Potent HIV-1 miniprotein inhibitors targeting highly conserved gp41 epitopes

The viral envelope glycoprotein (Env) mediates HIV entry to the cell. During this process, its gp41 subunits must assemble into a highly stable six-helix bundle (6HB) by association between their N-terminal and C-terminal heptad repeats (NHR or HR1 and CHR or HR2, respectively), bringing the viral and cell membranes into close proximity. Further interactions involving the gp41 fusion peptide and the membrane proximal external region (MPER) facilitate membrane fusion. Disrupting 6HB formation is a strategy to inhibit HIV. Previously, we reported chimeric miniproteins (termed covNHR-N) that mimic the first half of gp41 NHR and potently inhibit HIV-1. Stabilization of these miniproteins with disulfide bonds was essential for high inhibitory activity. Here, we introduce newly designed covNHR-N miniproteins, further stabilized by polar-to-hydrophobic mutations. Moreover, we incorporated additional structural motifs that interact with the MPER, a target of broadly neutralizing antibodies (bNAbs). These novel miniproteins showed increased binding affinity for gp41-derived peptides and improved HIV-1 inhibitory activity, particularly against infectious primary viruses on peripheral blood mononuclear cells (PBMC). Furthermore, they exhibited strong synergy with bNAbs and reduced HIV-1 replication in ex vivo experiments with cells from infected donors. These miniproteins could be developed as part of drug compositions against HIV-1.