Transient kinetic analyses of the ribonuclease H cleavage activity of HIV-1 reverse transcriptase in complex with efavirenz and/or a β-thujaplicinol analogue

Herman, Brian D.; Sluis‐Cremer, Nicolas

doi:10.1042/bj20130850

Cited by 11 publications

(11 citation statements)

References 17 publications

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“…However, the recent approval of boceprevir and telaprevir for the hepatitis C virus NS3-4A serine protease demonstrates that effective small molecules can be designed for shallow binding sites (34 -36). We and others have demonstrated that competition with the nucleic acid substrate is a related potential obstacle in this context (10,23,31). Order-of-addition experiments revealed that ␤-thujaplicinol does not bind to a pre-formed complex composed of HIV-1 RT and substrate (10).…”

Section: Discussionmentioning

confidence: 99%

“…The structure of RT in complex with ␤-thujaplicinol, along with a modeled RNA/DNA hybrid indeed point to a steric conflict between the inhibitor and the intact RNA/DNA substrate (22). Notably, ␤-thujaplicinol and derivatives appear to be able to bind to the nicked product of the primary cleavage reaction as subsequent secondary, polymerase-independent cuts are effectively inhibited (31).…”

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confidence: 99%

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Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

Beilhartz

Ngure

Johns³

et al. 2014

Journal of Biological Chemistry

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Background:The RNase H activity of HIV-1 reverse transcriptase (RT) is an under-explored target. Results: GSK5750 is a novel RNase H active site inhibitor that displays slow dissociation kinetics. Conclusion: Tight binding may compensate for the inability of active site inhibitors to access the RT-substrate complex. Significance: The GSK5750 scaffold may lead to the development of clinically relevant RNase H inhibitors.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

Beilhartz

Ngure

Johns³

et al. 2014

Journal of Biological Chemistry

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“…This same T/P substrate was used in the gel shift assays, however in these experiments neither the template nor primer was covalently labeled with a fluorescence dye. For the DNA polymerase-independent RNase H assays, we used an 18 nt template (5’-GAUCUGAG CCUGGGAGCU-3’) annealed to an 18 nt primer (5’-AGCTCC CAGGCTCAGATC-3’), as described previously [17]. For DNA polymerase-dependent RNase H cleavage assays we used a 26 nt DNA primer (5’-CCTGTTCGGGCGCCACT GCTAGAGAT-3’) annealed to a 35 nt RNA template (5’-GAAUGGAAAAUCUCUAGCA GUGGCGCCCGAACAG-3’) [17].…”

Section: Methodsmentioning

confidence: 99%

“…For the DNA polymerase-independent RNase H assays, we used an 18 nt template (5’-GAUCUGAG CCUGGGAGCU-3’) annealed to an 18 nt primer (5’-AGCTCC CAGGCTCAGATC-3’), as described previously [17]. For DNA polymerase-dependent RNase H cleavage assays we used a 26 nt DNA primer (5’-CCTGTTCGGGCGCCACT GCTAGAGAT-3’) annealed to a 35 nt RNA template (5’-GAAUGGAAAAUCUCUAGCA GUGGCGCCCGAACAG-3’) [17]. Both RNase H assays were FRET based and fluorescein and IowaBlack FQ TM were covalently attached to the 3’- and 5’-termini of the RNA and DNA oligonucleotides, respectively.…”

Section: Methodsmentioning

confidence: 99%

Novel high-throughput screen identifies an HIV-1 reverse transcriptase inhibitor with a unique mechanism of action

Sheen

Alptürk

Sluis‐Cremer

2014

Biochemical Journal

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HIV-1 resistance to zidovudine (AZT) is associated with selection of M41L, D67N, K70R, L210W, T215F/Y and K219Q/E in reverse transcriptase (RT). These mutations decrease HIV-1 susceptibility to AZT by augmenting RT’s ability to excise the chain-terminating AZT-monophosphate (AZT-MP) moiety from the chain-terminated DNA primer. Although AZT-MP excision occurs at the enzyme’s polymerase activ e site, it is mechanistically distinct from the DNA polymerase reaction. Consequently, this activity represents a novel target for drug discovery, and inhibitors that target this activity may increase the efficacy of nucleosid(t)e analogs, and may help to delay the onset of drug resistance. Here, we developed a Förster resonance energy transfer based high throughput screening assay for the AZT-MP excision activity of RT. This assay is sensitive and robust, and demonstrates a signal to noise ratio of 3.3 and a Z’ factor of 0.69. We screened 3 chemical libraries (7265 compounds) using this assay, and identified 3,3'-[(3-carboxy-4-oxo-2,5-cyclohexadien-1-lidene)methylene]bis[6-hydroxy-benzoic acid] (APEX57219) as the most promising hit. APEX57219 displays a unique activity profile against wild-type and drug-resistant HIV-1 RT, and was found to inhibit virus replication at the level of reverse transcription. Mechanistic analyses revealed that APEX57219 blocked the interaction between RT and the nucleic acid substrate.

