Human TRPV1 structure and inhibition by the analgesic SB-366791

Abstract

Pain therapy has remained conceptually stagnant since the opioid crisis, which highlighted the dangers of treating pain with opioids. An alternative addiction-free strategy to conventional painkiller-based treatment is targeting receptors at the origin of the pain pathway, such as transient receptor potential (TRP) ion channels. Thus, a founding member of the vanilloid subfamily of TRP channels, TRPV1, represents one of the most sought-after pain therapy targets. The need for selective TRPV1 inhibitors extends beyond pain treatment, to other diseases associated with this channel, including psychiatric disorders. Here we report the cryo-electron microscopy structures of human TRPV1 in the apo state and in complex with the TRPV1-specific nanomolar-affinity analgesic antagonist SB-366791. SB-366791 binds to the vanilloid site and acts as an allosteric hTRPV1 inhibitor. SB-366791 binding site is supported by mutagenesis combined with electrophysiological recordings and can be further explored to design new drugs targeting TRPV1 in disease conditions.

Fig. 1. Apo-state structure of human TRPV1.

a Representative current trace obtained at –40 mV in response to a temperature ramp from 25 °C to 45 °C recorded in the whole-cell mode from an HEK 293 cell expressing hTRPV1. b Current-temperature dependence obtained from recordings as in a showing temperature activation of hTRPV1. c Exemplar whole-cell current traces evoked by a voltage ramp in HEK 293 cells expressing hTRPV1 by capsaicin from 0.01 to 10 µM at 25 °C. d Concentration-dependence of hTRPV1 activation by capsaicin measured at +80 mV and normalized to the current at 10 µM of capsaicin. Data are shown as mean ± SEM from 7 cells and fitted with the Hill equation. Source data are provided as a Source Data file. e 3D cryo-EM reconstruction of hTRPV1 in the apo state viewed parallel to the membrane (left) or extracellularly (right), with density for subunits colored green, yellow, pink and blue, and lipids in purple. f hTRPV1_Apo structure viewed parallel to the membrane (left) or extracellularly (right), with subunits colored similarly to e and lipid molecules shown in sticks. g, h Exemplar cryo-EM densities (purple mesh) for lipid molecules PC (g) and PI (h), shown as stick models (yellow).

Fig. 2. Structure of human TRPV1 in complex with the inhibitor SB-366791.

a Exemplar whole-cell current recorded at –40 mV from HEK 293 cell expressing hTRPV1 in response to application of 1 µM of capsaicin (Cap) and 10 µM of SB-366791. b Quantification of the inhibitory effect of 10 µM SB-366791 on hTRPV1 peak current amplitude in the presence of 1 µM capsaicin, measured 10 s after SB-366791 application. Statistical analysis: two-tailed paired t-test. ***P = 0.0003. Data points represent paired recordings from 5 individual cells. c Quantification of capsaicin-stimulated hTRPV1 peak current amplitude recovery after termination of SB-366791 application. The cell was washed for at least 2 min before the second application of Cap. Data are shown as mean ± SEM. Data points represent recordings from 5 individual cells. d, e hTRPV1_SB-366791 structure viewed parallel to the membrane (d) or extracellularly (e), with subunits colored green, yellow, pink, and blue, and lipids shown as purple sticks. f Chemical structure (left) and molecular model (right) of SB-366791, with the cryo-EM density for the inhibitor shown as red mesh. g Close-up view of the binding pocket, with SB-366791 and residues that contribute to its binding shown in sticks. h Exemplar whole-cell currents recorded from hTRPV1-expressing HEK 293 cell in response to a voltage ramp in the presence of different concentrations of SB-366791 at pH 5. i Concentration-dependencies of SB-366791 inhibition of wild-type and mutant hTRPV1 channels at pH 5, measured at +80 mV and normalized to the value of pH5-induced current. Data are shown as mean ± SEM. Lines represent fits to the Hill equation (WT, n = 7; Y511A, n = 7) or connect data points (L515A, n = 7; L547R, n = 7, T550A, n = 7). Source data are provided as a Source Data file.

Fig. 3. Closed pore of hTRPV1_Apo and hTRPV1_SB-366791 structures.

a Pore-forming domain in hTRPV1_SB-366791 with the residues contributing to pore lining shown as sticks. Only two of four subunits are shown, with the front and back subunits omitted for clarity. The pore profile is shown as a space-filling model (gray). The region that undergoes α-to-π transition in S6 is labeled (π). b Pore radius for hTRPV1_Apo in synthetic (blue) and soybean (pink) lipids, hTRPV1_SB-366791 (green) and rTRPV1_Open (orange; PDB ID: 7RQW) calculated using HOLE. The vertical dashed line denotes the radius of a water molecule, 1.4 Å.

Fig. 4. Structural changes accompanying competitive inhibition of hTRPV1 by SB-366791.

a–d Gray (no changes) to red (strong changes) gradient of the r.m.s.d. (a, b) or translation (c, d) calculated between the inhibited (hTRPV1_SB-366791) and open (rTRPV1, PDB ID: 7RQW) states and mapped on the hTRPV1_SB-366791 TMD. Only two of four subunits are shown in a and c, with the front and back subunits omitted for clarity. Molecules of SB-366791 are shown in sticks (green). The r.m.s.d. values were calculated for C_α atoms along the entire TRPV1 sequence with a sliding window of 10 residues. C_α atom translations were calculated after aligning the TMDs of the corresponding structures. Regions of the greatest structural changes are labeled in red.

Fig. 5. Mechanism of hTRPV1 inhibition by SB-366791.

a Closeup view of the vanilloid site in superposed RTX-bound open rTRPV1 (orange; PDB ID: 7RQW) and inhibited hTRPV1_SB-366791 (green) structures. Relative movements of domains are indicated by pink arrows. b, c Intracellular view of the gate region in RTX-bound open rTRPV1 (b) and inhibited hTRPV1_SB-366791 (c) structures. d Schematic mechanism of TRPV1 activation and inhibition by SB-366791.