A fluorescence-based high-throughput screening assay for inhibitors of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H activity

Parniak, Michael A.; Min, Kyoungdoug; Budihas, Scott R.; Grice, Stuart F. J. Le; Beutler, John A.

doi:10.1016/j.ab.2003.06.001

Cited by 126 publications

(166 citation statements)

References 19 publications

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“…Another implication of these studies is that inhibitors of HIV-1 RNase H that are currently being identified as antiretroviral agents (30)(31)(32)(33)(34)(35) will enhance resistance to some NRTIs. It will be important to determine whether the anti-RNase H inhibitors under development will antagonize the activity of some NRTIs so that effective antiretroviral combination therapies can be devised.…”

Section: Discussionmentioning

confidence: 99%

Mechanism for nucleoside analog-mediated abrogation of HIV-1 replication: Balance between RNase H activity and nucleotide excision

Nikolenko

Palmer

Maldarelli

et al. 2005

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

120

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Understanding the mechanisms of HIV-1 drug resistance is critical for developing more effective antiretroviral agents and therapies. Based on our previously described dynamic copy-choice mechanism for retroviral recombination and our observations that nucleoside reverse transcriptase inhibitors (NRTIs) increase the frequency of reverse transcriptase template switching, we propose that an equilibrium exists between (i) NRTI incorporation, NRTI excision, and resumption of DNA synthesis and (ii) degradation of the RNA template by RNase H activity, leading to dissociation of the template-primer and abrogation of HIV-1 replication. As predicted by this model, mutations in the RNase H domain that reduced the rate of RNA degradation conferred high-level resistance to 3 -azido-3 -deoxythymidine and 2,3-didehydro-2,3-dideoxythymidine by as much as 180-and 10-fold, respectively, by increasing the time available for excision of incorporated NRTIs from terminated primers. These results provide insights into the mechanism by which NRTIs inhibit HIV-1 replication and imply that mutations in RNase H could significantly contribute to drug resistance either alone or in combination with NRTI-resistance mutations in reverse transcriptase.drug resistance ͉ recombination ͉ NRTI ͉ reverse transcriptase ͉ dynamic copy choice

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Section: Discussionmentioning

confidence: 99%

Mechanism for nucleoside analog-mediated abrogation of HIV-1 replication: Balance between RNase H activity and nucleotide excision

Nikolenko

Palmer

Maldarelli

et al. 2005

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

120

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“…1). In our initial design, we used a similar Fret pair as that used by parniak and coworkers 17 to measure rnase h activity (i.e., fluorescein donor and dabcyl quencher), except using the more photostable Alexa Fluor 488 (AF488) dye as the donor. to test the potential of this concept, we first synthesized a primer representing a fully polymerized dabcyl-labeled product.…”

Section: Discussionmentioning

confidence: 99%

A High-Throughput Continuous Assay for Screening and Characterization of Inhibitors of HIV Reverse-Transcriptase DNA Polymerase Activity

et al. 2011

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the authors have devised a continuous fluorescence-based assay to measure hIV reverse transcriptase (rt) polymerase activity for both high-throughput screening (htS) and mechanistic characterization of inhibitors. the designed substrate is composed of a recessed DnA primer annealed to a DnA template that is labeled at the 5′-terminus with a donor fluorophore (AlexaFluor 488). rt-catalyzed incorporation of an acceptor-labeled deoxyuridine (dUtp-AlexaFluor 555) at the 3′-terminus of the fully extended DnA primer juxtaposes donor and acceptor fluorophores, resulting in robust fluorescence resonance energy transfer that can be monitored kinetically in real time. the assay is sensitive, permitting the use of low enzyme concentrations (<0.5 nm), and can be miniaturized for use in 384-well htS mode. the authors further show that this assay is capable of evaluating inhibitor mechanism of action by confirming the binding mechanism of a set of nonnucleoside rt inhibitors. Given the versatility and the lack of requirement for costly platforms or radioactivity, this assay may serve to accelerate and streamline the discovery and characterization process for future antiviral agents.

