Large library docking and biophysical analysis of small molecule TMPRSS2 inhibitors

Transmembrane protease, serine -2 (TMPRSS2) is an essential host entry factor in human airways for SARS-CoV-2 and influenza A/B and has presented as a target for antiviral drug development; however, no clinically viable, oral small molecule TMPRSS2 inhibitors have been developed to date. Here we perform two large-scale docking campaigns to identify covalent and noncovalent TMPRSS2 small molecule inhibitors from a homology model and crystal structure. We establish a pipeline to rapidly screen TMPRSS2 inhibitors, then interrogate the potency, specificity and biophysical properties of covalent and noncovalent inhibition using enzyme kinetics on synthetic peptide and protein substrates and differential scanning fluorimetry. Furthermore, we established a readily crystallizable form of TMPRSS2 protein that produced high resolution crystal structures with nafamostat, ‘157, and 6-amidino-2-naphthol. A novel noncovalent inhibitor scaffold is biochemically and biophysically validated as a potential avenue to develop TMPRSS2-selective inhibitors.