Guanidine alkaloid analogs as inhibitors of HIV-1 Nef interactions with p53, actin, and p56
            <sup>lck</sup>

Olszewski, Allison; Sato, Kyosuke; Aron, Zachary D.; Cohen, Frederick; Harris, A.; McDougall, Brenda R.; Robinson, William E.; Overman, Larry E.; Weiss, Gregory A.

doi:10.1073/pnas.0406040101

Cited by 63 publications

(46 citation statements)

References 45 publications

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“…Nef is a key early gene in HIV-1 replication, and blockade of an essential step in its cellular signalling pathway will disrupt downstream synthesis of late-stage viral proteins, greatly affecting viral replication. A recent study indicated that disruption of interactions of Nef with host-cell proteins might be one means of inhibiting HIV-1 infectivity (Olszewski et al, 2004). Our findings expand this idea by suggesting that targeting Nef myristoylation by NMT2 may be a novel strategy in preventing the spread of infectious virions from host cells.…”

Section: Discussionsupporting

confidence: 72%

N-Myristoyltransferase isozymes exhibit differential specificity for human immunodeficiency virus type 1 Gag and Nef

Seaton

Smith

2008

Journal of General Virology

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Myristoylation of the human immunodeficiency virus type 1 (HIV-1) proteins Gag and Nef by Nmyristoyltransferase (NMT) is a key process in retroviral replication and virulence, yet remains incompletely characterized. Therefore, the roles of the two isozymes, NMT1 and NMT2, in myristoylating Gag and Nef were examined using biochemical and molecular approaches. Fluorescently labelled peptides corresponding to the N terminus of HIV-1 Gag or Nef were myristoylated by recombinant human NMT1 and NMT2. Kinetic analyses indicated that NMT1 and NMT2 had 30-and 130-fold lower K m values for Nef than Gag, respectively. Values for K cat indicated that, once Gag or Nef binds to the enzyme, myristoylation by NMT1 and NMT2 proceeds at comparable rates. Furthermore, the catalytic efficiencies for the processing of Gag by NMT1 and NMT2 were equivalent. In contrast, NMT2 had approximately 5-fold higher catalytic efficiency for the myristoylation of Nef than NMT1. Competition experiments confirmed that the Nef peptide acts as a competitive inhibitor for the myristoylation of Gag. Experiments using full-length recombinant Nef protein also indicated a lower K m for Nef myristoylation by NMT2 than NMT1. Small interfering RNAs were used to selectively deplete NMT1 and/or NMT2 from HEK293T cells expressing a recombinant Nef-sgGFP fusion protein. Depletion of NMT1 had minimal effect on the intracellular distribution of Nef-sgGFP, whereas depletion of NMT2 altered distribution to a diffuse, widespread pattern, mimicking that of a myristoylation-deficient mutant of Nef-sgGFP. Together, these findings indicate that Nef is preferentially myristoylated by NMT2, suggesting that selective inhibition of NMT2 may provide a novel means of blocking HIV virulence. INTRODUCTIONAIDS is a growing problem worldwide, with UNAIDS estimating that 40 million individuals are currently infected by human immunodeficiency virus type 1 (HIV-1). No cure for AIDS exists, and new therapies are needed to combat the growing problem of HIV-1 drug resistance. Two important HIV-1 proteins, Gag and Nef, contain Nterminal myristic acid, which is added to the proteins during viral processing (Guy et al., 1987;Veronese et al., 1988). This protein lipidation occurs via transfer of a myristate moiety from myristoyl-CoA to the N terminus of the proteins through the action of the N-myristoyltransferase isozymes (NMT1 and NMT2). This involves recognition of an 8 aa target sequence at the N terminus of HIV-1 Gag and Nef proteins by NMT activity in the host cell.Gag is a structural protein that binds tightly to the plasma membrane upon myristoylation (Bryant & Ratner, 1990;Zhou et al., 1994). Plasma membrane binding of Gag initiates recruitment of Gag molecules, RNA and various viral proteins, ultimately supporting the budding of newly formed viral particles (Bouamr et al., 2003;Gottlinger et al., 1989). Importantly, it has been demonstrated that nonmyristoylated Gag mutants fail to assemble into active viral particles and are unable to bud from the cell surface (Bryant & Ratner, ...

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Section: Discussionsupporting

confidence: 72%

N-Myristoyltransferase isozymes exhibit differential specificity for human immunodeficiency virus type 1 Gag and Nef

Seaton

Smith

2008

Journal of General Virology

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“…29 So far, only a few Nef inhibitors have been described. Chemical compounds capable of interfering with the ability of Nef to interact with SH3 domains [30][31][32] or activate the Hck tyrosine kinase 33,34 have been identified. Although some inhibitors were too cytotoxic for cellular assays, 30 others were only confirmed in cellular and biochemistry-based assays, but critical functions of Nef such as CD4 down-regulation and infectivity increase were not investigated.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

