A Low-Molecular-Weight Entry Inhibitor of both CCR5- and CXCR4-Tropic Strains of Human Immunodeficiency Virus Type 1 Targets a Novel Site on gp41

Murray, Edward J.; Leaman, Daniel P.; Pawa, Nishant; Perkins, Hannah; Pickford, Chris; Perros, Manos; Zwick, Michael B.; Butler, Scott L.

doi:10.1128/jvi.00535-10

Cited by 25 publications

(30 citation statements)

References 62 publications

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“…Indeed, one such inhibitor that binds to the viral envelope protein gp160 in a region proximal to the furin-cleavage site has been implicated as a possible treatment for HIV-1. 59 In this manner, the inhibitor interacts exclusively with the target antigen, preventing furin processing; thus, potential side effects arising from furin inhibition activity would be minimized. This avenue may be worth pursuing with anthrax toxin, as furin-loop accessibility can affect the rate of processing (Fig.…”

Section: Discussionmentioning

confidence: 99%

Domain Flexibility Modulates the Heterogeneous Assembly Mechanism of Anthrax Toxin Protective Antigen

Feld

Kintzer

Tang

et al. 2012

Journal of Molecular Biology

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Individually, the three protein components of anthrax toxin are nontoxic, but when they assemble, they form active holotoxin complexes. The role of the protective antigen (PA) component of the toxin is to deliver the two other enzyme components, lethal factor (LF) and edema factor (EF), across the plasma membrane and into the cytoplasm of target cells. PA is produced as a proprotein, which must be proteolytically activated, and generally cell-surface activation is mediated by a furin-family protease. Activated PA can then assemble into one of two non-interconverting oligomers, a homoheptamer and homooctamer, which have unique properties. Herein we describe molecular determinants that influence the stoichiometry of PA in toxin complexes. By tethering PA Domain 4 to Domain 2 with two different length cross-links, we can control the relative proportions of PA heptamers and octamers. The longer cross-link favors octamer formation, whereas the shorter one favors formation of the heptamer. X-ray crystal structures of PA (up to 1.45 Å resolution), including these cross-linked PA constructs, reveal that a hinge-like movement of Domain 4 correlates with the relative preference for each oligomeric architecture. Furthermore, we report the conformation of the flexible loop containing the furin-cleavage site and show that for efficient processing, the furin site cannot be moved ~5 or 6 residues within the loop. We propose that there are different orientations of Domain 4 relative to the main body of PA that favor the formation of either the heptamer or the octamer.

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Section: Discussionmentioning

confidence: 99%

Domain Flexibility Modulates the Heterogeneous Assembly Mechanism of Anthrax Toxin Protective Antigen

Feld

Kintzer

Tang

et al. 2012

Journal of Molecular Biology

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“…tion, the disulfide loop (DSL) region of gp41 has been shown to have membrane-interacting properties that may contribute to its role during fusion [232]. A novel low molecular weight inhibitor to HIV-1, PF-68742 [233], which targets this disulfide loop could be modified with a lipid tag to determine whether membrane-targeting can enhance its activity. Moreover, certain non-neutralizing antibodies to gp41 can potently inhibit HIV-1 entry if the antibody is first anchored in the host cell membrane [234], or is trapped via ectopic expression of the Fc RI receptor on the host cell [235].…”

Section: Membrane-targeting To Potentiate Gp41 Inhibitors: a Generalimentioning

confidence: 98%

Targeting HIV-1 gp41 in Close Proximity to the Membrane Using Antibody and Other Molecules

