Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Combination therapy with reverse transcriptase and protease inhibitors greatly reduces morbidity and mortality in HIV-1-infected individuals. However, current anti-retroviral treatment cannot eradicate the virus from infected individuals and is often limited by the emergence of drug-resistant HIV-1 strains and long-term toxicity. These problems emphasize the need to develop new anti-HIV-1 drugs targeting different steps in the viral replication cycle. HIV-1 entry into host cells represents a complex sequence of events involving several viral and cellular proteins that are potential drug targets. In particular, HIV-1 entry requires a sequential interaction of the viral envelope glycoprotein gp120 with CD4 and a co-receptor on the host cell plasma membrane. The CC-chemokine receptor 5 (CCR5) and the CXC-chemokine receptor 4 (CXCR4) are the primary HIV-1 co-receptors in vivo, and are attractive targets for the development of new anti-HIV-1 drugs. CCR5 and CXCR4 belong to the protein superfamily of G protein-coupled receptors (GPCRs). Many orally bioavailable small-molecules interact with specific GPCRs and many existing drugs are orally bioavailable small-molecule agonists or antagonists of GPCRs. Several small-molecule antagonists of CCR5 and CXCR4 that block chemokine binding and HIV-1 entry have been identified in recent years and are now in pre-clinical or clinical development as drug candidates. This review discusses structural and functional aspects of these compounds and summarizes recent insights into how small-molecule antagonists interact with CCR5 and CXCR4, focusing on drug development programs that are well documented in the scientific literature.
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PMID:Small-molecule antagonists of CCR5 and CXCR4: a promising new class of anti-HIV-1 drugs. 1527 44

The plant lectins from Hippeastrum hybrid (HHA) and Galanthus nivalis (GNA) are 50,000-D tetramers showing specificity for alpha-(1,3) and/or alpha-(1,6)-mannose oligomers. They inhibit HIV-1 infection at a 50% effective concentration of 0.2 to 0.3 microg/ml. Escalating HHA or GNA concentrations (up to 500 microg/ml) led to the isolation of three HIV-1(III(B)) strains in CEM T cell cultures that were highly resistant to HHA and GNA, several other related mannose-specific plant lectins, and the monoclonal antibody 2G12, modestly resistant to the mannose-specific cyanovirin, which is derived from a blue-green alga, but fully susceptible to other HIV entry inhibitors as well as HIV reverse transcriptase inhibitors. These mutant virus strains were devoid of up to seven or eight of 22 glycosylation sites in the viral envelope glycoprotein gp120 because of mutations at the Asn or Thr/Ser sites of the N-glycosylation motifs. In one of the strains, a novel glycosylation site was created near a deleted glycosylation site. The affected glycosylation sites were predominantly clustered in regions of gp120 that are not involved in the direct interaction with either CD4, CCR5, CXCR4, or gp41. The mutant viruses containing the deleted glycosylation sites were markedly more infectious in CEM T-cell cultures than wild-type virus.
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PMID:Marked depletion of glycosylation sites in HIV-1 gp120 under selection pressure by the mannose-specific plant lectins of Hippeastrum hybrid and Galanthus nivalis. 1571 24

Introduction of amino acids substitutions in murine leukemia virus genome is a powerful method to determine the relative importance of various viral factors in pathogenesis. However, introduction of such amino acids substitution could result in viruses at a selective disadvantage, and eventual selection of revertants. It is thus essential to verify if the mutation is maintained stably in replicating virus and in infected tumor cells. In the present study, viral nucleic acid sequences from diseased animals were determined using different approaches. Small blood samples were found adequate for direct RNA extraction and reverse transcriptase-PCR amplification followed by automated DNA sequencing. Alternatively, replication-competent viruses were recovered specifically by applying blood samples onto permissive cells; viral RNA is then extracted from tissue culture medium and similarly sequenced. Tissue samples were also used to amplify viral sequences from tumors DNA while small pieces of tumors tissues were applied onto permissive cells to isolate replicating viruses. The combined experimental approach was used to show sequence conservation using a mutant altered in the intracytoplasmic region of viral envelope glycoprotein. No difference was observed between viruses recovered directly from the animal and those amplified onto cultured cells.
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PMID:Sequence analysis of murine leukemia virus envelope gene from inoculated mice. 1579 90

