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

ABSTRACT: Under the selective pressure of therapy, HIV-1 protease mutants resistant to inhibitors evolve to confer drug resistance. Such mutations can impact both the dynamics and structures of the bound and unbound forms of the enzyme. Flap+ is a multidrug-resistant variant of HIV-1 protease with a combination of primary and secondary resistance mutations (L10I, G48V, I54V, V82A) and a strikingly altered thermodynamic profile for darunavir (DRV) binding relative to the wild-type protease. We elucidated the impact of these mutations on protein dynamics in the DRV-bound state using molecular dynamics simulations and NMR relaxation experiments. Both methods concur in that the conformational ensemble and dynamics of protease are impacted by the drug resistance mutations in Flap+ variant. Surprisingly this change in ensemble dynamics is different from that observed in the unliganded form of the same variant (Cai, Y. et al. J. Chem. Theory Comput.2012, 8, 3452-3462). Our comparative analysis of both inhibitor-free and bound states presents a comprehensive picture of the altered dynamics in drug-resistant mutant HIV-1 protease and underlies the importance of incorporating dynamic analysis of the whole system, including the unliganded state, into revealing drug resistance mechanisms.

SUBMITTER: Cai Y

PROVIDER: S-EPMC4132871 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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

Cai Yufeng Y Myint Wazo W Paulsen Janet L JL Schiffer Celia A CA Ishima Rieko R Kurt Yilmaz Nese N

Journal of chemical theory and computation 20140612 8

Under the selective pressure of therapy, HIV-1 protease mutants resistant to inhibitors evolve to confer drug resistance. Such mutations can impact both the dynamics and structures of the bound and unbound forms of the enzyme. Flap+ is a multidrug-resistant variant of HIV-1 protease with a combination of primary and secondary resistance mutations (L10I, G48V, I54V, V82A) and a strikingly altered thermodynamic profile for darunavir (DRV) binding relative to the wild-type protease. We elucidated t ...[more]

PMID: 25136270

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Project description:BackgroundStrategies to control HIV for improving the quality of patient lives have been aided by the Highly Active Anti-Retroviral Therapy (HAART), which consists of a cocktail of inhibitors targeting key viral enzymes. Numerous new drugs have been developed over the past few decades but viral resistances to these drugs in the targeted viral enzymes are increasingly reported. Nonetheless the acquired mutations often reduce viral fitness and infectivity. Viral compensatory secondary-line mutations mitigate this loss of fitness, equipping the virus with a broad spectrum of resistance against these drugs. While structural understanding of the viral protease and its drug resistance mutations have been well established, the interconnectivity and development of structural cross-resistance remain unclear. This paper reports the structural analyses of recent clinical mutations on the drug cross-resistance effects from various protease and protease inhibitors (PIs) complexes.MethodsUsing the 2015 updated clinical HIV protease mutations, we constructed a structure-based correlation network and a minimum-spanning tree (MST) based on the following features: (i) topology of the PI-binding pocket, (ii) allosteric effects of the mutations, and (iii) protease structural stability.Results and conclusionAnalyis of the network and the MST of dominant mutations conferring resistance to the seven PIs (Atazanavir-ATV, Darunavir-DRV, Indinavir-IDV, Lopinavir-LPV, Nelfinavir-NFV, Saquinavir-SQV, and Tipranavir-TPV) showed that cross-resistance can develop easily across NFV, SQV, LPV, IDV, and DRV, but not for ATV or TPV. Through estimation of the changes in vibrational entropies caused by each reported mutation, some secondary mutations were found to destabilize protease structure. Our findings provide an insight into the mechanism of PI cross-resistance and may also be useful in guiding the selection of PI in clinical treatment to delay the onset of cross drug resistance.

Project description:BackgroundHIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors.Methods and findingsWe have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy.ConclusionsHIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.

Project description:BackgroundViral resistance to antiretroviral therapy threatens our best methods to control and prevent HIV infection. Current drug resistance genotyping methods are costly, optimized for subtype B virus, and primarily detect resistance mutations to protease and reverse transcriptase inhibitors. With the increasing use of integrase inhibitors in first-line therapies, monitoring for integrase inhibitor drug resistance mutations is a priority. We designed a universal primer pair to PCR amplify all major group M HIV-1 viruses for genotyping using Illumina MiSeq to simultaneously detect drug resistance mutations associated with protease, nucleoside reverse transcriptase, non-nucleoside reverse transcriptase, and integrase inhibitors.ResultsA universal primer pair targeting the HIV pol gene was used to successfully PCR amplify HIV isolates representing subtypes A, B, C, D, CRF01_AE and CRF02_AG. The universal primers were then tested on 62 samples from a US cohort of injection drug users failing treatment after release from prison. 94% of the samples were successfully genotyped for known drug resistance mutations in the protease, reverse transcriptase and integrase gene products. Control experiments demonstrate that mutations present at ≥ 2% frequency are reliably detected and above the threshold of error for this method. New drug resistance mutations not found in the baseline sample were identified in 54% of the patient samples after treatment failure. 86% of patients with major drug resistance mutations had 1 or more mutations associated with drug resistance to the treatment regimen at the time point of treatment failure. 59% of the emerging mutations were found at frequencies between 2% and 20% of the total sequences generated, below the estimated limit of detection of current FDA-approved genotyping techniques. Primary plasma samples with viral loads as low as 799 copies/ml were successfully genotyped using this method.ConclusionsHere we present an Illumina MiSeq-based HIV drug resistance genotyping assay. Our data suggests that this universal assay works across all major group M HIV-1 subtypes and identifies all drug resistance mutations in the pol gene known to confer resistance to protease, reverse transcriptase and integrase inhibitors. This high-throughput and sensitive assay could significantly improve access to drug resistance genotyping worldwide.

Project description:BackgroundMaturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance.ResultsOur resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all in vitro selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different proteases.ConclusionsThese findings highlight the complicated interactions between the viral protease and its substrate. By providing a better understanding of these interactions, we aim to help guide the development of second generation maturation inhibitors.

Dataset Information

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

Publications

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

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