Larry V. Pearce

Structural insights into transient receptor potential vanilloid type 1 (TRPV1) from homology modeling, flexible docking, and mutational studies

The transient receptor potential vanilloid subtype 1 (TRPV1) is a non-selective cation channel composed of four monomers with six transmembrane helices (TM1–TM6). TRPV1 is found in the central and peripheral nervous system, and it is an important therapeutic target for pain relief. We describe here the construction of a tetrameric homology model of rat TRPV1 (rTRPV1). We experimentally evaluated by mutational analysis the contribution of residues of rTRPV1 contributing to ligand binding by the prototypical TRPV1 agonists, capsaicin and resiniferatoxin (RTX). We then performed docking analysis using our homology model. The docking results with capsaicin and RTX showed that our homology model was reliable, affording good agreement with our mutation data. Additionally, the binding mode of a simplified RTX (sRTX) ligand as predicted by the modeling agreed well with those of capsaicin and RTX, accounting for the high binding affinity of the sRTX ligand for TRPV1. Through the homology modeling, docking and mutational studies, we obtained important insights into the ligand-receptor interactions at the molecular level which should prove of value in the design of novel TRPV1 ligands.

Evodiamine functions as an agonist for the vanilloid receptor TRPV1

Evodiamine, a quinozole alkaloid constituent of Evodia rutaecarpa, has been reported previously to induce several responses comparable to capsaicin in animal systems. Here, we characterize evodiamine as an agonist for rat TRPV1 expressed heterologously in CHO cells. Evodiamine bound to rat TRPV1 with a Ki of 5.95 +/- 0.87 microM, as measured by inhibition of [3H] RTX binding (capsaicin, Ki = 1.8 +/- 0.3 microM). Evodiamine was a full agonist for induction of 45Ca2+ uptake, with an EC50 of 856 +/- 43 nM (capsaicin, EC50 = 45 +/- 4 nM) and was competitively antagonized by capsazepine, as revealed by a Schild plot. The pattern of cellular response, as determined by calcium imaging, was similar to that with capsaicin and yielded an EC(50) of 1.03 +/- 0.21 [micro sign]M. Molecular modeling suggested a consistent pattern of overlap between evodiamine and TRPV1 agonists. We conclude that evodiamine represents a novel class of agonists for rat TRPV1, albeit 3-19-fold less potent than capsaicin, and thus represents a new potential class of lead molecules for drug development.

Structural Insight into Tetrameric hTRPV1 from Homology Modeling, Molecular Docking, Molecular Dynamics Simulation, Virtual Screening, and Bioassay Validations

The transient receptor potential vanilloid type 1 (TRPV1) is a heat-activated cation channel protein, which contributes to inflammation, acute and persistent pain. Antagonists of human TRPV1 (hTRPV1) represent a novel therapeutic approach for the treatment of pain. Developing various antagonists of hTRPV1, however, has been hindered by the unavailability of a 3D structure of hTRPV1. Recently, the 3D structures of rat TRPV1 (rTRPV1) in the presence and absence of ligand have been reported as determined by cryo-EM. rTRPV1 shares 85.7% sequence identity with hTRPV1. In the present work, we constructed and reported the 3D homology tetramer model of hTRPV1 based on the cryo-EM structures of rTRPV1. Molecular dynamics (MD) simulations, energy minimizations, and prescreen were applied to select and validate the best model of hTRPV1. The predicted binding pocket of hTRPV1 consists of two adjacent monomers subunits, which were congruent with the experimental rTRPV1 data and the cyro-EM structures of rTRPV1. The detailed interactions between hTRPV1 and its antagonists or agonists were characterized by molecular docking, which helped us to identify the important residues. Conformational changes of hTRPV1 upon antagonist/agonist binding were also explored by MD simulation. The different movements of compounds led to the different conformational changes of monomers in hTRPV1, indicating that TRPV1 works in a concerted way, resembling some other channel proteins such as aquaporins. We observed that the selective filter was open when hTRPV1 bound with an agonist during MD simulation. For the lower gate of hTRPV1, we observed large similarities between hTRPV1 bound with antagonist and with agonist. A five-point pharmacophore model based on several antagonists was established, and the structural model was used to screen in silico for new antagonists for hTRPV1. By using the 3D TRPV1 structural model above, the pilot in silico screening has begun to yield promising hits with activity as hTRPV1 antagonists, several of which showed substantial potency.

