Virtual Screening And Activity Evaluation Of TRPC5 Inhibitors

TRPC5 function as either homo-or heterotetramers regulating intracellular Ca²⁺concentration in response to numerous physiological or pathological stimuli.TRPC5plays a crucial role in human physiology and pathology,for instance,anxiety,depression,epilepsy,pain,memory,and chronic kidney disease and cancer.Since there are few effective subtype-specific inhibitors,the exact role of TRPC5 channels in physiological and pathophysiological processes has not been elucidated.In the study,we designed a special virtual screening strategy for TRPC5 by using different virtual screening methods.The candidate compounds were tested for biological activity in order to find novel TRPC5 inhibitors.In the second part of the paper,based on mouse TRPC5 structure,the 3D structure of human TRPC5 was constructed through homology modeling,and used molecular dynamics simulation to optimize its structure.Then,the constructed 3D structure and amino acid conservation analysis of TRPC family were used to predict the potential active site.In addition,the best binding sites were selected by the docking hit rate,and the reported inhibitors were docked into the potential active sites to further identify the key interactions between small molecule inhibitors and TRPC5 channels.The study provides basis for further site-directed mutagenesis of amino acid and design of novel compounds as TRPC5 receptor antagonists.In the third part of the paper,the reported inhibitors of various structures were collected to construct Hip Hop and Hypo Gen pharmacophore models and execute virtual screening.The 9 inhibitors with higher activities and diversity of chemical structures were selected as a training set to generate Hip Hop model,and the optimal model Hip Hop-7 was obtained.Simultaneously,26 inhibitors with activity span over 5 orders of magnitude were selected as a training set based on activity and structural diversification with IC₅₀ values ranging from 1.14 n M to 18μM to construct Hypo Gen model,and Hypo1 selected as best model.Hip Hop-7 and Hypo1 were used to screen Specs database and top mapped compounds were selected.They were processed for Lipinski and Veber filter,ADMET analysis,PAINS analysis,cluster analysis and docking studies.We have obtained ten hits with comparable binding mode to the reported inhibitors.In the fourth part of study,,in the transfected HEK293_{h TRPC5} cells,using TRPC5agonist CCh,antagonist ML204,Ca²⁺-sensitive dye Fluo-4 AM and Flexstation 3intracellular calcium flow detection technology,biological assay of 10 candidate molecules were tested.It is found that the compounds Hit 1,Hit 3,Hit 4,Hit 6,and Hit10 have inhibitory effects on ion channels at 50μM concentration,and the IC₅₀value of Hit 4 is 41.14+1.13μM.