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Structural basis of TRPV3 inhibition by an antagonist

Nature Chemical Biology volume 19, pages 81–90 (2023)Cite this article

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Abstract

The TRPV3 channel plays vital roles in skin physiology. Dysfunction of TRPV3 causes skin diseases, including Olmsted syndrome. However, the lack of potent and selective inhibitors impedes the validation of TRPV3 as a therapeutic target. In this study, we identified Trpvicin as a potent and subtype-selective inhibitor of TRPV3. Trpvicin exhibits pharmacological potential in the inhibition of itch and hair loss in mouse models. Cryogenic electron microscopy structures of TRPV3 and the pathogenic G573S mutant complexed with Trpvicin reveal detailed ligand-binding sites, suggesting that Trpvicin inhibits the TRPV3 channel by stabilizing it in a closed state. Our G573S mutant structures demonstrate that the mutation causes a dilated pore, generating constitutive opening activity. Trpvicin accesses additional binding sites inside the central cavity of the G573S mutant to remodel the channel symmetry and block the channel. Together, our results provide mechanistic insights into the inhibition of TRPV3 by Trpvicin and support TRPV3-related drug development.

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Fig. 1: Trpvicin inhibits TRPV3.
Fig. 2: The detailed VSLD-PD-binding site for Trpvicin in TRPV3.
Fig. 3: Trpvicin binds to a VSLD-PD pocket.
Fig. 4: G573S mutation expands the ion path of hTRPV3.
Fig. 5: Symmetry remodeling and pore blocking of G573S-C2 by Trpvicin.

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Data availability

The cryo-EM maps for hTRPV3apo, hTRPV3Trpvicin, hTRPV3-G573S-C4Trpvicin and hTRPV3-G573S-C2Trpvicin have been deposited in the Electron Microscopy Data Bank with accession codes EMD-33218, EMD-33214, EMD-33217 and EMD-33216. Atomic coordinates for hTRPV3apo, hTRPV3Trpvicin, hTRPV3-G573S-C4Trpvicin and hTRPV3-G573S-C2Trpvicin have been deposited in the Protein Data Bank under accession codes 7XJ3, 7XJ0, 7XJ2 and 7XJ1. Source data are provided with this paper.

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Acknowledgements

We thank X. Huang, B. Zhu, X. Li and L. Chen at the Center for Biological Imaging, Core Facilities for Protein Science, at the Institute of Biophysics, Chinese Academy of Sciences, for support in cryo-EM data collection. Cryo-EM data collection was also supported by the Cryo-EM platform of Peking University with the assistance of Z. Guo, X. Pei, G. Wang, C. Qin and N. Li. We thank W. Guo, Q. Luo and Y. Huang for helpful discussion as well as N. Zheng, M. Liao, L. Chen and H. Hu for proofreading the manuscript. This work is funded by grants from the National Key Research and Development Program of China (2017YFA0505200 to X.L.), the National Natural Science Foundation of China (21625201, 21961142010, 21661140001, 91853202 and 21521003 to X.L.; 82130091 and 81673044 to Y.Y.; and 22177006 to J.F.), the CAMS Innovation Fund for Medical Sciences (2021-1-I2M-018 to Y.Y.), the Beijing Outstanding Young Scientist Program (BJJWZYJH01201910001001 to X.L.) and the Institute of Physics, Chinese Academy of Sciences (E0VK101 to D.J.).

Author information

Author notes

  1. These authors contributed equally: Junping Fan, Linghan Hu, Zongwei Yue, Daohong Liao.

  2. These authors jointly supervised this work: Daohua Jiang, Yong Yang, Xiaoguang Lei.

Authors and Affiliations

  1. Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Department of Chemical Biology, College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China

    Junping Fan, Zongwei Yue, Fusheng Guo, Han Ke & Xiaoguang Lei

  2. Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, China

    Linghan Hu & Yong Yang

  3. Iongen Therapeutics Co. Ltd., Nanjing, China

    Daohong Liao

  4. Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China

    Daohua Jiang

  5. University of Chinese Academy of Sciences, Beijing, China

    Daohua Jiang

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Contributions

X.L. and Y.Y. initiated the project. X.L., Y.Y. and D.J. supervised the project. J.F. and Z.Y. prepared the samples for the cryo-EM study and made the mutation constructs. D.J. and J.F. collected the cryo-EM data. D.J. calculated the EM maps. D.J. and J.F. built and refined the atomic model. L.H. performed the electrophysiological assays. L.H. and F.G. conducted the high-throughput chemical screening and animal studies. D.L. synthesized all of the compounds. H.K. did the MM/GBSA calculations. X.L., Y.Y., D.J., J.F. and L.H. analyzed the data. All authors contributed to the manuscript preparation.

