STRUCTURE-GUIDED RECEPTOR/INHIBITOR TRIMERIZATION AND RELATED STRATEGIES AGAINST CORONAVIRUSES

Project Summary The current SARS-CoV-2 pandemic poses an immediate and global public health threat. This is the third major zoonotic-born coronavirus outbreak in humans in the past twenty years, after SARS-CoV-1 and MERS-CoV. Because mainstream antiviral approaches such as vaccines and neutralizing antibodies have to be specifically developed for each virus, there has always been a lag in effective medical remedy, which resulted in grave human death toll and economic disruption. Since viruses rarely switch their receptor specificity, here we propose a therapeutic strategy that utilizes the trimeric receptor as a decoy to neutralize the virus. SARS-CoV- 1 and SARS-CoV-2 target the angiotensin-converting enzyme 2 (ACE2) to gain cell entry. The "tri-ACE2" decoys are designed in a structure-guided fashion to match the symmetry and geometry of the viral spike (S), which maximizes the binding affinity through the trimer avidity effect. Additional functional domains are then introduced into the tri-ACE2 platform to achieve better antiviral activity. We present strong preliminary data demonstrating the effectiveness of such designs. Tri-ACE2 decoys lock the viral spikes in a symmetric "3-UP" receptor binding domain (RBD) conformation, neutralizing various ACE2-tropic coronaviruses with nanomolar concentrations, ~100-fold better than monomeric ACE2. Replacing ACE2 with ab initial designed minibinders (miniature-ACE2s) further improved the IC50 to ~20 pM by enabling ultrafast S-binding. The advantage of tri- ACE2 over neutralizing antibodies lies in its potential broad-spectrum activity against all ACE2-tropic coronaviruses, and in its expected resistance against evader mutations in the viral receptor. Therefore, this line of inhibitors can potentially serve as an off-the-shelf therapeutic in future outbreaks caused by unknown ACE2- tropic coronaviruses. We propose to explore the full potential of the structure-guided receptor multimerization as a general antiviral strategy. These efforts will also generate new mechanistic insight about how the coronavirus spike protein functions in general. The specific aims include: 1) Produce potent and evader- resistant tri-ACE2 inhibitors against SARS-CoV-2; 2) Produce tri-miniatureACE2 with picomolar inhibitory activity and novel antiviral mechanism; 3) Determine the efficacy of tri-ACE2 against SARS-CoV-2 and related viruses in primary airway cell cultures and animal models; 4) Determine the prophylactic and therapeutic efficacy of tri-ACE2 based inhibitors against SARS-CoV-2 infection in a Golden Syrian hamster model. Upon the completion of this project, we expect that we will have developed safe, highly effective, and broad- spectrum anti-coronavirus drugs that can directly lead to trials in nonhuman primates or humans.