We investigated roles for chemoattractants in dissemination of HIV-1 by examining the induction of T cell-active chemokines in HIV-1-infected human monocyte-derived macrophages and dendritic cells. Of the 12 chemokines analyzed, mRNAs for two, CXCL10 and CXCL11, ligands for the chemokine receptor CXCR3, were up-regulated in both cell types upon infection by HIV-1. Induction of these chemokine genes in infected cultures was dependent on both viral entry and reverse transcriptase activity, but not on the HIV-1 envelope glycoprotein. Conditioned medium from infected cells was chemotactic for freshly isolated human CD4+ T cells, and chemotaxis was abolished by pretreatment with an Ab against CXCR3. A lymph node from an HIV-1-infected individual expressed CXCL10 and CXCL11 mRNAs in the paracortex, including venules, as detected by in situ hybridization, whereas neither mRNA was detected after highly active antiretroviral therapy. Because CCR5 on CD4+ T cells is found predominantly on cells that also express CXCR3, these data implicate CXCL10 and CXCL11 in the recruitment of susceptible T cells to HIV-1-infected lymph nodes, macrophages, and dendritic cells. This recruitment might enhance the sequestration of T cells in infected lymphoid organs and the spread of infection between cells, contributing to the immunopathology of AIDS.
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PMID:Roles for CXC chemokine ligands 10 and 11 in recruiting CD4+ T cells to HIV-1-infected monocyte-derived macrophages, dendritic cells, and lymph nodes. 1581 16

HIV-1 strains have diversified extensively through mutation and recombination since their initial transmission to human beings many decades ago in central Africa. The high error rate of HIV reverse transcriptase combined with the estimated in vivo HIV-1 replication rate of ten billion new virions each day leads to extraordinary genetic diversity of HIV. Twenty seven circulating genetic forms of the HIV-1 group M are presently recognized, including 11 subtypes and sub-subtypes, and 16 circulating recombinant forms (CRF). Genotypic analyses have provided a better understanding of the molecular diversity of HIV-1, enabling the detection of emerging HIV-1 variants and improving the tracking of the epidemic worldwide. The rapid evolution of HIV within infected hosts contributes significantly to the elusiveness of this pathogen from host antiviral responses. The complex nature of HIV envelope glycoprotein that is inherently resistant to neutralization, the selective infection, progressive destruction and impaired regeneration of CD4+ T helper cells, generation of cytotoxic T lymphocyte (CTL) escape mutants, together with high genetic diversity with continually evolving HIV variants worldwide, makes design of an effective vaccine a formidable task. Given the rapidity and unpredictability with which HIV-1 genetic forms may propagate in future, a vaccine protective against all major HIV-1 circulating genetic forms is desirable, which could require multivalent formulations. Understanding the kinetics and directions of this continuing adaptation and its impact on viral fitness, immunogenicity and pathogenicity are crucial to the successful design of effective HIV vaccines. In this review, we focus on extensive diversity of HIV-1, emergence of recombinant forms and their impact on diagnosis, antiretroviral therapy, disease progression, transmission, and vaccine development.
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PMID:Impact of genetic diversity of HIV-1 on diagnosis, antiretroviral therapy & vaccine development. 1581 45