Corresponding authors

Correspondence to Daohua Jiang, Yong Yang or Xiaoguang Lei.

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Competing interests

X.L. and Y.Y. are co-founders of Iongen Therapeutics Co. Ltd., and D.L. is currently a full-time employee of Iongen. The remaining authors declare no competing interests.

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Nature Chemical Biology thanks Jun Chen, Zhiguang Yuchi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Trpvicin inhibits TRPV3 in contrast to other representative members of TRP family.

a-g, The representative current traces of wild type hTRPV1 (a), mTRPV2 (b), hTRPV4 (c), hTRPV5 (d), hTRPV6 (e), hTRPA1 (f), and hTRPM8 (g) inhibited by increasing concentrations of Trpvicin (black bar), at ±80 mV. AITC, allyl-isothiocyanate. h-i, Curve fitting of dose-dependent inhibition of agonist-evoked currents by Trpvicin on hTRPA1 (h) (300 µM AITC, at −80 mV; logIC50 = 3.01 ± 0.75, n = 4), and hTRPM8 (i) (100 µM menthol, at +80 mV; logIC50 = 1.67 ± 0.10, n = 3). Data are presented as mean values + /- SEM.

Source data

Extended Data Fig. 2 Trpvicin relieves itch and hair loss in mice.

a, The representative current traces of mTRPV3-WT, mTRPV3-WT/G568V, and mTRPV3-G568V in response to 2-APB (300 μM, red bar) and co-application of increasing concentrations of Trpvicin (from 10 nM to 100 μM, black bar), at ±80 mV. 10 μM RR was used to assess the whole amplitudes of leak currents. b, Curve fitting of dose-dependent inhibition of 300 μM 2-APB-evoked mTRPV3 WT, WT/G568V, and G568V currents at −80 mV by Trpvicin (mTRPV3-WT, logIC50 = −0.42 ± 0.08, n = 5; mTRPV3-WT/G568V, logIC50 = −0.02 ± 0.10, n = 5; mTRPV3-G568V, logIC50 = 0.42 ± 0.03, n = 7). Data are presented as mean values + /- SEM. c, Acute itch behaviors of WT mice induced by intradermal injections of SLIGRL alone or co-application of Trpvicin. Two-sided paired t-tests with the corresponding vehicle-treated groups (10 μM, P = 0.0216; 100 μM, P = 0.0049).n = 7. Data are presented as mean values + /- SEM. SLIGRL, peptide SLIGRL-NH2. d,e, The scratching bouts (d) and percentage of ear thickness increase (e) of WT mice treated with MC903 alone or co-application of Trpvicin for 7 days. Two-way ANOVA (d, P = 0.0219; e, P = 0.0021) followed by Bonferroni post-tests with the vehicle-treated group (+100 mg/kg Trpvicin, n = 5; +30 mg/kg Trpvicin, n = 8; +Vehicle, n = 7). Data are presented as mean values + /- SEM. MC903, calcipotriol. f, Trpv3+/G568V mice were topically treated with 1 wt% Trpvicin or vehicle once per day starting from P50. After 16 days of treatment, for both genders at P66, the Trpvicin-treated mice showed apparently longer hair shafts in comparison to the vehicle-treated group. g, The inverted mean grey values of the back area of Trpv3+/G568V mice topically treated with 1 wt% Trpvicin or vehicle starting from P50. Two-way ANOVA compared with the vehicle-treated group, P = 0.0002. n = 3. Data are presented as mean values + /- SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Source data

Extended Data Fig. 3 Cryo-EM data processing of hTRPV3apo and hTRPV3Trpvicin.

