinase activities without affecting tyrosine kinases and tyrosine-like kinases. Applicant next screened STA-013 against 140 kinases via premier kinomc profiling assay to assess its potential to inhibit EphB 1, EphB2, and EphB4 tyrosine kinases without affecting other kinases.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0106] Next, Applicant profiled the in vitro phenotypic activity of STA-013 to inhibit lipid accumulation after differentiation to adipocytes in the embryonic mouse 3T3-L1 fibroblast in a dose-dependent manner. STA-013 inhibited lipid accumulation in differentiated 3T3-L1 cells compared to vehicle-treated one. The STA-013-induced elevation in glycolysis and glycolytic capacity supports a reprogramming toward anaerobic metabolism, possibly compensating for reduced oxidative phosphorylation. Additionally, it showed suppression of palmitate oxidation that may indicate a targeted downregulation of 0-oxidation, further supporting STA-013’ s role in redirecting energy metabolism away from lipid-dependent pathways. Collectively, these findings suggest that STA-013 promotes a metabolically flexible phenotype characterized by enhanced glycolysis and increased glycolytic capacity, reduced fatty acid oxidation, and decreased mitochondrial oxidative activity. This metabolic profile may contribute to a lowered oxidative burden and improved bioenergetic balance.

[0107] Based on the in vitro findings, Applicant evaluated the potential of STA-013 to restore glucose homeostasis and insulin sensitivity in an HFD-induced obesity male mice model. It is worth noting that this Example has a limitation to the generalizability of the Example is that it did not consider gender / sex issues. Intraperitoneal administration of STA-013 (25 mg / kg / day) improved glucose clearance and insulin sensitivity, increased BAT-specific thermogenesis, inhibited liver fat deposition, and decreased total cholesterol and free fatty acid levels in the plasma compared to vehicle-treated HFD-obese mice.

[0108] Most of the reported kinase inhibitors exhibit higher potency with ICsos in the nM range; however, it seems that STA-013 managed to modulate EphB tyrosine kinase signaling (EphBl (IC50= 0.69±0.08 pM), EphB2 (IC50= 1.73±0.37 pM), and EphB4 (1.02±0.06 pM)) rather than full inhibition. This resulted in less off-target effects associated with sufficient targeting to mitigate metabolic syndrome. The PK profiles of STA-013 were executed for IV bolus and IP administration. STA-013 showed a large apparent volume of distribution at steady state (Vss 88.8 L / kg) and a systemic clearance CL of 10.8 L h’1kg’1. STA-013 was almost completely eliminated 12h after or IP injection or 24h after an IV bolus. Therefore, no significant accumulation would bePCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049 expected with repeated once-daily dosing in mice. IP administration displayed rapid absorption, reaching peak plasma concentrations (Cmax) within less than 30 min, which are close to initial concentrations after IV bolus injection of an equivalent dose. The systemic bioavailability after IP injection is high (75%). Neither liver- specific knockout of EphB4 nor pharmacological inhibition of EphB4 tyrosine kinase showed a reduction of whole-body weight in db / db mouse models. Surprisingly, STA-013 induced whole-body weight reduction associated with a significant decrease in the fat mass while maintaining the lean muscle mass, compared to vehicle-treated HFD-obese mice. This was associated with reduction in the RER rates suggests the potential of STA-013 to induce fatty acid oxidation with no effect on the cumulative food intake. Next, Applicant investigated the molecular mechanism associated with STA-013 anti-diabctic activity in the liver and BAT isolated from HFD-induced obese mice to suggest its potential to inhibit EphB phosphorylated signal associated with increased p-AKT / AKT signaling to suggest activation of insulin signaling. Most importantly, STA-013 showed an elevated signal for InsR- to validate its potential to reverse the effect of insulin on InsR degradation, allowing the restoration of glucose homeostasis and mitigating insulin resistance.

[0109] The transcriptomic signature for STA-013 -treated livers isolated from HFD-obese mice showed upregulation of AK1, Fabp5, lrs2, Rtn2, and Nr4a3, which are involved in carbohydrates and glucose transmembrane transport and import. Rnasel, Pgam2, Pfkm, Dynlll, and CD38 were also upregulated, which regulate insulin secretion and cellular response to insulin stimulus. Interestingly, Lepr, Stat3, Sirtl, and Inhbb were elevated to regulate leptin and cellular response to leptin stimulus. In contrast, it showed the downregulation of Fasn, CD36, Acatl, Acaalb, Aldob, and mtor, which regulate fatty acids and lipids synthesis and metabolism.

