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
using a homology model and crystal structure. We establish a pipeline
to rapidly screen TMPRSS2 inhibitors and then interrogate the potency,
selectivity, 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 validated
as a potential avenue for developing TMPRSS2-selective inhibitors.