Dual‐Locking the SARS‐CoV‐2 Spike Trimer: An Amphipathic Molecular “Bolt” Stabilizes Conserved Druggable Interfaces for Coronavirus Inhibition

Abstract The SARS‐CoV‐2 spike (S) protein, a trimeric structure comprising three receptor binding domains (RBDs) and three N‐terminal domains (NTDs), undergoes substantial conformational changes to a fusion‐prone open state for angiotensin‐converting enzyme 2 (ACE2) binding and host cell infection. Stabilizing its closed state is a key antiviral strategy but remains challenging. Here, we introduce S416, a novel amphipathic molecule acting as a “molecular bolt”. Cryo‐EM study reveals that S416 binds concurrently to six sites across two distinct druggable interfaces: three molecules at the RBD‐RBD interfaces and three at the NTD‐RBD interfaces. This unique “dual‐locking” mechanism, driven by S416's polar carboxyl head and nonpolar phenylthiazole tail, robustly stabilizes the spike trimer in a locked, closed conformation through strong inter‐domain interactions, reducing structural flexibility and atomic fluctuations compared to the apo structure resolved synchronously. Crucially, these RBD‐RBD and NTD‐RBD interfaces are conserved across human‐infecting coronaviruses, suggesting potential as broad‐spectrum antiviral targets. Our findings demonstrate that the highly dynamic spike trimer can be effectively stabilized by an amphipathic molecular bolt targeting both the inter‐ and intra‐monomer interfaces, offering a promising strategy against emerging coronaviruses.