[0110] In summary, the current Example spans several disciplines, providing a molecular understanding of newly synthesized analogs not related to tetracyclines that can be used to develop selective pan-EphB tyrosine kinase inhibitors. Applicant demonstrates that STA-013 induced weight loss and improved glycaemic control. These results highlight the translational potential of reversing the effect of insulin on InsR degradation via pharmacological inhibition of EphBl,PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049EphB2, and EphB4 tyrosine kinases. The experimental studies featured the utility of STA-013 as a molecular modality to target EpB 1 / 2 / 4 tyrosine kinase signaling to mitigate metabolic syndrome preclinically.

[0111] Example 1.15. Zn silica molecular simulations

[0112] A virtual library of thienopyridine-based analogs was designed, and energy minimized using MMFF94 force field. The energy minimized library underwent semi-flexible docking using Omega and FRED along with the box assigned for the receptor (PDB ID: 6UMW). Scoring functions were sorted based on Chemgauss scores. Three dimensional diagrams were generated using Vida and PyMOL Molecular Graphics System, Version 1.2r3pre, Schrodinger, LLC.

[0113] Example 1,16. Chemistry

[0114] All reagents and solvents were obtained from commercial suppliers and used without further purification.JH NMR and13C NMR spectra were recorded on 400 MHz from Bruker (Bruker Avance 400). Chemical shifts are reported in parts per million (ppm, 5) relative to the reference signal. Spin multiplicities are described as s (singlet), d (duplet), t (triplet), q (quartet), and m (multiplet). Coupling constants (J) are reported in hertz (Hz). NMR data were analysed with ACD Labs Spectrus Processor software. Final reaction mixtures or residues were purified by flash silica column chromatography. A combination of TLC, flash silica column chromatography, NMR, HRMS, and HPLC analytical techniques were employed to characterize and to establish the purity of all synthesized compounds. AU Compounds are >95% pure by I iPLC analysis.

[0115] Example 1,17. IC50 profiling against EphB 1 / B2 / B3 / B4 activity

[0116] All the compounds were prepared in 7 serial dilutions ranging from 103to 10’3p M to be assayed against the kinase activity of EphBl / B2 / B3 / B4. The reactions were performed in white 96-well plates in a 50 pl volume reaction. The reactions were performed using EphBl kinase system (Promega VA7141), EphB4 kinase system (Promega VA7450), Recombinant mouse Ephrin B2 protein (His tag) (Abeam ab276883), and ADP-GLO kinase assay (Promega V6930).PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049The reaction was performed in four steps. First, 5 u L of tyrosine kinase assay buffer containing the kinase enzymes at a concentration of 10 ng / 50 pL and 2.5 pL of tested compounds (in 10% DMSO) were incubated at room temperature for 30 minutes. Second, 5 pL of substrate / ATP mixture composed of 0.125 pg / 50 pL of Poly (4:1 Glu, Tyr) Peptide Substrate and 1 pM ATP were added and incubated for 60 minutes at room temperature. Third, the reaction was stopped by adding 12.5 pL of ADP-Glo™ reagent and incubated for 40 minutes at room temperature. Finally, 25 pL of kinase detection reagent was added and incubated for 30 minutes at room temperature. The reaction was measured in luminescence with an integration time of 0.5-1 second.

[0117] Example 1,18. Kinome profiling

[0118] STA-013 (10 mM in 100% DMSO) was sent to MRC PPU International Center for premier kinase profiling at the University of Dundee, UK, to test compound selectivity against 140 kinase enzymes at a concentration of 5 pM. The results were presented as mean percentage activity with standard deviation for the test compound.

