Compositions and methods for mode-selective modulation of transient receptor potential vanilloid-1 (TRPV1)

Abstract

TRPV1 is an integral cation channel widely expressed in various tissues and involved in critical biological functions, such as thermosensation. It is polymodally activated by distinct stimuli, including chemical ligands, heat, and protons (pH). However, dozens of clinical trials targeting various types of TRPV1 -mediated pain have generally failed, primarily due to hyperthermia (increased body temperature). Meta-analysis of clinical data suggests that mode-selectivity is essential to realizing the therapeutic potential of TRPV1. TRPV1 is regulated allosterically, with vanilloids and other chemical ligands binding to the voltage-sensing-like domain (VSLD), resulting in the gating of the pore domain. Therefore, compositions and methods for modulating TRPV1 to treat human pathophysiologies, such as pain, are described herein.

Description

COMPOSITIONS AND METHODS FOR MODE-SELECTIVE MODULATION OF TRANSIENT RECEPTOR POTENTIAL VANILLOID-1 (TRPV1)CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 664,626 filed June 26, 2024, the specification of which is incorporated herein in their entirety by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. R01 NS119505 awarded by National Institutes of Health. The government has certain rights in the inventionFIELD OF THE INVENTION

[0003] The present invention features compositions and methods for mode selective modulation of transient receptor potential vanilloid-1 (TRPV1).BACKGROUND OF THE INVENTION

[0004] Chronic pain is a complex worldwide problem with tremendous negative social burdens and staggering economic impacts. Pain is estimated to affect -30% of the world's population. In the U.S., it costs more to treat pain than cancer and diabetes combined. Pain treatment is particularly challenging considering that current analgesics are often accompanied by addiction and withdrawal. This has given rise to a tenacious opioid epidemic, one that has worsened since the COVID-19 pandemic. Taken together, there is a dire need for more efficacious pain treatments.

[0005] Transient Receptor Potential Vanilloid-1 (TRPV1) is a widely expressed nociceptor and has garnered interest as a non-addictive analgesic target since its discovery in 1997. A major challenge in TRPV1 therapeutic intervention is its polymodal activation profile. TRPV1 transmits nociceptive (pain) signals from a variety of noxious stimuli including heat, chemical ligands, like the pungent compound capsaicin (CAP) from chili peppers, and low pH (protons) associated with inflammatory pathways. Attempts at inhibiting TRPV1 -mediated pain pathways have generally led to a common adverse effect - issues in thermosensing and body temperature misregulation. The challenges in antagonizing TRPV1 are thought to arise from two intertwined effects: 1) allosteric cross-talk between the polymodal activation mechanisms and 2) mechanistic and physiological differences between the better-studied rodent TRPV1 and the clinically relevant human ortholog.

[0006] Antagonists that are TRPV1 -selective, but not activation mode-selective, most commonly cause increased body temperature (hyperthermia), highlighting the molecular ties between ligand binding and TRPV1 -mediated temperature regulation. Cross-talk between the three canonical TRPV1 activation modes (e.g., heat, CAP, and protons) offers a complex landscape for identifyingdiscrete TRPV1 activation mechanisms and for developing mode-selective antagonists that are suitable for clinical use. TRPV1 is reported to function in physiologically distinct roles in humans and rodents, further convoluting drug discovery. Understanding human TRPV1 (hTRPVI) polymodal activation depends on the ability to identify mode-specific allosteric mechanisms and quantify the molecular origins of mode cross-talk.BRIEF SUMMARY OF THE INVENTION

[0007] It is an objective of the present invention to provide compounds, compositions, and methods that allow for mode selective modulation of transient receptor potential vanilloid-1 (TRPV1), as specified in the independent claims. Embodiments of the invention are given in the dependent claims. Embodiments of the present invention can be freely combined with each other if they are not mutually exclusive.

[0008] In some embodiments, the present invention features compounds and / or compositions comprising a structural analog of a compound according to the formula:

[0009] In other embodiments, the present invention features a composition for modulating Transient Receptor Potential Vanilloid-1 (TRPV1) ligand activation model, wherein the composition is according to the formula: [A]-L-[B], In some embodiments, [A] is a cyclic moiety or a bicyclic moiety comprising R1 and R2 groups and optionally substituted by one or more heteroatoms, [B] is a cyclic moiety or a bicyclic moiety, optionally substituted by one or more heteroatoms or substituents, and L is a linker.

[0010] In some embodiments, the present invention further includes methods for modulating transient receptor potential vanilloid-1 (TRPV1). In other embodiments, the present invention relates to methods for isolating, detecting and screening for novel drugs. In some other embodiments, the present invention features a method of modulating TRPV1 ligand activation mode. In some embodiments, the method comprises contacting a composition as described herein with the TRPV1. In some embodiments, the composition does not modulate TRPV1 pH activation mode and / or TRPV1 temperature activation mode

[0011] Moreover, prior references teach away from the present invention. For example, the present invention selectively modulates specific modes of TRPV1. The compositions and methodsdescribed herein specifically target the ligand binding mode without affecting the TRPV1 pH activation mode and / or TRPV1 temperature activation mode. In contrast, prior references are not mode-specific and often modulate multiple modes of TRPV1.

[0012] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0013] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0014] FIGs. 1A, 1 B, 1C, 1D, and 1E show the voltage sensing like domain of TRPV1 is fundamentally involved in TPRV1 polymodal activation. FIG. 1A shows the transmembrane region of a single TRPV1 subunit consisting of the voltage sensing-like domain (VSLD) and pore domain (PD). FIG. 1 B shows the native TRPV1 channel is arranged in a lipid bilayer through tetrameric oligomerization in which the four PD subunits form the ion channel pore. FIG. 1C shows the isolated VSLD demonstrates significant thermosensitive properties and FIG. 1D also shows houses the canonical capsaicin (CAP) binding pocket. Residues Y511 , S512, L547, and T550 in hTRPVI are all implicated in vanilloid binding. FIG. 1 E shows the VSLD allosterically regulates proton (low pH) sensing that occurs in the PD of an adjacent subunit. Protonation of residue E600 eliminates the interdomain salt-bridge between R455, which couples to movement of the VSLD that disrupts intradomain interactions, such as between K466 and E458.

[0015] FIGs. 2A, 2B, 2C, and 2D show capsaicin activates hTRPVI via binding in the VSLD. FIG. 2A shows whole-cell patch clamp electrophysiology of the human TRPV1 (hTRPVI) channel expressed in 293 cells measures robust currents in response to 1000 nM capsaicin (CAP). FIG. 2B shows the open probability of hTRPVI results in a CAP EC50 = 44 ± 7 nM measured at -80 mV and fit to a single binding site Hill equation. Data in FIGs. 2A and 2B are from n = 3 cells and error bars represent s.e.m. FIG. 2C shows CAP sensitivity is also observed in the isolated hTRPVI -VSLD measured by NMR spectroscopy. FIG. 2D shows CAP binds with a high affinity to the WT-VSLD (Kd= 1 .5 ± 0.3 mmol%). The Y511 A mutation decreases affinity and specificity for CAP (Kd= 7.5 ± 3.9 mmol%).

[0016] FIGs. 3A, 3B, and 3C show the hTRPVI -VSLD-Y511 A mutant is capsaicin insensitive. FIG. 3A shows CAP elicited concentration-dependent currents in 293 cells expressing hTRPVI . FIG. 3B shows that while 1 M CAP is sufficient to elicit cellular currents in WT hTRPVI , FIG. 3Cshows cells expressing the hTRPV1-Y511A mutant remain inactive at the same CAP concentration. Error bars represent s.e.m. in all panels.

[0017] FIGs. 4A, 4B, 4C, 4D, 4E, 4F, and 4G show ligand binding studies that reveal a latent proton allosteric network in the isolated TRPV1 VSLD. FIG. 4A shows the chemical shift perturbation (A5) of hTRPV1-VSLD residues in response to CAP binding. Dashed lines represent 2a and 3o. FIG. 4B shows rat TRPV1 extracellular residues E458 (equivalent to hTRPV1-D459) and K466 (equivalent to hTRPV1-E467) are highlighted in the conformational change between the rTRPV1-VSLD at neutral pH (7L2H) and FIG. 4C shows the proton-activated structure at pH 6 (7L2I). FIG. 4D, 4E, 4F, and 4G show NMR resonance intensities were measured, binned, and fit to a Gaussian curve to determine the average thermosensitivity (AHavg) of hTRPV1-VSLD- D459A; AHa.a= 21.7 ± 0.1 kcal / mol (FIG. 4D); -E467A; AHavg= 20.1 ± 0.3 kcal / mol (FIG. 4E); hTRPV1-VSLD in the presence of saturating CAP; AHavg = 17.1 ± 0.1 kcal / mol (FIG. 4F) and - Y511A in the presence of saturating CAP (FIG. 4G); AHavg= 21.3 ± 0.1 kcal / mol. A AHa g shifts were measured relative to WT-hTRPV1-VSLD (AHavg = 22.4 ± 0.1 kcal / mol; plotted in dark gray).

[0018] FIGs. 5A and 5B show capsaicin binding in the vanilloid binding pocket affects residues in the S1-S2 loop. FIG. 5A shows that the NMR CSP of the CAP binding residue Y511A and SI- 82 loop residue E467 were evaluated and compared in response to the binding CAP in the VSLD. Despite E467 being located across the membrane from the CAP binding site, it exhibits greater CSP than any other residue, including Y511 , which directly interacts with CAP. FIG. 5B shows mutations of extracellular residues D459 and E467 in the S1-S2 loop decrease CAP affinity allosterically (Kd = 10.4 ± 4.0 mmol% and 8.5 ± 1.1 , respectively).

[0019] FIGs. 6A, 6B, and 6C show NMR temperature titrations measure hTRPV1-VSLD thermosensitivity. FIG. 6A shows an overlay of 1 H-15N HSQC experiments collected over a range of temperatures of the hTRPV1-VSLD. Resonance intensities were fit to a two-state sigmoidal model as a function of temperature to evaluate the change in enthalpy (AH), or the slope of the fit at the midpoint. FIG. 6B shows the measured enthalpy changes were binned and fit to a Gaussian distribution to measure the average thermosensitivity (AHavg). The hTRPV1-VSLD in isolation measured a AHavg= 22.4 ± 0.1 kcal / mol. FIG. 6C shows the hTRPV1-VSLD-Y511A mutant measured a AHavg= 19.7 ± 0.1 kcal / mol.

[0020] FIGs. 7A, 7B, and 7C show single and double mutants in the hTRPV1-VSLD reveal altered CAP sensitivities. FIG. 7A shows although residue R557 is outside the vanilloid binding pocket, the R557A mutant decreases CAP sensitivity nearly 5-fold relative to WT. FIG. 7B shows the combined effects of the Y511 A / R557A double mutant appear to be additive, with CAP affinity only incrementally impacted relative to the R557A mutation alone. FIG. 7C shows double mutant E467A / Y511 A measured a CAP affinity of Kd = 6.0 ± 0.8 mmol%, greater than the Y511 A mutant alone but less than E467A.

[0021] FIGs. 8A, 8B, 8C, and 8D show Double Mutant Cycle Analysis (DMCA) of hTRPVI - VSLD mutants confirms an allosteric network that spans the membrane. FIG. 8A shows CAP binding affinities were used to derive the difference in free energy of binding ( AG; cycle edges) between the WT-VSLD, the binding mutant Y511A, the coupling mutant R557A, and the double mutant Y511A / R557A. FIG. 8B shows similar analysis was completed with the S1-S2 mutant E467A, and double mutant E467A / Y511A to evaluate for potential coupling between residues. FIG. 8C shows quantifying the coupling constant for each cycle suggests that Y511 is coupled to R557 (AGcoupiing = 0.8 ± 0.4) and E467 (AGcoupnng = 0.6 ± 0.2). FIG. 8D shows a double mutant cube extends the analysis to free energies derived from AH measurements for the WT-VSLD, single mutants, double mutant, and for each at saturating CAP concentrations. Coupling is again measured between E467 and Y511 based on thermosensing alone (front face). The coupling relationship is emphasized in the presence of CAP (back face*).

