Active site dynamics and combined quantum mechanics/molecular mechanics (QM/MM) modelling of a HIV-1 reverse transcriptase/DNA/dTTP complex

Conventional macromolecular crystallographic refinement relies on often dubious stereochemical restraints, the preparation of which often requires human validation for unusual species, and on rudimentary energy functionals that are devoid of nonbonding effects owing to electrostatics, polarization, charge transfer or even hydrogen bonding. While this approach has served the crystallographic community for decades, as structure-based drug design/discovery (SBDD) has grown in prominence it has become clear that these conventional methods are less rigorous than they need to be in order to produce properly predictive protein–ligand models, and that the human intervention that is required to successfully treat ligands and other unusual chemistries found in SBDD often precludes high-throughput, automated refinement. Recently, plugins to thePython-based Hierarchical ENvironment for Integrated Xtallography(PHENIX) crystallographic platform have been developed to augment conventional methods with thein situuse of quantum mechanics (QM) applied to ligand(s) along with the surrounding active site(s) at each step of refinement [Borbulevychet al.(2014),Acta CrystD70, 1233–1247]. This method (Region-QM) significantly increases the accuracy of the X-ray refinement process, and this approach is now used, coupled with experimental density, to accurately determine protonation states, binding modes, ring-flip states, water positions and so on. In the present work, this approach is expanded to include a more rigorous treatment of the entire structure, including the ligand(s), the associated active site(s) and the entire protein, using a fully automated, mixed quantum-mechanics/molecular-mechanics (QM/MM) Hamiltonian recently implemented in theDivConpackage. This approach was validated through the automatic treatment of a population of 80 protein–ligand structures chosen from the Astex Diverse Set. Across the entire population, this method results in an average 3.5-fold reduction in ligand strain and a 4.5-fold improvement inMolProbityclashscore, as well as improvements in Ramachandran and rotamer outlier analyses. Overall, these results demonstrate that the use of a structure-wide QM/MM Hamiltonian exhibits improvements in the local structural chemistry of the ligand similar to Region-QM refinement but with significant improvements in the overall structure beyond the active site.

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Two Coselected Distal Mutations in HIV-1 Reverse Transcriptase (RT) Alter Susceptibility to Nonnucleoside RT Inhibitors and Nucleoside Analogs

Journal of Virology ◽

10.1128/jvi.00224-19 ◽

2019 ◽

Vol 93 (11) ◽

Author(s):

Paul L. Boyer ◽

Kevin Melody ◽

Steven J. Smith ◽

Linda L. Dunn ◽

Chris Kline ◽

...

Keyword(s):

Nucleic Acid ◽

Reverse Transcriptase ◽

Active Site ◽

Nucleoside Analogs ◽

Transcriptase Inhibitor ◽

Drug Resistant ◽

Resistance Mutations ◽

Hydrophobic Region ◽

Nnrti Resistance ◽

Hiv 1

ABSTRACTTwo mutations, G112D and M230I, were selected in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) by a novel nonnucleoside reverse transcriptase inhibitor (NNRTI). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs, while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the “primer grip” region and could be influenced by the M230I mutation.IMPORTANCEAlthough antiretroviral therapy (ART) is highly successful, drug-resistant variants can arise that blunt the efficacy of ART. New inhibitors that are broadly effective against known drug-resistant variants are needed, although such compounds might select for novel resistance mutations that affect the sensitivity of the virus to other compounds. Compound 13 selects for resistance mutations that differ from traditional NNRTI resistance mutations. These mutations cause increased sensitivity to NRTIs, such as AZT.