[0119] Example 1.19. 3T3-L1 cell lines and Oil Red O staining

[0120] 3T3-L1 mouse fibroblast (ATCC CL- 173) were cultured in a growth media composed of Dulbecco’s modified Eagle’s medium (ATCC 30-2002), 10 % calf bovine serum (ATCC 30- 2030), and 1% penicillin-streptomycin solution 100X (Corning 30-002-C1) at 37°C, 5% CO2 incubator. For differentiation, the cells were treated after reaching confluence with growth media containing Dulbecco’s modified Eagle’s medium, 10 % fetal bovine serum (Sigma- Aldrich F2442), % penicillin-streptomycin solution 100X, 0.5 mM 3-isobutyl-l -methylxanthine (IBMX) (Sigma-Aldrich 15879), 1 pM dexamethasone (DEX) (Sigma-Aldrich D4902), 10 pg / ml insulin (Sigma- Aldrich 19278) for 48 hours. After that, the media were replaced with a growth medium containing 10 pg / ml insulin alone for another 48 hours and then every 3 days with a growth medium only. The cells were treated with either STA-013 at different serial concentrations or just by vehicle (DMSO not exceeding 0.5%) starting from the differentiation steps.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0121] For Oil Red O staining, the cells were washed with PBS solution twice after removing the growth medium and then fixed with 10% formaldehyde (Sigma-Aldrich F8775) for 30 minutes at room temperature. Then washed with PBS and 60% isopropyl alcohol for 5 mins. The cells were stained with diluted Oil Red O solution (Sigma- Aldrich 01391) (3 Oil Red O solution: 2 dFFO) for 15 minutes in the dark at room temperature and then washed 4 times with dH O). The nuclei were counterstained blue with Haematoxylin Gill III solution (Sigma-Aldrich 65067) for 30 seconds and then washed with running tap water to remove excess stains.

[0122] Example 1.20, Seahorse mitochondria stress test

[0123] The Mito Cell Stress test was used to explore the effect of STA-013 on different mitochondrial respiration-related parameters. Oxygen consumption rates were obtained before and after the sequential injection of 1 p oligomycin, 1 p FCCP, and simultaneous addition of 1 pM antimycin A and 2 pM rotenone according to the manufacturer’s instructions.

[0124] Example 1.21, Seahorse Palmitate Oxidation Assay

[0125] Cells were pre -cultured for 24 hours in substrate-limited media (DMEM supplemented with 1% FBS, 0.5 mM glucose, ImM glutamine, ImM pyruvate, and 0.5 mM carnitine). Before measurement, cells were incubated for one hour at 37 °C in CO2-free Seahorse assay media containing ImM pyruvate and 2.5mM glucose then pretreated with either 1 pM STA-013 or 0.1% DMSO for 30 minutes. During the mitochondrial stress test, sequential injections were administered, introducing 200pM Palmitate-BSA conjugate or BSA alone, along with IpM oligomycin, IpM FCCP, IpM rotenone, and 2pM antimycin A. Oxygen consumption rate (OCR) was then used to quantify exogenous palmitate oxidation.

[0126] Example 1 ,22, Seahorse glycolysis stress test

[0127] 3T3-L1 cells were cultured and differentiated in 24 well Agilent Seahorse XF FLUXPACK. Cells were incubated in Seahorse assay media with 2mM glutamine for 1 hour at 37°C non-CO2 incubator. Injectors were loaded with seahorse media with either 1 pM STA-013PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049 or 0.1 % DMSO, 20 Mm glucose, 1 pM oligomycin, and 50 Mm 2 deoxy-glucose (2-DG) according to the manufacturer’s instruction (Seahorse XFp glycolysis stress kit 103017-100). Extracellular acidification rate (ECAR) was used for calculating glycolysis and glycolytic capacity.

[0128] Example 1,23. Animals

[0129] Ethical approval for all animal-related activities was obtained from the Institutional Animal Care and Use Committee at Texas Tech University Health Sciences Center and the experiments protocols complied with the WMA Statement on animal use in biomedical research adhered to the National Research Council’s directives for the welfare and utilization of animals. All mice were kept under pathogen-free conditions, 12-h light-dark cycle, controlled temperature (20-22°C), and fed either a normal chow diet (70% CHO, 20% proteins, and 10% fats by kcal) or high-fat diet (60% fats by kcal Research Diets Inc, D 12492) and water ad libitum. Mice were obtained from Jackson lab (C57BL / 6J 000664, C57BL / 6J DIO 380050). The HFD mice (n=10 / group; 16-20 weeks old) were randomized into three groups: (1) HFD + vehicle (10 % DMSO, 5% Tween-20, and 85% lx PBS), (2) HFD + 12.5 mg / kg STA-013, and (3) HFD + 25 mg / kg STA- 013. Body weight was recorded twice weekly. Tissues were harvested between 10:00 am and 12:00 pm.