Gach¹,

Leaman²,

Zwick³

2011

CTMC

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HIV-1 envelope glycoprotein (Env) spikes are supported at the viral membrane interface by a highly conserved and hydrophobic region of gp41, designated the membrane-proximal external region (MPER). The MPER is mandatory for infection of host cells by HIV-1, and is the target of some of the most broadly neutralizing antibodies described to date. As such, the MPER is also of considerable interest for HIV vaccine design. However, structural models indicate that the MPER assumes distinct conformations prior to and leading up to Env-mediated fusion. Thus, the more of these distinct conformations that antibodies and inhibitors can recognize will likely be the better for antiviral potency. In addition to its flexibility, the MPER is lipophilic and its accessibility to bulky macromolecules is limited by steric and kinetic blocks that present particular challenges for eliciting HIV-1 neutralizing antibodies. Moreover, the ability of the MPER and viral membrane to combine as a complex has critical mechanistic implications for molecules that target lipid-bound and/or unbound states. Interestingly, membrane affinity frequently appears to enhance the potency of both fusion inhibitors and antibodies to different sites on gp41. We therefore highlight mechanisms to be harnessed in targeting membraneproximal sites on HIV gp41 for both vaccine and fusion inhibitor design. Such design efforts will likely need to draw upon knowledge of MPER structure and function, and may in turn inform analogous approaches to MPERs of other enveloped viruses and systems.

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“…In vitro screening has identified competitive inhibitors of assembly of the gp41 HR1-and HR2-derived peptides into the 6HB. [13][14][15][16][17][18][19][20][21][22][23][24] HTS for smallmolecule inhibitors competing with the chemokine (RANTES) binding to CCR5 has led to the identification of identified coreceptor antagonists that effectively blocked fusion of CCR5-tropic viruses: maraviroc, Sch-C, and TAK-779. [10][11][12] These narrowly focused readouts provide a powerful means to identify specific inhibitors of a given step of the virus entry, but exclude all other targets for inhibition of HIV-1 fusion.…”

Section: Introductionmentioning

confidence: 99%

“…In the final 6-helix bundle structure (6HB), three HR1 and three HR2 coalesce forming a highly stable antiparallel helical bundle. A number of small-molecule inhibitors of HIV-1 fusion that interfere with CD4-induced conformational changes in gp120, 8,9 coreceptor binding, [10][11][12] and the gp41 6HB formation [13][14][15][16][17][18][19][20][21][22][23][24] have been identified by high-throughput screening (HTS). Currently, only two HIV-1 fusion inhibitors (enfuvirtide and maraviroc) have been approved for clinical use.…”

Section: Introductionmentioning

confidence: 99%

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

Marin

Giroud

et al. 2015

ASSAY and Drug Development Technologies

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HIV-1 initiates infection by merging its envelope membrane with the target cell membrane, a process that is mediated by the viral Env glycoprotein following its sequential binding to CD4 and coreceptors, CXCR4 or CCR5. Although HIV-1 fusion has been a target for antiviral therapy, the virus has developed resistance to drugs blocking the CCR5 binding or Env refolding steps of this process. This highlights the need for novel inhibitors. Here, we adapted and optimized an enzymatic HIV-cell fusion assay, which reports the transfer of virus-encapsulated b-lactamase into the cytoplasm, to high-throughput screening (HTS) with a 384-well format. The assay was robustly performed in HTS format and was validated by the pilot screen of a small library of pharmacologically active compounds. Several hits identified by screening included a prominent cluster of purinergic receptor antagonists. Functional studies demonstrated that P2X1 receptor antagonists selectively inhibited HIV-1 fusion without affecting the fusion activity of an unrelated virus that enters cells through an endocytic route. The inhibition of HIV-cell fusion by P2X1 antagonists was not through downmodulation of the cell surface expression of CD4 or coreceptors, thus implicating P2X1 receptor in the HIV-1 fusion step. The ability of these antagonists to inhibit viruses regardless of their coreceptor (CXCR4 or CCR5) preference indicates that fusion is blocked at a late step downstream of coreceptor binding. A future large-scale screening campaign for HIV-1 fusion inhibitors, using the above functional readout, will likely reveal novel classes of inhibitors and suggest potential targets for antiviral therapy.

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A Low-Molecular-Weight Entry Inhibitor of both CCR5- and CXCR4-Tropic Strains of Human Immunodeficiency Virus Type 1 Targets a Novel Site on gp41

Cited by 25 publications

References 62 publications

Domain Flexibility Modulates the Heterogeneous Assembly Mechanism of Anthrax Toxin Protective Antigen

Domain Flexibility Modulates the Heterogeneous Assembly Mechanism of Anthrax Toxin Protective Antigen

Targeting HIV-1 gp41 in Close Proximity to the Membrane Using Antibody and Other Molecules

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

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