[0022] FIGs. 9A, 9B, and 9C show the antagonist ABT-102 blocks CAP response but not temperature in hTRPVI . FIG. 9A shows whole-cell patch clamp electrophysiology measurements in 293 cells, and ABT-102 sufficiently attenuates CAP response but does not inactivate cellular response to a temperature stimulus of 45 °C. Currents recorded at 100 mV, error bars are s.e.m. FIG. 9B shows ABT-102 specifically binds to the hTRPVI -VSLD with 19 ± 4 nmol% affinity. FIG. 9C shows hTRPVI- VSLD in the presence of 200 nmol% ABT-102 measures a thermosensitivity equivalent to WT alone; AHavll = 22.6 ± 0.2 kcal / mol.

[0023] FIG. 10 shows the chemical structures of TRPV1 agonists and antagonists. Chemical structures of compounds that bind to the TRPV1-VSLD and were evaluated for temperaturedependent ligand binding include agonists Capsaicin (CAP) and Resiniferatoxin (RTx) and Antagonists Capsazepine (CPZ), SB-366791 , and ABT-102.

[0024] FIGs. 11 A and 11 B show NMR ligand titrations measured as a function of temperature. FIG. 11A shows a representative series of ligand binding isotherms used to measure the binding equilibrium (Kd) at increasing temperatures ranging from 15 °C to 50 °C. Each data point (circle) represents a singular NMR experiment. FIG. 11 B shows overlays of binding isotherms (as in FIG. 11 A) measured from the ligand-temperature dual titrations. Each agonist panel, CAP (19F), CAP, and RTx, represents 72, 21 , and 28 experiments, respectively, while each antagonist panel, CPZ, SB-366791 , and ABT-102, represents 48, 36, and 45 experiments, respectively.19F experiments were included in analysis for CAP only, though an additional 80 and 72 points were collected by19F for SB-366791 and ABT-102.

[0025] FIGs. 12A, 12B, 12C, 12D, and 12E show thermodynamic analysis of ligand binding in the hTRPVI -VSLD quantifies coupling between ligand and temperature activation modes. FIG. 12A shows temperature-based ligand binding data for agonist CAP (circles), general antagonist SB366791 (triangles), and first-generation antagonist ABT- 102 (squares) are fit to linear and non-linear van’t Hoff models. For ABT-102, open squares represent Kdmeasurements from two separate samples. For CAP, solid circles were measured from a19F titration series, while open circles were measured from a HSQC titration series. FIG. 12B shows enthalpy- and entropy-driven binding in the VSLD are highlighted to distinguish antagonist from antagonist binding, respectively. Agonist RTx (light gray circle) and general antagonist CPZ (light gray triangle) were included in the analysis. FIG. 12C shows quantifying the change in entropy AS°, enthalpy AH°, heat capacity ACP°, and free energy (AG°) of binding at 310.15 K reveals stark differences in the thermodynamic binding profile for TRPV1 ligands. FIG. 12D shows the chemical shift perturbation (Ab) per-residue in the hTRPVI -VSLD in response to first-generation antagonist ABT-102; FIG. 12E shows general antagonist SB-366791. Dashed lines represent 2a and 3o.

[0026] FIGs. 13A and 13B show thermodynamic discrimination of ligand binding in the vanilloid binding pocket. FIG. 13A shows thermodynamic discrimination between agonists and antagonists is observed for binding in the hTPRV1-VSLD. In the temperature range investigated, the agonist CAP (circles) measures mostly an entropy-driven binding, while the general antagonist SB- 366791 (triangles) is mostly enthalpy-driven. FIG. 13B shows mode-selective antagonist ABT-102 measures high affinity to three residues identified for TRPV1 ligand binding: L547, T550, and L553.

[0027] FIGs. 14A and 14B show ligand binding in human TRPV1 induces distinct conformational changes in the VSLD that lead to activation-mode coupling or inactivation. FIG. 14A shows differences in Ab between ABT-102 and temperature mode-coupling ligands CAP and SB- 366791. FIG. 14B shows full-length TRPV1 functionality can be largely attributed to the VSLD which accounts for only ~18% of the entire channel. NMR CSP analysis highlights regions in the S1-S2 loop that are key to coupling proton activation: Y454, R456, F464, and E467. The VSLD is key to dissecting temperature from ligand activation, where ABT-102 measures a distinct CSP profile, relative to CAP and SB-366791 , as a thermal neutral antagonist. Residue L547 may be key in uncoupling ligand binding to Y511 with temperature sensing. The width and tint of each putty structure is proportional to the magnitude of Ab for CAP, SB-366791 , and ABT-102. Gray residues represent amino acids with no CSP information for either ligand. For the CAP representation, each segment is labelled with upward or downward arrows indicating the directionality of the VSLD.

[0028] FIG. 15A, 15B, and 15C shows NMR studies of PVH-100 TRPV1 binding show direct binding and mode selectivity. FIG. 15A shows an NMR-detected titration of the TRPV1 binding domain shows resonance peak perturbation upon the addition of PVH-100. FIG.15B shows an evaluation of the binding data from FIG. 15A and demonstrations specific and tight binding of PVH-100 to TRPV1 . FIG. 15C shows a further analysis of the data in FIG. 15A and indicates that a region of TRPV1 between the S1 and S2 transmembrane helices, that is known to impact protonactivation is not modulated by PVH-100. This is in comparison to ABT-102 and SB-366791 , which are known to impact proton activation and result in thermoregulatory issues in animals, including humans, for ABT-102.

[0029] FIG. 16 shows PVH-100 inhibits TRPV1 expressed in human cells. Recombinantly expressing 293 human cells enables whole-cell patch-clamp electrophysiology studies of TRPV1 . Data shows basal TRPV1 currents recorded at -80 mV, which are increased with 500 nM capsaicin stimulation. Co-application of capsaicin and PVH-100 results in the inhibition of capsaicin- activated currents. Thus, in some embodiments, PVH-100 is a mode-specific inhibitor of TRPV1 .

[0030] FIG. 17A and 17B show PVH-100 attenuates chronic pancreatitis induced hyperalgesia. FIG. 17A shows treatment with PVH-100 (2.5 mg / kg, intraperitoneally) for 7 days attenuated responses to VFF in rats with chromic pancreatitis (CP). FIG. 17B shows the same PVH-100 treatment also attenuated electric stimulation (ES) in rats with chronic pancreatitis. *P < .05 compared with CP / vehicle rats, #P < .05 compared with control / vehicle rats. n=7-8 rats.DETAILED DESCRIPTION OF THE INVENTION

[0031] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which a disclosed invention belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0032] The term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. Stated another way, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly the same meanings. In one respect, the technology described herein related to the herein described compositions, systems, methods, and respective component(s) thereof, as essential to the invention, yet open to the inclusion of unspecified elements, essential or not ("comprising").

[0033] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[0034] The term “about,” as used herein, can mean relative to the recited value, e.g., amount, dose temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1 %.

[0035] As used herein, the terms SIS3 and PVH-100 are used interchangeably; both refer to acompound according to the following formula:

[0036] The term “analog,” as used herein, refers to a chemical compound that is structurally similar to another compound (i.e. , a so-called “reference” compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.

[0037] The term “isomer,” as used herein, refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.

[0038] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the present invention. Unless otherwise stated, all tautomeric forms of the compounds of the present invention are within the scope of the present invention. Unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a13C- or14C-enriched carbon are within the scope of the present invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention. In certain embodiments, a warhead moiety of a provided compound comprises one or more deuterium atoms.

[0039] The term “ester,” as used herein, refers to a compound which is produced by modifying a functional group (e.g., hydroxyl, carboxyl, amino, or the like group). Examples of an “ester”include “esters formed with a hydroxyl group” and “esters formed with a carboxyl group.” The term “ester” can mean an ester whose ester residue is a “conventional protecting group” ora “protecting group removable in vivo by a biological method such as hydrolysis.” In some embodiments, the term “conventional protecting group” can mean a protecting group removable by a chemical method such as hydrogenolysis, hydrolysis, electrolysis, or photolysis. In other embodiments, the term “protecting group removable in vivo by a biological method such as hydrolysis” can mean a protecting group removable in vivo after administration to a subject such as by hydrolysis to produce a free acid or its salt.

[0040] The term “salt,” “pharmaceutically acceptable salt,” as used herein, refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, and allergic response, commensurate with a reasonable benefit to risk ratio, and effective for their intended use. A “pharmacologically acceptable salt” can refer to a salt, which can be formed when a compound herein has an acidic group such as carboxyl or a basic group such as amino or imino. In some embodiments, a salt of a compound disclosed herein can be formed with an acidic group, and can include, but is not limited to alkali metal salts such as a sodium salt, potassium salt, or lithium salt, alkaline earth metal salts such as a calcium salt or magnesium salt, metal salts such as an aluminum salt or iron salt; amine salts, e.g., inorganic salts such as an ammonium salt and organic salts such as a t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzylphenethylamine salt, piperazine salt, tetramethylammonium salt or tris(hydroxymethyl)aminomethane salt; and amino acid salts such as a glycine salt, lysine salt, arginine salt, ornithine salt, glutamate or aspartate. In some embodiments, a salt derivative of a compound disclosed herein formed with a basic group can include, but is not limited to, hydro-halides such as a hydrofluoride, hydrochloride, hydrobromide or hydroiodide, inorganic acid salts such as a nitrate, perchlorate, sulfate or phosphate; lower alkanesulfonates such as a methane sulfonate, trifluoromethanesulfonate or ethanesulfonate, arylsulfonates such as a benzenesulfonate or p-toluenesulfonate, organic acid salts such as an acetate, malate, fumarate, succinate, citrate, ascorbate, tartrate, oxalate or maleate; and amino acid salts such as a glycine salt, lysine salt, arginine salt, histidine salt, ornithine salt, glutamate or aspartate. In certain embodiments, when a pharmacologically acceptable salt of a compound disclosed herein remains exposed to the atmosphere or is recrystallized, it can absorb water to form a hydrate of use in formulations disclosed herein.

[0041] The term “intermediary,” or “intermediary compound,” as used herein, refers to amolecular entity (e.g., atom, ion, molecule, etc.) formed directly or indirectly from a reactant converted to a product in a multistep chemical reaction. A reactive intermediate may be reactive, short-lived, and high-energy and will typically react further to give a final product.

[0042] The term “tagged compound,” as used herein, refers to a compound tagged or labeled with a probe to aid in the detection and / or tracking of a biomolecule. Examples of tags may include, but are not limited to biotin, fluorescent tags, radioisotopes, and hydrophobic tags.

[0043] The term “prodrug,” as used herein, refers to a compound that is made more active in vivo through metabolism of a precursor drug. The compounds and compositions described herein can exist as prodrugs, as described in, for example, Hydrolysis in Drug and Prodrug Metabolism: Chemistry, Biochemistry, and Enzymology (Testa, Bernard and Mayer, Joachim M. Wiley-VHCA, Zurich, Switzerland 2003). Prodrugs described herein are structurally modified forms of the compound that readily undergo chemical changes under physiological conditions to provide the active compound. Additionally, prodrugs can be converted to the active compound by chemical or biochemical methods in an ex vivo environment.