[0130] Example 1.24, Body composition, food intake, and energy expenditure measurements

[0131] Body composition was determined using a non-invasive EchoMRI-700™ whole body composition analyser (EchoMRI LLC, Houston, Texas, USA). C57BL / 6J HFD-obese mice were singly housed and acclimated to metabolic cages (Comprehensive Lab Animal Monitoring System, CLAMS®) at close to thermoneutrality (25°C) for five days with ad libitum access to food and water. Food intake, respiratory quotient, and energy expenditure (EE) by indirect calorimetry system were measured for 22 hours (from 1230 h previous day to 1030 h next day) in all mice by a 16-unit CLAMS® indirect calorimetry metabolic cages following previous procedures. Food spillage was recorded manually and used to correct the daily food intakes before performing statistical analysis. Briefly, the volume of oxygen consumed (VO2, ml / kg body weight / h) andPCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049 carbon dioxide produced (VCO2, ml / kg body weight / h) were measured using Oxymax Zirconia 02 sensor, Oxymax universal gas conditioner and universal flow controllers, at frequent intervals (CLAMS setup: 0.8-0.9L / min flow, 70 kPa pressure, sampling for 1 minute at every 18 minutes interval). RER was calculated with VCO2:VO2 ratio. Total EE was computed with the following equation: Calorific value= VO2X[3.815+(1.232xRER)] and data were reported as kcal / h. On the last day of CLAMS®, to assess brown fat thermogenesis, non-invasive and rapid temperature measurements of interscapular brown adipose tissue (BAT) were recorded using a high-resolution FLIR T560® infra-red thermal imaging camera and associated software (FLIR® Systems Inc.).

[0132] Example 1.25. Glucose and insulin tolerance test

[0133] Glucose and insulin tolerance tests were performed in mice fasted overnight for glucose tolerance test and after a 4 h fast for insulin tolerance test, following which IP glucose (1 g / kg) or insulin (0.5 U / kg) was administered. Blood glucose measurements were assessed via tail wholeblood at the end of the fast (0 min), followed by samples at 15, 30, 60, 90, and 120 min postglucose or insulin administration using a blood glucose monitoring system.

[0134] Example 1,26. Lipid Profiling

[0135] For serum collection, tail blood was collected in tubes and centrifuged at 3000 xg for serum separation. A colorimetric enzymatic assay was used to determine total cholesterol (Sigma- Aldrich MAK043) and free fatty acid (Sigma- Aldrich MAK044) and performed according to the manufacturer’s instructions.

[0136] Example 1.27. PK studies

[0137] STA-013 was dissolved in 10%DMSO + 5% Tween20 + 85% PBS. Male C57bl / 6 mice (age 2-3 months, body weight 23-27 g, fed on standard rodent chow) received the drug at a dose of 25 mg / kg by either IV bolus or intraperitoneal (IP) injection. For IV injection a mouse tail vein catheter was introduced while the animals were short term restrained in a plexiglass. Repeated blood sampling (20-30 pL per sample) after injection was performed by tail nicking. Samples werePCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049 taken 5 min, 15 min, 30 min, Ih, 2h, 4h, 8h, 12h after IP administration and between 5 min and 24h after IV injection and collected in heparinized microcentrifuge vials. Plasma was obtained by centrifugation and stored at -80°C until processing for LC-MS / MS analysis. Pharmacokinetic evaluation and calculation of derived parameters was performed by non-compartmental analysis in PKanalix 2024R1, Lixoft SAS, a Simulations Plus company under an academic license.

