TRPA1 modulation by piperidine carboxamides suggests an evolutionarily conserved binding site and gating mechanism

Abstract

The transient receptor potential ankyrin 1 (TRPA1) channel functions as an irritant sensor and is a therapeutic target for treating pain, itch, and respiratory diseases. As a ligand-gated channel, TRPA1 can be activated by electrophilic compounds such as allyl isothiocyanate (AITC) through covalent modification or activated by noncovalent agonists through ligand binding. However, how covalent modification leads to channel opening and, importantly, how noncovalent binding activates TRPA1 are not well-understood. Here we report a class of piperidine carboxamides (PIPCs) as potent, noncovalent agonists of human TRPA1. Based on their species-specific effects on human and rat channels, we identified residues critical for channel activation; we then generated binding modes for TRPA1–PIPC interactions using structural modeling, molecular docking, and mutational analysis. We show that PIPCs bind to a hydrophobic site located at the interface of the pore helix 1 (PH1) and S5 and S6 transmembrane segments. Interestingly, this binding site overlaps with that of known allosteric modulators, such as A-967079 and propofol. Similar binding sites, involving π-helix rearrangements on S6, have been recently reported for other TRP channels, suggesting an evolutionarily conserved mechanism. Finally, we show that for PIPC analogs, predictions from computational modeling are consistent with experimental structure–activity studies, thereby suggesting strategies for rational drug design.

Dual regulation of TRPV1 channels by phosphatidylinositol via functionally distinct binding sites

“…To do so, we used the “Residue-Scanning and Mutation” protocol available in BioLuminate ( 44 ) (Schrödinger, LLC, 2018), as described in ref. ( 45 ). All residues (D509, S510, K571, L574, I696, L699, I703) were mutated to alanine sidechains; upon mutation, protein sidechains were minimized to optimize interactions with the bound lipid.…”

Remedia Sternutatoria over the Centuries: TRP Mediation

“…Agonists activate TRPA1 via covalent modifications of the cysteine and lysine residues located in the ankyrin repeat motifs within the cytoplasmic N -terminus of the channel. Thus, TRPA1 activators can be categorized into thiol-reactive electrophilic compounds, which activate TRPA1 via covalent modification, and non-electrophilic compounds that activate TRPA1 via ligand binding [ 144 , 145 ]. Allyl isothiocyanate, an electrophile, was shown to form adduct with thiols and primary amines, proposing covalent modification.…”

The Enigmas of Surfaces

“…There is a binding site (A) located in the region of S5, S6 and pore helix 1 (PH1) of the pore domain. [27][28][29][30] For instance, a known TRPA1 antagonist, A-967079 (3) was shown to bind in this pocket, undergoing interactions with residues F909 (Figure 2), S873, T874 and M912. [7,27] Piperidine carboxamides (PIPCs) 1 (4) and 2 are fitted in a pocket by residues F909, M912, and L913 of PH1 and L881, F877, M953 and I957 of S5 and S6.…”

“…PIPC1 and 2 are stabilized by residues of S6 (F938, V942 and I946) and residues of S5 (L870, S873, T874, I878 and L881). [28] Decanol (5), phenethyl butanoate (6) and 2-ethyl-1-hexanol (7) interacted with hydrophobic residues L881 and F909 and hydrophilic residue T874. The conformational re-arrangement of S5, S6 and PH1 was associated with channel opening, especially the conformational movement of S6 was linked with the opening of the lower and upper gates.…”

“…[29] PIPCs (10) have been identified as potent, noncovalent agonists of human TRPA1. [28] The most potent agonist (PIPC1 (4)) was estimated to have an EC50 = 6.5 nM towards human TRPA1. The binding site studies exploited the homology models of the open and closed states for TRPA1.…”