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Active site labeling of HIV-1 reverse transcriptase

Biochemistry ◽

10.1021/bi00069a031 ◽

1993 ◽

Vol 32 (18) ◽

pp. 4938-4942 ◽

Cited By ~ 6

Author(s):

Nijing Sheng ◽

Don Dennis

Keyword(s):

Reverse Transcriptase ◽

Active Site ◽

Hiv 1

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The Phenylmethylthiazolylthiourea Nonnucleoside Reverse Transcriptase (RT) Inhibitor MSK-076 Selects for a Resistance Mutation in the Active Site of Human Immunodeficiency Virus Type 2 RT

Journal of Virology ◽

10.1128/jvi.78.14.7427-7437.2004 ◽

2004 ◽

Vol 78 (14) ◽

pp. 7427-7437 ◽

Cited By ~ 13

Author(s):

Joeri Auwerx ◽

Miguel Stevens ◽

An R. Van Rompay ◽

Louise E. Bird ◽

Jingshan Ren ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Binding Site ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Active Site ◽

Resistance Mutation ◽

Site Directed Mutagenesis ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 μM) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 μM. As was also found for HIV-1 RT, MSK-076 inhibited HIV-2 RT in a noncompetitive manner with respect to dGTP and poly(rC)·oligo(dG) as the substrate and template-primer, respectively. MSK-076 selected for A101P and G112E mutations in HIV-2 RT and for K101E, Y181C, and G190R mutations in HIV-1 RT. The selected mutated strains of HIV-2 were fully resistant to MSK-076, and the mutant HIV-2 RT enzymes into which the A101P and/or G112E mutation was introduced by site-directed mutagenesis showed more than 50-fold resistance to MSK-076. Mapping of the resistance mutations to the HIV-2 RT structure ascertained that A101P is located at a position equivalent to the nonnucleoside RT inhibitor (NNRTI)-binding site of HIV-1 RT. G112E, however, is distal to the putative NNRTI-binding site in HIV-2 RT but close to the active site, implying a novel molecular mode of action and mechanism of resistance. Our findings have important implications for the development of new NNRTIs with pronounced activity against a wider range of lentiviruses.

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MOLECULAR DYNAMICS, DOCKING, DENSITY FUNCTIONAL, AND ADMET STUDIES OF HIV-1 REVERSE TRANSCRIPTASE INHIBITORS

Journal of Theoretical and Computational Chemistry ◽

10.1142/s0219633606002441 ◽

2006 ◽

Vol 05 (03) ◽

pp. 579-586 ◽

Cited By ~ 10

Author(s):

CARLOS H. T. P. DA SILVA ◽

IVONE CARVALHO ◽

CARLTON A. TAFT

Keyword(s):

Molecular Dynamics ◽

Reverse Transcriptase ◽

Active Site ◽

Density Functional ◽

Docking Studies ◽

Reverse Transcriptase Inhibitors ◽

Hydrogen Bond Interaction ◽

Receptor Interactions ◽

Similar Mode ◽

Hiv 1

Molecular dynamics, density functional with correlation, as well as docking studies of inhibitors of HIV-1 reverse transcriptase (RT) are reported. We propose in this work a novel potential HIV-1 RT inhibitor (RTI), which theoretically appears to bind in a similar mode as other nucleoside reverse transcriptase inhibitors, and in addition, it introduces a new hydrogen bond interaction with Trp229. Our novel RTI has high docking scores and the molecular dynamics studies, as well as the analysis of the ligand-receptor interactions in the active site and the ADMET properties suggest advantages and specificities for this potential RTI.

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Interactions of Conformationally Biased North and South 2‘-Fluoro-2‘,3‘-dideoxynucleoside 5‘-Triphosphates with the Active Site of HIV-1 Reverse Transcriptase

Biochemistry ◽

10.1021/bi001090n ◽

2000 ◽

Vol 39 (37) ◽

pp. 11205-11215 ◽

Cited By ~ 19

Author(s):

Lan Mu ◽

Stefan G. Sarafianos ◽

Marc C. Nicklaus ◽

Pamela Russ ◽

Maqbool A. Siddiqui ◽

...

Keyword(s):

Reverse Transcriptase ◽

Active Site ◽

North And South ◽

Hiv 1

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Nonnucleoside Inhibitor Binding Affects the Interactions of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with DNA

Journal of Virology ◽

10.1128/jvi.78.7.3387-3397.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3387-3397 ◽

Cited By ~ 24

Author(s):

Elena N. Peletskaya ◽

Alex A. Kogon ◽

Steven Tuske ◽

Edward Arnold ◽

Stephen H. Hughes

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Active Site ◽

Cross Linking ◽

Immunodeficiency Virus ◽

Dntp Binding ◽

Hiv 1

ABSTRACT Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the “open” configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.

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