Keywords
GFB-887
binding site
ADMET
molecular docking
smooth muscle
Abstract
The transient receptor potential (TRP) channel family, in particular the canonical TRPC4 and TRPC5 channels, plays a key role in the regulation of calcium signaling, membrane excitability, and smooth muscle contractile activity. Pharmacological modulation of these channels is considered a promising approach for influencing gastrointestinal function and other physiological processes. The small-molecule inhibitor GFB- 887 is a clinically investigated compound; however, the molecular details of its interaction with TRPC4 remain insufficiently characterized. The aim of thе study – to elucidate the possible mode of interaction between GFB-887 and the TRPC4 channel using structural modeling and to correlate the obtained in silico results with the functional effects of the compound on carbachol-induced contractility of mouse small intestinal smooth muscle. The methods included rational identification of a potential binding site in the TRPC4 structure by combining geometry-based cavity prediction (Fpocket) with literature-driven prioritization of amino acid residues using analysis of scientific publications. Molecular docking of GFB-887 was performed using AutoDock 4.2.6 based on the cryo-EM structure of mouse TRPC4 (PDB ID: 5Z96). In silico assessment of ADMET properties was carried out using machine-learning models trained on publicly available datasets from the Therapeutics Data Commons platform. Functional effects of GFB-887 were evaluated by isometric tensometry on isolated segments of the mouse ileum. The results demonstrated that the most probable binding site of GFB-887 is localized within the transmembrane S1–S4 region (VSL domain) of TRPC4, which is consistent with cryo-EM data of TRPC4 complexes with other inhibitors of the GFB series. The selected docking pose was characterized by a binding energy of − 8.11 kcal/mol and was stabilized by a network of hydrophobic-aromatic and polar interactions with functionally significant amino acid residues. ADMET predictions indicated a high probability of efficient intestinal absorption and a pharmacokinetic profile compatible with available clinical observations. In functional experiments, preincubation with GFB-887 (10 μmol/L) reduced the carbachol-induced contractile response of the mouse ileum to approximately 40–50% of the control level. The obtained structural and functional data consistently indicate the involvement of TRPC4-mediated mechanisms in cholinergic excitation of intestinal smooth muscle and support the hypothesis that GFB- 887 exerts its inhibitory action through binding within the VSL domain of the channel. The combination of in silico and in vitro approaches is appropriate for further elucidation of the molecular mechanisms of action of TRPC modulators and for assessing their translational relevance.
References
2. Structure of the mouse TRPC4 ion channel. J. Duan, Z. Li, J. Li et al. Nature Communications. 2018. V. 9. P. 3102.
3. Structural basis of TRPC4 regulation by calmodulin and small-molecule inhibitors. D. Vinayagam, D. Qiu, J. Yang et al. eLife. 2020. V. 9. P. e60603.
4. Structural basis for human TRPC5 channel inhibition by two distinct inhibitors. K. Song, M. Wei, W. Guo et al. eLife. 2021. V. 10. P. e63429.
5. Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels. M. Miller, J. Shi, Y. Zhu et al. Journal of Biological Chemistry. 2011. V. 286 (38). P. 33436–33446.
6. (−)-Englerin A is a potent and selective activator of TRPC4 and TRPC5 calcium channels. Y. Akbulut, H. J. Gaunt, K. Muraki et al. Angewandte Chemie International Edition. 2015. V. 54 (12). P. 3787–3791.
7. Pico145 inhibits TRPC4-mediated mICAT and postprandial small intestinal motility. D. O. Dryn, M. I. Melnyk, R. S. Bon et al. Biomedicine & Pharmacotherapy. 2023. V. 168. P.115672.
8. Safety and efficacy of GFB-887, a TRPC5 channel inhibitor, in patients with focal segmental glomerulosclerosis, treatment-resistant minimal change disease, or diabetic nephropathy: TRACTION-2 trial design. L. Walsh, J. F. Reilly, C. Cornwall et al. Kidney International Reports. 2021. V. 6 (10). P. 2575–2584.
9. Leveraging large language models for literature-driven prioritization of protein binding pockets. R. Stratiichuk, M. Melnychenko, I. Koleiev et al. Bioinformatics. 2025. V. 41 (8). P. btaf449.
10. Le GuillouxV., Schmidtke P., Tuffery P. Fpocket: an open source platform for ligand pocket detection. BMC Bioinformatics. 2009. V. 10. P. 168.
11. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. G. M. Morris, R. Huey, W. Lindstrom et al. Journal of Computational Chemistry. 2009. V. 30 (16). P. 2785–2791.
12. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. G. M. Morris, D. S. Goodsell, R. S. Halliday et al. Journal of Computational Chemistry. 1998. V. 19 (14). P. 1639–1662.
13. Improving ADMET prediction with descriptor augmentation of Mol2Vec embeddings. R. Stratiichuk, N. Shevchuk, R. Kyrylenko et al. bioRxiv. 2025. https://doi.org/10.1101/2025.07.14.664363.
14. Therapeutics Data Commons: Machine learning datasets and tasks for drug discovery and development. K. Huang, T. Fu, W. Gao et al. arXiv. 2021. V. 2102. P. 09548. https://arxiv.org/abs/2102.09548.
15. Electron cryo-microscopy structure of the canonical TRPC4 ion channel. D. Vinayagam, T. Mager, A. Apelbaum et al. eLife. 2018. V. 7. P. e36615.
16. Artificial intelligence foundation for therapeutic science. K. Huang, T. Fu, W. Gao et al. Nature Chemical Biology. 2022. V. 18 (10). P. 1033–1036.
17. Transplanted organoids empower human preclinical assessment of drug candidate for the clinic. A. D. Westerling-Bui, E. M. Fast, T. W. Soare et al. Science Advances. 2022. V. 8 (27). P. eabj5633.
18. ClinicalTrials.gov. (n.d.). A first-in-human, phase 1, placebo-controlled study to evaluate the safety, tolerability, and pharmacokinetics of GFB-887 (Identifier: NCT03970122). ClinicalTrials.gov. RLU: https://clinicaltrials.gov/study/NCT03970122.
19. Excitation-contraction coupling in gastrointestinal and other smooth muscles. T. B. Bolton, S. A. Prestwich, A. V. Zholos, D. V. Gordienko. Annual Review of Physiology. 1999. V. 61. P. 85–115.
20. Deletion of TRPC4 and TRPC6 in mice impairs smooth muscle contraction and intestinal motility in vivo. V. V. Tsvilovskyy, A. V. Zholos, T. Aberle et al. Gastroenterology. 2009. V. 137 (4). P. 1415–1424.
21. Zholos A. V., Melnyk M. I., Dryn D. O. Molecular mechanisms of cholinergic neurotransmission in visceral smooth muscles with a focus on receptor-operated TRPC4 channel and impairment of gastrointestinal motility by general anaesthetics and anxiolytics. Neuropharmacology. 2024. V. 242. P. 109776.
22. Acute treatment with a novel TRPC4/C5 channel inhibitor produces antidepressant and anxiolytic-like effects in mice. L. P. Yang, F. J. Jiang, G. S. Wu et al. PLOS ONE. 2015. V. 10 (8). P. e0136255.
23. Treatment with HC-070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice. S. Just, B. L. Chenard, A. Ceci et al. PLOS ONE. 2018. V. 13 (1). P. e0191225.