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“…RT RNH activity was measured using a FRET-based microplate fluorescence assay that we have recently described (41). Briefly, 50 µL of a 0.5 µM solution of RNA-DNA hybrid duplex in 50 mM Tris, pH 8.0, containing 60 mM KCl, was added to individual wells of a 96-well microplate.…”

Section: Assay Of Rt Rnh Activitymentioning

confidence: 99%

HIV-1 Reverse Transcriptase Structure with RNase H Inhibitor Dihydroxy Benzoyl Naphthyl Hydrazone Bound at a Novel Site

et al. 2006

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The rapid emergence of drug-resistant variants of human immunodeficiency virus, type 1 (HIV-1), has limited the efficacy of anti-acquired immune deficiency syndrome (AIDS) treatments, and new lead compounds that target novel binding sites are needed. We have determined the 3.15 Å resolution crystal structure of HIV-1 reverse transcriptase (RT) complexed with dihydroxy benzoyl naphthyl hydrazone (DHBNH), an HIV-1 RT RNase H (RNH) inhibitor (RNHI). DHBNH is effective against a variety of drug-resistant HIV-1 RT mutants. While DHBNH has little effect on most aspects of RT-catalyzed DNA synthesis, at relatively high concentrations it does inhibit the initiation of RNAprimed DNA synthesis. Although primarily an RNHI, DHBNH binds >50 Å away from the RNH active site, at a novel site near both the polymerase active site and the non-nucleoside RT inhibitor (NNRTI) binding pocket. When DHBNH binds, both Tyr181 and Tyr188 remain in the conformations seen in unliganded HIV-1 RT. DHBNH interacts with conserved residues (Asp186, Trp229) and has substantial interactions with the backbones of several less well-conserved residues. On the basis of this structure, we designed substituted DHBNH derivatives that interact with the NNRTI-binding pocket. These compounds inhibit both the polymerase and RNH activities of RT.Human immunodeficiency virus, type 1 (HIV-1), reverse transcriptase (RT) is essential for HIV replication. RT converts the single-stranded viral genomic RNA into a linear doublestranded DNA that can be integrated into the host chromosomes (reviewed in ref 1). The enzyme has two activities, (i) a DNA polymerase that can use either RNA or DNA as a template and (ii) an RNase H (RNH) that selectively degrades the RNA strand of an RNA-DNA heteroduplex. The RNH activity of RT is required for virus replication; cellular RNH cannot substitute for the retroviral enzyme (2). The RNH activity degrades the genomic RNA during first-strand ("minus-strand") DNA synthesis, which allows the newly synthesized DNA to be used as a template for second-strand ("plus-strand") DNA synthesis.HIV-1 RT is a heterodimer consisting of 66 kDa (p66) and 51 kDa (p51) subunits. The two polypeptide chains have 440 N-terminal amino acid residues in common. These comprise four polymerase subdomains: the thumb, palm, fingers, and connection (3,4). The C-terminus of p66 contains an additional 120 amino acid residues that form the bulk of the RNH domain. Despite having identical N-terminal sequences, the arrangement of the subdomains in the two subunits differs dramatically. The p66 subunit contains a large cleft formed by the fingers, palm, and thumb subdo-mains that can accommodate double-stranded nucleic acid templateprimers (3-6). Although the p51 subunit contains the same four subdomains, it does not form a nucleic acid binding cleft.Because of its pivotal role in the HIV life cycle, HIV RT is a primary target for antiretroviral agents. All RT inhibitors currently approved for the treatment of acquired immune deficiency syndrome (AIDS) inhibit...

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A fluorescence-based high-throughput screening assay for inhibitors of human immunodeficiency virus-1 reverse transcriptase-associated ribonuclease H activity

Cited by 126 publications

References 19 publications

Mechanism for nucleoside analog-mediated abrogation of HIV-1 replication: Balance between RNase H activity and nucleotide excision

Mechanism for nucleoside analog-mediated abrogation of HIV-1 replication: Balance between RNase H activity and nucleotide excision

A High-Throughput Continuous Assay for Screening and Characterization of Inhibitors of HIV Reverse-Transcriptase DNA Polymerase Activity

HIV-1 Reverse Transcriptase Structure with RNase H Inhibitor Dihydroxy Benzoyl Naphthyl Hydrazone Bound at a Novel Site

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