Insight into the molecular mode of action of a family of selective pan-coronavirus inhibitors

A family of antiviral molecules against human coronaviruses was identified using a phenotypic cell-based screening approach. Optimized preclinical candidates with optimal therapeutic windows in different cell culture models were characterized in cell culture, revealing interference with the formation of functional replication organelles (ROs). Coronavirus ROs are characterized by a series of cytoplasmic membranous structures, among which double-membrane vesicles (DMVs) are the main site of RNA replication.

To further characterize the lead compounds, we produced recombinant DMVs through overexpression of its minimal components (nsp3/nsp4) in a replication-independent manner to test the ability of the compounds to interfere with their formation. Viral protein overexpression is sufficient to observe numerous DMVs using transmission electron microscopy (TEM), together with less characterized structures such as multiple membrane vesicles (MMVs) or zippered membranes (ZM) in the cell cytoplasm. Nsp3/nsp4 polyprotein overexpression and processing was not affected by the presence of the lead antiviral compounds. However, DMV frequency per explored surface was significantly reduced, suggesting that the compounds indeed interfere with DMV assembly. No significant changes in the diameter of the DMVs were observed.

These results are supported by the study of resistance-associated mutations, which preferentially map in the ECTO-domain of nsp4, a region of nsp4 involved in the interaction with nsp3, interaction that is essential for DMV formation. Resistance-associated mutations from three different lineages were mapped into an atomic 3D model obtained via cryo-electron tomography of the DMV pore, a key structure for the functionality of the DMVs. All nsp4 mutations are located in the luminal side of the pore, close the ECTO-domain of nsp3.

Overall, these results support a model in which nsp3/nsp4 interaction or nsp4 oligomerization are molecular targets for this family of pan-coronavirus antivirals, constituting a first-in-class antiviral family, optimal for combination therapies.