Interrogating a compound library in search of an inhibitor for TREM-like transcript-1 to fibrinogen binding.

Abstract

Cardiovascular disease (CVD) remains one of the leading causes of death worldwide. Aberrant platelet function mediates fibrin(ogen)-rich thrombi that lead to occlusive thrombi associated with mortality. The receptor, TREM-like transcript-1 (TLT-1), stored in the platelet α-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 designed an assay to screen a compound library of small molecules inhibitors. Human embryonic kidney (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. We demonstrated 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 and spreading assays. SIGNIFICANCE STATEMENT: Triggering receptor expressed in myeloid cells-like transcript-1 (TLT-1) is a key platelet receptor that binds fibrinogen and mediates clot formation. The developed assay successfully screened 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 cardiovascular disease-related thrombosis.