Identification of novel intervention targets of severe acute respiratory syndrome coronavirus 2 infection

Abstract

Coronavirus disease 2019 (COVID-19) is an emerging infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite global joint efforts, there are still limited SARS-CoV-2 therapeutic options for COVID-19 patients. Thus there is an urgent need to explore novel antiviral drug targets for developing the next generation of anti-COVID-19 agents. In this study, two strategies were used to discover new antiviral drug targets: (1) host-targeting antivirals (HTAs) and (2) virus-targeting antivirals (VTAs). COVID-19 significantly improves our understanding of the host metabolic response to SARS-CoV-2 infection. The study of SARS-CoV-2 host interaction can provide a rich array of antiviral targets. In the first study of this thesis, we investigated the plasma lipidome in a cohort of COVID-19 patients over their disease course, and successfully identify triacylglycerol (TG) as the dominant lipid class of SARS-CoV-2-induced lipidomic dysregulation. In particular, lipid droplet (LD) formation enzyme triglyceride synthesizing enzyme diacylglycerol acyltransferase (DGAT) and LD stabilizer adipocyte differentiation-related protein (ADRP) were found to be essential host factors for SARS-CoV-2 replication. Mechanistically, the viral nucleoprotein drives DGAT1/2 gene expression to facilitate LDs formation. Moreover, the association of ADRP on the LD surface which facilitates the completion of the viral replication cycle. DGAT gene depletion was found to reduce SARS-CoV-2 protein synthesis without compromising viral genome replication/transcription. Xanthohumol is an orally available DGAT inhibitor. We found that Xanthohumol can suppress SARS-CoV-2 replication and the associated pulmonary inflammation in a Syrian hamster model. These findings uncover the mechanistic role of SARS-CoV-2 nucleoprotein that exploits the LD-oriented network to increase metabolic demand associated with viral infection, and shows that the LD-synthetase DGAT and LDs-stabilizer ADRP are potential therapeutic targets for COVID-19. Virus-targeting antivirals such as therapeutic antibodies and enzyme inhibitors are generally highly potent. For example, nanobodies (single-domain antibodies) derived from single‑chain camelid antibodies have shown potent antiviral activities against different viruses. In the second part of this thesis, we screened nanobodies that block the interaction between SARS-CoV-2 spike receptor-binding domain (RBD) and the human angiotensin-converting enzyme 2 (hACE2) receptor. Among the selected nanobodies, Nb54 demonstrated the best neutralization activity against spike pseudotyped virus in vitro (and inhibited authentic SARS-CoV-2 infection without causing antibody-dependent enhancement (ADE). In the Syrian hamster model, intranasal administration of Nb54 exhibited potent antiviral activities in both prophylactic and therapeutic regimens. Moreover, human Fc-tagged Nb54 (Nb54-Fc) was constructed to improve its neutralizing capacity and in vivo bioavailability. Intraperitoneal administration of Nb54-Fc decreased SARS-CoV-2 replication by >10 folds in the nasal turbinate and lungs of SARS-CoV-2-infected hamsters. In summary, this thesis demonstrated that TG hyperproduction is involved in SARS-CoV-2-induced metabolic dysregulation. The findings provide a basis for further development of DGAT- or ADRP-targeting therapeutics for COVID-19. A novel neutralizing antibody Nb54 is found by screening an alpaca nanobody phage display library against SARS-CoV-2 spike RBD. Importantly, Nb54 showed antiviral effects against virus with binding affinity. Additional structural analysis may help to further decipher the interactions between SARS-CoV-2 spike and Nb54.