Development of lipopeptide-based HIV-1/2 fusion inhibitors targeting the gp41 pocket site with a new design strategy

Emerging studies demonstrate that lipid conjugation is a vital strategy for designing peptide-based viral fusion inhibitors, and the so-called lipopeptides exhibit greatly improved antiviral activity. In the design of lipopeptides, a flexible linker between the peptide sequence and lipid molecule is generally required, mostly with a short polyethylene glycol or glycine-serine sequence. Very recently, we discovered that the helix-facilitating amino acid sequence "EAAAK" as a rigid linker is a more efficient method in the design of SARS-CoV-2 fusion inhibitory lipopeptides. In this study, we comprehensively characterized the functionalities of different linkers in HIV fusion inhibitors. A short-peptide inhibitor 2P23, which mainly targets the gp41 pocket site, was used as a design template, generating a group of cholesterol-modified lipopeptides. In the inhibition of HIV-1 infection, the lipopeptide inhibitors with a rigid linker were much superior than those with the flexible linkers, as indicated by LP-37 with the "EAAAK" linker and LP-39 with the repeated "EP" amino acid sequences. Both lipopeptides were very potent inhibitors of HIV-2 and simian immunodeficiency (SIV) either. Promisingly, LP-37 displayed high α-helicity, thermostability and binding ability to a target-mimic peptide, and it was metabolically stable when treated with temperature, proteolytic enzymes or human sera. Taken together, our studies have verified a universal strategy for designing viral fusion inhibitors and offered a novel HIV fusion inhibitor for drug development.