a, Flow chart of cryo-EM data processing of hTRPV3apo. A total of 1,451,587 particles were picked from 2,199 micrographs. A representative motion-corrected micrograph of the dataset is shown here (Bar = 400 Å). Several rounds of 2D and 3D classifications were conducted to clean particles, followed by Bayesian Polish to improve image quality. The final map was refined at 3.54 Å according to the GSFSC criterion. b, Local resolution distribution of the final sharpened map of the hTRPV3apo. c, Particle angular distribution calculated in cryoSPARC for the final reconstruction. d, Fourier Shell Correlations (FSC) of the final map of the hTRPV3apo structure, calculated between two independently refined half-maps before (blue) and after (red) post-processing, overlaid with an FSC curve calculated between the cryo-EM density map and the structural model shown in black. e, Flow chart of cryo-EM data processing of hTRPV3Trpvicin. A total of 1,491,046 particles were picked from 3,203 micrographs. A representative motion-corrected micrograph is shown here (Bar = 400 Å). Several rounds of 2D and 3D classifications were conducted to clean particles, followed by Bayesian Polish and CTF Refine to improve image quality. The final map was refined at 2.53 Å according to the GSFSC criterion. f, Sharpened map of the hTRPV3Trpvicin, colored based on the local resolution values. g, Particle angular distribution calculated in cryoSPARC for the final reconstruction. h, Fourier Shell Correlations (FSC) of the final map of the hTRPV3Trpvicin, calculated between two independently refined half-maps before (blue) and after (red) post-processing, overlaid with an FSC curve calculated between the cryo-EM density map and the structural model shown in black.

Extended Data Fig. 4 B-factor distribution in hTRPV3 structures.

a-d, The Cα B-factors are depicted on the refined structure from dark blue (lowest B-factor) to red (highest B-factor) for hTRPV3apo (a), hTRPV3Trpvicin (b), hTRPV3-G573S-C4Trpvicin (c) and hTRPV3-G573S-C2Trpvicin (d) respectively.

Extended Data Fig. 5 Structural comparison of TRPV3 structures determined in open and closed states.

a,b, Superposition of hTRPV3Trpvicin (light pink) with mouse TRPV3 structure (PDB code: 6LGP, grey, (a)) and human TRPV3 structure (PDB code: 6UW4, grey, (b)) at closed states viewed parallel to the membrane. c,d, Superposition of hTRPV3-G573S-C4Trpvicin (wheat) with mouse TRPV3 structure (PDB code: 7MIO, grey, (c)) and human TRPV3-K169A structure (PDB code: 6UW6, grey, (d)) at open states viewed parallel to the membrane. On the right, showing the close-up views for the comparisons of S6 and pore-helices.

Extended Data Fig. 6 The binding sites function evaluation.

a, The representative current traces of hTRPV3 mutants in the VSLD-PD binding sites in response to 2-APB (300 μM, red bar) and co-application of increasing concentrations of Trpvicin (from 1 nM to 100 μM, black bar), at ±80 mV. b, The representative current traces of hTRPV3-G573S mutants in the pore binding sites and the VSLD-PD binding sites inhibited by increasing concentrations of Trpvicin at ±80 mV. 10 μM RR was used to assess the whole amplitudes of leak currents. c, The representative current traces of hTRPV3 mutants in the pore binding sites in response to 2-APB and co-application of increasing concentrations of Trpvicin at ±80 mV. d, Per-residues binding energy calculated by MM-GBSA. Data are presented as mean values +/− SD of triplicates. e, The hTRPV3 mutants function similarly to the hTRPV3-WT in response to 300 μM 2-APB.The current density at −80 mV of hTRPV3 mutant channels activated by 300 μM 2-APB, compared with hTRPV3-WT. One-way ANOVA followed by Bonferroni post-tests (WT, n = 6; A556V, n = 4; A560T, n = 4; F597Y, n = 4; F601A, n = 4; T660A, n = 5; T665A, n = 5; F666A, n = 5; F666Y, n = 5). Data are presented as mean values + /- SEM. WT, wild type. f, The double mutation variants function similarly to the hTRPV3-G573S. The current density of leak currents at −80 mV of hTRPV3 double mutation variants, compared with hTRPV3-G573S. One-way ANOVA followed by Bonferroni post-tests (hTRPV3-G573S, n = 5; G573S-A556V, n = 4, G573S-A560T, n = 4; G573S-T665A, n = 4; G573S-F666A, n = 5; G573S-F666Y, n = 4). Data are presented as mean values + /- SEM; n.s., not significant.

