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

Significance The TRPA1 channel functions as an irritant sensor and is a therapeutic target for treating pain, itch, and respiratory diseases. TRPA1 can be activated by electrophilic compounds via covalent modification or activated by noncovalent agonists via ligand binding. However, how covalent modification leads to channel opening and, importantly, how noncovalent binding activates TRPA1 are not well-understood. Here we identified a group of noncovalent agonists and used them to explore TRPA1 gating through iterative functional analyses, molecular modeling, and structure–activity relationship studies. We show that TRPA1 possesses an evolutionarily conserved ligand binding site common to other TRP channels. The combination of computational modeling and experimental structure–activity data lays the foundations for rational drug design. 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.