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.

Keywords: TRPA1; agonist; binding; gating.

Fig. 1.

Piperidines activate human TRPA1. (A) Structure and activity of PIPC1 and PIPC2. PIPC1 and PIPC2 evoked Ca²⁺ influx in HEK-293F cells expressing human TRPA1, as represented by an increase in fluorescence signal (RFU, relative fluorescence unit). In contrast, A-967079 did not evoke Ca²⁺ response and blocked AITC-evoked responses. Of note, the responses to 100 nM PIPC1 or 400 nM PIPC2 decayed over time and prevented subsequent response to 100 μM AITC. n = 4 to 8. (B) PIPC1- (0.4 μM) and PIPC2- (1 μM) induced Ca²⁺ influx was blocked by preincubation of A-967079 (1 μM). n = 4 to 8. (C) Concentration dose–response of PIPC1 and PIPC2. PIPC1- and PIPC2-induced Ca²⁺ signals were normalized against the effect of 300 μM AITC. n = 4. Hill slopes were 1.5, 1.4, and 1.4 for PIPC1, PIPC2, and AITC, respectively. (D) In representative cells expressing human TRPA1, PIPC1 (10 nM) or PIPC2 (30 nM) evoked currents that were sensitive to block by A-967079 (10 μM). At high concentrations, PIPC1 (300 nM) and PIPC2 (1.2 μM) evoked fast-onset currents, followed by current decay and insensitivity to AITC (100 μM). Dark trace: current at +80 mV; gray trace: current at −80 mV; dotted line: 0-current level. n = 4 to 8. (E) Relative current amplitudes at baseline (Base) and in response to varying concentrations of PIPC1 or PIPC2. Each cell was stimulated with a single concentration of compounds, and currents were normalized against peak currents of 100 μM AITC obtained from a different group of cells. The asterisk indicates significantly different (*P < 0.001) from baseline. n = 13 to 25. Representative traces are shown. The apparent potency of PIPC1 and PIPC2 from patch-clamp experiments could be overestimated, due to desensitization and the use of divalent-free solution. However, qualitatively, these patch-clamp experiments confirmed the agonist effect of PIPC1 and PIPC2.

Scheme 1.

Structure–activity relationship for PIPC1 to PIPC4.

Fig. 2.

Pore domain underlies the species-specific effect of PIPC1. (A) AITC activated TRPA1 from human, guinea pig, chicken, rat, and dog in a concentration-dependent manner, whereas PIPC1 only activated the human TRPA1 channel. Hill slope values for AITC were 1.4 to 1.7 for all species, and the Hill slope for PIPC1 was 1.6. (B) PIPC1 (20, 4, and 0.8 μM) evoked robust Ca²⁺ responses on hTRPA1, rN, rPreS1, rVSD, and rC but not on rS5P, rS6, or rTRPA1. Traces were normalized to the response evoked by 300 μM AITC. n = 4 to 8 for all experiments, and representative traces are shown.

Fig. 3.

V875 and I946 are critical for PIPC1 and PIPC2 activation of human TRPA1. (A) Divergent residues between human and rat channel in the S5, S5–S6 linker, and S6 segments. Each of the human TRPA1 residues was substituted by the equivalent residue in rat TRPA1 either individually or in combination; the fold change in PIPC1 potency is indicated below the sequences. V875G and I946M abolished response to PIPC1 whereas other mutations retained PIPC1 responses. (B) Representative Ca²⁺ influx traces in response to 20, 4, 0.8, and 0 μM PIPC1. I946M eliminated response to PIPC1, whereas rG878V/I949M restored response. n = 4 to 8. (C) Concentration–response relationships of PIPC1. EC₅₀ and Hill slope values were 0.0065 ± 0.0003 μM and 1.5 for human TRPA1, 0.031 ± 0.04 μM and 1.4 for I890V, and 0.123 ± 0.006 μM and 1.5 for rG878V/M949I; E_max values (relative to AITC) were 0.41 ± 0.03 for human, 0.39 ± 0.04 for I890V, and 0.38 ± 0.06 for rG878V/M949I. n = 4. (D) Representative currents in response to PIPC1 and 100 μM AITC stimulation. The voltage protocol was the same as in Fig. 1D and currents at +80 mV are plotted. The dotted line indicates 0-current level. PIPC1 evoked currents in hTRPA1 and rG878V/M949I at 3 and 10 nM, respectively, but failed to induce current in I946M at 300 nM. n = 6 to 8. (E) Representative Ca²⁺ influx traces in response to PIPC2. n = 4 to 8. (F) Concentration–response relationships for PIPC2. EC₅₀ and Hill slope values of PIPC2 were 0.026 ± 0.003 μM and 1.4 for hTRPA1, and 0.035 ± 0.06 μM and 1.4 for I890V. For rG878V/M949I, EC₅₀ was not determined due to relatively small E_max (∼0.16). n = 4. (G) Representative currents in response to PIPC2 and 100 μM AITC stimulation. PIPC2 (30 nM) activated currents in human TRPA1 and rG878V/M949I but had no effect on I949M. Representative traces are shown for all experiments.

