Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti–HIV-1 drugs

Crespillo, Sara; Cámara-Artigas, A.; Casares, Salvador; Morel, Bertrand; Cobos, Eva S.; Mateo, Pedro L.; Mouz, Nicolas; Martin, Christophe E.; Roger, Marie G.; Habib, Raphaëlle El; Su, Bin; Moog, Christiane; Conejero‐Lara, Francisco

doi:10.1073/pnas.1413592112

Cited by 34 publications

(39 citation statements)

References 37 publications

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“…The crystal structures of diverse 6-HBs have universally revealed a deep hydrophobic pocket on the C-terminal portion of NHR helices, which is docked by hydrophobic residues from the N-terminal pocket-binding domain (PBD) of the CHR helix (2)(3)(4). We and others have recently identified a subpocket located immediately downstream of the deep pocket, which further stabilizes the interactions of the NHR and CHR helices (5)(6)(7). Both pockets offer ideal targets for developing anti-HIV therapeutics.…”

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confidence: 98%

Mechanism of HIV-1 Resistance to an Electronically Constrained α-Helical Peptide Membrane Fusion Inhibitor

Liu

Ding

et al. 2018

J Virol

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SC29EK is an electronically constrained α-helical peptide HIV-1 fusion inhibitor that is highly effective against both wild-type and enfuvirtide (T20)-resistant viruses. In this study, we focused on investigating the mechanism of HIV-1 resistance to SC29EK by two approaches. First, SC29EK-escaping HIV-1 variants were selected and characterized. Three mutant viruses, which possessed two (N43K/E49A) or three (Q39R/N43K/N126K and N43K/E49A/N126K) amino acid substitutions in the N- and C-terminal repeat regions of gp41 were identified as conferring high resistance to SC29EK and cross-resistance to the first-generation (T20 and C34) and newly designed (sifuvirtide, MT-SC29EK, and 2P23) fusion inhibitors. The resistance mutations could reduce the binding stability of SC29EK, impair viral Env-mediated cell fusion and entry, and change the conformation of the gp41 core structure. Further, we determined the crystal structure of SC29EK in complex with a target mimic peptide, which revealed the critical intra- and interhelical interactions underlying the mode of action of SC29EK and the genetic pathway to HIV-1 resistance. Taken together, the present data provide new insights into the structure and function of gp41 and the structure-activity relationship (SAR) of viral fusion inhibitors. T20 is the only membrane fusion inhibitor available for treatment of viral infection, but it has relatively low anti-HIV activity and genetic barriers for resistance, thus calling for new drugs blocking the viral fusion process. As an electronically constrained α-helical peptide, SC29EK is highly potent against both wild-type and T20-resistant HIV-1 strains. Here, we report the characterization of HIV-1 variants resistant to SC29EK and the crystal structure of SC29EK. The key mutations mediating high resistance to SC29EK and cross-resistance to the first and new generations of fusion inhibitors as well as the underlying mechanisms were identified. The crystal structure of SC29EK bound to a target mimic peptide further revealed its action mode and genetic pathway to inducing resistance. Hence, our data have shed new lights on the mechanisms of HIV-1 fusion and its inhibition.

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confidence: 98%

Mechanism of HIV-1 Resistance to an Electronically Constrained α-Helical Peptide Membrane Fusion Inhibitor

Liu

Ding

et al. 2018

J Virol

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“…A prominent feature of 6-HB structures is the deep pocket on the C-terminal portion of NHR helices, which is penetrated by three hydrophobic residues (Trp628, Trp631, and Ile635) from the pocket-binding domain (PBD) of the CHR helix (3)(4)(5)(6). Recently, a subpocket, located immediately downstream of the deep pocket, has been identified (7)(8)(9). Both pockets play critical roles in the stability of the 6-HB core and offer ideal target sites for anti-HIV agents (6,7,(9)(10)(11).…”

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confidence: 99%

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

Ding

Zhang

Chong

et al. 2017

J Virol

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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 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. 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.

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“…Crespillo et al engineered a single-chain construct to mimic the native gp41 N-segment homotrimer. [66] The resulting protein, which is ~170 residues long, has an up-up-down arrangement of helices combined in a single chain to present a binding surface similar to the native homotrimer. The engineered pseudo-trimer is active as a fusion inhibitor.…”

Section: Other Targetsmentioning

confidence: 99%

Designing helical peptide inhibitors of protein–protein interactions

Araghi

Keating

2016

Current Opinion in Structural Biology

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Short helical peptides combine characteristics of small molecules and large proteins and provide an exciting area of opportunity in protein design. A growing number of studies report novel helical peptide inhibitors of protein-protein interactions. New techniques have been developed for peptide design and for chemically stabilizing peptides in a helical conformation, which frequently improves protease resistance and cell permeability. We summarize advances in peptide crosslinking chemistry and give examples of peptide design studies targeting coiled-coil transcription factors, Bcl-2 family proteins, MDM2/MDMX, and HIV gp41, among other targets.

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Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti–HIV-1 drugs

Cited by 34 publications

References 37 publications

Mechanism of HIV-1 Resistance to an Electronically Constrained α-Helical Peptide Membrane Fusion Inhibitor

Mechanism of HIV-1 Resistance to an Electronically Constrained α-Helical Peptide Membrane Fusion Inhibitor

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

Designing helical peptide inhibitors of protein–protein interactions

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