ABSTRACT
The risk of zoonotic coronavirus spillover into the human population, as highlighted by the SARS-CoV-2 pandemic, demands the development of pan-coronavirus antivirals. The efficacy of existing antiviral ribonucleoside/ribonucleotide analogs, such as remdesivir, is decreased by the viral proofreading exonuclease NSP14-NSP10 complex. Here, using a novel assay and in silico modeling and screening, we identified NSP14-NSP10 inhibitors that increase remdesivir's potency. A model compound, sofalcone, both inhibits the exonuclease activity of SARS-CoV-2, SARS-CoV, and MERS-CoV in vitro, and synergistically enhances the antiviral effect of remdesivir, suppressing the replication of SARS-CoV-2 and the related human coronavirus OC43. The validation of top hits from our primary screenings using cellular systems provides proof-of-concept for the NSP14 complex as a therapeutic target.
Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Exoribonucleases/metabolism , SARS-CoV-2/drug effects , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , A549 Cells , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Antiviral Agents/pharmacology , Humans , SARS-CoV-2/enzymology , Virus Replication/drug effectsABSTRACT
Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a "microbody," in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of ß coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses.