Cathepsin K as a Key SARS-CoV-2 Cell Entry Protease and Dual-Inhibition Target

SARS-CoV-2 relies on host proteases to cleave and activate its spike protein for cell entry through either the endosomal or plasma membrane pathway. Although cysteine cathepsins are known to mediate the endosomal route, the identity of the dominant enzyme has remained unclear. Here, we identify human Cathepsin K (hCatK), a lysosomal cysteine protease, as a previously unrecognized but functionally important mediator of viral entry. While human Cathepsin L (hCatL) has long been regarded as the principal endosomal protease, inhibition of hCatK with the selective inhibitor Odanacatib suppressed viral propagation in endothelial cells as effectively as the broad-spectrum cysteine protease inhibitor E-64d, implicating hCatK as a key driver of endosomal entry. Enzymatic profiling further revealed that hCatK exhibits 25- to 70-fold higher catalytic efficiency at the Furin cleavage site (FCS) compared to hCatL and displays a distinct cleavage pattern in the Omicron FCS relative to the Wuhan variant. We also demonstrate that hCatK is a submicromolar (0.6 ± 0.1 μM) off-target of Nirmatrelvir, a clinically approved 3CL-Mpro inhibitor, and present a high-resolution crystal structure of the hCatK–Nirmatrelvir complex that informs rational design of dual-acting antivirals. Collectively, these findings redefine the landscape of host proteases in SARS-CoV-2 entry and establish hCatK as an overlooked yet strategic target for antiviral intervention.