Engineering protease-resistant antiviral peptide inhibitors for SARS-CoV-2

No vaccines or treatments for SARS-CoV-2 are yet available. A simple prophylactic antiviral strategy would protect naïve individuals from infection now. In the future, when vaccines should be available, a prophylactic antiviral will be essential for individuals who do not mount a suitable immune response. Antivirals that target viral entry into the host cell have been proven effective against a wide range of viral diseases. The entry/fusion process for CoV (including SARS-CoV-2) is mediated by the viral envelope glycoprotein (S). Concerted action by the receptor-binding domain and the fusion domain is required for fusion. Upon viral attachment (and uptake in certain cases), large-scale conformational rearrangements occur in the fusion domain, driven by formation of a structure that couples protein refolding directly to membrane fusion. The formation of this structure can be targeted by fusion inhibitory peptides (C-terminal heptad repeat or HRC peptides) that prevent proper apposition of the HRC and HRN domains in S. We have found that conjugation of a lipid to an inhibitory peptide directs the peptide to cell membranes and increases antiviral efficacy. Analogous lipo-peptides prevent infection by several viruses (measles, Nipah, parainfluenza, influenza), and can be administered via the airway. Treatment is effective for some of these even several days after infection. In addition, we have shown that modifying the backbone of an HRC peptide via periodic replacement of ?-amino acid residues with ?- amino acid residues generates ?/?-peptides that retain antiviral potency (toward HIV or parainfluenza) but are highly resistant to proteolysis. We recently generated an HRC lipopeptide that is effective against both SARS- CoV2 and MERS live viruses in vitro, blocks spread of SARS-CoV2 in human airway tissue, and inhibits transmission of SARS-CoV-2 between ferrets in direct contact. Here we propose to combine the lipid conjugation and backbone-modification strategies to generate potent inhibitors of SARS-CoV2 infection that display a long half-life in vivo. 1. Optimize the antiviral potency and bioavailability of SARS-CoV-2 HRC peptide fusion inhibitors via rational molecular engineering. Antiviral efficacy of ?/?-lipopeptide candidates will be measured in quantitative in vitro assays, in authentic virus infection, and in a human airway model. 2. Evaluate the protection afforded by new backbone-modified ?/?-lipopeptide fusion inhibitors against SARS-CoV-2 infection in hamsters. Analysis of in vivo biodistribution and toxicity of backbone modified S- CoV-2 ?/?-lipopeptide fusion inhibitors and assessment of in vivo potency and resistance mechanisms will lay the foundation for a safe and effective SARS CoV-2 fusion inhibitor for coronavirus prevention and therapy.