scholarly journals A Suite of Modular Fluorescence Assays Interrogate the Human Immunodeficiency Virus Glycoprotein-41 Coiled Coil and Assist in Determining Binding Mechanism of Low Molecular Weight Fusion Inhibitors

2012 ◽  
Vol 10 (5) ◽  
pp. 407-416 ◽  
Author(s):  
Miriam Gochin
Keyword(s):  
Molecular Weight ◽  
Human Immunodeficiency Virus ◽  
Coiled Coil ◽  
Low Molecular Weight ◽  
Binding Mechanism ◽  
Virus Glycoprotein ◽  
Fusion Inhibitors ◽  
Immunodeficiency Virus ◽  
Fluorescence Assays

scholarly journals A Novel Fluorescence Intensity Screening Assay Identifies New Low-Molecular-Weight Inhibitors of the gp41 Coiled-Coil Domain of Human Immunodeficiency Virus Type 1

2007 ◽  
Vol 51 (7) ◽  
pp. 2388-2395 ◽  
Author(s):  
Lifeng Cai ◽  
Miriam Gochin
Keyword(s):  
Molecular Weight ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Coiled Coil ◽  
Heptad Repeat ◽  
Low Molecular Weight ◽  
Hydrophobic Pocket ◽  
Fusion Inhibitors ◽  
Immunodeficiency Virus

ABSTRACT A metallopeptide-based fluorescence assay has been designed for the detection of small-molecule inhibitors of human immunodeficiency virus type 1 gp41, the viral protein involved in membrane fusion. The assay involves two peptides representing the inner N-terminal-heptad-repeat (HR1) coiled coil and the outer C-terminal-heptad-repeat (HR2) helical domains of the gp41 six-helix bundle which forms prior to fusion. The two peptides span a hydrophobic pocket previously defined in the literature. The HR1 peptide is modified with a metal-ligated dye complex, which maintains structural integrity and permits association with a fluorophore-labeled HR2 peptide to be followed by fluorescence quenching. Compounds able to disrupt six-helix bundle formation can act as fusion inhibitors, and we show that they can be detected in the assay from an increase in the fluorescence that is correlated with the potency of the compound. Assay optimization and validation have resulted in a simple quantitative competitive inhibition assay for fusion inhibitors that bind in the hydrophobic pocket. The assay has an assay quality factor (Z′) of 0.88 and can rank order inhibitors at 10 μM concentration with Ki s in the range of 0.2 μM to 30 μM, an ideal range for drug discovery. Screening of a small peptidomimetic library has yielded three new low-molecular-weight gp41 inhibitors. In vitro syncytium inhibition assays confirmed that the compounds inhibited cell-cell fusion in the low micromolar range. These lead compounds provide a new molecular scaffold for the development of fusion inhibitors.

scholarly journals 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

2010 ◽  
Vol 84 (14) ◽  
pp. 7288-7299 ◽  
Author(s):  
Edward J. Murray ◽  
Daniel P. Leaman ◽  
Nishant Pawa ◽  
Hannah Perkins ◽  
Chris Pickford ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Entry Inhibitor ◽  
Low Molecular Weight ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A low-molecular-weight human immunodeficiency virus type 1 (HIV-1) inhibitor, PF-68742 (molecular weight, 573), has been identified in a high-throughput screen for compounds that block HIV-1 envelope glycoprotein (Env)-mediated fusion. The compound is shown to be potent against R5 and X4 isolates in both cell-cell fusion and antiviral assays (50% effective concentrations of ∼0.1 to 1 μM). Postfusion and HIV-1 pseudotyping control experiments confirm that PF-68742 is an entry inhibitor with Env as the specific target for antiviral action. PF-68742 was not able to block binding of monomeric gp120 to soluble CD4 or the binding of gp120:CD4 complexes to cell-associated CCR5, thus distinguishing PF-68742 from described gp120 antagonists and coreceptor binders. Escape variants of HIV-1NL4-3 were selected, and all resistant viruses were found to contain a common G514R (HxB2 numbering) mutation in Env, located proximal to the furin cleavage site in the fusion peptide of gp41. When introduced into wild-type NL4-3 gp41, G514R conferred resistance to PF-68742. Resistance via G514R is shown to be associated with enhancement of virion infectivity by PF-68742 that may result from altered properties of inhibitor-bound Env, rather than from a loss of compound binding. Wild-type viruses and those with substitutions in the disulfide loop (DSL) region of gp41 were also examined for PF-68742 sensitivity. Here, complete resistance to PF-68742 was found to occur through changes outside of position 514, including in the gp41 DSL region. The results highlight PF-68742 as a starting point for novel therapies against HIV-1 and provide new insights into models of Env-mediated fusion.

