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Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition.

ABSTRACT: The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and in vivo stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket-binding sequence and targets a different site. First, the C-terminal tryptophan-rich motif (TRM) of T20 was verified to be essential for its target binding and inhibition; then, a novel lipopeptide, termed LP-40, was created by replacing the TRM with a fatty acid group. LP-40 showed markedly enhanced binding affinity for the target site and dramatically increased inhibitory activity on HIV-1 membrane fusion, entry, and infection. Unlike LP-11 and LP-19, which required a flexible linker between the peptide sequence and the lipid moiety, addition of a linker to LP-40 sharply reduced its potency, implying different binding modes with the extended N-terminal helices of gp41. Also, interestingly, LP-40 showed more potent activity than LP-11 in inhibiting HIV-1 Env-mediated cell-cell fusion while it was less active than LP-11 in inhibiting pseudovirus entry, and the two inhibitors displayed synergistic antiviral effects. The crystal structure of LP-40 in complex with a target peptide revealed their key binding residues and motifs. Combined, our studies have not only provided a potent HIV-1 fusion inhibitor, but also revealed new insights into the mechanisms of viral inhibition.IMPORTANCE T20 is the only membrane fusion inhibitor available for treatment of viral infection; however, T20 requires high doses and has a low genetic barrier for resistance, and its inhibitory mechanism and structural basis remain unclear. Here, we report the design of LP-40, a T20-based lipopeptide inhibitor that has greatly improved anti-HIV activity and is a more potent inhibitor of cell-cell fusion than of cell-free virus infection. The binding modes of two classes of membrane-anchoring lipopeptides (LP-40 and LP-11) verify the current fusion model in which an extended prehairpin structure bridges the viral and cellular membranes, and their complementary effects suggest a vital strategy for combination therapy of HIV-1 infection. Moreover, our understanding of the mechanism of action of T20 and its derivatives benefits from the crystal structure of LP-40.

SUBMITTER: Ding X

PROVIDER: S-EPMC5571253 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition.

Ding Xiaohui X Zhang Xiujuan X Chong Huihui H Zhu Yuanmei Y Wei Huamian H Wu Xiyuan X He Jinsheng J Wang Xinquan X He Yuxian Y

Journal of virology 20170824 18

The peptide drug enfuvirtide (T20) is the only viral fusion inhibitor used in combination therapy for HIV-1 infection, but it has relatively low antiviral activity and easily induces drug resistance. Emerging studies demonstrate that lipopeptide-based fusion inhibitors, such as LP-11 and LP-19, which mainly target the gp41 pocket site, have greatly improved antiviral potency and <i>in vivo</i> stability. In this study, we focused on developing a T20-based lipopeptide inhibitor that lacks pocket- ...[more]

PMID: 28659478

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Project description:Peptide T20, which targets the HIV protein gp41, represents the first approved member of the class of HIV drugs known as membrane fusion inhibitors. However, mechanisms which lead to resistance through clinical use of T20 are not well-understood because the structure of the bound complex remains undetermined. In this report, an atomic-level model of a T20-gp41 complex embedded in an explicit DOPC membrane was constructed, and molecular dynamics simulations, followed by binding energy analysis (MM-GBSA method), were performed to delineate structural and energetic features that contribute to drug resistance. Per-residue binding footprints for T20 with wild-type gp41 reveal strong intermolecular van der Waals, Coulombic, and H-bond interactions in striking agreement with clinically observed resistance patterns. In addition, seven deleterious gp41 point mutations (L33Q, L33S, G36V, I37K, V38E, Q40H, and Q40K) were simulated, and all correctly exhibited decreases in the level of binding, including the fact that L33Q and Q40K are most detrimental. Six of the seven simulations yield good quantitative agreement (r(2) = 0.72; N = 6) with available experimental fold resistance data. Results from energy decomposition, heat map analysis, and differential (mutant minus wild-type) footprinting indicate the following. (1) Mutations disrupt intermolecular H-bonding and reduce the level of favorable contact with gp41 at M19. (2) Charged mutations (I37K, Q40K, and V38E) lead to significant Coulombic changes that weaken favorable van der Waals interactions. (3) Q40K is more detrimental than I37K because of interaction differences with a polar/charged patch on T20 in the initial (wild-type) state. (4) Resistance for L33S versus L33Q likely involves side chain packing differences in the final (mutated) state. A valuable finding of the work involves identification of favorable interactions among the C-terminal end of T20 (WNWF motif), residues on gp41 (including the fusion peptide), and headgroups in the adjacent membrane. The results suggest a complete T20 binding site would contribute to a stable complex, which could help to explain why prior studies, which employed truncated gp41 constructs, reported that C-terminal T20 residues may not interact with gp41. A hypothesis resulting from this study is that peptides could be designed to increase the level of favorable contact with both the membrane and gp41 which would lead to enhanced activity.

Project description:Peptides derived from the C-terminal heptad repeat (CHR) region of the human immunodeficiency virus type 1 (HIV-1) fusogenic protein gp41 are potent viral entry inhibitors, and currently, enfuvirtide (T-20) is the only one approved for clinical use; however, emerging drug resistance largely limits its efficacy. In this study, we generated a novel lipopeptide inhibitor, named LP-19, by integrating multiple design strategies, including an N-terminal M-T hook structure, an HIV-2 sequence, intrahelical salt bridges, and a membrane-anchoring lipid tail. LP-19 showed stable binding affinity and highly potent, broad, and long-lasting antiviral activity. In in vitro studies, LP-19 efficiently inhibited HIV-1-, HIV-2-, and simian immunodeficiency virus (SIV)-mediated cell fusion, viral entry, and infection, and it was highly active against diverse subtypes of primary HIV-1 isolates and inhibitor-resistant mutants. Ex vivo studies demonstrated that LP-19 exhibited dramatically increased anti-HIV activity and an extended half-life in rhesus macaques. In short-term monotherapy, LP-19 reduced viral loads to undetectable levels in acutely and chronically simian-human immunodeficiency virus (SHIV)-infected monkeys. Therefore, this study offers an ideal HIV-1/2 fusion inhibitor for clinical development and emphasizes the importance of the viral fusion step as a drug target.IMPORTANCE The peptide drug T-20 is the only viral fusion inhibitor in the clinic, which is used for combination therapy of HIV-1 infection; however, it requires a high dosage and easily induces drug resistance, calling for a new drug with significantly improved pharmaceutical profiles. Here, we have developed a short-lipopeptide-based fusion inhibitor, termed LP-19, which mainly targets the conserved gp41 pocket site and shows highly potent inhibitory activity against HIV-1, HIV-2, and even SIV isolates. LP-19 exhibits dramatically increased antiviral activity and an extended half-life in rhesus macaques, and it has potent therapeutic efficacy in SHIV-infected monkeys, highlighting its high potential as a new viral fusion inhibitor for clinical use.

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Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition.

Publications

Enfuvirtide (T20)-Based Lipopeptide Is a Potent HIV-1 Cell Fusion Inhibitor: Implications for Viral Entry and Inhibition.

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