APP and APLP2 Kunitz Domains Are Potent Endogenous Inhibitors of TMPRSS2 and Respiratory Virus Infection

<jats:p>Respiratory viruses depend on host proteases for activation of viral fusion proteins, making these enzymes attractive targets for broad-spectrum antiviral strategies. We previously identified Trypstatin, a human Bikunin-derived Kunitz domain, as a potent endogenous inhibitor of the airway serine protease TMPRSS2. Here, we investigated whether TMPRSS2 inhibition is shared by additional human Kunitz domains. Kunitz domains with high sequence similarity to Trypstatin were synthesized, refolded, and functionally characterized. Domains derived from amyloid precursor protein (APP) and amyloid precursor-like protein 2 (APLP2) potently inhibited TMPRSS2, with APP displaying subnanomolar activity comparable to camostat mesylate. APP and APLP2 selectively blocked SARS-CoV-2 Spike-mediated entry without affecting VSV-G-mediated entry or cell viability and inhibited infection by multiple coronaviruses and influenza viruses, but not TMPRSS2-independent rhinovirus. In primary human airway epithelial cultures, APP and Trypstatin reduced replication of SARS-CoV-2, endemic coronaviruses, and influenza A virus, and remained stable in airway mucus. These findings identify APP and APLP2 Kunitz domains as potent endogenous inhibitors of TMPRSS2-dependent respiratory virus infection and promising scaffolds for host-directed broad-spectrum antivirals.</jats:p>