[0138] Example 1,28. LC-MS / MS Analysis

[0139] STA-013 was extracted from plasma using the protein precipitation technique. Briefly, the plasma sample was treated with ice-cold acetonitrile for protein precipitation, vortexed thoroughly, and centrifuged at 13,000 rpm for 10 minutes at 4°C. The supernatant was collected and transferred to an autosampler vial. Chromatographic separation was performed on a Shimadzu LC-40 system equipped with a Phenomenex Kinetex 2.6 pm C18 100A column (50 x 2.1 mm). A 2ul of sample was injected into a column. A mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) was used at a flow rate of 0.4 mL / min and a column temperature of 45 °C. The following linear gradient elution program was applied: 0.0 min: 50% B; 0.0- 1 .0 min: 95% B; 1 .0-1.5 min: 95% B; 1 .6-2.5 min: 50% B. STA-013 eluted at 1 .0 minutes, and the total run time was 2.5 minutes per injection. Detection and quantification were performed on a SCIEX 5500 QTRAP mass spectrometer operating in positive ion mode using multiple reaction monitoring (MRM). The QI and Q3 transitions for STA-013 were 412.1 Da and 233.9 Da, respectively, at a collision energy (CE) of 23 volts. A matrix-matched calibration curve was used to determine STA-013 plasma concentrations. An in-house synthesized compound of STA-013 served as an internal standard to account for variability during sample preparation and analysis.

[0140] Example 1,29. Immunoblotting

[0141] Snap-frozen tissues (liver and brown adipose tissue) were homogenized in tissue protein extraction reagent buffer (T-PER catalog no. 78 10) containing protease and phosphatase inhibitor cocktail (Sigma) at a concentration of 1:100. Total protein concentration was quantified using Pierce BCA protein assay kit. The samples were prepared in 4x Laemmli protein sample buffer forPCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049SDS-PAGE (BIO-RAD) and separated via lx Tris / Glycine / SDS (BIO-RAD). Proteins were transferred to PVDF or nitrocellulose membranes (BIO-RAD) and blocked in 5% non-fat dry milk / TBST for one hour at room temperature. All membranes were incubated with the primary antibodies prepared in 5% BSA / TBST at 4°C overnight. The following primary antibodies were used; goat polyclonal anti-EphB2 (R & D systems AF467, 1:2000), rabbit polyclonal anti- phospho-EphB Pan (Tyr600, Tyr602, Tyr614, Tyr596) (Thermofisher Scientific PA5-64578, 1:1000), rabbit monoclonal anti-INSR P (Thermofisher Scientific MA5-42446, 1:1000), rabbit polyclonal anti-AKT (Cell signaling 9272, 1:1000), rabbit polyclonal anti-phospho-Akt (Ser473) (Cell signaling 4060, 1:1000), rabbit polyclonal anti-GADPH (Sigma- Aldrich SAB4300645, 1:5000), rabbit monoclonal anti- -actin (Sigma- Aldrich SAB5600204, 1:4000). Horseradish peroxidase-conjugated anti-rabbit (Cell signaling 7074, 1:2000) or Alexa Flour 680 goat antirabbit (Thermofisher Scientific A21109, 1:5000) was used as secondary antibody and incubated for one hour at room temperature. The membranes were incubated with SuperSignal West Femto (Thermofisher) chemiluminescent substrate for 5 mins and then explored using LICOR Odyssey Fc for chemiluminescent or fluorescence detection and quantified by image studio software version 5.5.

[0142] Example 1,30. Liver Oil Red O staining

[0143] Snap-frozen liver tissues (n=4) in liquid nitrogen were sent to Histowiz for sectioning and staining. The slides were stained using an Oil Red O stain kit (American MasterTech Scientific KTORO). Results were presented as a percentage of Oil Red O positive tissue.

[0144] Example 1.31. RNA sequencing

[0145] Snap-frozen liver tissues (n=3) in liquid nitrogen were sent to Novogene for mRNA extraction and sequencing. The RNA extraction performed was a column-based method. The libraries were non-directional (un-stranded) mRNA (poly-A enriched) libraries that were sequenced on the NovaSeq 6000, paired-end 150. GO and KEGG terms with padj < 0.05 are significant enrichment.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0146] Example 1.32, Statistics

