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A series of α-methylated analogues of the potent sRTX thiourea antagonists were investigated as rTRPV1 ligands in order to examine the effect of α-methylation on receptor activity. The SAR analysis indicated that activity was stereospecific with the (R)-configuration of the newly formed chiral center providing high binding affinity and potent antagonism while the configuration of the C-region was not significant., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

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.