A SARS-CoV-2 entry inhibitor trimerizes to lock the spike protein in a closed conformation

Mou, H.; Gao, B.; Ye, G.; Zhang, L.; Saxena, D.; Bu, F.; Vishwakarma, J.; Zhou, S.; Bayona, C. R.; Li, L.; Getmanenko, Y.; Bhandari, J.; Bailey, C. C.; Crynen, G. C.; Kitzmiller, C. E.; Li, H.; Otsuka, Y.; Wang, C.; Batra, L.; Weston, S.; Frieman, M. B.; Meyerholz, D. K.; Scampavia, L.; Spicer, T.; Harris, R. S.; Cameron, M.; Bannister, T.; Zheng, J.; Farzan, M.; Li, F.; Choe, H.

doi:10.1101/2025.11.19.689107

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bioRxiv · 10.1101/2025.11.19.689107

A SARS-CoV-2 entry inhibitor trimerizes to lock the spike protein in a closed conformation

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Abstract

The SARS-CoV-2 spike protein binds its receptor ACE2 to initiate target-cell infection. To engage ACE2, at least one of the three receptor-binding domains (RBDs) of the spike must adopt the up orientation. Here we describe S22, a potent, bioavailable, and non-toxic inhibitor of BA.2 and all subsequent Omicron variants. Cryo-EM analyses showed that S22 assembled as a trimer in a previously uncharacterized pocket of the spike apex, stabilizing all three RBDs in the down orientation, thereby preventing ACE2 association. Binding studies, especially those using mixed S22-sensitive and -resistant spikes, imply a cooperative assembly of three S22 molecules with three RBDs, resulting in an unusually slow S22 off-rate. Consistent with its slow dissociation and favorable pharmacokinetics, S22 suppressed viral replication 100-fold in the lungs of XBB.1.5-infected mice. Thus, S22 potently inhibits Omicron entry through a distinct mechanism whereby a small compound assembles cooperatively as a trimer to stabilize spike in an inactive conformation.

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Mou, H., Gao, B., Ye, G., Zhang, L., Saxena, D., Bu, F., Vishwakarma, J., Zhou, S., Bayona, C. R., Li, L., Getmanenko, Y., Bhandari, J., Bailey, C. C., Crynen, G. C., Kitzmiller, C. E., Li, H., Otsuka, Y., Wang, C., Batra, L., Weston, S., Frieman, M. B., Meyerholz, D. K., Scampavia, L., Spicer, T., Harris, R. S., Cameron, M., Bannister, T., Zheng, J., Farzan, M., Li, F., Choe, H.. 2025-11-20. A SARS-CoV-2 entry inhibitor trimerizes to lock the spike protein in a closed conformation. https://doi.org/10.1101/2025.11.19.689107

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