[0044] The term “drug conjugate,” as used herein, refers to covalently linking drugs or prodrugs to a natural or synthetic molecule carrier for a specific application. Conjugation of a drug can be used to control drug release, target drug delivery, improve drug stability (e.g., pharmacokinetics and pharmacodynamics), enhance drug solubility, and alter toxicity profiles. Drug conjugation can occur with polymers, proteins, antibodies, etc. Examples of drug conjugates include small molecule drug conjugates (SMDC’s), nanoparticles, antibodies, and peptide sequences. SMDC’s allow for targeted therapy and are composed of a low molecular weight, high affinity targeting ligand; a linker; and a drug payload.

[0045] The term “alkyl,” as used herein, refers to a saturated aliphatic hydrocarbon group including C1-C20 straight chain and branched chain groups. Representative examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n- pentyl, 1 ,1-dimethyl propyl, 1 ,2-dimethyl propyl, 2,2-dimethyl propyl, l-ethyl propyl, 2-methylbutyl,3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1 ,1 ,2-trimethylpropyl, 1 ,1 -dimethylbutyl, 1 ,2- dimethylbutyl, 2,2-dimethylbutyl, 1 ,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl,4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5- methylhexyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2- ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2- dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2- methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3- ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and the isomers of iobranched chain thereof. An alkyl group can be a lower alkyl having 1 to 6 carbon atoms. Representative examples include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1 ,1 -dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1 -ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1 ,1 ,2-trimethylpropyl, 1 ,1 -dimethylbutyl, 1 ,2-dimethylbutyl, 2,2-dimethylbutyl, 1 ,3-dimethylbutyl, 2-ethylbutyl, 2- methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl and etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent group(s) can have one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy I, alkylsulfo, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxyl, heterocyclic alkoxyl, cycloalkylthio, heterocyclic alkylthio, carbonyl, carboxy or carboxylic ester.

[0046] The term “alkylsulfo,” as used herein, refers to esters of alkane sulfonic acids. The term “alkylamino,” as used herein, refers to alkyl substitutes attached to an amino group. Representative examples include phenylalanino, threonine, tryptophano, tyrosine, valino, N2- glutamino, N2-histidino, N4-asparagino, and the like.

[0047] The term “cycloalky I," as used herein, refers to saturated and / or partially unsaturated monocyclic or polycyclic hydrocarbon groups that have 3 to 20 carbon atoms. Representative examples of monocyclic cycloalkyl include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl etc. A polycyclic cycloalkyl can include the cycloalkyl having Spiro ring, fused ring, and bridged ring. Representative examples of polycyclic cycloalkyl include but are not limited to bicyclo[1 .1 .1 ]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.2]octyl. Cycloalkyls herein can be substituted or unsubstituted. When substituted, the substituent group(s) can be one or more groups independently selected from of alkyl, alkenyl, alkynyl, alkoxyl, alkylsulfo, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxyl, heterocyclic alkoxyl, cycloalkylthio, heterocyclic alkylthio, carbonyl, carboxy or carboxylic ester. The term “cycloal kylth io,” as used herein refers to a cycloalkyl ring attached to a sulfur group. The term “heterocyclic alkyl,” as used herein, refers to a cycloalkyl group derived from a cycloalkane by removal of a hydrogen atom from a ring with atoms of at least two different elements as members of its ring(s). The term “heterocyclic alkylthio,” as used herein refers to a cycloalkyl group derived from a cycloalkane by removal of a hydrogen atom from a ring with a sulfur atom as a member of its ring(s). Cycloalkyls, heterocyclic alkyls, and heterocyclic alkylthios herein can be substituted or unsubstituted. When substituted, the substituent group(s) can be one or more groups independently selected from of alkyl, alkenyl, alkynyl, alkoxyl, alkylsulfo, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxyl, heterocyclic alkoxyl, cycloalkylthio, heterocyclic alkylthio, carbonyl, carboxy or carboxylic ester.

[0048] The term “alkoxyl” as used herein, refers to an alkyl group which is singularly bonded to oxygen. Representative examples of alkoxyl groups include, but are not limited to, methoxyl, ethoxyl, and the like. The term “cycloalkoxyl” as used herein, refers to a cycloalkyl group bonded to an oxygen. Representative examples of cycloalkoxyl groups include but are not limited to, cyclomethoxyl, cycloethoxyl, and the like.

[0049] The term “heterocyclic alkoxyl,” as used herein, refers to a cycloalkoxyl group with atoms of at least two different elements as members of its ring(s). Cycloalkoxyls and heterocyclic alkoxyls herein can be substituted or unsubstituted. When substituted, the substituent group(s) can be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxyl, alkylsulfo, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxyl, heterocyclic alkoxyl, cycloalkylthio, heterocyclic alkylthio, carbonyl, carboxy or carboxylic ester.

[0050] The term “alkenyl,” as used herein, refers to a fragment formed from an alkene, i.e., double bond, by the removal of one hydrogen atom from any carbon atom. Representative examples of alkenyl groups include but are not limited to, allyl, isopropenyl, oleyl, phytyl, prenyl, vinyl, and the like. The term “alkynyl,” as used herein, refers to a fragment formed from an alkyne, i.e. triple bond, by the removal of one hydrogen atom from any carbon atom.

[0051] The term “aryl” as used herein, refers to an organic group derived from an aromatic ring where one hydrogen atom is removed from the ring. Representative examples of aryl groups are phenyl, naphthyl, tolyl, xylyl, and the like. The term “heteroaryl,” as used herein, refers to a 5 to 14 membered aryl having 1 to 4 heteroatoms selected from O, S, and N as ring atoms, the remaining ring atoms being C. Examples of heteroaryl groups are furan, thiophene, pyridine, pyrrole, N-alkyl pyrrole, pyrimidine, pyrazine, imidazole, tetrazolyl, and the like. Heteroaryl herein can be fused to aryl, heterocyclic alkyl, or cycloalkyl, wherein the ring connected with parent structure is heteroaryl. Heteroaryls herein can be substituted or unsubstituted. When substituted, the substituent group(s) can be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxyl, alkylsulfo, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxyl, heterocyclic alkoxyl, cycloalkylthio, heterocyclic alkylthio, carbonyl, carboxy or carboxylic ester.

[0052] The term “hydroxyl,” as used herein, refers to an — OH group. As used herein, “hydroxyalkyl” refers to -alkyl-OH, wherein alkyl is defined above. As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo, or iodo. As used herein, “thiol” refers to an organosulfur compound according to the form R-SH, where R represents an alkyl or other organic substituent. As used herein, “carbonyl” refers to — C(=O) — . As used herein, “nitro” refers to — NO2. As used herein, “cyano” refers to — CN. As used herein, “amino” refers to — NH2. As used herein, “carboxy”refers to — C(=O)OH. As used herein, “carboxylic ester” refers to — C(=O)O-alky.

[0053] The term “heterocyclyl,” as used herein, refers to a univalent group formed by removing a hydrogen atom from any ring atom of a heterocyclic compound. Representative examples of heterocyclyls include non-aromatic monocyclic, bicyclic, tricyclic, or spirocyclic ring systems comprising up to 7 atoms in each ring. Heterocyclyls herein can be substituted or unsubstituted. When substituted, the substituent group(s) can be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxyl, alkylsulfo, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxyl, heterocyclic alkoxyl, cycloalkylthio, heterocyclic alkylthio, carbonyl, carboxy or carboxylic ester.

[0054] The term “bicyclic,” or “bicyclic ring,” as used herein, refers to a polycyclic molecule featuring two joined rings with at least two common atoms. Examples of bicyclic rings include fused bicyclic rings and bridged bicyclic rings. Non-limiting examples of fused bicyclic rings include but are not limited to, bicyclo[4.3.0]nonane, bicyclo[3.3.0]octane, bicyclo[4.2.0]octane, and bicyclo[3.2.0]heptane. As used herein, the term “bridged bicyclic” or “bridged bicyclic ring” refers to a molecule featuring two rings that are joined sharing three or more atoms, with two bridgeheads separated by “bridges” containing at least one atom. Bicyclic compounds containing a bridge are typically in a rigid formation with little flexibility. Non-limiting examples of bridged bicyclic rings include but are not limited to, bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, bicyclo[2.2.2]octane, 5-oxaspiro[3.4]octane. Bicyclic rings herein can be substituted or unsubstituted. When substituted, the substituent group(s) can be one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxyl, alkylsulfo, alkylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxyl, heterocyclic alkoxyl, cycloalkylthio, heterocyclic alkylthio, carbonyl, carboxy or carboxylic ester.

[0055] The term “optionally substituted,” as used herein, indicates that a group can be unsubstituted or can be substituted with one or more substituents as provided herein or known in the art. As used herein, "substituted" in reference to a group indicates that a hydrogen atom attached to a member atom within a group is replaced. It should be understood that the term "substituted" includes the provision that such substitution be in accordance with the permitted valence of the substituted atom and the substituent and that the substitution results in a stable compound (e.g., one that does not spontaneously undergo transformation such as by rearrangement, cyclization, or elimination). In certain embodiments, a single atom can be substituted with more than one substituent as long as such substitution is in accordance with the permitted valence of the atom. Suitable substituents are defined herein for each substituted or optionally substituted group.

[0056] The term “therapeutically effective amount” may refer to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for administration purposes. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days, weekly, twice weekly, etc. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

[0057] The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the disorder being treated, the particular composition used, its mode of administration, and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0058] In some examples, the dosage can be administered to a subject once daily or in divided dosages throughout a day, depending on a subject's clinical response to the medication, as determined by methods known in the art. This dosage can be administered to a subject for one day, one a week, or a number of days, and then stopped if the subject responds immediately, or the dosage can be administered on a daily basis until a clinical response is noted. A person of skill can monitor a subject's clinical response to the administration of the composition and administer additional dosages as needed. It is contemplated that the composition can be administered to a subject on a daily basis, on an alternating daily basis, on a weekly basis, or at any interval in between.

[0059] In some examples, it may be advantageous to formulate the compositions in dosage units for ease of administration and uniformity of dosage. Dosage units refer to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predeterminedquantity of the compound calculated to produce the desired therapeutic effect.

[0060] The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A dose may be formulated in animal models to achieve a concentration range that includes the IC50 (i.e. , the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information may be used to more accurately determine useful doses in humans.

[0061] The term “pharmaceutically acceptable,” can refer to compounds, formulations, and compositions, in any dosage form within the scope of sound medical judgment, suitable for use in contact with a human subject or tissues thereof and as appropriate, in animals without excessive toxicity, irritation, with reduced side effect or complication as a consumable or for administration thereof, commensurate with a reasonable benefit / risk ratio.

[0062] The terms “individual,” “subject,” “host,” “animal,” and “patient,” as used herein, can be used interchangeably and refer to any subject or any mammalian regarding diagnosis, treatment, prophylaxis, or therapy as desired; for example, humans (e.g., adults, adolescents, toddlers, senior adults, children, infants and a fetus), companion animals (e.g., pets, horses), livestock, or other animals.

[0063] The term “treat,” “treating,” and “treatment,” as used herein, can refer to both therapeutic treatment and prophylactic or preventative measures, with the objective of preventing, reducing, slowing down (lessen), inhibiting, or eliminating an undesired physiological change, symptom, disease, or disorder (e.g., pain, obesity, or diabetes).

[0064] For purposes of this invention, beneficial or desired clinical results include but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented or onset delayed. Optionally, the subject or patient may be identified (e.g., diagnosed) as one suffering from the disease or condition prior to administration of the compositions of the invention. Subjects at risk for the disease or disorder can be identified by, for example, any or a combination of appropriate diagnostic or prognostic assays known in the art.

[0065] The term “administration,” as used herein, refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment to a subject or in vivo, in vitro, or ex vivo cells, tissues, andorgans. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

[0066] The term “co-administration” or “co-administering,” as used herein, refers to the administration of more than one active ingredient at the same time, just prior to, or just after the administration of one or more additional therapies. The compounds of the disclosure can be administered alone or can be co-administered to the subject along with another compound or a standard agent known in the art. Co-administration is meant to include simultaneous or sequential administration of the compounds individually or in combination. Dosage amounts and intervals can be adjusted individually to provide levels of the administered compound effective for the particular clinical indication being treated. This will provide a therapeutic regimen that is commensurate with the severity of the individual's disease state.

