Drug Resistance Mutations Alter Dynamics of Inhibitor-Bound HIV-1 Protease

Cai, Yufeng; Myint, Wazo; Paulsen, Janet L.; Schiffer, Celia A.; Ishima, Rieko; Yilmaz, Neşe Kurt

doi:10.1021/ct4010454

Cited by 36 publications

(53 citation statements)

References 43 publications

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“…4,8,9,12–14 One well characterized extremely drug resistant isolate of PR bearing 19 mutations (PR20) 10,15–17 exhibits 3–4 orders of magnitude weaker binding to clinical PIs relative to the wild-type enzyme. However, it mediates Gag processing only about 4-fold more slowly and retains the same order of cleavage as the wild-type enzyme.…”

Section: Introductionmentioning

confidence: 99%

Binding of Clinical Inhibitors to a Model Precursor of a Rationally Selected Multidrug Resistant HIV-1 Protease Is Significantly Weaker Than That to the Released Mature Enzyme

et al. 2016

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We have systematically validated the activity and inhibition of a HIV-1 protease (PR) variant bearing 17 mutations (PRS17), selected to represent high resistance by machine learning on genotype-phenotype data. Three of five mutations in PRS17 correlating with major drug resistance, M46L, G48V and V82S, and five of eleven natural variations, differ from two clinically derived extreme mutants, PR20 and PR22 bearing 19 and 22 mutations, respectively. PRS17, which forms a stable dimer (<10 nM), is ~10- and 2-fold less efficient in processing the Gag polyprotein relative to the wild-type and PR20, respectively, but maintains the same cleavage order. Isolation of a model precursor of PRS17 flanked by the 56 amino acid transframe region (TFP-p6pol) at its N-terminus, which is impossible when expressing an analogous PR20 precursor, allowed systematic comparison of inhibition of TFP-p6pol-PRS17 and mature PRS17. Resistance of PRS17 to 8 protease inhibitors (PIs) relative to PR ranges from 1.5 to 5 orders of magnitude increase in Ki from 0.01 to 8.4 μM. Amprenavir, darunavir, atazanavir and lopinavir, the most effective of the 8 PIs, inhibit precursor autoprocessing at the p6pol/PR site with IC50 ranging from ~7.5 to 60 μM. Thus this process, crucial for stable dimer formation, shows ~200 to 800-fold weaker inhibition than the mature PRS17. TFP/p6pol cleavage, which occurs faster, is inhibited even more weakly by all PIs except darunavir (IC50 of 15 μM); amprenavir shows a 2-fold increase in IC50 (~15 μM), and atazanavir and lopinavir show increased IC50 of >42 μM and >70 μM, respectively.

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Section: Introductionmentioning

confidence: 99%

Binding of Clinical Inhibitors to a Model Precursor of a Rationally Selected Multidrug Resistant HIV-1 Protease Is Significantly Weaker Than That to the Released Mature Enzyme

et al. 2016

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“…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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“…Furthermore, experimental studies have shown that there is a relationship between conformational sampling and drug resistance in HIV-PR (Cai et al, 2014;Cai et al 2012Foulkes-Murzycki et al, 2013Gibbs, 2014 andde Vera et al, 2013). Therefore, understanding functional dynamics of the flaps of HIV-PR is of great importance to the development of new HIV-PR inhibitors.…”

Section: Introductionmentioning

confidence: 99%

Unraveling HIV protease flaps dynamics by Constant pH Molecular Dynamics simulations

Soares

Tôrres

Silva

et al. 2016

Journal of Structural Biology

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a b s t r a c tThe active site of HIV protease (HIV-PR) is covered by two flaps. These flaps are known to be essential for the catalytic activity of the HIV-PR, but their exact conformations at the different stages of the enzymatic pathway remain subject to debate. Understanding the correct functional dynamics of the flaps might aid the development of new HIV-PR inhibitors. It is known that, the HIV-PR catalytic efficiency is pHdependent, likely due to the influence of processes such as charge transfer and protonation/deprotonation of ionizable residues. Several Molecular Dynamics (MD) simulations have reported information about the HIV-PR flaps. However, in MD simulations the protonation of a residue is fixed and thus it is not possible to study the correlation between conformation and protonation state. To address this shortcoming, this work attempts to capture, through Constant pH Molecular Dynamics (CpHMD), the conformations of the apo, substrate-bound and inhibitor-bound HIV-PR, which differ drastically in their flap arrangements. The results show that the HIV-PR flaps conformations are defined by the protonation of the catalytic residues Asp25/Asp25 0 and that these residues are sensitive to pH changes. This study suggests that the catalytic aspartates can modulate the opening of the active site and substrate binding.

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Drug Resistance Mutations Alter Dynamics of Inhibitor-Bound HIV-1 Protease

Cited by 36 publications

References 43 publications

Binding of Clinical Inhibitors to a Model Precursor of a Rationally Selected Multidrug Resistant HIV-1 Protease Is Significantly Weaker Than That to the Released Mature Enzyme

Binding of Clinical Inhibitors to a Model Precursor of a Rationally Selected Multidrug Resistant HIV-1 Protease Is Significantly Weaker Than That to the Released Mature Enzyme

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

Unraveling HIV protease flaps dynamics by Constant pH Molecular Dynamics simulations

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