Publication date: Jul 23, 2026
SARS-CoV-2 continues to circulate with the emergence of variants that evade existing immunity. However, all strains rely on conserved host factors for entry, making them attractive targets for host-directed antivirals. SARS-CoV-2 engages the ACE2 receptor and can enter cells through two alternative pathways depending on cell type: Spike cleavage at the plasma membrane by TMPRSS2, or within endocytic compartments by cathepsins. Cleavage triggers Spike-mediated membrane fusion and release of the viral genome. To discover small-molecule inhibitors of entry, we first screened compounds against live virus and active candidates were then tested using recombinant VSV expressing SARS-CoV-2 Spike, with VSV expressing its native glycoprotein serving as a control. This approach identified known and novel TMPRSS2 inhibitors, as well as Staurosporine and Retro-2. 1. Both compounds inhibited infection in both TMPRSS2-dependent and -independent cell types. Entry bypass studies revealed that Staurosporine acts upstream of Spike cleavage, while Retro-2. 1 functions downstream. Mechanistic studies in Calu-3 cells showed that Staurosporine, a pan-PKC inhibitor, blocks entry via PKCη, a pro-viral factor acting before Spike cleavage. Retro-2. 1 targets NF1, which promotes infection downstream of Spike cleavage. Together, our screening pipeline identified inhibitors that block SARS-CoV-2 entry at distinct stages and revealed host factors that may inform the development of current and novel antiviral strategies.
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| Concepts | Keywords |
|---|---|
| Antivirals | Cleavage |
| Host | Cov |
| Recombinant | Entry |
| Staurosporine | Factors |
| Viral | Host |
| Infection | |
| Inhibitors | |
| Membrane | |
| Nf1 | |
| Pkc | |
| Retro | |
| Sars | |
| Spike | |
| Staurosporine | |
| Targets |