The association of a drug with its target protein has the effect of blocking the protein activity and is termed a promiscuous function to distinguish from the protein's native function (Tawfik and associates, Nat. Genet. 37, 73-6, 2005). Obviously, a protein has not evolved naturally for drug association or drug resistance. Promiscuous protein functions exhibit unique traits of evolutionary adaptability, or evolvability, which is dependent on the induction of novel phenotypic traits by a small number of mutations. These mutations might have small effects on native functions, but large effects on promiscuous function; for example, an evolving protein could become increasingly drug resistant while maintaining its original function. Ariel Fernandez, in his opinion piece, notes that drug-binding "promiscuity" can hardly be dissociated from native functions; a dominant approach to drug discovery is the protein-native-substrate transition-state mimetic strategy. Thus, man-made ligands (e.g. drugs) have been successfully crafted to restrain enzymatic activity by focusing on the very same structural features that determine the native function. Using the successful inhibition of HIV-1 protease as an example, Fernandez illustrates how drug designers have employed naturally evolved features of the protein to suppress its activity. Based on these arguments, he dismisses the notion that drug binding is quintessentially promiscuous, even though in principle, proteins did not evolve to associate with man made ligands. In short, Fernandez argues that there may not be separate protein domains that one could term promiscuous domains. While acknowledging that drugs may bind promiscuously or in a native-like manner a la Fernandez, Tawfik maintains the role of evolutionary adaptation, even when a drug binds native-like. In the case of HIV-1 protease, drugs bind natively, and the initial onset of mutations results in drug resistance in addition to a dramatic decline in enzymatic activity and fitness of the virus. A chain of compensatory mutations follows this, and then the virus becomes fully fit and drug resistant. Ben Berkhout and Rogier Sanders subscribe to the evolution of new protein functions through gene duplication. With two identical protein domains, one domain can be released from a constraint imposed by the original function and it is thus free to move in sequence space toward a new function without loss of the original function. They emphasize that the forced evolution of drug-resistance differs significantly from the spontaneous evolution of an additional protein function. For instance, the latter process could proceed gradually on an evolutionary time scale, whereas the acquisition of drug-resistance is an all or nothing process for a virus, leading to the failure or success of therapy. They find no evidence to the thesis that resistance-mutations appear more rapidly in promiscuous domains than native domains. Berkhout and Sanders illustrate the genetic plasticity of HIV-1 by citing examples in which well-conserved amino acid residues of catalytic domains are forced to mutate under drug-pressure. HIV drug resistance biology is very complex. Instead of a viral protein, a drug can be targeted at a cellular protein. For example, Berkhout and Sanders claim, a drug targeted at the cellular protein CCR5 inhibits the binding of the viral envelope glycoprotein (Env) to CCR5. However, Env mutates so that it binds to the CCR5-drug complex and develops drug resistance. Interestingly, CCR5 has not evolved to bind to Env, but to a series of chemokines. Andrzej Kloczkowski, Taner Sen, and Bob Jernigan point out the importance of protein motions for binding. They believe it is likely that different ligands can bind to the diverse protein conformations sampled in the course of normal protein conformational fluctuations. They have been applying simple elastic network models to extract the motions as normal modes, which yield relatively small numbers of conformations that are useful for developing protein mechanisms; while these are typically small motions, for some proteins they can be quite large in scale. One of the major advantages of the approach is that only relatively small numbers of modes are important contributors to the overall motion -- so the approach provides a way to systematically map out a protein's motions. These models successfully represent the conformational fluctuations manifested in the crystallographic B-factors, and often suggest motions related to protein functional behaviors, such as those observed for reverse transcriptase, where two dominant hinges clearly relate to the processing steps -- one showing anti-correlation between the polymerase and ribonuclease H sites related to the translation and positioning of the nucleic acid chain, and another for opening and closing the polymerase site. Disordered proteins represent a more extreme case where the set of accessible conformations is much larger; thus they could offer up a broader range of possible binding forms. Whether evolution controls the functional motions for proteins remains little studied. Intriguingly, buried in the existing databases of protein-protein interactions may be information that can shed light on the extent of promiscuous binding among proteins themselves. Within these data there are cases where large numbers of diverse proteins have been shown to interact with a single protein; some of these could represent promiscuous protein-protein binding. Uncovering these promiscuous behaviors could be important for comprehending the details of how proteins can bind promiscuously to one another, and can exhibit even greater promiscuity in their binding to small molecules. The evolutionary routes, the dynamics of the target protein, and the many other aspects that need to be addressed while designing a drug that may dodge drug resistance, indicate the complexity and multi-disciplinary nature of the issue of drug resistance.
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PMID:Protein promiscuity: drug resistance and native functions--HIV-1 case. 1584 67

