P2Y12-oriented computational screening and functional validation identify antiplatelet compounds with antithrombotic activity

INTRODUCTION: Arterial thrombosis is driven by excessive platelet activation, in which the P2Y12 receptor is a key amplifier of ADP-dependent signaling and a clinically validated antiplatelet target. P2Y12-oriented computational screening may facilitate the discovery of structurally diverse compounds with antiplatelet activity and provide new starting points for antithrombotic intervention. OBJECTIVES: This study aimed to identify antiplatelet lead candidates using a P2Y12-oriented computational screening strategy and to evaluate their antiplatelet and antithrombotic activities. METHODS: A hybrid screening pipeline integrating ligand-similarity filtering, receptor pocket-based docking, and molecular dynamics-guided stability assessment was applied to prioritize candidate molecules from the TargetMol Approved Drug Library and the TargetMol Bioactive Compound Library, comprising approximately 3,075 and 19,650 compounds, respectively. Selected compounds were tested for inhibition of ADP-induced platelet aggregation and activation in human platelets, and for antithrombotic efficacy using a zebrafish laser-injury thrombosis model. Association with P2Y12-expressing membrane fractions was assessed using a membrane-based ligand-binding assay, and effects on ADP-responsive signaling were evaluated by measuring AKT and VASP phosphorylation. RESULTS: Among more than forty computationally prioritized compounds, VS-5584 and GNE-493 were selected as lead-like antiplatelet candidates. Both compounds suppressed ADP-induced platelet aggregation and activation in human platelets and reduced thrombus formation in vivo in zebrafish. Membrane-based binding assays showed enrichment of both compounds in P2Y12-expressing membrane fractions, and signaling studies demonstrated reduced ADP-induced AKT phosphorylation together with detectable modulation of the VASP response. CONCLUSION: These results support a practical computational-experimental strategy for identifying biologically active antiplatelet leads through P2Y12-oriented screening. VS-5584 and GNE-493 were identified as bioactive antiplatelet candidates with antithrombotic activity, whose effects may involve both P2Y12-associated activity and modulation of downstream PI3K/AKT/mTOR signaling.