Selection with a Peptide Fusion Inhibitor Corresponding to the First Heptad Repeat of HIV-1 gp41 Identifies Two Genetic Pathways Conferring Cross-Resistance to Peptide Fusion Inhibitors Corresponding to the First and Second Heptad Repeats (HR1 and HR2) of gp41

ABSTRACTT-20EK is a novel fusion inhibitor designed to have enhanced α-helicity over T-20 (enfuvirtide) through engineered electrostatic interactions between glutamic acid (E) and lysine (K) substitutions. T-20EK efficiently suppresses wild-type and T-20-resistant variants. Here, we selected T-20EK-resistant variants. A combination of L33S and N43K substitutions in gp41 were required for high resistance to T-20EK. While these substitutions also caused resistance to T-20, they did not cause cross-resistance to other known fusion inhibitors.

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Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques

Journal of Virology ◽

10.1128/jvi.00384-20 ◽

2020 ◽

Vol 94 (15) ◽

Cited By ~ 1

Author(s):

Danwei Yu ◽

Jing Xue ◽

Huamian Wei ◽

Zhe Cong ◽

Ting Chen ◽

...

Keyword(s):

Membrane Fusion ◽

Therapeutic Efficacy ◽

Rhesus Macaques ◽

Heptad Repeat ◽

Fusion Inhibitor ◽

Resistance Mutations ◽

Fusion Inhibitors ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance. IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.

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

Journal of Virology ◽

10.1128/jvi.02044-17 ◽

2018 ◽

Vol 92 (7) ◽

Cited By ~ 9

Author(s):

Xiyuan Wu ◽

Zixuan Liu ◽

Xiaohui Ding ◽

Danwei Yu ◽

Huamian Wei ◽

...

Keyword(s):

Crystal Structure ◽

Cross Resistance ◽

Fusion Inhibitor ◽

Viral Fusion ◽

Wild Type ◽

Genetic Pathway ◽

Fusion Inhibitors ◽

Helical Peptide ◽

Mimic Peptide ◽

Hiv 1

ABSTRACTSC29EK 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.IMPORTANCET20 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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Mechanism of HIV-1 Resistance to Short-Peptide Fusion Inhibitors Targeting the Gp41 Pocket

Journal of Virology ◽

10.1128/jvi.00373-15 ◽

2015 ◽

Vol 89 (11) ◽

pp. 5801-5811 ◽

Cited By ~ 23

Author(s):

Yang Su ◽

Huihiui Chong ◽

Zonglin Qiu ◽

Shengwen Xiong ◽

Yuxian He

Keyword(s):

Structure Function ◽

Heptad Repeat ◽

Cross Resistance ◽

Short Peptide ◽

Fusion Inhibitors ◽

Structure Function Relationship ◽

Underlying Mechanisms ◽

Function Relationship ◽

Relationship Of ◽

Hiv 1

ABSTRACTThe deep hydrophobic pocket on the N trimer of HIV-1 gp41 has been considered an ideal drug target. On the basis of the M-T hook structure, we recently developed short-peptide-based HIV-1 fusion inhibitors (MTSC22 and HP23), which mainly target the pocket site and possess highly potent antiviral activity. In this study, we focused on investigating their resistance pathways and mechanisms by escape HIV-1 mutants to SC22EK, a template peptide for MTSC22 and HP23. Two substitutions, E49K and N126K, located, respectively, at the N- and C-heptad repeat regions of gp41, were identified as conferring high resistance to the inhibitors targeting the pocket and cross-resistance to enfuvirtide (T20) and sifuvirtide (SFT). The underlying mechanisms of SC22EK-induced resistance include the following: (i) significantly reduced binding affinity of the inhibitors, (ii) dramatically enhanced interaction of the viral six-helix bundle, and (iii)severely damaged functionality of the viral Env complex. Our data have provided important information for the structure-function relationship of gp41 and the structure-activity relationship of viral fusion inhibitors.IMPORTANCEEnfuvirtide (T20) is the only HIV-1 fusion inhibitor in clinical use, but the problem of resistance significantly limits its use, calling for new strategies or concepts to develop next-generation drugs. On the basis of the M-T hook structure, short-peptide HIV-1 fusion inhibitors specifically targeting the gp41 pocket site exhibit high binding and antiviral activities. Here, we investigated the molecular pathway of HIV-1 resistance to the short inhibitors by selecting and mapping the escape mutants. The key substitutions for resistance and the underlying mechanisms have been finely characterized. The data provide important information for the structure-function relationship of gp41 and its inhibitors and will definitely help our future development of novel drugs that block gp41-dependent fusion.

