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

Xu, Weia,*; Pu, Jinga,*; Su, Shana,*; Hua, Chena; Su, Xiaojiea; Wang, Qiana,†; Jiang, Shiboa,b,†; Lu, Lua,†

doi: 10.1097/QAD.0000000000002208

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.

aKey Laboratory of Medical Molecular Virology of MOE/MOH, School of Basic Medical Sciences and Shanghai Public Health Clinical Center, Fudan University, Shanghai, China

bLindsley F. Kimball Research Institute, New York Blood Center, New York, New York, USA.

Correspondence to Lu Lu, PhD, Fudan University, Shanghai, China. E-mail:

Received 21 January, 2019

Revised 25 February, 2019

Accepted 4 March, 2019

Copyright © 2019 Wolters Kluwer Health, Inc.