Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

Balázs Nemes; Szabolcs László; Balázs Zoltán Zsidó; Csaba Hetényi; Adam Feher; Ferenc Papp; Zoltan Varga; Éva Szőke; Zoltán Sándor; Erika Pintér

doi:10.3389/fphys.2023.1180896

Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor

Front Physiol. 2023 Jun 7:14:1180896. doi: 10.3389/fphys.2023.1180896. eCollection 2023.

Authors

Affiliations

¹ Department of Pharmacology and Pharmacotherapy, Medical School, University of Pécs, Pécs, Hungary.
² Department of Inorganic and Analytical Chemistry, Faculty of Chemical Technology and Biotechnology, Budapest University of Technology and Economics, Budapest, Hungary.
³ Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary.

Abstract

Introduction: Previous studies have established that endogenous inorganic polysulfides have significant biological actions activating the Transient Receptor Potential Ankyrin 1 (TRPA1) receptor. Organic polysulfides exert similar effects, but they are much more stable molecules, therefore these compounds are more suitable as drugs. In this study, we aimed to better understand the mechanism of action of organic polysulfides by identification of their binding site on the TRPA1 receptor. Methods: Polysulfides can readily interact with the thiol side chain of the cysteine residues of the protein. To investigate their role in the TRPA1 activation, we replaced several cysteine residues by alanine via site-directed mutagenesis. We searched for TRPA1 mutant variants with decreased or lost activating effect of the polysulfides, but with other functions remaining intact (such as the effects of non-electrophilic agonists and antagonists). The binding properties of the mutant receptors were analyzed by in silico molecular docking. Functional changes were tested by in vitro methods: calcium sensitive fluorescent flow cytometry, whole-cell patch-clamp and radioactive calcium-45 liquid scintillation counting. Results: The cysteines forming the conventional binding site of electrophilic agonists, namely C621, C641 and C665 also bind the organic polysulfides, with the key role of C621. However, only their combined mutation abolished completely the organic polysulfide-induced activation of the receptor. Discussion: Since previous papers provided evidence that organic polysulfides exert analgesic and anti-inflammatory actions in different in vivo animal models, we anticipate that the development of TRPA1-targeted, organic polysulfide-based drugs will be promoted by this identification of the binding site.

Keywords: DADS; DATS; DMTS; binding site; electrophilic TRPA1 agonist; human TRPA1; organic polysulfide; site-directed mutagenesis.

Grants and funding

EP, ZS, and BN were funded by National Brain Research Program 3.0 (NAP 3.0); RRF-2.3.1-21-2022-00015 National Laboratory for Drug Research and Development; TKP2021-EGA-16 National Research, Development, and Innovation Fund of Hungary and the ELKH Network. BZ and CH were supported by the Medical School, University of Pécs (PTE ÁOK KA-2022-26), and by the European Union, co-financed by the European Social Fund (project name and code: Comprehensive Development for Implementing Smart Specialization Strategies at the University of Pécs, EFOP-3.6.1-16-2016-00004). SL was funded by OTKA_NKFI-K-134214. Project no. TKP2021-EGA-13 was implemented with the support provided from the National Research, Development, and Innovation Fund of Hungary, financed under the EGA-13 funding scheme; National Research, Development and Innovation Office (Pharma-Lab, RRF-2.3.1-21-2022-00015). ZV was funded by OTKA_NKFI-K-132906, and AF was supported by the UNKP-22-3-II-DE-38 New National Excellence Program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation Fund.