[0147] Statistical analysis was performed using GraphPad Prism (vlO.2.3). Differences between groups were examined for statistical significance using an unpaired two-sided Student’s / -test (between 2 groups), one-way ANOVA (among 3 groups), and two-way ANOVA. Total energy expenditure was analyzed by repeated measures linear mixed models using body lean mass and 20% fat mass as a covariate using IBM SPSS Statistics version 29 for statistical analysis. All bar graphs represent mean ± s.e.m. Statistical significance was assessed as *P < 0.05, **P < 0.01 and *** P < 0.001. P values are shown in graphs.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0148] Example 2. STA-013 as a novel therapeutic strategy for diabetic cardiomyopathy

[0149] Preliminary pharmacological studies were conducted using the streptozotocin-high-fat diet (HFD) mouse model of diabetic cardiomyopathy. The results are summarized in FIGS. ISA- 151. Eight-week-old male C57BL / 6J mice were subjected to a Type 2 diabetes (T2D) model through 12 weeks of HFD supplementation combined with low-dose streptozotocin (75 mg / kg, intraperitoneally for three consecutive days) administered at week 4. Following the induction of diabetic cardiomyopathy, mice were treated with either STA-013 (25 mg / kg / day) or vehicle for 4 weeks.

[0150] STA-013 treatment resulted in a significant reduction in whole-body weight, accompanied by a decrease in fat mass, improved glucose homeostasis, and enhanced insulin sensitivity. Echocardiographic analysis revealed marked improvements in cardiac function, including, but not limited to, all parameters related to ejection fraction and fractional shortening (FIG. 15C). Furthermore, Masson’s trichrome staining demonstrated a significant reduction in myocardial collagen deposition in STA013-treated mice compared to vehicle controls, indicating attenuation of cardiac fibrosis (FIGS. 15G-15H). Taken together, these results suggest that STA- 013 not only improved systemic metabolic functions associated with T2D but also directly mitigated structural and functional deterioration of the diabetic heart. These findings provide strong proof-of-concept evidence that pharmacological targeting of EphB signaling with STA-013 could represent a novel therapeutic strategy for diabetic cardiomyopathy, with dual benefits on both whole-body metabolic homeostasis and cardiac fibrosis associated with diabetic cardiomyopathy.PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049

[0151] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the ail without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

1. PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049WHAT IS CLAIMED IS:

1. A method of treating or preventing a disease in a subject, said method comprising: administering to the subject an inhibitor of an EphB receptor, wherein the inhibitor is selected from the group consisting of:derivatives thereof, salts thereof, or combinations thereof, wherein each of Ri, R2, R3, R4 and R5 is independently selected from the group consisting of aromatic groups, cyclic groups, heterocyclic groups, polycyclic groups, polyaromatic groups, phenyl groups, pyridyl groups, 2-pyridyl groups, 3-pyridyl groups, 4-pyridyl groups, morpholine groups, piperidine groups, pyrrolidine groups, oxazolidine groups, thiazolidine groups, n-methyl piperazine, methanamine groups, (lH-Indol-4-yl) methanamine, (lH-Indol-5-yl) methanamine, (lH-Indol-6-yl) methanamine, derivatives thereof, salts thereof, or combinations thereof.524823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-0492. The method of claim 1, wherein the inhibitor comprises the following structure:

3. The method of claim 2, wherein Rs is selected from the group consisting of phenyl groups, 2- pyridyl groups, 3-pyridyl groups, 4-pyridyl groups, morpholine groups, piperidine groups, n- methyl piperazine, derivatives thereof, salts thereof, or combinations thereof.

4. The method of claim 2, wherein the inhibitor comprises the following structure:

5. The method of claim 1, wherein the inhibitor comprises the following structure:4823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-0496. The method of claim 5, wherein the inhibitor is selected from the group consisting of4823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049554823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049derivatives thereof, salts thereof, or combinations thereof.

7. The method of claim 5, wherein the inhibitor comprises the following structure:

8. The method of claim 1, wherein the inhibitor comprises the following structure:

9. The method of claim 8, wherein the inhibitor is selected from the group consisting of:564823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-0494823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049derivatives thereof, salts thereof, or combinations thereof.

10. The method of claim 1, wherein the inhibitor comprises the following structure:584823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-04911. The method of claim 10, wherein the inhibitor is selected from the group consisting of:594823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049604823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049derivatives thereof, salts thereof, or combinations thereof.