Low molecular weight proteinuria in human immunodeficiency virus-infected patients

1996 ◽  
Vol 27 (6) ◽  
pp. 803-808 ◽  
Author(s):  
André Kabanda ◽  
Bernard Vandercam ◽  
Alfred Bernard ◽  
Robert Lauwerys ◽  
Charles van Ypersele de Strihou
Keyword(s):  
Molecular Weight ◽  
Human Immunodeficiency Virus ◽  
Low Molecular Weight ◽  
Low Molecular Weight Proteinuria ◽  
Immunodeficiency Virus

scholarly journals Stable Extended Human Immunodeficiency Virus Type 1 gp41 Coiled Coil as an Effective Target in an Assay for High-Affinity Fusion Inhibitors

2009 ◽  
Vol 53 (6) ◽  
pp. 2444-2449 ◽  
Author(s):  
Lifeng Cai ◽  
Edina Balogh ◽  
Miriam Gochin
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Metal Ion ◽  
Coiled Coil ◽  
Hydrophobic Pocket ◽  
High Affinity ◽  
Fusion Inhibitors ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) gp41 coiled-coil domain is an important target for fusion inhibitors, including the peptide T20, which has been approved as a drug against HIV-1. Research into nonpeptide fusion inhibitors has focused primarily on a hydrophobic pocket located within the coiled coil and has so far yielded compounds with relatively weak fusion inhibitory activity. Here, we describe metal ion-assisted stabilization of an extended 39-residue construct of gp41, which includes residues of the hydrophobic pocket and also of an extended groove N terminal to the hydrophobic pocket. We show that the presence of a metal ion and the high-affinity interaction between the receptor construct and cognate C-peptides result in a simple and highly selective assay for fusion inhibitors that may be used to scan large compound libraries. The long construct presents multiple potential binding sites along the extended coiled-coil groove. We demonstrate the modular use of assay probes to detect whether compounds bind in the hydrophobic pocket or elsewhere along the groove. Rapid detection and quantitation of hits can lead to the discovery of compounds binding to different sites along the groove and provide structure-activity relationship data for optimization. Compounds binding to adjacent sites could be linked to form more potent fusion inhibitors.

scholarly journals Inhibition of Human Immunodeficiency Virus Envelope Glycoprotein- Mediated Single Cell Lysis by Low-Molecular-Weight Antagonists of Viral Entry

2006 ◽  
Vol 81 (2) ◽  
pp. 532-538 ◽  
Author(s):  
Navid Madani ◽  
Amy M. Hubicki ◽  
Ana Luisa Perdigoto ◽  
Martin Springer ◽  
Joseph Sodroski
Keyword(s):  
Molecular Weight ◽  
Human Immunodeficiency Virus ◽  
Single Cell ◽  
Single Cells ◽  
Cell Lysis ◽  
Envelope Glycoproteins ◽  
Low Molecular Weight ◽  
Virus Envelope ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The coexpression of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins and receptors leads to the lysis of single cells by a process that is dependent upon membrane fusion. This cell lysis was inhibited by low-molecular-weight compounds that interfere with receptor binding or with receptor-induced conformational transitions in the envelope glycoproteins. A peptide, T20, potently inhibited cell-cell fusion but had no effect on single cell lysis mediated by the HIV-1 envelope glycoproteins. Thus, critical events in the lysis of single cells by the HIV-1 envelope glycoproteins occur in intracellular compartments accessible only to small inhibitory compounds.

scholarly journals Pradimicin A, a Carbohydrate-Binding Nonpeptidic Lead Compound for Treatment of Infections with Viruses with Highly Glycosylated Envelopes, Such as Human Immunodeficiency Virus

2006 ◽  
Vol 81 (1) ◽  
pp. 362-373 ◽  
Author(s):  
Jan Balzarini ◽  
Kristel Van Laethem ◽  
Dirk Daelemans ◽  
Sigrid Hatse ◽  
Antonella Bugatti ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Human Immunodeficiency Virus ◽  
Prolonged Exposure ◽  
Glycosylation Site ◽  
Carbohydrate Binding ◽  
Genetic Barrier ◽  
Drug Pressure ◽  
Immunodeficiency Virus ◽  
Virus Strains ◽  
Hiv 1

ABSTRACT Pradimicin A (PRM-A), an antifungal nonpeptidic benzonaphtacenequinone antibiotic, is a low-molecular-weight (molecular weight, 838) carbohydrate binding agent (CBA) endowed with a selective inhibitory activity against human immunodeficiency virus (HIV). It invariably inhibits representative virus strains of a variety of HIV-1 clades with X4 and R5 tropisms at nontoxic concentrations. Time-of-addition studies revealed that PRM-A acts as a true virus entry inhibitor. PRM-A specifically interacts with HIV-1 gp120 and efficiently prevents virus transmission in cocultures of HUT-78/HIV-1 and Sup T1 cells. Upon prolonged exposure of HIV-1-infected CEM cell cultures, PRM-A drug pressure selects for mutant HIV-1 strains containing N-glycosylation site deletions in gp120 but not gp41. A relatively long exposure time to PRM-A is required before drug-resistant virus strains emerge. PRM-A has a high genetic barrier, since more than five N-glycosylation site deletions in gp120 are required to afford moderate drug resistance. Such mutated virus strains keep full sensitivity to the other known clinically used anti-HIV drugs. PRM-A represents the first prototype compound of a nonpeptidic CBA lead and, together with peptide-based lectins, belongs to a conceptually novel type of potential therapeutics for which drug pressure results in the selection of glycan deletions in the HIV gp120 envelope.

