Development of broad-spectrum coronavirus therapeutics: identification of potent inhibitors of SARS-CoV-2 binding and entry

Abstract

Coronavirus outbreaks have been increasing in both frequency and magnitude for the last 20 years. Prior to 2002, there were two identified human coronaviruses. Now in 2022 there are seven, potentially eight. From severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, to Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 and SARS-CoV-2 in 2019, coronavirus outbreaks have continued to escalate, causing more infections and more deaths with each successive outbreak. SARS-CoV-2 was declared a pandemic on March 11, 2020, by the World Health Organization. To date, there have been around 460 million cases and over 6 million deaths, worldwide. The need for therapeutic interventions for SARS-CoV-2 is continuing, but even greater is the need for broad spectrum coronavirus therapeutics that may be used for this and other coronavirus outbreaks. Due to the critical role of ACE2 in the replication of SARS-CoV, SARS-CoV-2 and human coronavirus (HCoV)-NL63, ACE2 has become a prime target for therapeutic intervention. Groups have tested chimerics of ACE2 fused to an Fc peptide: ACE2-Fc as a decoy molecule that would bind the receptor binding domain (RBD) of SARS-CoV-2 with the same or greater affinity than ACE2 on the surface of cells. Here is described the production and evaluation of seven ACE2-Fc chimerics, and the down-selection to one lead candidate as a potent inhibitor of SARS-CoV-2 cell entry. All ACE2-Fc chimerics were sequence verified, their expression was confirmed in HEK293Ts using western blots and their relative neutralization capacity was tested in in vitro neutralization assays. Evaluation of these constructs led to the identification of a lead construct, which was a potent inhibitor of a SARS-CoV-2 D614G pseudotype luciferase reporter virus. Additional testing of the lead ACE2-Fc construct in wild-type SARS-CoV-2 also demonstrated potent neutralization. Further evaluation of this protein in vivo will allow for determination of dose range, and how well this protein is tolerated in a live host. ACE2-Fc would fill a potential gap in early treatment and would have the unique benefit of being a broad-spectrum therapeutic able to target infection with multiple different coronaviruses.