The transmembrane serine protease 2 (TMPRSS2) primes the SARS-CoV-2 Spike (S) protein for host cell entry and represents a promising target for COVID-19 therapy. Here we describe the in silico development and in vitro characterization of peptidomimetic TMPRSS2 inhibitors. Molecular docking studies identified peptidomimetic binders of the TMPRSS2 catalytic site, which were synthesized and coupled to an electrophilic serine trap. The compounds inhibit TMPRSS2 while demonstrating good off-target selectivity against selected coagulation proteases. Lead candidates are stable in blood serum and plasma for at least ten days. Finally, we show that selected peptidomimetics inhibit SARS-CoV-2 Spike-driven pseudovirus entry and authentic SARS-CoV-2 infection with comparable efficacy as camostat mesylate. The peptidomimetic TMPRSS2 inhibitors also prevent entry of recent SARS-CoV-2 variants of concern Delta and Omicron BA.1. In sum, our study reports antivirally active and stable TMPRSS2 inhibitors with prospects for further preclinical and clinical development as antiviral agents against SARS-CoV-2 and other TMPRSS2-dependent viruses.
© 2022. The Author(s).