High-throughput screening using pseudotyped lentiviral particles: A strategy for the identification of HIV-1 inhibitors in a cell-based assay (original) (raw)
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Antimicrobial Agents and Chemotherapy, 2003
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Antiviral Research, 2011
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Journal of Biomolecular Screening, 2006
There has been increasing interest in the identification of novel HIV entry inhibitors. For the discovery of these entry inhibitors, robust surrogate anti-HIV assays are highly desired. The authors report a novel anti-HIV assay system using Moloney murine leukemia viruses (MMLVs) pseudotyped with cytoplasmic tail-truncated HIV envelope protein gp140. These pseudotyped MMLV-HIVgp140 viral particles carry luciferase transcripts; therefore, robust luciferase signal can be detected in cells infected by these pseudotypes. Polycationic agent polybrene and spinoculation markedly enhanced the infection efficiency of these pseudotypes. It was demonstrated that the tropism of these pseudotypes is dependent on the pseudotyped HIV envelope proteins. MMLV viruses pseudotyped with gp140 from an R5 HIV virus specifically infect CCR5-expressing cells, and viruses pseudotyped with gp140 from an X4 HIV virus specifically infect CXCR4-expressing cells. Furthermore, CCR5 antagonists inhibited only MMLV-gp140(R5) infections, and CXCR4 antagonists inhibited only MMLV-gp140(X4) infections. A variety of known HIV entry inhibitors were tested in both R5-and X4-dependent pseudotype antiviral assays, and the IC 50 values generated were consistent with published results. The pseudotype antiviral assay was also used in the characterization of hundreds of novel CCR5 antagonists. The IC 50 values determined in this assay were compared with those determined in HIV antiviral and cell-cell fusion (CCF) assays, and good correlation was found between pseudotype antiviral assay and HIV antiviral assay (R 2 = 0.9) or CCF assay (R 2 = 0.8). (Journal of Biomolecular Screening 2006:652-663)
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Journal of Laboratory Automation, 2013
Current antiretroviral treatments target multiple pathways important for human immunodeficiency virus (HIV) multiplication, including viral entry, synthesis and integration of the DNA provirus, and the processing of viral polyprotein precursors. However, HIV is becoming increasingly resistant to these "combination therapies." Recent findings show that inhibition of HIV Gag protein cleavage into its two structural proteins, matrix (MA) and capsid (CA), has a devastating effect on viral production, revealing a potential new target class for HIV treatment. Unlike the widely used HIV protease inhibitors, this new class of inhibitor would target the substrate, not the protease enzyme itself. This approach offers a distinct advantage in that inhibitors of MA/CA would only need to affect a subset of the Gag molecules to disable viral replication. To discover MA/CA-specific inhibitors, we constructed a modified MA/CA fusion peptide (MA/CAΔ) that contains the HIV protease (PR) cleavage site as well as a tetracysteine motif for fluorescent labeling. The HIV PR cleavage of MA/CAΔ can then be monitored via fluorescence polarization (FP). We have adapted this FP assay for high-throughput screening and validated it according to industry standards using a 384-well plate format. We have currently tested 24,000 compounds in this assay and here detail the screening methodology and the results of this screening campaign.
AIDS Research and Therapy, 2013
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Anti-Hiv Agents: A Step Towards Future
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Novel targets for the management of HIV infection have become increasingly relevant in view of extensive drug resistance, side effects and high pill burden of some of the conventional anti-retroviral agents. These agents include chemokine receptor antagonists and the integrase inhibitors which were recently approved for HIV treatment, as well as numerous other agents directed to previously untested targets such as the maturation inhibitors, pharmacological CDK inhibitors, TateTAR interaction inhibitors, anti-CD4 monoclonal antibody, antisense oligonucleotides, Use of new agents with novel mechanism of action requires the development of new laboratory assays to detect viral tropism and new resistance mutations. This review discusses issues surrounding the development of these new agents as well as various traditional approaches for the treatment of AIDS.
A novel system for screening antiretroviral agents
Journal of Virological Methods, 1989
We propose a new screening system of drugs capable of inactivating retroviruses by interfering with the binding, entry into the cell, uncoating, reverse transcription, migration into the nucleus or integration of the retrovirus. It is based on the utilization of recombinant retroviruses which can be detected in single cells by the expression of a LacZ reporter gene. It allows simple and rapid quantification of the number of infectious viral particles. The screening system can then be used to precisely define the period sensitive to the drug. Antiretroviral agent; 3'-Azido-3'-deoxythymidine (AZT); LacZ gene; Retrovirus AntiretroviraI therapeutic strategy requires a simple and rapid test to identify agents capable of inactivating the viral particle or interfering with the replicative cycle or production (Mitsuya et al., 1987). We here propose a new screening system of such drugs based on the utilization of murine LacZ (Sanes et al., 1986; Bonnerot et al., 1987) recombinant retroviruses (Weiss et al., 1985; Nicolas et al., 1987) (RRV)-which allows enumeration of the virus. RRV share with replication competent retroviruses (including human immunodefi~iency viruses, HIV) (BarrC-Sinoussi et al., 1983; Popovic et al., 1984) a number of steps (Weiss et al., 1985)-binding, entry into the cell, uncoating, reverse transcription, migration into the nucleus and integration-each of these steps being a potential target for drugs. The proposed test is quantitative, can be performed in 48 h, gives the lethal dose for the virus as well as the toxicity on cells (in vitro therapeutic index). Furthermore, the system can be adapted to identify the step at which the agent is active.
EASY-HIT: HIV Full-Replication Technology for Broad Discovery of Multiple Classes of HIV Inhibitors
2010
yielded high Z scores (>0.9) and signal stabilities, confirming its robustness. Screening of the LOPAC 1280 library identified 10 compounds (0.8%), of which eight were known to inhibit HIV, validating the suitability of this assay for screening applications. Studies evaluating anti-HIV activities of natural products with the EASY-HIT technology led to the identification of three novel inhibitory compounds that apparently act at different steps of HIV-1 replication. Furthermore, we demonstrate successful evaluation of plant extracts for HIV-inhibitory activities, suggesting application of this technology for the surveillance of biological extracts with anti-HIV activities. We conclude that the EASY-HIT technology is a versatile tool for the discovery and characterization of HIV inhibitors.