12. The method of claim 1, wherein the inhibitor comprises the following structure:614823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-04913. The method of claim 12, wherein the inhibitor is selected from the group consisting of:624823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049634823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049, derivatives thereof, salts thereof, or combinations thereof.

14. The method of claim 1, wherein the disease comprises a metabolic disorder.

15. The method of claim 14, wherein the metabolic disorder is selected from the group consisting of obesity, insulin resistance, metabolic syndrome, hyperglycemia, diabetes, type 2 diabetes (T2D), dyslipidemia, diabetic cardiomyopathy, or combinations thereof.

16. The method of claim 14, wherein the metabolic disorder comprises obesity.

17. The method of claim 1, wherein the disease comprises pain.

18. The method of claim 17, wherein the pain is selected from the group consisting of chronic pain, acute pain, neuropathic pain, peripheral neuropathic pain (PNP), chemotherapy-induced neuropathic pain, or combinations thereof.

19. The method of claim 1, wherein the subject is a human being.644823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-04920. An EphB receptor inhibitor selected from the group consisting of:derivatives thereof, salts thereof, or combinations thereof, wherein each of Ri, Ri, R3, R4 and R5 is independently selected from the group consisting of aromatic groups, cyclic groups, heterocyclic groups, polycyclic groups, polyaromatic groups, phenyl groups, pyridyl groups, 2-pyridyl groups, 3-pyridyl groups, 4-pyridyl groups, morpholine groups, piperidine groups, pyrrolidine groups, oxazolidinc groups, thiazolidinc groups, n-mcthyl piperazine, methanamine groups, (lH-Indol-4-yl) methanamine, (lH-Indol-5-yl) methanamine, (lH-Indol-6-yl) methanamine, derivatives thereof, salts thereof, or combinations thereof.

21. The inhibitor of claim 20, wherein the inhibitor comprises the following structure:654823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-04922. The inhibitor of claim 21, wherein Rs is selected from the group consisting of phenyl groups, 2-pyridyl groups, 3-pyridyl groups, 4-pyridyl groups, morpholine groups, piperidine groups, n- methyl piperazine, derivatives thereof, salts thereof, or combinations thereof.

23. The inhibitor of claim 21, wherein the inhibitor comprises the following structure:

24. The inhibitor of claim 20, wherein the inhibitor comprises the following structure:

25. The inhibitor of claim 24, wherein the inhibitor is selected from the group consisting of664823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-0494823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049684823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049derivatives thereof, salts thereof, or combinations thereof.

26. The inhibitor of claim 24, wherein the inhibitor comprises the following structure:

27. The inhibitor of claim 20, wherein the inhibitor comprises the following structure:

28. The inhibitor of claim 27, wherein the inhibitor is selected from the group consisting of:694823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049704823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049derivatives thereof, salts thereof, or combinations thereof.

29. The inhibitor of claim 20, wherein the inhibitor comprises the following structure:714823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-04930. The inhibitor of claim 29, wherein the inhibitor is selected from the group consisting of:724823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049734823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049derivatives thereof, salts thereof, or combinations thereof.

31. The inhibitor of claim 20, wherein the inhibitor comprises the following structure:744823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-04932. The method of claim 31, wherein the inhibitor is selected from the group consisting of:754823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-0494823-4406-5785V.3 13368-42PCT Application Attorney Docket No. AF13368.P068WOTexas Tech No. 2024-049, derivatives thereof, salts thereof, or combinations thereof.

33. The inhibitor of claim 20, wherein the inhibitor is suitable for use in treating or preventing a disease in a subject.

34. The inhibitor of claim 33, wherein the disease comprises a metabolic disorder.

35. The inhibitor of claim 34, wherein the metabolic disorder is selected from the group consisting of obesity, insulin resistance, metabolic syndrome, hyperglycemia, diabetes, type 2 diabetes (T2D), dyslipidemia, diabetic cardiomyopathy, or combinations thereof.

36. The inhibitor of claim 33, wherein the disease comprises pain.

37. The inhibitor of claim 36, wherein the pain is selected from the group consisting of chronic pain, acute pain, neuropathic pain, peripheral neuropathic pain (PNP), and chemotherapy- induced neuropathic pain.774823-4406-5785V.3 13368-42

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