[0067] In one example, the compositions of the present invention can be administered by oral administration including, but not limited to, powders or granules, suspensions or solutions in water or non-aqueous media, pills, lozenges, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable. A person of skill, monitoring a subject's clinical response, can adjust the frequency of administration and dosage of the medication according to methods known in the art.

[0068] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes. In case of conflict, the present specification, including explanations of terms, will control.

[0069] Briefly, the present invention features compounds and compositions for the treatment of health conditions (e.g., pain, obesity, and / or diabetes) associated with the modulation of Transient Receptor Potential Vanilloid-1 (TRPV1). In some embodiments, the compositions include one or more novel compounds, tagged compounds, prodrugs, drug conjugates, metabolites and intermediary compounds thereof.

[0070] Compositions and Compounds for mode selective modulation of transient receptor potential vanilloid-1 (TRPV1)

[0071] Referring now to FIGs. 1A-17B, the present invention features compositions and methods for mode selective modulation of transient receptor potential vanilloid-1 (TRPV1).

[0072] In the following sections, certain exemplary compositions and methods are described todetail certain embodiments of the invention. It will be obvious to one skilled in the art that practicing the certain embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times, and other specific details can be modified through routine experimentation. In some cases, well known methods, or components have not been included in the description.

[0073] In certain embodiments, the present disclosure provides compounds for use in treating, preventing, or ameliorating human pathophysiologies, e.g., pain, obesity, and diabetes, in a subject or of use as combination therapies in treating, reducing onset, or ameliorating a human pathophysiology in a subject in need thereof. Non-limiting examples of such pathophysiologies include: pain-related conditions (e.g., neuropathic pain such as diabetic neuropathy and postherpetic neuralgia, osteoarthritis pain, cancer-related pain including chemotherapy-induced peripheral neuropathy (CIPN), inflammatory pain such as that associated with rheumatoid arthritis, migraine and cluster headaches, chronic lower back pain, and fibromyalgia); thermoregulatory disorders (e.g., fever, heat stroke, and hot flashes); respiratory disorders (e.g., cough and asthma); inflammatory and autoimmune conditions (e.g., chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), and rheumatoid arthritis); metabolic disorders (e.g., obesity and type 2 diabetes); dental conditions (e.g., tooth pulpitis and temporomandibular joint (TMJ) disorders); urological and gastrointestinal conditions (e.g., overactive bladder and irritable bowel syndrome (IBS)); ocular disorders (e.g., glaucoma and dry eye disease); dermatological conditions (e.g., psoriasis, atopic dermatitis, and rosacea); and neurodegenerative diseases (e.g., Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis). However, these examples are intended to be illustrative and not limiting of the scope of the present disclosure.

[0074] For example, in some embodiments, the present invention features compounds and / or compositions comprising a structural analog of a compound according to the formula:

[0075] In some embodiments, the present invention features a composition for modulating Transient Receptor Potential Vanilloid-1 (TRPV1) ligand activation model, wherein the composition is according to the formula: [A]-L-[B]. In some embodiments, [A] is a cyclic moiety or a bicyclic moiety comprising R1 and R2 groups and optionally substituted by one or more heteroatoms, [B] is a cyclic moiety or a bicyclic moiety, optionally substituted by one or moreheteroatoms or substituents, and L is a linker.

[0076] In some embodiments, the cyclic moiety comprises a 3-, 4-, 5-, 6- membered ring. In some embodiments, the cyclic moiety is an aromatic ring.

[0077] In some embodiments, the Ri and R2groups on [A] are on adjacent carbons. In other embodiments, the Ri and R2 groups on [A] are on non-adjacent carbons. In certain embodiments, R1 or R2 is an H, acyl, alkyl, alkylcarbonyl, carboxyl, carboxylic ester, carboalkoxy, ester, heterocyclic alkyl, aryl, heteroaryl, halo alkyl, hydrocarbyl, alkenyl, alkynyl, phosphate, acetyl, a cyclic hydrocarbon, or a combination thereof.

[0078] Non-limiting examples of [A] may include, but are not limited to:

[0079] As used herein, the squiggly or wavy lines depicted in the chemical structure of the compounds represent bonding with the linker [L]. In some embodiments, the linker [L] is bound to [A] through a cyclic moiety of linker [L] sharing a bond with the two carbons of the squiggly line in the cyclic moiety of [A], e.g. bridge. In other embodiments, the linker [L] is bound to [A] via a bond that may attach to either carbon at the ends of the squiggly line.

[0080] In some embodiments, [B] further comprises at least an R3 moiety attached thereto (e.g., to a carbon). In some embodiments, R3is an H, acyl, alkyl, alkylcarbonyl, carboxyl, carboxylic ester, carboalkoxy, ester, heterocyclic alkyl, aryl, heteroaryl, halo alkyl, hydrocarbyl, alkenyl, alkynyl, phosphate, acetyl, a cyclic hydrocarbon, or a combination thereof.

[0081] Non-limiting examples of [B] may include but are not limited to:

[0082] Again, as used herein, the squiggly or wavy lines depicted in the chemical structure of the compounds represent bonding with the linker [L], In some embodiments, the squiggly or wavy line represents where [B] is bound to the linker ([L]) by a single bond with the cyclic moiety of [B].

[0083] In some embodiments, L is a linker comprising at least one thioester, ester, -CONH-, or -SO2NH-, -NHCO-, or any combination thereof.

[0084] Without wishing to limit the present invention to any theory or mechanism it is believed that the compounds described herein bind to the orthosteric binding pocket of the TRPV1 voltage- sensor-like domain (VSLD). This pocket includes regions of the VSLD, specifically the transmembrane helices S3 and S4, as well as adjacent structural elements such as the S4-S5 linker. Additional interactions may occur with portions of the pore domain, including the S5 and S6 helices. [A] binds lower within the pocket, closer to the intracellular side, whereas [B] binds more centrally, within the mid-membrane region of the TRPV1 binding pocket.

[0085] In some embodiments, the composition does not modulate TRPV1 pH activation mode and / or TRPV1 temperature activation mode.

[0086] In some embodiments, compounds of the present disclosure can include isomers of any one of the compounds disclosed herein and be designated as being of a “cis” or “trans” configuration. In accordance with these embodiments, compounds of the present disclosure can be a cis or a trans isomer of any one of the compounds or formulas disclosed herein.

[0087] In some embodiments, compounds of the present disclosure can contain asymmetrically substituted carbon atoms in the R or S configuration, in which the terms “R” and “S” are as defined as known in the art. In accordance with some embodiments disclosed herein, compounds having asymmetrically substituted carbon atoms with equal amounts of R and S configurations can be racemic at those carbon atoms. In certain embodiments, the present disclosure can include racemic mixtures, relative and absolute stereoisomers, and / or mixtures of relative and absolute stereoisomers.

[0088] In certain embodiments, compounds of the present disclosure can include, but are not limited to, compounds In a solid or liquid form or state. In some embodiments, compounds of the present disclosure can be in an amorphous form, in other embodiments, compounds of the present disclosure can be in a crystalline form or a crystalline and amorphous form combination or mixture. In accordance with some embodiments disclosed herein, compounds in a solid state can exist in a crystalline, powder, or non-crystalline form, or as a mixture thereof. In some embodiments, compounds disclosed herein in crystalline form can be used to form pharmaceutically acceptable solvates. A skilled artisan can appreciate that pharmaceutically acceptable solvates can be formed where solvent molecules are incorporated into the crystalline lattice during crystallization. In accordance with some embodiments, solvates for uses disclosed herein can include nonaqueous solvents such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and EtOAc, or they can include water as the solvent incorporated into the crystalline lattice. Solvates, where water is the solvent that is incorporated into the crystalline lattice, are typically referred to as "hydrates." Hydrates can include stoichiometric hydrates as well as compositions containing variable amounts of water. The present disclosure encompasses all such solvates known in the art.

[0089] In certain embodiments, compounds of the present disclosure can exist in crystalline form, including various solvates thereof, and can exhibit polymorphism (e.g., the capacity to occur in different crystalline structures). These different crystalline forms are referred to herein as "polymorphs." Polymorphs have the same chemical composition but differ in packing, geometrical arrangement, and / or other descriptive properties of the crystalline solid state. Polymorphs, therefore, can have different physical properties such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, X- ray powder diffraction patterns, and NMR signatures, which can be used for identification. In certain embodiments, compounds of the present disclosure can be polymorphs. In certain embodiments, compounds of the present disclosure can be polymorphs that are identified by their melting points, IR spectra, X-ray powder diffraction patterns, NMR signatures, or any combination thereof. In some embodiments, different polymorphs of compounds herein can be produced by changing and / or adjusting the reaction conditions and / or reagents, used in making the compound. For example (but not limited to), changes in temperature, pressure, or solvent can result in polymorphs. In some embodiments, different polymorphs of compounds disclosed herein can spontaneously convert to another polymorph.

[0090] In certain embodiments and further to the previous paragraphs, compounds disclosed herein can have a sufficiently high solubility, which can be determined using kinetic or thermodynamic solubility approaches, to achieve desired bioavailability and concentrations insystemic circulation for a desired pharmacological response. In some embodiments, the kinetic solubility parameters of compounds disclosed herein can be determined. In some embodiments, compounds disclosed herein can have kinetic solubility indicative of the bioavailability of formulations having the compounds, such as oral, inhalable, topical, subcutaneous, and / or intravenous formulations.

[0091] In other embodiments, compounds disclosed herein can be in the form of an ester such as an ester prodrug. In some embodiments, the compound is a drug conjugate or a prodrug.

[0092] Methods of Use

[0093] In some embodiments, the compositions described herein allow for the modulation of Transient Receptor Potential Vanilloid-1 (TRPV1) ligand activation mode. In certain embodiments, the compositions do not modulate TRPV1 pH activation mode and / or TRPV1 temperature activation mode. The method may involve contacting a composition described herein with TRPV1.

[0094] In certain embodiments, the present disclosure provides methods for treating, preventing, reducing onset of, or ameliorating a human pathology, such as pain, diabetes, or obesity in a subject having, is suspected of developing, or is at risk of developing the human pathology. In accordance with these embodiments, the methods can include administering to the subject a therapeutically effective amount of a compound according to the instant disclosure alone or as combination therapies in treating, reducing onset or ameliorating a human pathology (e.g., pain, diabetes, or obesity) in a subject in need thereof. In accordance with these embodiments, the compound can be in the form of a therapeutic composition including at least one compound according to the instant disclosure or can be a dosage form including at least one compound according to the instant disclosure

[0095] In some embodiments, the present invention features a method for screening one or more of a potential drug candidate compound to determine whether it is therapeutically effective as compared to any one of the compounds disclosed herein as a reference compound. The method may comprise a) identifying one or more of an in vitro, ex vivo, or in vivo model for experimentation; b) contacting at least one of a cell, fluid, tissue, organ or animal with at least of one of the reference compound, the potential drug candidate compound, or a negative control compound; c) determining in parallel one or more drug parameter or characteristic from contacting the cell, fluid, tissue, organ, or animal with at least one of the reference compound, the potential drug candidate compound or the negative control compound; and d) comparing the drug parameter or characteristic of one or more of the negative control, the reference compound, or the drug candidate compound to determine whether the drug candidate compound is therapeutically effective.

[0096] In other embodiments, the present invention may feature a method for using artificial intelligence (Al) to generate one or more candidate compounds, wherein the one or more candidate compounds are derived from SIS3. The method may comprise a) providing an Al model comprising one or more neural networks, trained with a training data set comprising chemical training data, wherein training the Al model comprises feeding the training data set as input into the Al model, wherein the Al model is trained to generate the one or more candidate compounds as output; inputting chemical data into the Al model, wherein the chemical data comprises data unique to SIS3; and b) generating, by the Al model, the one or more candidate compounds.