Source data

Extended Data Fig. 7 Cryo-EM data processing of hTRPV3-G573S.

a, Flow chart of cryo-EM data processing. A total of 691,221 particles were picked from 2,594 micrographs. A representative motion-corrected micrograph of the dataset is shown here (Bar = 400 Å). Several rounds of 2D and 3D classifications were conducted to clean particles, followed by Bayesian Polish and CTF Refine to improve image quality. The final map was refined at 3.64 Å and 2.93 Å for hTRPV3-G573S-C4 and hTRPV3-G573S-C2 according to the GSFSC criterion, respectively. (b & e) Local resolution distribution of the sharpened map of the hTRPV3-G573S-C4Trpvicin and hTRPV3-G573S-C2Trpvicin. (c & f) Particle angular distribution calculated in cryoSPARC for the final reconstruction of hTRPV3-G573S-C4Trpvicin and hTRPV3-G573S-C2Trpvicin. (d & g) Fourier Shell Correlations (FSC) of the final map of the hTRPV3-G573S-C4Trpvicin and hTRPV3-G573S-C2Trpvicin, calculated between two independently refined half-maps before (blue) and after (red) post-processing, overlaid with an FSC curve calculated between the cryo-EM density map and the structural model shown in black.

Extended Data Fig. 8 Comparison of the Trpvicin VSLD-PD site and fenestrations of hTRPV3Trpvicin, G573S-C4Trpvicin, and G573S-C2Trpvicin.

a, Side view of hTRPV3-G573S-C4 in complex with Trpvicin colored in yellow and wheat. The Trpvicin is shown in spheres. b-c, Close-up views for the comparison of the Trpvicin VSLD-PD binding site in G573S-C4Trpvicin (wheat), hTRPV3Trpvicin (light pink), and G573S-C2Trpvicin (cyan). The Trpvicin is shown in sticks. d, Side-views of fenestrations formed by two adjacent pore domains from hTRPV3apo, hTRPV3-G573S-C4Trpvicin, hTRPV3-G573S-C2Trpvicin-chainA/chainB, and hTRPV3-G573S-C2Trpvicin-chainB/chainC, respectively.

Extended Data Fig. 9 Representative cryo-EM density of S6 helix and Trpvicin.

a, Cryo-EM density for S6 helix of hTRPV3apo, hTRPV3Trpvicin, hTRPV3-G573S-C4Trpvicin, and hTRPV3-G573S-C2Trpvicin. The π- and α-helix on S6 helices are highlighted and related resides are labeled accordingly. b, Cryo-EM density for Trpvicin bound to hTRPV3Trpvicin, hTRPV3-G573S-C4Trpvicin, and hTRPV3-G573S-C2Trpvicin at the VSLD-PD site. c, Cryo-EM density for Trpvicin bound to the central cavity of hTRPV3-G573S-C2Trpvicin.

Extended Data Fig. 10 A cartoon model for the antagonist inhibition mechanism of TRPV3 and G573S mutant.

a, The hTRPV3apo at closed-state. The hTRPV3apo VSLD domain, linker region, and TRP helix from the two opposing subunits and the ion-conduction pores from the adjacent subunit are shown. b, Conformational changes induced by the Trpvicin bound to TRPV3. The Trpvicin wedges into the VSLD-PD site between the VSLD and the PD from the adjacent subunit, inducing conformational changes of the entire channel into a more closed state. The movements of individual domains are indicated by arrows. c, Conformational changes induced by G573S mutation expand the ion path at both the SF and the activation gate in the C4 symmetry. d, Trpvicin molecules enter the central cavity of the G573S mutant channel, inducing structural rearrangements to form C2 symmetry and block ion permeation. The pore-lining S6 helices undergo π- to α-helix transitions for the G573S structures. Two gear shapes stand for the interaction between the S4-S5 linker and TRP helix. In the hTRPV3Trpvicin structure, the S4-S5 linker and TRP helix interaction was strengthened compared to hTRPV3Apo because of the shrinking caused by Trpvicin binding while in the G573S mutant, the interaction was interrupted, showing loosely interaction in hTRPV3-G573S-C4Trpvicin structure and even less in hTRPV3-G573S-C2Trpvicin structure because of the twisting between the two domains. The G573S mutation position is indicated by a black asterisk.

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Fan, J., Hu, L., Yue, Z. et al. Structural basis of TRPV3 inhibition by an antagonist. Nat Chem Biol 19, 81–90 (2023). https://doi.org/10.1038/s41589-022-01166-5

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