Structural and Functional Characterization of Membrane Fusion Inhibitors with Extremely Potent Activity against Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus

Abstract

T-20 (enfuvirtide) is the only membrane fusion inhibitor available for the treatment of viral infection; however, it has low anti-human immunodeficiency virus (anti-HIV) activity and a low genetic barrier for drug resistance. We recently reported that T-20 sequence-based lipopeptides possess extremely potent and efficacies (X. Ding, Z. Zhang, H. Chong, Y. Zhu, H. Wei, X. Wu, J. He, X. Wang, Y. He, 2017, J Virol 91:e00831-17, https://doi.org/10.1128/JVI.00831-17; H. Chong, J. Xue, Y. Zhu, Z. Cong, T. Chen, Y. Guo, Q. Wei, Y. Zhou, C. Qin, Y. He, 2018, J Virol 92:e00775-18, https://doi.org/10.1128/JVI.00775-18). Here, we focused on characterizing the structure-activity relationships of the T-20 derivatives. First, a novel lipopeptide termed LP-52 was generated with improved target-binding stability and anti-HIV activity. Second, a large panel of truncated lipopeptides was characterized, revealing a 21-amino-acid sequence core structure. Third, it was surprisingly found that the addition of the gp41 pocket-binding residues in the N terminus of the new inhibitors resulted in increased binding but decreased antiviral activities. Fourth, while LP-52 showed the most potent activity in inhibiting divergent HIV-1 subtypes, its truncated versions, such as LP-55 (25-mer) and LP-65 (24-mer), still maintained their potencies at very low picomolar concentrations; however, both the N- and C-terminal motifs of LP-52 played crucial roles in the inhibition of T-20-resistant HIV-1 mutants, HIV-2, and simian immunodeficiency virus (SIV) isolates. Fifth, we verified that LP-52 can bind to target cell membranes and human serum albumin and has low cytotoxicity and a high genetic barrier to inducing drug resistance. Development of novel membrane fusion inhibitors against HIV and other enveloped viruses is highly important in terms of the peptide drug T-20, which remains the only one for clinical use, even if it is limited by large dosages and resistance. Here, we report a novel T-20 sequence-based lipopeptide showing extremely potent and broad activities against HIV-1, HIV-2, SIV, and T-20-resistant mutants, as well as an extremely high therapeutic selectivity index and genetic resistance barrier. The structure-activity relationship (SAR) of the T-20 derivatives has been comprehensively characterized, revealing a critical sequence core structure and the target sites of viral vulnerability that do not include the gp41 pocket. The results also suggest that membrane-anchored inhibitors possess unique modes of action relative to unconjugated peptides. Combined, our series studies have not only provided drug candidates for clinical development but also offered important tools to elucidate the mechanisms of viral fusion and inhibition.

Monoclonal Antibody 2C6 Targets a Cross-Clade Conformational Epitope in gp41 with Highly Active Antibody-Dependent Cell Cytotoxicity

“…The epitope of Ab QA255.067 (from 952-606; LLGIWGCSGKLICTT), identified by binding to similarly constructed VLPs (32), is near 2C6 but is also dependent on the disulfide bridge of the C-C loop sequence. Besides the fusion peptide-targeting Abs, 2C6 also maps near the hydrophobic pocket targeted by enfuvirtide (T20) and other fusion inhibitors in development (33). A number of our QtAbs can interfere with enfuvirtide in a fusion assay, but 2C6 failed to interfere with the function of T20 (34).…”

Design and Characterization of Cholesterylated Peptide HIV-1/2 Fusion Inhibitors with Extremely Potent and Long-Lasting Antiviral Activity

“…Cholesterylated inhibitors exhibit extremely potent activity against HIV-2 and SIV isolates. In the previous works, we also demonstrated that the fatty acidconjugated inhibitors possess highly potent activity against HIV-2 and SIV isolates (25)(26)(27). Here, we also characterized the inhibitory activities of cholesterol-modified inhibitors in comparison to T-20, LP-80, and C34-Chol.…”

“…By adding a cholesterol group to the template peptide C34, the lipopeptide C34-Chol was reported as the most potent HIV-1 fusion inhibitor (17), and it is currently being evaluated in clinical trials (28). However, we recently found that the T-20-based sequence is a more efficient template, resulting in the inhibitors conjugated with a fatty acid group (24)(25)(26)(27). To create an ideal candidate for clinical development and to exploit the structure-function relationship of diverse lipopeptide inhibitors, here we generated a group of T-20 backbone-based fusion inhibitors by cholesterol conjugation.…”

“…Cholesterylated inhibitors exhibit extremely potent activity against T-20resistant mutants. We previously found that fatty acid-modified inhibitors such as LP-51, LP-52, and LP-80 were highly active in inhibiting diverse HIV-1 mutants that are resistant to T-20, but these lipopeptides still displayed greatly decreased potencies relative to their inhibitions on T-20-sensitive viruses (26,27). Therefore, we sought to characterize the newly generated cholesterol-modified inhibitors using the same panel of T-20-resistant mutants.…”

“…By substituting the TRM sequence with a fatty acid group, we generated a T-20-based lipopeptide (LP-40) with greatly increased potency (24). It was more surprising that LP-40 could be dramatically improved by further sequence optimization, resulting in several lipopeptides that inhibited divergent HIV-1/2 and simian immunodeficiency virus (SIV) isolates at very low picomolar concentrations, including LP-50, LP-51, and LP-52 (25,26). Very recently, a stearic acid-modified lipopeptide, termed LP-80, was generated with exceptionally potent in vitro inhibitory activity and in vivo therapeutic efficacy (27).…”