[0097] In some embodiments, the present invention features a system for using artificial intelligence (Al) to generate one or more candidate compounds. The one or more candidate compounds may be derived from SIS3, incorporate SIS3, or a combination thereof. In some embodiments, the system may comprise a processor configured to execute computer-readable instructions, and a memory component communicatively coupled to the processor. The memory component may comprise an Al model comprising one or more neural networks, trained with a training data set comprising chemical training data. Training the Al model may comprise feeding the training data set as input into the Al model. The Al model may be trained to generate the one or more candidate compounds as output. The memory component may further comprise computer-readable instructions. The computer-readable instructions may comprise inputting chemical data into the Al model. The chemical data may comprise data unique to SIS3. The computer-readable instructions may further comprise generating, by the Al model, the one or more candidate compounds.

[0098] In some embodiments, the present invention features a method for using artificial intelligence (Al) to generate one or more candidate compounds. The one or more candidate compounds may be derived from SIS3, incorporate SIS3, or a combination thereof. The method may comprise providing an Al model comprising one or more neural networks, trained with a training data set comprising chemical training data. Training the Al model may comprise feeding the training data set as input into the Al model. The Al model may be trained to generate the one or more candidate compounds as output. The method may further comprise inputting chemical data into the Al model, wherein the chemical data comprises data unique to SIS3, and generating, by the Al model, the one or more candidate compounds.

[0099] In some embodiments, the one or more candidate compounds may comprise one or more candidate drug compounds. In some embodiments, the data unique to SIS3 may comprise one or more structural properties, one or more physical properties, one or more interactions between SIS3 and one or more other chemical compounds, one or more molecular pathways, one or more molecular cell profiles, or a combination thereof.

[0100] In some embodiments, the present invention features a method for training an artificial intelligence (Al) model to generate one or more candidate compounds. The one or more candidate compounds may be derived from SI S3 , incorporate SIS3, or a combination thereof. The Al model may comprise one or more neural networks. The training method may comprise feeding a training data set comprising chemical training data unique to SIS3 as input into the Al model. The method may further comprise inputting chemical data into the Al model, wherein the chemical data comprises data unique to SIS3. The Al model may be trained to generate the one or more candidate compounds as output.

[0101] In some embodiments, the chemical training data may comprise data pertaining to one or more chemical compounds. The data pertaining to the one or more chemical compounds may comprise one or more structural properties of the one or more chemical compounds, one or more physical properties of the one or more chemical compounds, one or more interactions between the one or more chemical compounds, one or more molecular pathways of the one or more chemical compounds, one or more molecular cell profiles of the one or more chemical compounds, or a combination thereof.

[0102] In some embodiments, the chemical training data may comprise one or more vector representations of the one or more chemical compounds. In some embodiments, the chemical training data may comprise one or more knowledge graphs representing the one or more chemical compounds. In some embodiments, the chemical training data may comprise any single or multidimensional representation of the one or more chemical compounds.

[0103] In some embodiments, the chemical training data may comprise one or more training labels associated with the one or more chemical compounds. In some embodiments, the one or more training labels may comprise one or more label elements having predetermined values. In some embodiments, the one or more training labels may comprise bioassay results, toxicity, cross-reactivity, pharmacokinetics, pharmacodynamics, bioavailability, solubility data, or a combination thereof.

[0104] In some embodiments, the chemical data may comprise data pertaining to one or more chemical compounds. The data pertaining to the one or more chemical compounds may comprise one or more structural properties of the one or more chemical compounds, one or more physical properties of the one or more chemical compounds, one or more interactions between the one or more chemical compounds, one or more molecular pathways of the one or more chemical compounds, one or more molecular cell profiles of the one or more chemical compounds, or a combination thereof.

[0105] In some embodiments, the chemical data may comprise one or more vector representations of the one or more chemical compounds. In some embodiments, the chemical data may comprise one or more knowledge graphs representing the one or more chemical compounds. In some embodiments, the chemical data may comprise any single or multidimensional representation of the one or more chemical compounds.

[0106] In some embodiments, the chemical data may comprise one or more labels associated with the one or more chemical compounds. In some embodiments, the one or more labels may comprise one or more label elements having predetermined values. In some embodiments, the one or more labels may comprise bioassay results, toxicity, cross-reactivity, pharmacokinetics, pharmacodynamics, bioavailability, solubility data, or a combination thereof.

[0107] The Al model may be stored, trained, and / or executed entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer, or entirely on the remote computer or server. The Al model may be stored in the form of program code, as described above. The one or more neural networks of the Al model, in some embodiments, may comprise a perceptron neural network, a feed-forward neural network, a multilayer perceptron neural network, a radial basis functional neural network, a recurrent neural network, a long short-term memory neural network, a sequence-to-sequence neural network model, a modular neural network, a graph-based convolutional neural network, an instance-based model, a feature attribution model, or the like.

[0108] In a non-limiting example, the Al model of the presently claimed invention may comprise a perceptron neural network, configured to accept the chemical data as input, execute one or more functions on the input, multiply the output of the one or more functions by a plurality of weights, and generating a final output comprising the one or more candidate compounds. In another non-limiting example, the Al model of the presently claimed invention may comprise a multilayer perceptron neural network comprising a plurality of layers, each layer configured to execute the process of a single perceptron network, each layer linked to an input and output feed such that the output of one layer is the input of a subsequent layer. The input to the first layer may be the chemical data and the output of the last layer may be the one or more candidate compounds.

[0109] In another non-limiting example, the Al model may comprise a graph-based convolutional neural network comprising a plurality of layers. Each layer of the plurality of layers may be linked to an input and output feed such that the output of one layer is the input of a subsequent layer. The input to the first layer may be the chemical data and the output of the last layer may be the one or more candidate compounds. The chemical data may be converted into one or more graphstructures before being transmitted to the plurality of layers. In some embodiments, the plurality of layers and the functions therein may be configured to extract one or more subgraphs from the input graph comprising one or more relevant node features and apply one or more attention-based functions to the subgraphs and relevant node features to assign importance to certain molecules, connections, atoms, etc. such that the model is able to generate and / or predict new candidate compounds based on the input comprising RGN6024, utilizing the most relevant patterns determined by the one or more attention-based functions. Determining which patterns are relevant and important for discovery may be a component of the training data set.[001 10] The one or more candidate compounds may comprise one or more chemical compounds linked to SIS3, one or more chemical compounds reassembled from the components of SIS3, one or more chemical compounds deriving from SIS3 by way of a chemical reaction, one or more chemical compounds related to SIS3, or a combination thereof.[001 11] With regards to the methods for implementing Al for discovering chemical compounds related to SIS3 and the Al-based systems thereof, the specifications of U.S. Patent No. 10,776,712, issued September 15, 2020, and U.S. Patent No. 11 ,462,304, issued October 4, 2022, are incorporated herein in their entirety by reference.[001 12] EXAMPLE[001 13] The following is a non-limiting example of the present invention. It is to be understood that said example is not intended to limit the present invention in any way. Equivalents or substitutes are within the scope of the present invention.[001 14] TRPV1 structural and biophysical characterization have alluded to the important role of allostery in heat, CAP, and proton activation, where the voltage sensing-like domain (VSLD; FIG. 1A) integrates functional stimuli. While certain details of TRPV1 heat activation remain opaque, NMR studies identified temperature-dependent conformational changes in the VSLD that couple to heat activation (FIG. 1 B and 1 C). Cryo-EM studies further supported the role of the VSLD in TRPV1 heat-dependent activation. Likewise, CAP activation originates in the VSLD, where it binds in the orthosteric vanilloid ligand binding pocket which couples to TRPV1 pore opening (FIG. 1 D). While pH sensing and proton activation begin in the TRPV1 outer pore, protonation couples to conformational changes in the VSLD that lead to lower gate opening (FIG. 1 E). Though the VSLD is relatively diminutive, ~18% of the channel by mass, it is implicated in all three canonical TRPV1 activation modes.[001 15] Capsaicin sensitivity in the hTRPV1-VSLD reflects cellular function. Given the physiological and functional differences between human and rodent TRPV1 , these studies focuson the clinically relevant human ortholog. The cognate TRPV1 agonist capsaicin (CAP) elicited robust current densities in 293 cells measured by manual whole-cell patch-clamp electrophysiology (FIG. 2A and FIG. 3A). The half-maximum effective concentration (EC5Q) of hTRPVI activation by CAP was measured to be 44 ± 7 nM (FIG. 2B), and is in the realm of reported rat EC50 values (50-800 nM;). The orthosteric TRPV1 vanilloid binding site residues are located in voltage-sensing like domain (VSLD; FIG. 1A). NMR and cryo-EM studies have also identified that the VSLD functions as an allosteric hub in TRPV1 polymodal activation.[001 16] NMR spectroscopy structural characterization showed that the isolated hTRPVI -VSLD retains structural (overall fold, secondary structure, and membrane topology) and functional attributes, including significant thermosensitivity (AHavB= 21.2 ± 0.1 kcal / mol) and CAP binding (Kd = 3.4 ± 0.4 mmol%). The CAP affinity (Kd) was measured from the average of vanilloid binding site residues, including Y511 , S512, and T550. Here, the global binding affinity was quantified from NMR data analyzed with principal component analysis (PCA). PCA is an unsupervised dimensionality reduction method that transforms the NMR HSQC-based titration series to weighted components from the data variance (FIG. 2C). The measured global CAP K<t in this study is 1.5 ± 0.3 mmol% (FIG. 2D). While traditional chemical shift perturbation (CSP) analysis gives detailed insight into binding affinities on a per-residue basis, PCA provides an unsupervised global analysis. This allows for comparative binding studies even when precise ligand binding residues have not yet been identified or between mutants, including residues that are directly involved in binding. The combination of CSP analysis and PCA methods provides a robust toolkit for quantifying and characterizing ligand binding in the isolated hTRPV1-VSLD.[001 17] Capsaicin and other vanilloid compounds like resiniferatoxin (RTx) bind in the orthosteric hTRPVI vanilloid binding pocket located in the inner membrane leaflet of the VSLD (FIG. 1 D). Residues Y511 , S512, L547, and T550 have been ascribed to bind specifically to critical moieties in this agonist class. For example, a loss of CAP activation occurs in the Y511A rat TRPV1 (rTRPVI) mutant. Manual whole-cell patch-clamp electrophysiology measurements of the human ortholog were measured with similar outcomes where the hTRPV1-Y511A mutant is insensitive to 1 pM CAP (FIG. 3B and 3C), consistent with an interaction between the Y511 phenol and the CAP vanilloid moiety. To validate the relationship between NMR-based biophysical and cellular functional data, the isolated VSLD was mutated to Y511A and assessed for CAP affinity. The affinity and specificity for CAP decreased relative to WT from 1 .5 ± 0.3 mmol% to 7.5 ± 3.9 mmol% (FIG. 2D). Given that NMR data from the isolated hTRPVI -VSLD agree with cellular electrophysiology measurements, this combination of methods was used to probe and establish a framework to evaluate and quantify the canonical TRPV1 activation modes.[001 18] Extracellular residues in the S1-S2 loop allosterically regulate CAP binding andthermosensing: Analysis of the NMR-detected hTRPVI -VSLD CAP titration identifies residues distal to the vanilloid binding site that exhibit substantial CSP (FIG. 4A). This is most pronounced in the S1-S2 loop opposite the membrane from the orthosteric CAP binding site. pH-induced conformational changes in the rTRPVI VSLD S1-S2 loop couple pH activation from the extracellular pore domain to the intracellular gate resulting in proton-dependent channel activation. Cryo-EM structures of rTRPVI at neutral pH show that the proton-sensing residue E600 in the pore loop forms a salt bridge with R455 in the S1-S2 loop of a neighboring VSLD subunit; upon E600 protonation, the interaction is disrupted and the VSLD tilts away from the pore loop of the adjacent unit. An intradomain salt-bridge is also observed in the S1-S2 loop between rTRPVI -E458 and -K46646; the distance between the carboxylate oxygen and the ammonium nitrogen at neutral pH increases from ~4 A to ~8 A in the pH 6.0-activated state (FIG. 4B and 4C). The hTRPVI equivalent residue E467 in the isolated VSLD exhibits significant CSP in response to CAP; greater in magnitude than any other assigned VSLD residue, including Y511 (FIG. 5A). Functionally, protons (i.e., acidic pH) potentiate the CAP response in rTRPVI -expressing cells. Mutations in the TRPV1 S1-S2 loop are expected to couple to CAP binding by disrupting the allosteric network that enables pH and CAP cross-talk. Based on the intradomain interaction in rTRPVI , the equivalent mutants in hTRPVI , -D459A and -E467A, were assessed for CAP binding. Both mutants exhibited diminished CAP affinity (Kd = 10.4 ± 4.0 and 8.5 ± 1.1 mmol%, respectively; FIG. 5B). Though vanilloid binding occurs in the inner leaflet side of the hTRPVI VSLD S3 and S4 helices, residues across the membrane in the S1-S2 extracellular loop modulate CAP affinity (FIG. 1 A). The isolated VSLD did not respond directly to changes in pH, yet the latent pH allosteric network that is retained in the VSLD modulates CAP affinity and underlies mode specific crosstalk.[001 19] Given the established cooperativity between TRPV1 pH and temperature activation, D459A and E467A mutations in the S1 -S2 loop should impact hTRPVI -VSLD thermosensitivity. The average WT hTRPVI -VSLD NMR measured thermosensitivity (A / - / avg) to be 22.4 ± 0.1 kcal / mol (FIG. 6A and 6B). The D459A hTRPVI -VSLD mutant thermosensitivity is on par with WT (FIG. 4D; AF / avg = 21.7 ± 0.1 kcal / mol); whereas the E467A mutant caused a decrease in thermosensitivity of ~2.3 kcal / mol (FIG. 4E; AHavg= 20.1 ± 0.3 kcal / mol). That mutations in the S1-S2 loop result in a detectable decrease in thermosensitivity provides strong evidence for a latent pH allosteric network that is retained in the isolated hTRPVI -VSLD and is the source of mode-specific coupling between temperature and CAP activation - a feature seen in the cellular studies of the full length channel and ascribed to complicate clinical intervention of TRPV.

