Structural implications of drug‐resistant mutants of HIV‐1 protease: High‐resolution crystal structures of the mutant protease/substrate analogue complexes

Mahalingam, Bhuvaneshwari; Louis, John M.; Hung, Jason; Harrison, Robert W.; Weber, Irene T.

doi:10.1002/prot.1057

Cited by 125 publications

(189 citation statements)

References 30 publications

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“…Protease mutations are classified by position relative to the active site: substrate cleft mutations, protease flap mutations, and those at other conserved residues (23,54). All single mutations studied exhibited the same pattern, however, showing modest change in IC 50 but decreased slope.…”

Section: Analysis Of Resistance Mutations Using Primary Cells Vs Tramentioning

confidence: 99%

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Dose–response curve slope is a missing dimension in the analysis of HIV-1 drug resistance

Sampah¹,

Shen

Jilek³

et al. 2011

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

129

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HIV-1 drug resistance is a major clinical problem. Resistance is evaluated using in vitro assays measuring the fold change in IC 50 caused by resistance mutations. Antiretroviral drugs are used at concentrations above IC 50 , however, and inhibition at clinical concentrations can only be predicted from IC 50 if the shape of the dose-response curve is also known. Curve shape is influenced by cooperative interactions and is described mathematically by the slope parameter or Hill coefficient (m). Implicit in current analysis of resistance is the assumption that mutations shift dose-response curves to the right without affecting the slope. We show here that m is altered by resistance mutations. For reverse transcriptase and fusion inhibitors, single resistance mutations affect both slope and IC 50 . For protease inhibitors, single mutations primarily affect slope. For integrase inhibitors, only IC 50 is affected. Thus, there are fundamental pharmacodynamic differences in resistance to different drug classes. Instantaneous inhibitory potential (IIP), the log inhibition of single-round infectivity at clinical concentrations, takes into account both slope and IC 50 , and thus provides a direct measure of the reduction in susceptibility produced by mutations and the residual activity of drugs against resistant viruses. The standard measure, fold change in IC 50 , does not correlate well with changes in IIP when mutations alter slope. These results challenge a fundamental assumption underlying current analysis of HIV-1 drug resistance and suggest that a more complete understanding of how resistance mutations reduce antiviral activity requires consideration of a previously ignored parameter, the dose-response curve slope.HIV/AIDS | pharmacology | virology | highly active antiretroviral therapy | evolution

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Section: Analysis Of Resistance Mutations Using Primary Cells Vs Tramentioning

confidence: 99%

“…At the molecular level, resistance often results from mutations that interfere with drug binding to the target enzyme or protein (20)(21)(22)(23). Additional mechanisms may also contribute.…”

mentioning

confidence: 99%

Dose–response curve slope is a missing dimension in the analysis of HIV-1 drug resistance

Sampah¹,

Shen

Jilek³

et al. 2011

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

129

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“…The PR 1M mutant was expressed in Escherichia coli BL21 (DE3) and the protein was purified from inclusion bodies as described. 33 The presence of the appropriate mutations was confirmed by DNA sequencing. PR 2 was prepared as described.…”

Section: Preparation Of Proteasesmentioning

confidence: 99%

Critical differences in HIV‐1 and HIV‐2 protease specificity for clinical inhibitors

Tie¹,

Wang²,

Boross³

et al. 2012

Protein Science

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Clinical inhibitor amprenavir (APV) is less effective on HIV-2 protease (PR 2 ) than on HIV-1 protease (PR 1 ). We solved the crystal structure of PR 2 with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR 1 mutant (PR 1M ) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR 2 . PR 1M more closely resembled PR 2 than PR 1 in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR 1M with APV, DRV, and SQV were compared with available PR 1 and PR 2 complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR 1M and PR 1 , however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR 1M . Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR 1M and PR 2 relative to the strong hydrogen bonds observed in PR 1 , consistent with 15-and 19-fold weaker inhibition, respectively. Overall, PR 1M partially replicates the specificity of PR 2 and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV-2.

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“…[1][2][3] Although studies have investigated the substrate binding and cleavage properties of proteases, [4][5][6][7][8] the factors that determine the specificity of substrate binding remain largely uncharacterized. Studies of HIV-1 protease in complex with substrates and inhibitors indicate that the drug-resistant mutants of this enzyme have altered molecular recognition abilities; they still recognize the enzyme's substrates but binding to inhibitors is disrupted.…”

Section: Introductionmentioning

confidence: 99%

“…For example, replacing glycine with glutamic acid at position 48 in the flap region affects the specificity of HIV-1 protease. 8,21 Several structural, [4][5][6]11,13 kinetic, 7,[22][23][24] and binding studies conducted on both active and inactive protease variants in complex with different substrate peptides [25][26][27][28] have provided insights into the mode of substrate binding and the role of primary and compensatory mutations in altered molecular recognition by resistant proteases. However, a thorough understanding of substrate recognition would require the ability to predict and modulate substrate specificity and computational protein design can be used as a technique to probe what determines specificity.…”

Section: Introductionmentioning

confidence: 99%

Structural, kinetic, and thermodynamic studies of specificity designed HIV‐1 protease

et al. 2012

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HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

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Structural implications of drug‐resistant mutants of HIV‐1 protease: High‐resolution crystal structures of the mutant protease/substrate analogue complexes

Cited by 125 publications

References 30 publications

Dose–response curve slope is a missing dimension in the analysis of HIV-1 drug resistance

Dose–response curve slope is a missing dimension in the analysis of HIV-1 drug resistance

Critical differences in HIV‐1 and HIV‐2 protease specificity for clinical inhibitors

Structural, kinetic, and thermodynamic studies of specificity designed HIV‐1 protease

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