Small-Molecule Disruptors of the Interaction between Calcium- and Integrin-Binding Protein 1 and Integrin αIIbß3 as Novel Antiplatelet Agents.

ABSTRACT

Thrombosis, a key factor in most cardiovascular diseases, is a major contributor to human mortality. Existing antithrombotic agents carry a risk of bleeding. Consequently, there is a keen interest in discovering innovative antithrombotic agents that can prevent thrombosis without negatively impacting hemostasis. Platelets play crucial roles in both hemostasis and thrombosis. We have previously characterized calcium- and integrin-binding protein 1 (CIB1) as a key regulatory molecule that regulates platelet function. CIB1 interacts with several platelet proteins including integrin αIIbß3, the major glycoprotein receptor for fibrinogen on platelets. Given that CIB1 regulates platelet function through its interaction with αIIbß3, we developed a fluorescence polarization (FP) assay to screen for potential inhibitors. The assay was miniaturized to 1536-well and screened in quantitative high-throughput screening (qHTS) format against a diverse compound library of 14,782 compounds. After validation and selectivity testing using the FP assay, we identified 19 candidate inhibitors and validated them using an in-gel binding assay that monitors the interaction of CIB1 with αIIb cytoplasmic tail peptide, followed by testing of top hits by intrinsic tryptophan fluorescence (ITF) and microscale thermophoresis (MST) to ascertain their interaction with CIB1. Two of the validated hits shared similar chemical structures, suggesting a common mechanism of action. Docking studies further revealed promising interactions within the hydrophobic binding pocket of the target protein, particularly forming key hydrogen bonds with Ser180. The compounds exhibited a potent antiplatelet activity based on their inhibition of thrombin-induced human platelet aggregation, thus indicating that disruptors of the CIB1- αIIbß3 interaction could carry a translational potential as antithrombotic agents.