pAAV-IRES-EGFP

Researchers have previously identified a polyethylene glycol-conjugated (PEGylated) lipopeptide, EK1C4, with potent pan-coronavirus fusion inhibitory activity. However, PEG linkers in peptide or protein drugs may reduce stability or induce anti-PEG antibodies in vivo. Here, researchers report the design and synthesis of a series of dePEGylated lipopeptide-based pan-CoV fusion inhibitors featuring the replacement of the PEG linker with amino acids in the heptad repeat 2 C-terminal fragment (HR2-CF) of HCoV-OC43. Among these lipopeptides, EKL1C showed the most potent inhibitory activity against infection by SARS-CoV-2 and its spike (S) mutant, as well as other HCoVs and some bat SARS-related coronaviruses (SARSr-CoV) tested.

In this study, 293T effector cells were transfected with plasmid pAAV-IRES-EGFP encoding SARS-CoV-2 S protein (293T/SARS-CoV-2/GFP), while Huh-7 cells, expressing various HCoV receptors on the membrane surface, were used as target cells. Researchers tested the potential inhibitory activity of lipopeptides against cell-cell fusion mediated by the SARS-CoV-2 S protein. EKL1C can completely inhibit cell-cell fusion at a concentration of 2.5 μmol/L and exhibits the strongest inhibitory activity among the lipopeptides tested, which is consistent with the results of SARS-CoV-2 PsV infection experiments. EKL1C inhibits cell-to-cell fusion mediated by SARS-CoV-2 S protein with an IC50 value of 0.008 μmol/L, which is approximately 40-fold stronger than EK1 but approximately 2.6-fold weaker than EK1C4.

Figure 1. Images of SARS-CoV-2 S protein-mediated cell-cell fusion in the presence of EK1 (I), EKL1C (II), EKL1 (III), and PBS (IV). (Zhou, Jie, et al., 2022)