Discovery of Highly Selective Noncovalent Macrocyclic Peptide Inhibitors Suppressing Both TMPRSS2 Protease Activity and Viral Receptor Function.

The type II transmembrane serine protease TMPRSS2 plays a critical role in respiratory virus entry, including SARS-CoV-2. Using an mRNA display platform with genetic code reprogramming, we identified macrocyclic peptide (MCP) inhibitors of TMPRSS2 that bound to the TMPRSS2 active site. The most potent MCPs displayed picomolar binding affinities and excellent selectivity across a panel of 22 trypsin-fold serine proteases. Although the lead MCP showed strong in vitro potency, proteolytic instability reduced its cellular activity. Rational optimization yielded a stability-enhanced variant, T2-MCP-19, which bound TMPRSS2 with high affinity (KD = 80 pM) and inhibited the uptake of virus-like particles pseudotyped with SARS-CoV-2 spike protein in lung epithelial Calu-3 cells with nanomolar potency. Notably, several MCPs exhibited dual functionality by inhibiting both the enzymatic and receptor functions of TMPRSS2, as demonstrated by their blockade of the HKU1 coronavirus spike protein binding. These results highlight MCPs as promising, highly selective TMPRSS2-directed antiviral lead compounds.