Interrogating a Compound Library in Search of an Inhibitor for TREM-like Transcript-1 to Fibrinogen Binding.

Abstract

Cardiovascular disease (CVD) remains one of leading causes of death worldwide. Aberrant platelet function mediate fibrin(ogen) rich thrombi that lead to occlusive thrombi associated with mortality. The receptor, TREM-like transcript-1 (TLT-1), stored in the platelet a-granules and released upon platelet activation, binds fibrinogen and von Willebrand factor. Once it is released from platelets TLT-1 is a potential therapeutic target to prevent the thrombosis associated with CVD. Here we design an assay to screen a compound library of small molecules inhibitors. HEK-293 cells stably transfected with a full length human treml-1 construct were used to screen library of 800 compounds, for inhibition of TLT-1 to fibrinogen binding in an attachment assay using crystal violet staining. The possible cytotoxicity of the best compounds was determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide MTT and calcein AM staining assays. Here we demonstrate that the addition of TLT-1 to HEK-293 cells increases cell adhesion by more than 2-fold. We identified ~80 compounds that inhibit binding by more than 80%. We further tested the top compounds and confirmed that reduction of hTLT-1 to fibrinogen bound in the top compounds was not caused by cytotoxicity, as per colorimetric and fluorescent viability assays. Four compounds were identified as potential small molecule inhibitors one of which, BM-8372, demonstrated significant effect in platelet aggregation assays. Significance Statement TLT-1 is a key platelet receptor that binds fibrinogen and mediates clot formation The developed assay successfully screens 800 small molecules, pinpointing ~80 potent inhibitors that reduce TLT-1 binding by over 80%. Importantly, the study rigorously rules out cytotoxicity concerns, affirming the therapeutic potential of the identified compounds. By elucidating TLT-1's role and presenting promising inhibitors, this research offers a significant stride toward developing novel strategies to combat CVD-related thrombosis.