2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent transient receptor potential vanilloid 1 (TRPV1) antagonists: structure-activity relationships of 2-amino derivatives in the N-(6-trifluoromethylpyridin-3-ylmethyl) C-region.

A series of N-(2-amino-6-trifluoromethylpyridin-3-ylmethyl)-2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were designed combining previously identified pharmacophoric elements and evaluated as hTRPV1 antagonists. The SAR analysis indicated that specific hydrophobic interactions of the 2-amino substituents in the C-region of the ligand were critical for high hTRPV1 binding potency. In particular, compound 49S was an excellent TRPV1 antagonist (K(i(CAP)) = 0.2 nM; IC(50(pH)) = 6.3 nM) and was thus approximately 100- and 20-fold more potent, respectively, than the parent compounds 2 and 3 for capsaicin antagonism. Furthermore, it demonstrated strong analgesic activity in the rat neuropathic model superior to 2 with almost no side effects. Compound 49S antagonized capsaicin induced hypothermia in mice but showed TRPV1-related hyperthermia. The basis for the high potency of 49S compared to 2 is suggested by docking analysis with our hTRPV1 homology model in which the 4-methylpiperidinyl group in the C-region of 49S made additional hydrophobic interactions with the hydrophobic region.

N-4-t-Butylbenzyl 2-(4-Methylsulfonylaminophenyl) Propanamide TRPV1 Antagonists : Structure Activity Relationships in the A-region

Structure-activity relationships for the A-region in a series of N-4-t-butylbenzyl 2-(4-methylsulfonylaminophenyl) propanamides as TRPV1 antagonists have been investigated. Among them, the 3-fluoro analogue 54 showed high binding affinity and potent antagonism for both rTRPV1 and hTRPV1 in CHO cells. Its stereospecific activity was demonstrated with marked selectivity for the (S)-configuration (54S versus 54R). A docking study of 54S with our hTRPV1 homology model highlighted crucial hydrogen bonds between the ligand and the receptor contributing to its potency.

TRPV1 Activation is Not An All-Or-None Event: TRPV1 Partial Agonism/Antagonism and Its Regulatory Modulation

TRPV1 has emerged as a promising therapeutic target for pain as well as a broad range of other conditions such as asthma or urge incontinence. The identification of resiniferatoxin as an ultrapotent ligand partially able to dissect the acute activation of TRPV1 from subsequent desensitization and the subsequent intense efforts in medicinal chemistry have revealed that TRPV1 affords a dramatic landscape of opportunities for pharmacological manipulation. While agonism and antagonism have represented the primary directions for drug development, the pharmacological complexity of TRPV1 affords additional opportunities. Partial agonism/partial antagonism, its modulation by signaling pathways, variable desensitization, and slow kinetics of action can all be exploited through drug design.

Differential modulation of agonist and antagonist structure activity relations for rat TRPV1 by cyclosporin A and other protein phosphatase inhibitors

