CryoEM structures of the SARS-CoV-2 spike bound to antivirals

(Póster 63) Background: Single-particle cryoelectron microscopy (cryoEM) has played a key role in the fight against COVID-19. The molecular mechanisms for the action of some of the currently approved drugs targeting the SARS-CoV-2 RNA-dependent RNA polymerase, the fast developments of the current available vaccines and antibody therapies are examples of the impact of the knowledge gained from the cryoEM structures of SARS-CoV-2 proteins in complex with proteins (ACE2 or antibodies/nanobodies) or small compounds. Our aim is to use this technology to understand structurally how certain antiviral compounds and proteins targeting the spike may inhibit viral entry. Methods: 1) Production of wild-type and mutated spike and ACE2 proteins using baculovirus/insect cells. 2) Spike binding kinetics: protein-protein and protein-small compound interactions measured by BLI Biolayer interferometry (BLI) and/or microscale Thermophoresis (MST). 3) Buffer optimization for cryoEM grid preparation of spike variants by thermal shift assays and negative-staining electron microscopy (NSEM). These techniques are also used to adjust the molar ratio of spike:ACE2 and spike:small-compound complexes. 4) Structural characterization by cryoEM. Results: At IBV-CSIC we have created a pipeline for the production and characterization of several spike variants and ACE2 decoys. While this pipeline is described in detail in other oral/poster communications, this communication is centered around one of the pillars within this pipeline; the structural characterization of possible drug candidates bound to the SARS-CoV-2 spike by cryoEM. In this way, we have successfully solved structures of the spike bound to: A) protein inhibitors as ACE2 decoys; B) a small inhibitory compound; C) mixtures of proteins and small-compound (nanobody-heparan derivative) working cooperatively as inhibitors. These protein/drug candidates were previously selected based on the results obtained in our interactomics platform, whereas their concentration and the buffer conditions for cryoEM grids preparation were established based on thermal shift assays and NSEM. Conclusion: CryoEM is a powerful tool to directly visualize the effect caused by a potential drug on a protein target. In a short period of time we have developed this technique in our institute to be applied to the SARS-CoV-2 spike protein, not only to obtain high-resolution structures of SARS- CoV-2 spike variants of concern (see WP4) but also to obtain the structures of complexes of the spike with various inhibitory compounds of very different nature.