scholarly journals Human Immunodeficiency Virus Type 1 Variants Resistant to First- and Second-Version Fusion Inhibitors and Cytopathic in Ex Vivo Human Lymphoid Tissue

2007 ◽  
Vol 81 (12) ◽  
pp. 6563-6572 ◽  
Author(s):  
Raghavan Chinnadurai ◽  
Devi Rajan ◽  
Jan Münch ◽  
Frank Kirchhoff
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Lymphoid Tissue ◽  
Ex Vivo ◽  
Fusion Inhibitors ◽  
Immunodeficiency Virus ◽  
Human Lymphoid Tissue ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) fusion inhibitors blocking viral entry by binding the gp41 heptad repeat 1 (HR1) region offer great promise for antiretroviral therapy, and the first of these inhibitors, T20 (Fuzeon; enfuvirtide), is successfully used in the clinic. It has been reported previously that changes in the 3-amino-acid GIV motif at positions 36 to 38 of gp41 HR1 mediate resistance to T20 but usually not to second-version fusion inhibitors, such as T1249, which target an overlapping but distinct region in HR1 including a conserved hydrophobic pocket (HP). Based on the common lack of cross-resistance and the difficulty of selecting T1249-resistant HIV-1 variants, it has been suggested that the determinants of resistance to first- and second-version fusion inhibitors may be different. To further assess HIV-1 resistance to fusion inhibitors and to analyze where changes in HR1 are tolerated, we randomized 16 codons in the HR1 region, including those making contact with HR2 codons and/or encoding residues in the GIV motif and the HP. We found that changes only at positions 37I, 38V, and 40Q near the N terminus of HR1 were tolerated. The propagation of randomly gp41-mutated HIV-1 variants in the presence of T1249 allowed the effective selection of highly resistant forms, all containing changes in the IV residues. Overall, the extent of T1249 resistance was inversely correlated to viral fitness and cytopathicity. Notably, one HIV-1 mutant showing ∼10-fold-reduced susceptibility to T1249 inhibition replicated with wild type-like kinetics and caused substantial CD4+-T-cell depletion in ex vivo-infected human lymphoid tissue in the presence and absence of an inhibitor. Taken together, our results show that the GIV motif also plays a key role in resistance to second-version fusion inhibitors and suggest that some resistant HIV-1 variants may be pathogenic in vivo.

scholarly journals N-Substituted Pyrrole Derivatives as Novel Human Immunodeficiency Virus Type 1 Entry Inhibitors That Interfere with the gp41 Six-Helix Bundle Formation and Block Virus Fusion

2004 ◽  
Vol 48 (11) ◽  
pp. 4349-4359 ◽  
Author(s):  
Shibo Jiang ◽  
Hong Lu ◽  
Shuwen Liu ◽  
Qian Zhao ◽  
Yuxian He ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Small Molecule ◽  
Cooh Group ◽  
Hydrophobic Pocket ◽  
Fusion Inhibitors ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A recently approved peptidic human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, T-20 (Fuzeon; Trimeris Inc.), has shown significant promise in clinical application for treating HIV-1-infected individuals who have failed to respond to the currently available antiretroviral drugs. However, T-20 must be injected twice daily and is too expensive. Therefore, it is essential to develop orally available small molecule HIV-1 fusion inhibitors. By screening a chemical library consisting of “drug-like” compounds, we identified two N-substituted pyrroles, designated NB-2 and NB-64, that inhibited HIV-1 replication at a low micromolar range. The absence of the COOH group in NB-2 and NB-64 resulted in a loss of anti-HIV-1 activity, suggesting that this acid group plays an important role in mediating the antiviral activity. NB-2 and NB-64 inhibited HIV-1 fusion and entry by interfering with the gp41 six-helix bundle formation and disrupting the α-helical conformation. They blocked a d-peptide binding to the hydrophobic pocket on surface of the gp41 internal trimeric coiled-coil domain. Computer-aided molecular docking analysis has shown that they fit inside the hydrophobic pocket and that their COOH group interacts with a positively charged residue (K574) around the pocket to form a salt bridge. These results suggest that NB-2 and NB-64 may bind to the gp41 hydrophobic pocket through hydrophobic and ionic interactions and block the formation of the fusion-active gp41 core, thereby inhibiting HIV-1-mediated membrane fusion and virus entry. Therefore, NB-2 and NB-64 can be used as lead compounds toward designing and developing more potent small molecule HIV-1 fusion inhibitors targeting gp41.

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