Abstract/Summary

Background and Purpose The C-type lectin-like receptor-2 (CLEC-2) is a platelet receptor for the endogenous ligand podoplanin. This interaction contributes to a number of (patho)physiological processes, such as lymphangiogenesis, preservation of blood and lymphatic vessel integrity, organ development and tumour metastasis. Activation of CLEC-2 leads to the phosphorylation of its cytoplasmic hemITAM domain and initiates a signalling cascade involving the kinase Syk. The aim of this study was to identify and characterise a novel small molecule inhibitor of CLEC-2. Experimental Approach An AlphaScreen-based high-throughput screening identified a small-molecule inhibitor of the CLEC-2-podoplanin interaction. Binding site interactions were assessed using in silico modelling. Functional assays, including light transmission aggregometry, platelet spreading and phosphorylation assays, were used to evaluate the effect of the small molecule on CLEC-2-mediated platelet activation. Key Results 18,476 small molecules were screened resulting in 14 candidates. Following secondary screening, one novel small molecule, MAS9, was taken forward for further characterisation. The binding sites of MAS9 to CLEC-2 were predicted to share binding sites with the CLEC-2 ligands podoplanin and rhodocytin. MAS9 inhibited CLEC-2 mediated platelet aggregation, spreading and signalling. MAS9 also resulted in inhibited fibrinogen binding. Conclusion and Implications MAS9 inhibits CLEC-2-mediated aggregation, platelet spreading and signalling, showing selectivity of CLEC-2 inhibition over GPVI. This study paves the way for future preclinical assays to test the potential of MAS9 as a novel therapeutic tool to treat pathologies such as thromboinflammation and cancer.