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“…In our functional form derivation of the basic probability of chain termination, we presupposed that RTIs do not significantly impact the interactions between RT and the T/P. Recent evidence has nevertheless suggested that NNRTIs trap HIV-1 RT in a polymerase-independent RNase H competent mode, preventing it from binding to the T/P in a polymerase-dependent mode (33,55). This may partially inhibit or accelerate RT polymerase-dependent or -independent RNase H activity, respectively (56)(57)(58).…”

mentioning

confidence: 99%

A Cell-Based Strategy To Assess Intrinsic Inhibition Efficiencies of HIV-1 Reverse Transcriptase Inhibitors

Abram¹,

Tsiang²,

White³

et al. 2015

Antimicrob Agents Chemother

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During HIV-1 reverse transcription, there are increasing opportunities for nucleos(t)ide (NRTI) or nonnucleoside (NNRTI) reverse transcriptase (RT) inhibitors to stop elongation of the nascent viral DNA (vDNA). In addition, RT inhibitors appear to influence the kinetics of vDNA synthesis differently. While cell-free kinetic inhibition constants have provided detailed mechanistic insight, these assays are dependent on experimental conditions that may not mimic the cellular milieu. Here we describe a novel cell-based strategy to provide a measure of the intrinsic inhibition efficiencies of clinically relevant RT inhibitors on a perstop-site basis. To better compare inhibition efficiencies among HIV-1 RT inhibitors that can stop reverse transcription at any number of different stop sites, their basic probability, p, of getting stopped at any potential stop site was determined. A relationship between qPCR-derived 50% effective inhibitory concentrations (EC 50 s) and this basic probability enabled determination of p by successive approximation. On a per-stop-site basis, tenofovir (TFV) exhibited 1.4-fold-greater inhibition efficiency than emtricitabine (FTC), and as a class, both NRTIs exhibited an 8-to 11-fold greater efficiency than efavirenz (EFV). However, as more potential stops sites were considered, the probability of reverse transcription failing to reach the end of the template approached equivalence between both classes of RT inhibitors. Overall, this novel strategy provides a quantitative measure of the intrinsic inhibition efficiencies of RT inhibitors in the natural cellular milieu and thus may further understanding of drug efficacy. This approach also has applicability for understanding the impact of viral polymerase-based inhibitors (alone or in combination) in other virus systems.T he HIV-encoded reverse transcriptase (RT) enzyme catalyzes the initiation, elongation, and termination of viral DNA (vDNA) synthesis through an ordered multistep process known as reverse transcription (Fig. 1A) (1, 2). This process, by which single-stranded HIV-1 RNA is converted to double-stranded HIV-1 DNA, follows a series of successive events whereby the product of each nucleoside incorporation serves as a substrate for the following reaction until the end of the genomic template is reached. First, minus-strand vDNA synthesis is initiated inefficiently from a primer-binding site (PBS) by a cell-derived tRNA 3Lys primer, resulting in the formation of minus-strand strong-stop vDNA product (3). Initiation is then followed by a processive mode of vDNA synthesis with continued elongation (4). Subsequent steps in reverse transcription involve selective degradation of genomic vRNA, minus-strand transfer, initiation of plus-strand vDNA from polypurine tracts (PPT and cPPT), formation of plus-strand strong-stop vDNA product, plus-strand transfer, and continued minus-and plus-strand vDNA synthesis until the end of the template is reached and full-length viral double-stranded DNA (dsDNA) is formed (5).Due to its essential role in ...

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Transient kinetic analyses of the ribonuclease H cleavage activity of HIV-1 reverse transcriptase in complex with efavirenz and/or a β-thujaplicinol analogue

Cited by 11 publications

References 17 publications

Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

Inhibition of the Ribonuclease H Activity of HIV-1 Reverse Transcriptase by GSK5750 Correlates with Slow Enzyme-Inhibitor Dissociation

Novel high-throughput screen identifies an HIV-1 reverse transcriptase inhibitor with a unique mechanism of action

A Cell-Based Strategy To Assess Intrinsic Inhibition Efficiencies of HIV-1 Reverse Transcriptase Inhibitors

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