[0120] hTRPVI ligand binding and temperature activation can be decoupled: Complex interplay between agonist binding and thermosensitivity has been observed in TRPV1 across cellularstudies for both RTx and CAP. Outside-out patch-clamp electrophysiology measurements have demonstrated that even in the presence of 10 nM CAP (~0.2x ECso) rTRPVI thermosensitivity is reduced by nearly half (from 110 ± 4 kcal / mol to 48 ± 2 kcal / mol). Similarly, inside-out patch clamp electrophysiology showed that 293 cells expressing rTRPVI measured a maximal current response from 4 pM CAP, yet when concurrently exposed to a 20-50°C temperature ramp, the measured current reduced to leak levels, presumably from irreversible heat inactivation. These results indicate that CAP reduces rTRPVI heat activation and thermosensitivity. NMR measurements of the isolated hTRPV1-VSLD reflect this phenotype, where 4 mol% CAP reduces the VSLD thermosensitivity by ~5 kcal / mol (FIG. 4F; AHava= 17.1 ± 0.1 kcal / mol). The thermosensitivity of the Y511 A mutant, which reduces CAP affinity (FIG. 2D), was evaluated as a control (FIG. 6C). In the presence of CAP, this mutation should partially recover the lost thermosensitivity due to the functional significance of Y511 in CAP binding. The Y511A mutant in the presence of saturating CAP increases the AHavgby ~4 kcal / mol over WT +CAP (FIG. 4G; AHavg= 21.3 ± 0.1 kcal / mol). These results show that the isolated hTRPV1-VSLD retains the allosteric network that underlies the coupling between CAP and heat activation modes, reflective of functional outcomes from cellular measurements.

[0121] Quantifying the allosteric coupling network that links the vanilloid binding pocket with the S1-S2 loop: As demonstrated herein, Y511A reduced the isolated VSLD CAP affinity by about five-fold. Y511 plays an integral role in binding CAP and other compounds within the vanilloid binding pocket. Functionally, agonist binding in the VSLD couples to pore opening through the S4-S5 linker. NMR spectroscopy, whole-cell patch-clamp electrophysiology, and cryo-electron microscopy have identified R557 as a key residue involved in the coupling between these two domains. Previous structures have indicated that in the closed, inactivated state, R557 forms a cation-pi interaction with Y554 that is disrupted upon ligand-based channel activation and replaced with a salt-bridge between R557-E570. In cellular electrophysiology experiments, hTRPV1-R557A mode-selectively abrogated heat and CAP activation while leaving proton activation intact. NMR data supports that R557 is integral for VSLD coupling to the TRPV1 PD because the R577A mutant had no meaningful impact of CAP affinity relative to WT. However, the reported affinity for hTRPVI -VSLD-R557A was determined exclusively from the T550 residue, as other CAP binding residues were precluded from analysis. Though the CAP affinity for T550 alone may not be significantly impacted by the R557A mutation, PCA reveals the global VSLD CAP affinity is decreased by this mutation (Kd = 9.4 ± 1.8 mmol%; FIG. 7A). This result begins to paint a more comprehensive picture of how ligand affinity is distally impacted by residues outside the vanilloid binding pocket.

[0122] Structures of hTRPVI show that Y511 and R557 are not in direct contact, with a Cadistance of -10.5 A (PDB: 8GF8). The hTRPV1-VSLD-Y511A / R557A double mutant further decreases CAP affinity by -10-fold (Kd = 11 .3 ± 1 .7 mmol%, FIG. 7B) relative to WT. The decrease in affinity indicates the relevance of these two residues in CAP binding and activation, as well as the additivity of the two mutational effects. A double mutant cycle analysis (DMCA) was employed to determine the thermodynamic coupling between these two amino acids. DMCA measures the energetic impact of a double mutant relative to independent, single mutations on a range of data types and experimental readouts, including ligand binding data. DMCA quantifies if two residues are coupled, either directly or allosterically, through a non-zero coupling energy. Binding free energies (AG°) were derived from the CAP Kd measured by NMR for each WT and mutant hTRPV1-VSLD construct to create a DMCA. Since CAP binding in the VSLD couples to channel activation via the S4-S5 linker, residues Y511 and R557 should be coupled. Indeed, Y511 and R557 show thermodynamic coupling (FIG. 8A and 8C; 0.8 ± 0.4 kcal / mol). A larger coupling magnitude is measured between the orthosteric vanilloid binding site and across the membrane in the S1-S2 loop. Residues Y511 and E467 have a coupling energy of 1.2 ± 0.4 kcal / mol (FIG. 8B and 8C). The E467A mutant reduces CAP affinity to the same degree as the ligand-binding mutant Y511 A. Despite a direct interaction between the phenol group of Y511 and the vanilloid group of CAP, allosteric regulation from the S1-S2 loop (residue E467) strongly modulates CAP affinity via a latent pH-activation mode network.

[0123] DMCA was extended to show coupling between heat, CAP, and proton sensing activation modes (FIG. 8D). Here, coupling between all three canonical TRPV1 activation modes was probed in a temperature-sensing context, where the AHavgwith and without CAP was measured for each mutant. E467 was used as a proxy for the VSLD latent proton allosteric network, where the front face of the cube (black text) demonstrates coupling between proton and vanilloid activation modes with respect to thermosensing. Again it was assessed that E467 and Y511 are coupled with a coupling energy of -2.6 ± 0.4 kcal / mol. This relationship is emphasized after CAP binding (back face, red text), where the double mutant measures a higher thermosensitivity than would be expected from the single mutants. The coupling between Y511 in the vanilloid binding pocket and E467 across the membrane in the S1 -S2 loop is a feature for both temperature sensing and CAP binding in the VSLD. These data highlight an allosteric network that spans the membrane for regulating ligand binding and thermosensitivity in TRPV1 and parallels what has been observed in functional measurements.

[0124] Predicting and quantifying the coupling between hTRPVI activation modes: TRPV1 polymodal activation imparts challenges for targeted therapeutic development. Meta-analysis of rodent and human clinical trial data exposes a path forward in unlocking TRPV1 therapeutic potential; where analgesics must have a mode-specific antagonistic profile which only inhibitsligand activation while leaving both temperature- and pH-sensing intact. However, predicted mode-specific requirements of human TRPV1 are distinct from those of rodent TRPV1. One partially mode selective early-generation TRPV1 antagonist, ABT-102, effectively decoupled temperature activation while inhibiting both ligand and proton activation modes. Clinical trials of ABT-102 resulted in increased body temperature (hyperthermia), which was later attributed to the inhibition of proton activation. The whole-cell patch-clamp electrophysiology measurements confirm that ABT-102 selectively antagonizes 500 nM CAP- evoked currents yet does not reduce current response to a heat (45°C) stimulus (FIG. 9A). ABT-102 binds specifically to the hTRPVI - VSLD with high affinity (Kd = 19 ± 4 nmol%, FIG. 9B), the VSLD thermosensitivity with saturating ABT-102 present was measured to be identical to WT-VSLD (A / - / avg = 22.6 ± 0.2), indicating that binding ABT-102 has no impact on hTRPVI- VSLD thermosensitivity (FIG. 9C). This combined data bolster the evidence that TRPV1 ligand and temperature sensitivities can be decoupled in a cellular context and are recapitulated in the isolated hTRPVI -VSLD. How an antagonist impacts hTRPVI thermosensing has been identified as a critical factor in therapeutic success. These data support that NMR characterization of the isolated hTRPVI -VSLD reflects cellular functional data, which has direct implications for pharmacological development.

[0125] While ligand binding impacts thermosensitivity (FIGs. 4A-4G), the influence of temperature on ligand binding has not yet been characterized. Temperature-dependent NMR binding studies of five ligands were investigated to evaluate this aspect of mode selectivity. Specifically, CAP (cognate agonist), resiniferatoxin (RTx; agonist), capsazepine (CPZ; polymodal pan antagonist), SB-366791 (polymodal pan antagonist), and ABT-102 (partially mode-selective antagonist). The chemical structures are shown in FIG. 10. The binding profiles should differ among these compounds, specifically between ligands that alter TRPV1 thermosensitivity (e.g. CAP) and those that do not (e.g. ABT-102). For every investigated ligand, a titration series of 2D 1 H-15N- HSQC spectra were collected over a temperature range of 15 to 50 °C to determine the binding dissociation constant (Kd) at distinct temperatures (FIG. 11A). In parallel, VSLD residue C443 was also labeled with a trifluoroacetone group as a probe for 19F experiments to allow for more efficient data collection. For example, a CAP titration series using 19F experiments was collected from 15 to 50°C and the resulting binding affinities were congruent with those measured from the 2D 1H-15N TROSY-HSQC experiments (FIG. 11 B). In the case of antagonists SB- 366791 and ABT-102, however, the measured affinities from 19F probes were significantly different, which aligns with the reported VSLD conformational changes induced by an agonist like CAP versus antagonists.

[0126] The temperature-dependent d values from 2D TROSY-HSQC data were fit to van’t Hoff first- and second-degree polynomials to evaluate the relationship between ligand binding andtemperature. Since the correlation coefficient alone is not sufficient to determine the best model of fit, the sum of squared residuals and F-statistic were also considered. The better model was determined by the combination of these parameters, and validated by a previously established framework to minimize bias in the evaluation. Although ~75 independent 1 H-15N TROSY- HSQC experiments were collected and analyzed, the better-suited model for two of the investigated ligands, RTx (28 HSQCs) and CPZ (48 HSQCs), could not be definitively distinguished (FIG. 1 1 B).