Fig. 4.

Structural comparisons of closed and open TRPA1 states. (A) Homology models of TRPA1 in the closed (blue) and open states (red; TRPV1 as template). Only TM domains (residues 446 to 1078) are shown. TRPA1 structures are rendered as a cartoon. (A, Left) Side views. (A, Middle) Zoom into the pore structures. (A, Right) Top views. LG, lower gate; UG, upper gate. Only backbones are shown, with the exception of upper (D915) and lower gates (I957 and V961). (B) Closed and open TRPA1 states are superimposed along the pore helices (PH1 and PH2) and the S6 segments. For each state, PH1 and PH2, along with TM helical segment 6, are shown for 2 opposing subunits. S5 segments were removed from the visualization. Residues of S6 involved in the π-bulge rearrangement are labeled (⁹⁴⁶IFVPI⁹⁵⁰). (C) Superimposition of the 2 TRPA1 open models using TRPV1 and TRPV6 as templates (shown in red and pink, respectively). (D) Superimposition of TRPA1 structures in the open and closed states over residues in S5 and S6 helices and PH1. Models obtained using TRPV6 as template are shown in pink (open) and cyan (closed), respectively. (E) Superimposition of S6 segments from TRPA1 models and TRPV6 experimental structures (colored as in A–D).

Fig. 5.

Binding of PIPC to the open TRPA1 channel. (A) The PIPC binding site lies right below PH1, at the interface of 1 helical segment S5 with 2 helical segments S6, the latter from an adjacent subunit. The receptor surface is rendered in gray. Protein atoms are rendered as cartoons, colored by residue position (S6, blue; S5 and PH1, green). PIPC1 is shown in ball-and-stick rendering; carbon, oxygen, nitrogen, and halogen atoms are colored in cyan, red, blue, and green, respectively. (B) Two-dimensional map of PIPC1 interactions with TRPA1. Critical binding residues (blue circles) are confirmed by mutagenesis study (SI Appendix, Table S1). Green, light blue, and gray lines indicate hydrophobic, polar, and contact interactions; purple arrows indicate hydrogen-bonding interactions; dot-connecting dark green lines indicate π-stacking. Green and light blue petals indicate hydrophobic and polar residues. “A” and “C” in petals indicate adjacent TRPA1 subunits. (C–E) Critical binding residues are rendered as volumetric Gaussian density maps (at 0.5 density isovalue) in transparent mode. Maps in different colors indicate regions of the binding site that account for specific interactions with the bound ligand (licorice mode). Protein atoms are rendered in cartoon representation, with gray and pink indicating distinct and adjacent subunits. Critical binding residues are labeled in blue. For PIPC1, ligand protonation state was determined experimentally to be neutral; carbon, nitrogen, oxygen and hydroxyl group, chlorine, and fluorine atoms are colored in gray, blue, red, purple, and light green, respectively. Binding modes were validated by mutagenesis and SAR explorations (Schemes 1 and 2).

Scheme 2.

Structure–activity relationship expansion for PIPC5 to PIPC13.

Fig. 6.

Rearrangement of an evolutionarily conserved π-bulge on S6 results in differential sets of residues to face the pore and the PIPC binding site in TRPA1. (A) Superimposition of S5, S6, and PH1 helices in the closed and open TRPA1 states. Models were built on TRPV6 as templates. Critical residues undergoing structural rearrangements, namely M953 and N954, are highlighted in licorice mode. M953 faces the pore or the PIPC binding site in the closed or open states, respectively. N954 faces outside or inside the pore in the closed or open states, respectively. Additional residues responsible for differential binding of PIPC1 to the open and closed states are also shown, including L870, L881, M912, and I950. Residues that were not previously found to interact with other ligands (L870) or were only partially characterized (M953) are included in a blue circle. (A, Inset) Zoom into PIPC1 binding from a different angle and highlight of M953, N954, and L870. Protein atoms are rendered as in Fig. 4. (B) Top and lateral views of TRPA1 in the closed and open states, respectively. (C) S6 in 1 subunit was removed for visualization purposes.