Dual engagement of Spike and ACE2 by annexin A5 contributes to pleiotropic SARS-CoV-2 inhibition.

COVID-19 is primarily a respiratory tract infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can enter host airway epithelial cells through angiotensin-converting enzyme 2 (ACE2) receptors. Emerging SARS-CoV-2 variants are a roadblock to irradicating the disease. Given recombinant human annexin A5 (Anx5) inhibits proinflammatory responses, improves survival in sepsis models and binds to several receptors and lipids, we hypothesized that Anx5 impedes SARS-CoV-2 viral entry and lessens disease severity. To assess SARS-CoV-2 Spike-receptor binding domain (RBD) and ACE2 interactions with Anx5, the recombinant proteins were expressed and isolated, and interactions were evaluated using solution NMR, microscale thermophoresis, steady-state fluorescence and size-exclusion chromatography coupled to multi-angle light scattering. Remarkably, we found that Anx5 binds to both the Spike-RBD and ACE2. Further, Anx5 induced extensive 15N-Spike-RBD amide resonance broadening, most concentrated in the region that engages with ACE2 and least enriched on the opposite face, highlighting the ACE2 interface as the primary site of Anx5 binding. Finally, we demonstrate that Anx5 inhibits SARS-CoV-2 pseudoviral entry and reduces viral burden while enhancing survival in SARS-CoV-2-infected mammalian cells. Collectively, our findings demonstrate that Anx5 directly engages both Spike-RBD and ACE2, inhibits SARS-CoV-2 pseudoviral entry and reduces viral burden while enhancing survival of infected mammalian cells, supporting further investigation of Anx5 as a multi-functional anti-SARS-CoV-2 therapeutic.