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Membrane-Anchored HIV-1 N-Heptad Repeat Peptides Are Highly Potent Cell Fusion Inhibitors via an Altered Mode of Action

PLoS Pathogens ◽

10.1371/journal.ppat.1000509 ◽

2009 ◽

Vol 5 (7) ◽

pp. e1000509 ◽

Cited By ~ 38

Author(s):

Yael Wexler-Cohen ◽

Yechiel Shai

Keyword(s):

Cell Fusion ◽

Mode Of Action ◽

Heptad Repeat ◽

Fusion Inhibitors ◽

Hiv 1

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Trimeric heptad repeat synthetic peptides HR1 and HR2 efficiently inhibit HIV-1 entry

Bioscience Reports ◽

10.1042/bsr20192196 ◽

2019 ◽

Vol 39 (9) ◽

Author(s):

Olfa Mzoughi ◽

Meritxell Teixido ◽

Rémi Planès ◽

Manutea Serrero ◽

Ibtissem Hamimed ◽

...

Keyword(s):

Antiviral Activity ◽

Heptad Repeat ◽

Fusion Activity ◽

Helical Structures ◽

Fusion Inhibitors ◽

Immunodeficiency Virus ◽

Antiviral Activities ◽

Physical Interactions ◽

Kinetics Of ◽

Hiv 1

Abstract The trimeric heptad repeat domains HR1 and HR2 of the human immunodeficiency virus 1 (HIV-1) gp41 play a key role in HIV-1-entry by membrane fusion. To develop efficient inhibitors against this step, the corresponding trimeric-N36 and C34 peptides were designed and synthesized. Analysis by circular dichroism of monomeric and trimeric N36 and C34 peptides showed their capacities to adopt α-helical structures and to establish physical interactions. At the virological level, while trimeric-C34 conserves the same high anti-fusion activity as monomeric-C34, trimerization of N36-peptide induced a significant increase, reaching 500-times higher in anti-fusion activity, against R5-tropic virus-mediated fusion. This result was associated with increased stability of the N36 trimer peptide with respect to the monomeric form, as demonstrated by the comparative kinetics of their antiviral activities during 6-day incubation in a physiological medium. Collectively, our findings demonstrate that while the trimerization of C34 peptide had no beneficial effect on its stability and antiviral activity, the trimerization of N36 peptide strengthened both stability and antiviral activity. This approach, promotes trimers as new promising HIV-1 inhibitors and point to future development aimed toward innovative peptide fusion inhibitors, microbicides or as immunogens.

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Structural and Functional Characterization of the Secondary Mutation N126K Selected by Various HIV-1 Fusion Inhibitors

Viruses ◽

10.3390/v12030326 ◽

2020 ◽

Vol 12 (3) ◽

pp. 326

Author(s):

Danwei Yu ◽

Yang Su ◽

Xiaohui Ding ◽

Yuanmei Zhu ◽

Bo Qin ◽

...

Keyword(s):

Cell Fusion ◽

Functional Characterization ◽

Glycosylation Site ◽

Heptad Repeat ◽

Viral Fusion ◽

Secondary Mutation ◽

Fusion Inhibitors ◽

Hiv 1

Peptides derived from the C-terminal heptad repeat (CHR) region of HIV-1 gp41 is potent viral membrane fusion inhibitors, such as the first clinically approved peptide drug T20 and a group of newly-designed peptides. The resistance profiles of various HIV-1 fusion inhibitors were previously characterized, and the secondary mutation N126K in the gp41 CHR was routinely identified during the in vitro and in vivo selections. In this study, the functional and structural relevance of the N126K mutation has been characterized from multiple angles. First, we show that a single N126K mutation across several HIV-1 isolates conferred mild to moderate cross-resistances. Second, the N126K mutation exerted different effects on Env-mediated HIV-1 entry and cell-cell fusion. Third, the N126K mutation did not interfere with the expression and processing of viral Env glycoproteins, but it disrupted the Asn126-based glycosylation site in gp41. Fourth, the N126K mutation was verified to enhance the thermal stability of 6-HB conformation. Fifth, we determined the crystal structure of a 6-HB bearing the N126K mutation, which revealed the interhelical and intrahelical interactions underlying the increased thermostability. Therefore, our data provide new information to understand the mechanism of HIV-1 gp41-mediated cell fusion and its resistance mode to viral fusion inhibitors.

