Revisiting the mechanism of enfuvirtide and designing an analog with improved fusion inhibitory activity by targeting triple sites in gp41

OBJECTIVE: To revisit the mechanism of action of enfuvirtide (T20) and based on the newly defined mechanism, design an analogous peptide of T20 with improved antiviral activity. DESIGN: We compared the inhibitory activity of T20 with that of T1144 on six-helix bundle (6HB) formation at different time after coculture of HIV type 1 (HIV-1) envelope (Env)-expressing Chinese hamster ovary (CHO-Env) cells and CD4-expressing MT-2 cells at 31.5 °C and with that of T20-SF, an analogous peptide of T20 with an additional tryptophan-rich motif, on hemolysis mediated by FP-P, which contains fusion peptide and fusion peptide (FP) proximal region (FPPR), and HIV-1 infection. METHODS: Inhibitory activity of peptides on 6HB formation was tested in a temperature-controlled cell-cell fusion assay by flow cytometry using 6HB-specific mAb 2G8; on HIV-1 infection and fusion was assessed by p24 and cell-cell fusion assays. Interaction between different peptides or peptide and antibody was evaluated by ELISA. RESULTS: T20 could inhibit 6HB formation at early, but not late, stage of HIV-1 fusion, whereas T1144 was effective at both stages. T20-SF is much more effective than T20 in binding to FP-P and inhibiting infection of HIV-1, including T20-resistant strains, and FP-P-mediated hemolysis. CONCLUSION: Results suggest that T20 has a double-target mechanism, by which its N-terminal and C-terminal portions bind to N-terminal heptad repeat and FPPR, respectively. T20-SF designed based on this new mechanism exhibits significantly improved anti-HIV-1 activity because it targets the triple sites in gp41, including N-terminal heptad repeat, FPPR, and fusion peptide. Thus, this study provides clues for designing novel HIV fusion inhibitors with improved antiviral activity.