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Optimization of peptidic HIV-1 fusion inhibitor T20 by phage display.

ABSTRACT: The HIV fusion inhibitor T20 has been approved to treat those living with HIV/AIDS, but treatment gives rise to resistant viruses. Using combinatorial phage-displayed libraries, we applied a saturation scan approach to dissect the entire T20 sequence for binding to a prefusogenic five-helix bundle (5HB) mimetic of HIV-1 gp41. Our data set compares all possible amino acid substitutions at all positions, and affords a complete view of the complex molecular interactions governing the binding of T20 to 5HB. The scan of T20 revealed that 12 of its 36 positions were conserved for 5HB binding, which cluster into three epitopes: hydrophobic epitopes at the ends and a central dyad of hydrophilic residues. The scan also revealed that the T20 sequence was highly adaptable to mutations at most positions, demonstrating a striking structural plasticity that allows multiple amino acid substitutions at contact points to adapt to conformational changes, and also at noncontact points to fine-tune the interface. Based on the scan result and structural knowledge of the gp41 fusion intermediate, a library was designed with tailored diversity at particular positions of T20 and was used to derive a variant (T20v1) that was found to be a highly effective inhibitor of infection by multiple HIV-1 variants, including a common T20-escape mutant. These findings show that the plasticity of the T20 functional sequence space can be exploited to develop variants that overcome resistance of HIV-1 variants to T20 itself, and demonstrate the utility of saturation scanning for rapid epitope mapping and protein engineering.

SUBMITTER: Chen G

PROVIDER: S-EPMC6635768 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Optimization of peptidic HIV-1 fusion inhibitor T20 by phage display.

Chen Gang G Cook Jonathan D JD Ye Wei W Lee Jeffrey E JE Sidhu Sachdev S SS

Protein science : a publication of the Protein Society 20190801 8

The HIV fusion inhibitor T20 has been approved to treat those living with HIV/AIDS, but treatment gives rise to resistant viruses. Using combinatorial phage-displayed libraries, we applied a saturation scan approach to dissect the entire T20 sequence for binding to a prefusogenic five-helix bundle (5HB) mimetic of HIV-1 gp41. Our data set compares all possible amino acid substitutions at all positions, and affords a complete view of the complex molecular interactions governing the binding of T20 ...[more]

PMID: 31228294

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Project description: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.

Project description:Background Middle East respiratory syndrome coronavirus (MERS-CoV), a new coronavirus that emerged in 2012, causes severe and fatal acute respiratory illness in man. Its high mortality (roughly 38%) has raised public fear worldwide, calling for the development of effective and safe therapeutics to combat MERS-CoV infection. On the basis of our previous work in development of peptide fusion inhibitors against HIV and SARS-CoV, and the crystal structure of the six-helix-bundle core of the MERS-CoV spike protein, we identified a peptide derived from the MERS-CoV S protein S2 subunit HR2 domain (HR2P) with a potent inhibitory activity against S-protein-mediated cell–cell fusion. Here, we tested an HR2P analogue with improved pharmaceutical properties, HR2P-M2, for its inhibitory activity against MERS-CoV infection in vitro and in vivo (appendix). Methods We assessed the anti-MERS-CoV activities of HR2P-M2 in vitro with pseudotyped MERS-CoVs in cell culture and in vivo with live MERS-CoVs in hDPP4-transgenic mice. The activities of HR2P-M2 to form six-helix bundles with HR1 peptides and to block fusion core formation between HR1 and HR2 peptides were studied using methods such as circular dichroism, N-PAGE, and FN-PAGE. Findings HR2P-M2 was highly effective in blockade of cell–cell fusion mediated by MERS-CoV S proteins and inhibition of infection by MERS pseudoviruses expressing MERS-CoV S protein with or without mutation in the HR1 region, with IC50 (the concentration causing 50% inhibition) less than 0·7 ?M. HR2P-M2 bound to the HR1 peptide to form stable six-helix bundles and blocked fusion core formation between the HR1 and HR2 peptides. Intranasal administration of HR2P-M2 before viral challenge fully protected hDPP4-transgenic mice (n=5) from MERS-CoV infection, whereas all the untreated mice (n=5) died 8 days after viral challenge. Interpretation Our findings suggest that the HR2P-M2 peptide merits further development as an effective and safe anti-MERS-CoV drug, alone or in combination with other antiviral drugs, to treat MERS-CoV-infected patients and to prevent the disease in high-risk populations, including health-care workers and family members of patients. Funding This work was supported by grants from the National Science Fund of China (81173098 and 81361120378 to SJ, 81373456 to LL), the National 973 Program of China (2012CB519001 to SJ), and the US National Biocontainment Laboratories Operations Support (5UC7AI094660-05 to C-TKT). The funders had no role in interpretation of the findings and writing of this abstract.

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.

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Optimization of peptidic HIV-1 fusion inhibitor T20 by phage display.

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Optimization of peptidic HIV-1 fusion inhibitor T20 by phage display.

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