We have identified two N-phenyl-N'-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamide analogs as a novel class of human immunodeficiency virus type 1 (HIV-1) entry inhibitors that block the gp120-CD4 interaction, using database screening techniques. The lead compounds, NBD-556 and NBD-557, are small molecule organic compounds with drug-like properties. These compounds showed potent cell fusion and virus-cell fusion inhibitory activity at low micromolar levels. A systematic study showed that these compounds target viral entry by inhibiting the binding of HIV-1 envelope glycoprotein gp120 to the cellular receptor CD4 but did not inhibit reverse transcriptase, integrase, or protease, indicating that they do not target the later stages of the HIV-1 life cycle to inhibit HIV-1 infection. These compounds were equally potent inhibitors of both X4 and R5 viruses tested in CXCR4 and CCR5 expressing cell lines, respectively, indicating that their anti-HIV-1 activity is not dependent on the coreceptor tropism of the virus. A surface plasmon resonance study, which measures binding affinity, clearly demonstrated that these compounds bind to unliganded HIV-1 gp120 but not to the cellular receptor CD4. NBD-556 and NBD-557 were active against HIV-1 laboratory-adapted strains including an AZT-resistant strain and HIV-1 primary isolates, indicating that these compounds can potentially be further modified to become potent HIV-1 entry inhibitors.
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PMID:Identification of N-phenyl-N'-(2,2,6,6-tetramethyl-piperidin-4-yl)-oxalamides as a new class of HIV-1 entry inhibitors that prevent gp120 binding to CD4. 1599 3

A central question in the pathogenesis of HIV-associated thrombotic microangiopathic (HIV-TMA) lesions is whether the HIV-1 envelope glycoprotein (HIV-1 Env) can interact directly with human glomerular endothelial cells (HGECs) through specific HIV-1 co-receptors. The goal of this study was to determine whether cultured primary HGECs express significant levels of the major HIV-1 co-receptors CD4, CXCR4, and/or CCR5 to allow fusion interactions with HIV-1. The expression of CD4, CXCR-4 and CCR-5 was assessed in cultured HGECs by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry using specific antibodies. The HIV-1 Env-mediated membrane fusion of target glomerular cells was evaluated by a fluorescent dye transfer-based cell-cell fusion microscopic method. HGECs express CXCR4 mRNA and protein as determined by RT-PCR and immunostaining with phycoerythrin-conjugated anti-CXCR4 Mab 12G5. CD4 and CCR5 were not detected in HGECs, either by RT-PCR or by surface immunostaining with specific antibodies. Incubation of HGECs with cells expressing a CD4-independent envelope strain (HIV-1IIIB-8x) and the CD4-dependent envelope strain (HIV-1IIIB) resulted in transfer of fluorescent dyes of approximately 20% after 8-16 h incubation at 37 degrees C. Incubation in the presence of inhibitors (C34, which blocks six-helix bundle formation, and AMD3100, which interacts with CXCR4) reduced dye transfer by 60%-80%, confirming that the dye transfer was specific with respect to gp120-gp41-mediated fusion. Cultured primary HGECs express CXCR4 but not CD4 or CCR5. The ability of HGECs to promote fusion by a CD4-independent HIV-1 envelope glycoprotein suggests that these cells may become a potential direct target of certain HIV-1 isolates.
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PMID:Fusion of HIV-1 envelope-expressing cells to human glomerular endothelial cells through an CXCR4-mediated mechanism. 1604 21

