Thermodynamics of HIV-1 Reverse Transcriptase in Action Elucidates the Mechanism of Action of Non-Nucleoside Inhibitors

Bec, Guillaume; Meyer, Benoît; Gérard, Marie-Aline; Steger, Jessica; Fauster, Katja; Wolff, Philippe; Burnouf, Dominique; Micura, Ronald; Dumas, Philippe; Ennifar, Eric

doi:10.1021/ja4018418

Cited by 56 publications

(46 citation statements)

References 46 publications

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“…The inability of the nucleic acid to reach the active site and inability of the active site to chelate metal ions suggests that a dNTP would not be able to bind at the N site when RT is bound to an NNRTI. A recent kinetic study using an incremental isothermal titration calorimetry (ITC) technique revealed that a dNTP does not bind to a RT/DNA/NNRTI complex 51 . Overall, analysis of our MD simulations revealed that binding of an NNRTI has multiple effects, primarily caused by repositioning of the primer grip and distortion of the active site triad; however, the fingers subdomain remained flexible in the RT-DNA-NVP complexes (Fig.…”

Section: Resultsmentioning

confidence: 99%

Molecular dynamics study of HIV‐1 RT‐DNA‐nevirapine complexes explains NNRTI inhibition and resistance by connection mutations

2013

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HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that is targeted by nucleoside analogs (NRTIs) and nonnucleoside inhibitors (NNRTIs). NNRTIs are allosteric inhibitors of RT, and constitute an integral part of the highly active antiretroviral therapy (HAART) regimen. Under selective pressure, HIV-1 acquires resistance against NNRTIs primarily by selecting mutations around the NNRTI pocket. Complete RT sequencing of clinical isolates revealed that spatially distal mutations arising in connection and the RNase H domain also confer NNRTI resistance and contribute to NRTI resistance. However, the precise structural mechanism by which the connection domain mutations confer NNRTI resistance is poorly understood. We performed 50-ns MD simulations, followed by essential dynamics, free-energy landscape analyses and network analyses of RT-DNA, RT-DNA-nevirapine, and N348I/T369I mutant RT-DNA-nevirapine complexes. MD simulation studies revealed altered global motions and restricted conformational landscape of RT upon nevirapine binding. Analysis of protein structure network parameters demonstrated a dissortative hub pattern in the RT-DNA complex and an assortative hub pattern in the RT-DNA-nevirapine complex suggesting enhanced rigidity of RT upon nevirapine binding. The connection subdomain mutations N348I/T369I did not induce any significant structural change; rather, these mutations modulate the conformational dynamics and alter the long-range allosteric communication network between the connection subdomain and NNRTI pocket. Insights from the present study provide a structural basis for the biochemical and clinical findings on drug resistance caused by the connection and RNase H mutations.

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Section: Resultsmentioning

confidence: 99%

Molecular dynamics study of HIV‐1 RT‐DNA‐nevirapine complexes explains NNRTI inhibition and resistance by connection mutations

2013

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“…These data are in line with previous studies showing that Nevirapine favors the RNase H orientation 29 and induces the opening of the finger and thumb region of RT, 50−52 preventing dNTP incorporation and primer extension. 51,53 In the second strategy, Nevirapine was used in competition with dNTPs. No change was observed in the progress curve as compared to the curve obtained in the absence of Nevirapine ( Figure 3A and Table 1).…”

Section: Analytical Chemistrymentioning

confidence: 99%

Reverse Transcriptase in Action: FRET-Based Assay for Monitoring Flipping and Polymerase Activity in Real Time

et al. 2015

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Reverse transcriptase (RT) of human immunodeficiency virus-1 (HIV-1) is a multifunctional enzyme that catalyzes the conversion of the single stranded viral RNA genome into double-stranded DNA, competent for host-cell integration. RT is endowed with RNA- and DNA-dependent DNA polymerase activity and DNA-directed RNA hydrolysis (RNase H activity). As a key enzyme of reverse transcription, RT is a key target of currently used highly active antiretroviral therapy (HAART), though RT inhibitors offer generally a poor resistance profile, urging new RT inhibitors to be developed. Using single molecule fluorescence approaches, it has been recently shown that RT binding orientation and dynamics on its substrate play a critical role in its activity. Currently, most in vitro RT activity assays, inherently end-point measurements, are based on the detection of reaction products by using radio-labeled or chemically modified nucleotides. Here, we propose a simple and continuous real-time Förster resonance energy transfer (FRET) based-assay for the direct measurement of RT's binding orientation and polymerase activity, with the use of conventional steady-state fluorescence spectroscopy. Under our working conditions, the change in binding orientation and the primer elongation step can be visualized separately on the basis of their opposite fluorescence changes and their different kinetics. The assay presented can easily discriminate non-nucleoside RT inhibitors from nucleoside RT inhibitors and determine reliably their potency. This one-step and one-pot assay constitutes an improved alternative to the currently used screening assays to disclose new anti-RT drugs and identify at the same time the class to which they belong.

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“…The exact binding site of INDOPY-1, which preferentially binds to the binary RT-T/P complex, is unknown (7). Although NNRTIs have recently been shown to prevent dNTP binding to the HIV-1 RT using thermodynamic (32) and single-molecule fluorescencebased assays (33), no studies have shown that NNRTIs compete with dNTP using enzyme kinetic assays as performed in this study. Our data suggest that oxime 5 is of the NcRTI class, but its activity against NNRTI-and NRTI-resistant RT indicates different drug resistance profiles to INDOPY-1 and DAVP-1.…”

Section: Discussionmentioning

confidence: 78%

Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

Latham

Tinetti

et al. 2015

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

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Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/ nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA-and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment.HIV | reverse transcriptase | fragment-based drug discovery | allosteric inhibitors | STD-NMR

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Thermodynamics of HIV-1 Reverse Transcriptase in Action Elucidates the Mechanism of Action of Non-Nucleoside Inhibitors

Cited by 56 publications

References 46 publications

Molecular dynamics study of HIV‐1 RT‐DNA‐nevirapine complexes explains NNRTI inhibition and resistance by connection mutations

Molecular dynamics study of HIV‐1 RT‐DNA‐nevirapine complexes explains NNRTI inhibition and resistance by connection mutations

Reverse Transcriptase in Action: FRET-Based Assay for Monitoring Flipping and Polymerase Activity in Real Time

Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

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