[0127] For ABT-102 a linear van’t Hoff relationship was observed, which arises when the binding enthalpy (AH°) and entropy (21S°) are temperature independent and the change in heat capacity (21Cp0) is null. CAP and SB-366791 exhibited a non-linear van’t Hoff relationship, indicating temperature dependence of AH° and 21S0that arises from a non-zero change in 21Cpo(FIG. 12A). Consequently, enthalpy-entropy compensation for binding agonist CAP and antagonist SB- 366791 in the VSLD (FIG. 12B) was observed. Notably, in the temperature range investigated CAP binding appears mostly entropy-driven, whereas SB-366791 binding is mostly enthalpy- driven (FIG. 12B; FIG. 13A). When comparing the thermodynamic changes that result from ligand binding at the physiologically relevant temperature 37 °C, CAP induces a positive ACP° while SB- 366791 measures a negative ACP°, 0.72 kcal / mol and -0.48 kcal / mol respectively. Additionally, the hTRPVI -VSLD presents a suitable model for discriminating SB-366791 (ACP°= 0.48 kcal / mol), a general antagonist, from ABT-102 (4CPO= 0), a mode-selective antagonist that does not inhibit hTRPVI thermosensing (FIG. 12C). These measurements uncover a metric for quantifying the coupling between ligand and temperature sensing in TRPV1 and pave the way for identifying mode-selective antagonism induced by binding in the hTRPVI- VSLD.

[0128] Molecular determinants of TRPV1 selective antagonism'. CSP analysis of the VSLD in response to CAP agonist binding (FIG. 4A) and antagonists ABT-102 and SB-366791 (FIG. 12D and 12E) unveils residues that are impacted directly by ligand binding and allosterically through conformational change. One general observation is that CAP binding in the VSLD elicits larger CSP than what is observed from ABT-102 or SB-366791 binding. This should be expected since the conformational changes induced by agonist binding are generally larger compared to an antagonist (FIG. 4A and FIG. 12D and 12E). While all three ligands produce substantial CSP in the S3 helix, the effect appears more pronounced from antagonist binding. These two antagonists also cause significant CSP for residues within the S1-S2 loop (FIG. 12D and 12E). A sequence alignment between human and rat TRPV1 orthologs (UniProt IDs Q8NER1 and 035433, respectively) shows the VSLD retains 94% sequence similarity, comparable to 92% similarity between full-length sequences, but 7 out of the 19 amino acid differences are located in S1-S2 loop only. One S1-S2 loop residue R456 exhibited exceptionally large CSP in response to binding SB-366791 (A6 = 0.067; >6o), so a second iteration of CSP analysis was conducted in whichR456 was excluded and resonance CSP were redistributed (empty bar; FIG. 12E). As is observed in other TRPV family members, intramolecular interactions between the extracellular PD of one subunit and the S1- S2 loop of a second subunit stabilize the apo state. In rTRPVI , residue R455 interacts with E600 via a salt-bridge that is disrupted during proton activation, suggesting that this loop and R455, in particular, are critically involved in pH-sensing. That the hTRPVI equivalent residue R456 and accompanying S1-S2 loop residues undergo substantial CSP upon ABT-102 and SB-366791 binding suggest that CSP of these residues may be a valuable molecular readout for identifying pH / proton mode specific antagonists.

[0129] Though the binding site for ABT-102 has not yet been structurally identified, specific binding were measured of ABT-102 to L547, T550, and L553, all of which have been attributed to binding cognate vanilloid ligands TRPV122, 32, 37 (FIG. 13B). Residues identified by structural investigations to bind SB-366791 , such as Y511 , L515, L547, T550, and L553, measure CSP that is smaller (L515 is not assigned nor included in CSP analysis for any ligand in this study). This likely suggests minimal conformational change for these residues in the VSLD, which is supported by the respective apo and SB-366791 -bound hTRPVI structures where the VSLD RMSD varies by only 1.425 A (PDB ID 8GF9 and 8GFA, respectively; residues 418-558). The CSP profiles for binding ABT-102 and SB-366791 are generally similar, apart from a few discreet VSLD regions. The S2-S3 loop does not exhibit substantial perturbation upon ABT-102 binding, yet this same region shows some of the largest CSP for SB-366791. This is not unlike what is observed for CAP binding where the same region undergoes relatively large CSP compared to any other subdomain (FIG. 4A). While the van’t Hoff ligand binding profile in the hTRPVI -VSLD provides insights to ligand-temperature coupling, CSP analysis reveals ligand-proton coupling.

[0130] Meta-analyses of TRPV1 clinical trial data infer that a successful analgesic targeting hTRPVI must only block ligand activation while leaving temperature- and pH-sensing modalities intact. Although repeated dosing of ABT-102 in rodent models is effective in treating a range of pain presentations, including inflammatory and osteoarthritic pain, the analgesic efficacy was accompanied by an increase in core body temperature. Similar outcomes were observed in human clinical trials where ABT-102 was effective against some pain modalities but also elicited hyperthermic effects - a phenotype that has since been linked to antagonizing proton activation. Identifying molecular components of proton activation that crosstalk with ligand binding is critical to successful therapeutic targeting of TRPV1.

[0131] The TRPV1 VSLD was originally proposed to remain stationary during vanilloid activation; however, ligand, proton, and heat activation causes VSLD structural changes that coordinate with channel opening In addition to the integral role of the pore domain in TRPV1 activation by protons and heat, this data builds on the role of the VSLD as a nexus of polymodalactivation with solution NMR and electrophysiology studies. Because solution NMR spectroscopy provides valuable insights into temperature-dependent atomic details and protein dynamics, it is well-suited for exploring the interplay among TRPV1 canonical activation modes. NMR measurements confirm that the isolated VSLD reflects full-length channel features from functional cellular studies. For instance, Y511A reduces current in cells and increases Ka in NMR experiments (FIG. 1D and 3A-3C, validating the VSLD as a tool to study TRPV1 polymodal function.

[0132] Intimate molecular ties between TRPV1 activation modes have complicated the therapeutic development of mode-selective antagonists. Crosstalk between heat and CAP activation is highlighted by a ~5 kcal / mol decrease in VSLD thermosensitivity in the presence of CAP (FIG. 4F). Although Y511 A partially recovers this loss, it does not fully separate ligands from temperature effects (FIG. 4G). The electrophysiology data confirm ABT-102 antagonizes hTRPVI currents elicited by CAP but not heat (FIG. 9A), a phenotype reproduced in the isolated VSLD, emphasizing the ability to disentangle TRPV1 activation modes - heat and CAP directly from VSLD NMR measurements.

[0133] Ligand-VSLD interactions present a particular thermodynamic profile, offering insights into ligand pharmacological mode-specificity. TRPV1 appears to share similarities with other receptors, such as GPCR p-adrenergic, nicotinic, GABA and 5-HT3receptors, in having distinct agonist and antagonist thermodynamic binding profiles. hTRPVI agonist binding (e.g., CAP, RTx) at physiological temperature is generally entropy-driven over the assessed temperature range, while antagonist binding (e.g., SB-36691 , CPZ) is predominantly enthalpy-driven. This profile is most similar to GABA and 5-HT3 receptors. Despite an emerging thermodynamic fingerprint of hTRPVI ligand regulation, its polymodal activation renders it more complicated than other receptor classes. Non-linear van’t Hoff plots reveal temperature-dependent enthalpy and entropy contributions that presumably arise from coupling between ligand- and temperature-sensing (FIG. 11 B).

[0134] Conformational selection by TRPV1 activation modes which alter the energetic landscape, biasing movements through dynamic allosteric networks that ultimately contribute to channel regulation and signaling. For example, the vanilloid binding pocket residue Y511A is energetically coupled to the S1-S2 loop across the membrane (FIG. 8B-8D), and mutations across the membrane distally decrease CAP affinity (FIG. 5A-5B). Allosteric intersubunit networks are present in TRPV1 , where proton activation in the extracellular PD destabilizes interactions in the VSLD S1-S2 loop. CSP analysis identifies VSLD components of the proton network are retained in isolation (FIG 4A-4G). Together this suggests that CSP measurements of S1- S2 serve as a molecular fingerprint capable of evaluating the crosstalk of candidate ligands with protonactivation.

[0135] This work suggests how mode-selective antagonists can be identified through NMR- detected binding studies of the isolated hTRPVI -VSLD. These data are consistent with the idea that the hTRPVI -VSLD, which consists of ~18% of the full-length channel, contains key motifs for integrating temperature, ligand, and proton activation modes. Coupled ligand and temperature NMR-detected titrations of the VSLD, should be sufficient for detecting mode- specific antagonists that discriminate ligand binding from temperature and pH activation modes. Promising candidates are anticipated to have a profile with minimal 4CP° values from van’t Hoff analysis and low CSP for S1-S2 loop residues (i.e. , Y454, R456, D459, F464, E467) upon binding in the hTRPVI -VSLD (FIG. 14B). The identification of an antagonist with this mode-specific profile is anticipated to be the key to unlocking TRPV1 therapeutic intervention, which could lead to more effective and personalized pain management strategies.

[0136] Whole-cell Patch-clamp Electrophysiology: Cell culture was carried out as described31 . Electrophysiology recordings were performed on HEK293 cells transfected with 0.4 ng / pL WT- hTRPVI or Y511A-hTRPV1. The full-length human TRPV1 gene was subcloned into a plRES-2 plasmid also containing the EGFP gene. This construct produces bicistronic mRNA containing an internal ribosome entry site (IRES) between the two genes, allowing for the independent translation of TRPV1 and the EGFP reporter. Y511 A-hTRPV1 was generated in the same plasmid using standard site-directed mutagenesis. Cells were transiently transfected with 0.4 pg DNA using FuGENE 6 transfection reagent (Promega) in a 1 :3 pg DNA:pL FuGENE ratio 48 h before electrophysiology measurements were performed.

[0137] Whole-cell voltage-clamp measurements were collected with an Axopatch 200B amplifier using Clampex 10.7 software (Molecular Devices). Data analysis was performed using Clampfit 11.1 (Axon Instruments). Cells were plated on 8 mm glass coverslips about 1 hour before data collection. At the time of measurement, the glass coverslips were placed in an extracellular buffer solution containing 140 mM NaCI, 5.4 mM KCI, 1.8 mM CaCI2, 1.0 mM MgCh, 5.5 mM glucose, and 5.0 mM HEPES. The extracellular solution pH was adjusted to 7.4 using NaOH, and osmolality adjusted to 310 mOsm with sucrose. The osmolality was measured using a Vapro 5600 vapor pressure osmometer (Wescor). The cells were acclimatized in the extracellular solution for 10 minutes prior to patching. Glass pipettes were pulled from borosilicate glass capillaries (World Precision Instruments) using a P-2000 laser puller (Sutter Instruments) and heat polished with an MF-830 micro forge (Narashige). Pipettes were filled with an intracellular buffer solution containing 125 mM potassium gluconate, 20 mM KCI, 5 mM potassium ATP, 10 mM HEPES, 10 mM EGTA. The pH of the solution was adjusted to 7.2 using KOH and osmolality was adjusted to 300 mOsm. The current magnitudes (pA) were normalized by the cell membrane capacitance(pF) to give current densities.

[0138] Temperature was controlled by perfusing preheated or cooled extracellular solution using an HCPC perfusion system and HCT-10 temperature controller (ALA Scientific), which heats or cools solution by supplying a specified voltage to a Peltier device through which perfusion solution flows. Temperature was calibrated by measuring the temperature of the solution exiting the HCPC at a given voltage.