Kaposi's sarcoma-associated herpesvirus (KSHV) in vitro target cell infection is characterized by the expression of the latency-associated genes ORF 73 (LANA-1), ORF 72, and K13 and by the transient expression of a very limited number of lytic genes such as lytic cycle switch gene ORF 50 (RTA) and the immediate early (IE) lytic K5, K8, and v-IRF2 genes. During the early stages of infection, several overlapping multistep complex events precede the initiation of viral gene expression. KSHV envelope glycoprotein gB induces the FAK-Src-PI3K-RhoGTPase (where FAK is focal adhesion kinase) signaling pathway. As early as 5 min postinfection (p.i.), KSHV induced the extracellular signal-regulated kinase 1 and 2 (ERK1/2) via the PI3K-PKCzeta-MEK pathway. In addition, KSHV modulated the transcription of several host genes of primary human dermal microvascular endothelial cells (HMVEC-d) and fibroblast (HFF) cells by 2 h and 4 h p.i. Neutralization of virus entry and infection by PI-3K and other cellular tyrosine kinase inhibitors suggested a critical role for signaling molecules in KSHV infection of target cells. Here we investigated the induction of ERK1/2 by KSHV and KSHV envelope glycoproteins gB and gpK8.1A and the role of induced ERK in viral and host gene expression. Early during infection, significant ERK1/2 induction was observed even with low multiplicity of infection of live and UV-inactivated KSHV in serum-starved cells as well as in the presence of serum. Entry of UV-inactivated virus and the absence of viral gene expression suggested that ERK1/2 induction is mediated by the initial signal cascade induced by KSHV binding and entry. Purified soluble gpK8.1A induced the MEK1/2 dependent ERK1/2 but not ERK5 and p38 mitogen-activated protein kinase (MAPK) in HMVEC-d and HFF. Moderate ERK induction with soluble gB was seen only in HMVEC-d. Preincubation of gpK8.1A with heparin or anti-gpK8.1A antibodies inhibited the ERK induction. U0126, a selective inhibitor for MEK/ERK blocked the gpK8.1A- and KSHV-induced ERK activation. ERK1/2 inhibition did not block viral DNA internalization and had no significant effect on nuclear delivery of KSHV DNA during de novo infection. Analyses of viral gene expression by quantitative real-time reverse transcriptase PCR revealed that pretreatment of cells with U0126 for 1 h and during the 2-h infection with KSHV significantly inhibited the expression of ORF 73, ORF 50 (RTA), and the IE-K8 and v-IRF2 genes. However, the expression of lytic IE-K5 gene was not affected significantly. Expression of ORF 73 in BCBL-1 cells was also significantly inhibited by preincubation with U0126. Inhibition of ERK1/2 also inhibited the transcription of some of the vital host genes such as DUSP5 (dual specificity phosphatase 5), ICAM-1 (intercellular adhesion molecule 1), heparin binding epidermal growth factor, and vascular endothelial growth factor that were up-regulated early during KSHV infection. Several MAPK-regulated host transcription factors such as c-Jun, STAT1alpha, MEF2, c-Myc, ATF-2 and c-Fos were induced early during infection, and ERK inhibition significantly blocked the c-Fos, c-Jun, c-Myc, and STAT1alpha activation in the infected cells. AP1 transcription factors binding to the RTA promoter in electrophoretic mobility shift assays were readily detected in the infected cell nuclear extracts which were significantly reduced by ERK inhibition. Together, these results suggest that very early during de novo infection, KSHV induces the ERK1/2 to modulate the initiation of viral gene expression and host cell genes, which further supports our hypothesis that beside the conduit for viral DNA delivery into the cytoplasm, KSHV interactions with host cell receptor(s) create an appropriate intracellular environment facilitating infection.
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PMID:ERK1/2 and MEK1/2 induced by Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) early during infection of target cells are essential for expression of viral genes and for establishment of infection. 1605 24

Although combinations of drugs that target the HIV reverse transcriptase and protease enzymes have clearly revolutionized the treatment of HIV/AIDS, problems with these agents, such as viral escape mutants, persistence of viral reservoirs, poor patient compliance due to complicated regimens, and toxic side effects, have emphasized the need for development of new drugs with novel mechanisms of action, as well as an HIV vaccine. Recently two new classes of drugs have been identified that interfere with the membrane fusion reaction required for HIV entry of target cells. Two such agents, T-20 (enfuvirtide) and T-1249, which have been approved by the Food and Drug Administration (FDA), block the action of the fusogenic envelope glycoprotein gp41. Others target the HIV coreceptors CCR5 and CXCR4, and are now in clinical trials. Also under development are novel agents that target the HIV integrase and HIV regulatory gene products as well as immunomodulators such as IL-12 and IL-2. This article will focus on these and other novel approaches to HIV therapeutics.
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PMID:Anti-HIV therapy: Current and future directions. 1678 46


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