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Identification of a Critical Motif for the Human Immunodeficiency Virus Type 1 (HIV-1) gp41 Core Structure: Implications for Designing Novel Anti-HIV Fusion Inhibitors

Journal of Virology ◽

10.1128/jvi.00319-08 ◽

2008 ◽

Vol 82 (13) ◽

pp. 6349-6358 ◽

Cited By ~ 63

Author(s):

Yuxian He ◽

Jianwei Cheng ◽

Jingjing Li ◽

Zhi Qi ◽

Hong Lu ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Core Structure ◽

Heptad Repeat ◽

Fusion Inhibitors ◽

Immunodeficiency Virus ◽

Anti Hiv ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry into the host cell involves a cascade of events and currently represents one of most attractive targets in the search for new antiviral drugs. The fusion-active gp41 core structure is a stable six-helix bundle (6-HB) folded by its trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR). Peptides derived from the CHR region of HIV-1 gp41 are potent fusion inhibitors that target the NHR to block viral and cellular membrane fusion in a dominant negative fashion. However, all CHR peptides reported to date are derived primarily from residues 628 to 673 of gp41; little attention has been paid to the upstream sequence of the pocket binding domain (PBD) in the CHR. Here, we have identified a motif (621QIWNNMT627) located at the upstream region of the gp41 CHR, immediately adjacent to the PBD (628WMEWEREI635). Biophysical characterization demonstrated that this motif is critical for the stabilization of the gp41 6-HB core. The peptide CP621-652, containing the 621QIWNNMT627 motif, was able to interact with T21, a counterpart peptide derived from the NHR, to form a typical 6-HB structure with a high thermostability (thermal unfolding transition [T m ] value of 82°C). In contrast, the 6-HB formed by the peptides N36 and C34, which has been considered to be a core structure of the fusion-active gp41, had a T m of 64°C. Different from T-20 (brand name Fuseon), which is the first and only HIV-1 fusion inhibitor approved for clinical use, CP621-652 could efficiently block 6-HB formation in a dose-dependent manner. Significantly, CP621-652 had potent inhibitory activity against HIV-1-mediated cell-cell fusion and infection, especially against T-20- and C34-resistant virus. Therefore, our works provide important information for understanding the core structure of the fusion-active gp41 and for designing novel anti-HIV peptides.

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Design of potent membrane fusion inhibitors against SARS-CoV-2, an emerging coronavirus with high fusogenic activity

10.1101/2020.03.26.009233 ◽

2020 ◽

Cited By ~ 4

Author(s):

Yuanmei Zhu ◽

Danwei Yu ◽

Hongxia Yan ◽

Huihui Chong ◽

Yuxian He

Keyword(s):

Membrane Fusion ◽

Cell Receptor ◽

Heptad Repeat ◽

Fusion Inhibitor ◽

Fusion Activity ◽

Global Public Health ◽

S Protein ◽

Entry Pathway ◽

Fusion Inhibitors ◽

A Cell

AbstractThe coronavirus disease COVID-19, caused by emerging SARS-CoV-2, has posed serious threats to global public health, economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we firstly verified that SARS-CoV-2 uses human ACE2 as a cell receptor and its spike (S) protein mediates high membrane fusion activity. Comparing to that of SARS-CoV, the heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR1) site. Then, we designed a HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly poent activities in inibibiting the SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus infection. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the strcuture and activity relationship (SAR) of IPB02 were characterzized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the presented results have provided important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step.

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Evolution of HIV-1 Resistance to a Cholesterol-Linked D-Peptide Fusion Inhibitor

Biophysical Journal ◽

10.1016/j.bpj.2012.11.539 ◽

2013 ◽

Vol 104 (2) ◽

pp. 90a

Author(s):

Amanda E. Siglin ◽

Nicholas Francis ◽

Michael S. Kay ◽

Micheal J. Root

Keyword(s):

Fusion Inhibitor ◽

Hiv 1 ◽

Peptide Fusion Inhibitor

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