Development of highly effective LCB1-based lipopeptides targeting the spike receptor-binding motif of SARS-CoV-2

LCB1 is a computationally designed 56-mer miniprotein targeting the spike (S) receptor-binding motif of SARS-CoV- 2 with high potent activity (Science, 2020; Cell host microbe, 2021); however, recent studies have demonstrated that emerging SARS-CoV-2 variants are highly resistant to LCB1's inhibition. In this study, we first identified a truncated peptide termed LCB1v8, which maintained the high antiviral potency. Then, a group of lipopeptides were generated by modifying LCB1v8 with diverse lipids, and of two lipopeptides, the C-terminally stearicacid-conjugtaed LCB1v17 and cholesterol-conjugated LCB1v18, were highly effective in inhibiting both S protein-pseudovirus and authentic SARS-CoV-2 infections. We further showed that LCB1-based inhibitors had similar α-helicity and thermostability in structure and bound to the target-mimic RBD protein with high affinity, and the lipopeptides exhibited greatly enhanced binding with the viral and cellular membranes, improved inhibitory activities against emerging SARS-CoV-2 variants. Moreover, LCB1v18 was validated with high preventive and therapeutic efficacies in K18-hACE2 transgenic mice against lethal SARS-CoV-2 challenge. In conclusion, our studies have provided important information for understanding the structure and activity relationship (SAR) of LCB1 inhibitor and would guide the future development of novel antivirals.