Structure-based design and characterization of novel fusion-inhibitory lipopeptides against SARS-CoV-2 and emerging variants

Abstract

The ongoing pandemic of COVID-19, caused by SARS-CoV-2, has severely impacted the global public health and socio-economic stability, calling for effective vaccines and therapeutics. In this study, we continued our efforts to develop more efficient SARS-CoV-2 fusion inhibitors and achieved significant findings. First, we found that the membrane-proximal external region (MPER) sequence of SARS-CoV-2 spike fusion protein plays a critical role in viral infectivity and can serve as an ideal template for design of fusion-inhibitory peptides. Second, a panel of novel lipopeptides was generated with greatly improved activity in inhibiting SARS-CoV-2 fusion and infection. Third, we showed that the new inhibitors maintained the potent inhibitory activity against emerging SARS-CoV-2 variants, including those with the major mutations of the B.1.1.7 and B.1.351 strains circulating in the United Kingdom and South Africa, respectively. Fourth, the new inhibitors also cross-inhibited other human CoVs, including SARS-CoV, MERS-CoV, HCoV-229E, and HCoV-NL63. Fifth, the structural properties of the new inhibitors were characterized by circular dichroism (CD) spectroscopy and crystallographic approach, which revealed the mechanisms underlying the high binding and inhibition. Combined, our studies provide important information for understanding the mechanism of SARS-CoV-2 fusion and a framework for the development of peptide therapeutics for the treatment of SARS-CoV-2 and other CoVs.

SARS-CoV-2 fusion-inhibitory lipopeptides maintain high potency against divergent variants of concern including Omicron

“…The antiviral activity of IPB02 could be greatly improved by sequence optimization and introduction of a flexible linker between the peptide sequence and lipid group, as evidenced by IPB02V3 [ 12 ]. Later, we found that the S protein membrane-proximal external region (MPER) critically determines the SARS-coV-2 infectivity and accordingly designed a panel of lipopeptides (IPB20∼IPB27) containing the MPER sequence [ 13 ]. As shown by IPB24, the newly developed inhibitors possessed highly potent activities in inhibiting SARS-CoV-2 bearing S protein with the mutations of D614G, E484K, N501Y, Δ69–70, N501Y/Δ69–70/P681H, or N501Y/E484K/K417N [ 13 ].…”

“…Later, we found that the S protein membrane-proximal external region (MPER) critically determines the SARS-coV-2 infectivity and accordingly designed a panel of lipopeptides (IPB20∼IPB27) containing the MPER sequence [ 13 ]. As shown by IPB24, the newly developed inhibitors possessed highly potent activities in inhibiting SARS-CoV-2 bearing S protein with the mutations of D614G, E484K, N501Y, Δ69–70, N501Y/Δ69–70/P681H, or N501Y/E484K/K417N [ 13 ]. Compared to the S1 subunit, the fusogenic S2 subunit of SARS-CoV-2 is more conserved during the virus evolution; however, three mutations (Q954H, N969K, and L981F) also exist in the HR1 site of Omicron.…”

“…Cells were cultured in complete growth medium consisting of Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin–streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate under 37°C and 5% CO2. Two SARS-CoV-2 fusion-inhibitory lipopeptides, IPB02V3 and IPB24, were synthesized and used in our previous studies [ 12 , 13 ].…”

“…The infectivity of divergent SARS-CoV-2 pseudoviruses in 293T/ACE2 or Huh-7 cells was determined by a single-cycle infection assay as described previously [ 13 ]. Briefly, SARS-CoV-2 pseudoviruses were generated by cotransfecting 293T cells with a wild-type (WT) or mutant S protein-expressing plasmid and a backbone plasmid (pNL4-3.luc.RE) that encodes an Env-defective, luciferase reporter-expressing HIV-1 genome.…”

“…The inhibitory activities of IPB02V3 and IPB24 against authentic SARS-CoV-2 (WT or Omicron variant) infection were determined as described previously [ 13 ]. In brief, a serially 3-fold diluted lipopeptide was incubated with 0.1 MOI of live WT or Omicron virus at 37°C for 1 h, and the lipopeptide-virus mixture was then added into Vero cells that were seeded in a 96-well plate one day before infection at a concentration of 1×10 4 cells/well.…”