The transient receptor potential V1 channel (vanilloid receptor, TRPV1) represents a promising therapeutic target for inflammatory pain and other conditions involving C-fiber sensory afferent neurons. Sensitivity of TRPV1 is known to be subject to modulation by numerous signaling pathways, in particular by phosphorylation, and we wished to determine whether TRPV1 structure activity relations could be differentially affected. We demonstrate here that the structure activity relations of TRPV1, as determined by 45Ca2 uptake, were substantially altered by treatment of the cells with cyclosporin A, an inhibitor of protein phosphatase 2B. Whereas the potency of resiniferatoxin for stimulation of 45Ca2 was not altered by cyclosporin A treatment, the potencies of some other agonists were increased up to 8-fold. Among the antagonists examined, potencies were reduced to a lesser extent, ranging from 1- to 2.5-fold. Finally, the efficacy of partial agonists was increased. In contrast to cyclosporin A, okadaic acid, an inhibitor of protein phosphatases 1 and 2A, had little effect on agonist potencies, and calyculin A, an inhibitor of protein phosphatases 1 and 2A but with somewhat different selectivity from that of okadaic acid, caused changes in structure activity relations distinct from those induced by cyclosporin A. Because phosphatase activity differentially modulates the structure activity relations of TRPV1 agonists and antagonists, our findings predict that it may be possible to design agonists and antagonists selective for TRPV1 in a specific regulatory environment. A further implication is that it may be desirable to tailor screening approaches for drug discovery to reflect the desired regulatory state of the targeted TRPV1.

Conformationally constrained analogues of diacylglycerol (DAG). Effect on protein kinase C (PK-C) binding by the isosteric replacement of sn-1 and sn-2 esters in DAG-lactones

In order to determine the importance of the two ester pharmacophores in high affinity, conformationally constrained DAG-lactones (Lac-1-5) as PK-C ligands, we have independently replaced the sn-1 and sn-2 carbonyl esters in these compounds by ketone (2, 10, 11), amide (3, 25-28), and hydroxyl (12, 13) isosteres. Although the ketone analogue of the sn-1 ester (2) exhibited comparable activity to the parent Lac-1 when taking into account the difference in lipophilicities, the other isosteres were significantly poorer PK-C alpha ligands compared to the parent DAG-lactones. This study demonstrates that the ester functionality in DAG-lactone plays an important role in the ligands capacity to form a strong hydrogen bond with Gly253 at the active site. The discrete K(i) analysis from the sn-1 and sn-2 isosteres further confirms that the DAG-lactones bind preferentially to the C1-domain in the sn-2 binding mode, as previously suggested.

Asymmetric synthesis and receptor activity of chiral simplified resiniferatoxin (sRTX) analogues as transient receptor potential vanilloid 1 (TRPV1) ligands

The chiral isomers of the two potent simplified RTX-based vanilloids, compounds 2 and 3, were synthesized employing highly enantioselective PTC alkylation and evaluated as hTRPV1 ligands. The analysis indicated that the R-isomer was the eutomer in binding affinity and functional activity. The agonism of compound 2R was comparable to that of RTX. Docking analysis of the chiral isomers of 3 suggested the basis for its stereospecific activity and the binding mode of 3R.

Non-vanillyl resiniferatoxin analogues as potent and metabolically stable transient receptor potential vanilloid 1 agonists.

A series of non-vanillyl resiniferatoxin analogues, having 4-methylsulfonylaminophenyl and fluorophenyl moieties as vanillyl surrogates, have been investigated as ligands for rat TRPV1 heterologously expressed in Chinese hamster ovary cells. Although lacking the metabolically problematic 4-hydroxy substituent on the A-region phenyl ring, the compounds retained substantial agonist potency. Indeed, the 3-methoxy-4-methylsulfonylaminophenyl analog (1) was modestly (2.5-fold) more potent than RTX, with an EC(50)=0.106 nM. Further, it resembled RTX in its kinetics and pattern of stimulation of the levels of intracellular calcium in individual cells, as revealed by imaging. Compound 1 displayed modestly enhanced in vitro stability in rat liver microsomes and in plasma, suggesting that it might be a pharmacokinetically more favorable surrogate of resiniferatoxin. Molecular modeling analyses with selected analogues provide evidence that the conformational differences could affect their binding affinities, especially for the ester versus amide at the B-region.