AI-based prediction of new binding site and virtual screening for the discovery of novel P2X3 receptor antagonists

Artificial intelligence (AI) has been recognized as a powerful technique that can accelerate drug discovery during the hit compound identification step. However, most simple deep learning models have been used for naive pre-filtering as the prediction result cannot be interpreted. Recently, our group developed a new deep learning model (Highlight on Target Sequence; HoTS) that can predict binding regions in a target protein sequence based on patterns learned from interactions between a target protein sequence and a ligand. In this study, we searched for new binding regions of the P2X3 receptor (P2X3R) using HoTS, and suggested a novel putative binding site of P2X3R by a cavity search on the predicted binding regions. The novel putative binding site was employed to generate pharmacophore features, and combinations of pharmacophore features were validated as queries. Two separate virtual screenings using the optimized pharmacophore query Q12 with docking-based scoring and HoTS-based prediction of ligand interactions enabled the initial selection of the compound library for in vitro screening. The screening of each set of 500 compounds from the two approaches (HoTS interaction prediction and Pharmacophore-LibDock cascade) resulted in the identification of 10 (HoTS-1 - 10) and 6 compounds (PD-1 - 6) with low micromolar IC₅₀ values. Remarkably, the hit rate was 10-fold higher than that from the previous random screening of 8364 compound library, and the chemical structures of all identified hit compounds were distinct from those of known P2X3R antagonists, indicating that novel chemical entities could be developed for P2X3R antagonists by targeting the binding site. Overall, this study suggests the discovery of a novel putative binding site for P2X3R using the AI deep learning protocol along with in silico MD simulation and experimental screening of targeted library compounds to successfully identify 16 unique and novel hit compounds. These results may accelerate the discovery of novel chemical-class drugs for P2X3R antagonists.