Drug Resistance Conferred by Mutations Outside the Active Site through Alterations in the Dynamic and Structural Ensemble of HIV-1 Protease

Ragland, Debra A.; Nalivaika, E.A.; Nalam, M.N.L.; Prachanronarong, Kristina L.; Cao, Hong; Bandaranayake, R.M.; Cai, Yufeng; KurtYilmaz, N.; Schiffer, Celia A.

doi:10.1021/ja504096m

Cited by 89 publications

(122 citation statements)

References 30 publications

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“…Analysis of crystal structures showed significant loss of interactions with DRV, while protease interactions were maintained with SQV. Molecular dynamic studies combined with crystal structures suggested L76V contributed to resistance by altering the overall enzyme dynamics [34]. Many distal mutations are classified as minor mutations that modulate the effects of major mutations.…”

Section: Diverse Mechanisms Of Resistancementioning

confidence: 99%

Highly Resistant HIV-1 Proteases and Strategies for Their Inhibition

2015

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The virally encoded protease is an important drug target for AIDS therapy. Despite the potency of the current drugs, infections with resistant viral strains limit the long-term effectiveness of therapy. Highly resistant variants of HIV protease from clinical isolates have different combinations of about 20 mutations and several orders of magnitude worse binding affinity for clinical inhibitors. Strategies are being explored to inhibit these highly resistant mutants. The existing inhibitors can be modified by introducing groups with the potential to form new interactions with conserved protease residues, and the flexible flaps. Alternative strategies are discussed, including designing inhibitors to bind to the open conformation of the protease dimer, and inhibition of the protease-catalyzed processing of the Gag-Pol precursor.

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Section: Diverse Mechanisms Of Resistancementioning

confidence: 99%

Highly Resistant HIV-1 Proteases and Strategies for Their Inhibition

2015

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“…4, 23, 24, 34–38 The mechanisms by which accumulated mutations affect the active site pocket and confer drug resistance are actively being studied. At present, the mechanism is believed to be multifaceted in that several aspects of protein function are altered such as protein flexibility through the hydrophobic sliding mechanism, 39, 40 protein stability, 41 or altered dynamics 42 and conformational sampling. 43 …”

Section: Introductionmentioning

confidence: 99%

“…This model also allows for the possibility that dynamics of each of the conformational states varies 67, 68 and, as mutations accumulate, the exchange rate among the states also varies. 42, 69 The four conformers of the proposed ensemble are shown in Figure 3 where the most noticeable change in protein structure involves a segmental motion of the β-hairpin flaps (also shown in Figure 1). Each conformer has been observed, to some degree, via X-ray crystallography.…”

Section: Introductionmentioning

confidence: 99%

Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

Liu

Casey

Blackburn

et al. 2016

Phys. Chem. Chem. Phys.

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The conformational landscape of HIV-1 protease (PR) can be experimentally characterized by pulsed-EPR double electron-electron resonance (DEER). For this characterization, nitroxide spin labels are attached to an engineered cysteine residue in the flap region of HIV-1 PR. DEER distance measurements from spin-labels contained within each flap of the homodimer provide a detailed description of the conformational sampling of apo-enzyme as well as induced conformational shifts as a function inhibitor binding. The distance distribution profiles are further interpreted in terms of a conformational ensemble scheme that consists of four unique states termed “curled/tucked”, “closed”, “semi-open” and “wide-open” conformations. Reported here are the DEER results for a drug-resistant variant clinical isolate sequence, V6, in the presence of FDA approved protease inhibitors (PIs) as well as a non-hydrolyzable substrate mimic, CaP2. Results are interpreted in the context of the current understanding of the relationship between conformational sampling, drug resistance, and kinetic efficiency of HIV-1PR as derived from previous DEER and kinetic data for a series of HIV-1PR constructs that contain drug-pressure selected mutations or natural polymorphisms. Specifically, these collective results support the notion that inhibitor-induced closure of the flaps correlates with inhibitor efficiency and drug resistance. This body of work also suggests DEER as a tool for studying conformational sampling in flexible enzymes as it relates to function.

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“…The protease variants with available DRV bound structures were SF-2 (PDB: 1T3R), L76V, V32I and V32I/L33F 17, 21 . The NL4-3 wild type and remaining variants were modeled based on the DRV-bound wild-type SF-2 structure.…”

Section: Resultsmentioning

confidence: 99%

“…This set included both intra-protease and protease–DRV hydrogen bonds. With an expanded panel of protease variants, relative to our earlier study 17 , the use of algorithms for detecting patterns of altered occupancies and identifying specific mutations that may underlie these alterations becomes essential. A combination of principal component analysis (PCA), to detect alterations, followed by hypothesis testing based on amino acid substitution at specific sites in the protease, was employed.…”

Section: Resultsmentioning

confidence: 99%

Elucidating the Interdependence of Drug Resistance from Combinations of Mutations

Ragland¹,

Whitfield²,

Lee

et al. 2017

J. Chem. Theory Comput.

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HIV-1 protease is responsible for the cleavage of 12 non-homologous sites within the Gag and Gag-Pro-Pol polyproteins in the viral genome. Under the selective pressure of protease inhibition, the virus evolves mutations within (primary) and outside of (secondary) the active site allowing the protease to process substrates while simultaneously countering inhibition. The primary protease mutations impede inhibitor binding directly, while the secondary mutations are considered accessory mutations that compensate for a loss in fitness. However, the role of secondary mutations in conferring drug resistance remains a largely unresolved topic. We have shown previously that mutations distal to the active site are able to perturb binding of darunavir (DRV) via the protein’s internal hydrogen-bonding network. In this study we show that mutations distal to the active site, regardless of context, can play an interdependent role in drug resistance. Applying eigenvalue decomposition to collections of hydrogen bonding and van der Waals interactions from a series of molecular dynamics simulations of 15 diverse HIV-1 protease variants, we identify sites in the protease where amino acid substitutions lead to perturbations in non-bonded interactions with DRV and/or the hydrogen-bonding network of the protease itself. While primary mutations are known to drive resistance in HIV-1 protease, these findings delineate the significant contributions of accessory mutations to resistance. Identifying the variable positions in the protease that have the greatest impact on drug resistance may aid in future structure-based design of inhibitors.

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Drug Resistance Conferred by Mutations Outside the Active Site through Alterations in the Dynamic and Structural Ensemble of HIV-1 Protease

Cited by 89 publications

References 30 publications

Highly Resistant HIV-1 Proteases and Strategies for Their Inhibition

Highly Resistant HIV-1 Proteases and Strategies for Their Inhibition

Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

Elucidating the Interdependence of Drug Resistance from Combinations of Mutations

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