[0139] Bacterial Transformation'. The hTRPV1-VSLD includes 141 residues with identical amino acid sequence to human TRPV1 residues 418-558. The synthetic gene was engineered from ATUM and modified to include an N-terminal 10* His tag and PCR primers between Ncol and BamHI restriction sites. Transformation of the construct used 20 ng of plasmid pET16b DNA in E. coli BL21 (DE3) competent cells. After heat shock and incorporation of the DNA, 50 pL of the mixture was plated on an AMP-LB agar plate to grow overnight at 37 °C. Starter cultures containing 50 mL of LB broth, 5 mg of ampicillin, and 6 colonies were incubated for 7 hours at 37 °C. Each liter of M9 minimal media was inoculated with 12 mL of culture. For 15N labeling, 15NH4CI (Cambridge Isotopes) was used as the nitrogen source. The cultures were grown at 18 °C, 230 RPM for about 14 hours to reach an ODeoo of 0.5-0.6. The cells were induced with 0.1 mM isopropyl -D- 1 -thiogalactopyranoside (IPTG, Research Products International Corp.) and continued to incubate under the same conditions for 36 hours. The cells were harvested by centrifugation at 6,000 xg for 20 min at 4 °C. The final cell pellets (~2.5 g / L) were frozen and stored at -80 °C.

[0140] Protein Purification: The protein was purified using previously established protocol31. Briefly, frozen cell pellets were thawed on ice then resuspended in 10 mL lysis buffer (75 mM Tris-HCI, 300 mM NaCI, 0.5 mM EDTA, pH 7.7) with lysozyme (0.2 mg / mL), DNase (0.2 mg / mL), RNase (0.2 mg / mL), PMSF (5 mg / mL), and 5 mM magnesium acetate per gram of cell pellet. After solubilizing and tumbling the pellet at room temperature, the cells were sonicated at 4 °C for 20 minutes at 50% power and 50% duty cycle with 5 sec on and 5 sec off. The lysate was then extracted into N,N-dimethyl-N-dodecylglycine (Empigen) (3% (v / v)) for 1 h at 4 °C. This mixture was centrifuged at 30,000 xg for 25 min at 4 °C and the supernatant was tumbled with preequilibrated Ni(ll)-NTA Superflow (Qiagen) resin ( 0.5 mL resin / 25 mL lysate) for 1 h at 4 °C. The bound resin was packed in a gravity column and washed with 10 CVs of Buffer A (40 mM HEPES, 300 mM NaCI, 2 mM BME pH 7.8) with 1.5% (v / v) Empigen, then with 10 CVs of wash buffer (40 mM HEPES, 300 mM NaCI, 60 mM Imidazole, 1.5% (v / v) Empigen, 2 mM BME, pH 7.8). The hTRPV1-VSLD was detergent exchanged with 10 CVs of detergent exchange buffer (25 mM NazHPO4, 0.05% (w / v) lyso-palmitoylphosphatidylglycerol (LPPG), 2 mM BME, pH 7.8). Finally, the protein was eluted with 5 CVs of elution buffer (25 mM Na2HPO4, 300 mM Imidazole, 0.1 %(w / v) LPPG, 2 mM BME, pH 7.8). Elution fractions containing hTRPV1-VSLD were buffer exchanged to thrombin cleavage buffer (25 mM Na2HPO4, 150 mM NaCI, pH 7.8) using an Amicon Ultra centrifugal ultrafiltration unit (Milipore, 10 kDa cutoff). After concentrating to ~500 pL, 3 units of thrombin were added and tumbled at room temperature for 24 h. The reaction mixture was incubated with 0.5 CV of pre-equilibrated Ni-NTA resin packed in a gravity column for 30 min. at room temperature. The flowthrough containing cleaved hTRPV1-VSLD was collected. The resin was washed with thrombin cleavage buffer containing 0.05% LPPG to collect residual hTRPVI- VSLD. Cleaved protein was concentrated to ~500 pL. For 19F labelling, the protein sample was tumbled with 50 x 3-Bromo-1 ,1 ,1 -trifluoroacetone (BTFA) for ~12 hours at room temperature and quenched with an equal amount of L-cysteine. The protein was then run over a XK 16 / 30 gel filtration column equilibrated with NMR buffer (25 mM Na2HPO4, pH 6.5) +0.05% LPPG and eluted in the same buffer. Fractions with the highest A280 values (generally fractions #35-39) were collected and concentrated to ~200 pL. Protein concentrations were measured using a BCA kit (ThermoFisher Scientific).

[0141] NMR Spectroscopy of Ligand Titrations: NMR samples were prepared in a 3 mm NMR tube (Bruker) using 5 pL (2.7% v / v) D2O (Sigma Aldrich) with a total sample volume of 180 pL. Protein concentrations ranged from 40-140 pM and final LPPG concentrations were ~1.2%. Ligand titrations were carried out by first preparing a stock solution in 200 proof ethanol (Thermo Scientific) and storing at -20 °C. Titration concentrations were measured in mol% and calculated as: moles ligand mol% ligand = - x 100 moles ligand + moles LPPG + moles hTRPVl — VSLD(Equation 1)

[0142] Ligand titration points were chosen and completed according to available data regarding Kd, EC50, or IC50. Ligands were added directly to the protein sample and allowed to equilibrate for a minimum of 30 min. at 37 °C before data collection. All NMR experiments were recorded with a Bruker 850 MHz 1 H spectrometer and Avance III HD console equipped with a 5 mm TCI CryoProbe. Each ligand titration set was collected using 1 H-15N TROSY experiments (b_trosyf3gpph) or 19F 1 D experiments at the calibrated temperature of 37 °C. For some ligands reported in this study, a ligand titration was carried out in conjunction with a temperature titration. For such data sets, a 1 H-15N TROSY experiment was collected at a series of temperatures for each ligand concentration. This allowed for protein / ligand concentrations to remain constant throughout all measured temperatures. The protein was given 15 minutes to equilibrate to each temperature point before data collection. Spectra were identically processed in NMRPipe.

[0143] NMR Analysis: TREND NMR was implemented using the protocols outlined by Xu andVan Doren. TREND software provides a simple means to summarize NMR spectra containing a multitude of reactions or exchanges. RCA was used to compare and categorize titration data, by way of PC1 , since it depicts the majority of the variances in measurements and is most representative of ligand binding. PCA data were fit to equations 2 and 3,

[0144] Where Bmaxis the maximum specific binding, Kdis the binding dissociation constant, and M is the non-specific binding factor. These models produced binding isotherms and ligand affinities that were used in thermodynamic calculations.

[0145] Resonance assignments were mapped and analyzed in CNMR3.0.4. Chemical shift perturbation (CSP) analysis of ligand titrations was calculated using, (Equation 4)

[0146] where 3H is the chemical shift change in the 1 H dimension and 5N is the chemical shift change in the 15N dimension, multiplied by a 0.2 scaling factor typically used for nitrogen. Plotting the CSP as a function of ligand concentration, and fitting to a single-site binding model (Equations 2 and 3) allowed for determination of the per-residue dissociation constant (Kd) and maximal value of CSP (A6 max).

[0147] For thermosensing analysis, NMR resonance intensities were evaluated as a function of temperature and fit two a temperature-dependent two-state model where the slope is a measure of the thermosensitivity (AH). These values were binned and fit to a Gaussian curve to determine the average thermosensitivity (AHave) of the hTRPV1-VSLD.

[0148] In the conventional linear van't Hoff equation (Equation 5, first-order polynomial), the slope is directly proportional to the change in enthalpy (AH"), which is generally considered to be independent of temperature over the biologically relevant temperature range and, therefore, the change in heat capacity (AC° ) is zero (Equation 6).(Equation 5) (Equation 6)

[0149] A non-linear van’t Hoff model for ligand binding (Equation 7, second-order polynomial) approximates that AH° and / or AS0varies with temperature, resulting in a non-zero AC0. In the caseof ligand binding in the TRPV1-VSLD, the AC° is a proxy for the coupling between temperature and ligand binding.(Equation 7)

[0150] As used herein, the term “about” refers to plus or minus 10% of the referenced number.

[0151] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase “comprising” includes embodiments that could be described as “consisting essentially of’ or “consisting of’, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase “consisting essentially of’ or “consisting of’ is met.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a structural analog of a compound according to the formula:

2. A composition for modulating Transient Receptor Potential Vanilloid-1 (TRPV1) ligand activation model, wherein the composition is according to the formula: [A]-L-[B], wherein [A] is a cyclic moiety or a bicyclic moiety comprising an Ri and R2groups and optionally substituted by one or more heteroatoms, wherein [B] is a cyclic or a bicyclic moiety, optionally substituted by one or more heteroatoms or substituents; wherein L is a linker.

3. The composition of claim 2, wherein the cyclic moiety comprises a 3-, 4-, 5-, 6- membered ring.

4. The composition of claim 2 or claim 3, wherein the cyclic moiety is an aromatic ring.

5. The composition of any one of claims 2-5, wherein the Ri and R2groups are on adjacent carbons.

6. The composition of any one of claims 2-5, wherein the Ri and R2groups are on non- adjacent carbons.

7. The composition of any one of claims 2-6, wherein Ri or R2is a H, acyl, alkyl, alkylcarbonyl, carboxyl, carboxylic ester, carboalkoxy, ester, heterocyclic alkyl, aryl, heteroaryl, halo alkyl, hydrocarbyl, alkenyl, alkynyl, phosphate, acetyl, a cyclic hydrocarbon, or a combination thereof.

8. The composition of any one of claims 1-7, wherein [A] is:

9. The composition of any one of claims 2-8, wherein [B] further comprises at least an R3 moiety attached to a carbon.

10. The composition of claim 9, wherein R3 is a H, acyl, alkyl, alkylcarbonyl, carboxyl, carboxylic ester, carboalkoxy, ester, heterocyclic alkyl, aryl, heteroaryl, halo alkyl, hydrocarbyl, alkenyl, alkynyl, phosphate, acetyl, a cyclic hydrocarbon, or a combination thereof.

11. The composition of any one of claims 2-10, wherein [B] comprises:

12. The composition of any one of claims 2-11 , L is a linker comprising at least one thioester, ester, -CONH-, or -SO2NH-, -NHCO-, or any combination thereof;13. The composition of any one of claims 2-12, wherein the composition does not modulateTRPV1 pH activation mode and / or TRPV1 temperature activation mode.

14. A method of modulating Transient Receptor Potential Vanilloid-1 (TRPV1) ligand activation mode, the method comprising contacting a composition according to any one of claims 1 - 13 with the TRPV1.

15. A method for screening one or more of a potential drug candidate compound to determine whether it is therapeutically effective as compared to any one of the compounds disclosed herein as a reference compound, wherein the method comprises: a) identifying one or more of an in vitro, ex vivo, or in vivo model for experimentation; b) contacting at least one of a cell, fluid, tissue, organ or animal with at least of one of the reference compound, the potential drug candidate compound, or a negative control compound; c) determining in parallel one or more drug parameter or characteristic from contacting the cell, fluid, tissue, organ, or animal with at least one of the reference compound, the potential drug candidate compound or the negative control compound; and d) comparing the drug parameter or characteristic of one or more of the negative control, the reference compound, or the drug candidate compound to determine whether the drug candidate compound is therapeutically effective.

16. A method for using artificial intelligence (Al) to generate one or more candidate compounds, wherein the one or more candidate compounds are derived from SIS3, the method comprising: a) providing an Al model comprising one or more neural networks, trained with a training data set comprising chemical training data, wherein training the Al model comprises feeding the training data set as input into the Al model, wherein the Al model is trained to generate the one or more candidate compounds as output; inputting chemical data into the Al model, wherein the chemical data comprises data unique to SIS3; and b) generating, by the Al model, the one or more candidate compounds.

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