Bouchet

Basmaciogullari

Chrobák

et al. 2011

Blood

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IntroductionThe advent of monoclonal antibodies was a major breakthrough for the development of antibody-based therapies against various diseases, including virus infection. 1 However, expression in the cytoplasm or nucleus of eukaryotic cells of full-length immunoglobulin G or single-chain antibody variable domain fragments aiming at blocking an intracellular target is poorly efficient because of the reducing nature of these compartments. The engineering of single-chain antibody variable domain fragments (scFvs) that consist in a tandem fusion of the VH and VL domains of a specific antibody partially overcame this limitation. Numerous scFvs have shown their potency for inhibition of the function of their target proteins in cells. In particular, a HIV-1 Tat-specific scFv was shown to neutralize the trans-activating function of this viral protein and to prevent full expression of the viral genome in infected cells. 2 ScFvs specific for HIV-1 matrix, integrase, or regulatory proteins such as Tat, Rev, and Vif are also able to interfere with HIV-1 replication. [3][4][5][6][7][8][9] However, disulfide bonds between VH and VL chains of scFvs are unlikely to form when scFvs are targeted to reducing compartments such as the cytoplasm or the nucleus, resulting in scFvs with lower or no affinity for their antigen. 10 Recently, a major improvement was brought by the use of single-domain antibodies (sdAbs) derived from camelids. In addition to conventional antibodies, camelids express antibodies composed of heavy chains only, with a single variable domain (VHH) capable of recognizing their cognate antigens. 11 These variable domains, called sdAbs, are endowed with many attractive features. 12 The absence of requirement for disulfide bond formation in sdAbs is particularly interesting when targeting of proteins found in reducing cell compartments is considered. 13 In contrast to conventional antibodies, these 13-kDa sdAb fragments can penetrate in cavities located on the surface of antigens (for review, see Nguyen et al 14 ). These cryptic sites are often more conserved than exposed epitopes on viral proteins and are a target of choice for blocking antibodies. Interestingly, sdAb fragments targeting the HIV-1 Rev or Vif proteins have been already characterized and have displayed antiviral activity in cell-culture assays. 15,16 In the present study, we characterized an sdAb from a llama immunized with a recombinant form of Nef, an HIV-1 nonstructural protein found both in the cytoplasm of infected cells and in association with cellular membranes. Considering Nef as a potential target for antiviral therapy arose from the findings that this HIV-1 protein is important for AIDS pathogenesis in vivo (for review, see Foster and Garcia 17 ). Nef is abundantly expressed early after virus infection and perturbs the trafficking of several membrane proteins through action on the endocytic pathway. This leads to the modulation of cell surface expression of some receptors, including CD4 and major histocompatibility complex class I (MHC-I...

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“…In designing synthetic routes for further elaboration of these chemical structures, we were inspired by naturally occurring guanidines (25) such as the batzellidines and derived analogs, inhibitors of HIV gp120-human CD4 binding (26) and HIV-1 Nef-protein interactions (27) and the muramycins, antimicrobial natural products active against Gram-positive bacteria (28,29). A synthetic route was devised converting the dihydropyrimidinone 15 to a collection of guanidines 17 via the thiourea 16 (Fig.…”

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confidence: 99%

Discovery of new antimalarial chemotypes through chemical methodology and library development

Brown

Cheng

Wei

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC 50 's for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.T he synthesis of compound libraries embodying structural diversity and complexity is one potential avenue to enabling the discovery of potent and selective bioactive agents (1-3). Synthetic methodology aimed at library design and distribution is the focus of the Center for Chemical Methodology and Library Development at Boston University (CMLD-BU). Currently the CMLD-BU maintains a collection of approximately 2,500 compounds derived from reaction methodologies developed in the Center and associated groups. The CMLD-BU has developed a workflow that leverages focused methodology development (4) and identification of new scaffolds and chemical reactions utilizing multidimensional reaction screening (5) for the synthesis of libraries.In a collaboration between the CMLD-BU and the NIH Chemical Genomics Center (NCGC), an effort to discover unique chemotypes which inhibit the viability of P. falciparum, the causative agent of malaria, is underway. Malaria not only inflicts tremendous suffering and morbidity on the human population, but has dramatically influenced the course of civilization with profound sociological and economic implications (6). In response, there have been significant screening efforts for new antimalarial agents (7,8). Effective treatments continue to be needed in part due to acquired resistance of P. falciparum. Identification of molecular targets affecting the viability of malaria is necessary for efficient drug development as is the discovery of chemotypes active across a variety of geographic isolates. With an aim to improve the process of identifying new compounds and targets, we have used quantitative high-throughput screening (qHTS) to achieve pharmacological fingerprints of chemical libraries (9). This technique tests each compound as a seven point titration covering four orders of magnitude in concentration thus allowing sufficient resolving power by virtue of IC 50 determinations to reveal subtle differences in compound activities between parasite lines. A compound with a differential chemical phenotype (DCP) can enable mapping of target genes using genetic recombination wherein the DCP emerges between two or more...

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Guanidine alkaloid analogs as inhibitors of HIV-1 Nef interactions with p53, actin, and p56 ^lck

Cited by 63 publications

References 45 publications

N-Myristoyltransferase isozymes exhibit differential specificity for human immunodeficiency virus type 1 Gag and Nef

N-Myristoyltransferase isozymes exhibit differential specificity for human immunodeficiency virus type 1 Gag and Nef

Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

Discovery of new antimalarial chemotypes through chemical methodology and library development

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Guanidine alkaloid analogs as inhibitors of HIV-1 Nef interactions with p53, actin, and p56 lck

Cited by 63 publications

References 45 publications

N-Myristoyltransferase isozymes exhibit differential specificity for human immunodeficiency virus type 1 Gag and Nef

N-Myristoyltransferase isozymes exhibit differential specificity for human immunodeficiency virus type 1 Gag and Nef

Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

Discovery of new antimalarial chemotypes through chemical methodology and library development

Contact Info

Product

Resources

About

Guanidine alkaloid analogs as inhibitors of HIV-1 Nef interactions with p53, actin, and p56 ^lck