Resistance to novel drug classes

Understanding the mechanisms that underlie resistance development to novel drugs is essential to a better clinical management of resistant viruses and to prevent further resistance development and spread. RECENT FINDINGS: Integrase inhibitors and CCR5 antagonists are the more recent antiretroviral classes developed. The HIV-1 integrase, responsible for the chromosomal integration of the newly synthesized double-stranded viral DNA into the host genomic DNA, represents a new and important target; and two integrase inhibitors (INIs), raltegravir and elvitegravir, have been shown promising results in clinical trials. Viral entry is also an attractive step for the development of new drugs against HIV variants resistant to current antiretroviral drugs, and two CCR5 antagonists have been designed to inhibit HIV-1 binding to R5 co-receptor and are under clinical investigation. SUMMARY: Drug resistance to INIs occurs through the selection of mutations within HIV integrase. The kinetic of selection seems rapid and one mutation alone is able to confer resistance to integrase inhibitor, suggesting that this class of drug has a low genetic barrier. Two ways could explain the failure of the CCR5 antagonist class: a rapid outgrowth of pre-existing archived X4 virus or the selection of a resistance to CCR5 antagonists through amino acid changes in V

Adaptive HIV-1 evolutionary trajectories are constrained by protein stability

Despite the use of combination antiretroviral drugs for the treatment of HIV-1 infection, the emergence of drug resistance remains a problem. Resistance may be conferred either by a single mutation or a concerted set of mutations. The involvement of multiple mutations can arise due to interactions between sites in the amino acid sequence as a consequence of the need to maintain protein structure. To better understand the nature of such epistatic interactions, we reconstructed the ancestral sequences of HIV-1's Pol protein, and traced the evolutionary trajectories leading to mutations associated with drug resistance. Using contemporary and ancestral sequences we modelled the effects of mutations (i.e. amino acid replacements) on protein structure to understand the functional effects of residue changes. Although the majority of resistance-associated sequences tend to destabilise the protein structure, we find there is a general tendency for protein stability to decrease across HIV-1's evolutionary history. That a similar pattern is observed in the non-drug resistance lineages indicates that non-resistant mutations, for example, associated with escape from the immune response, also impacts on protein stability. Maintenance of optimal protein structure therefore represents a major constraining factor to the evolution of HIV-1

The Impact of HIV-1 Drug Escape on the Global Treatment Landscape

The rising prevalence of HIV drug resistance (HIVDR) could threaten gains made in combating the HIV epidemic and compromise the 90-90-90 target proposed by United Nations Programme on HIV/AIDS (UNAIDS) to have achieved virological suppression in 90% of all persons receiving antiretroviral therapy (ART) by the year 2020. HIVDR has implications for the persistence of HIV, the selection of current and future ART drug regimens, and strategies of vaccine and cure development. Focusing on drug classes that are in clinical use, this Review critically summarizes what is known about the mechanisms the virus utilizes to escape drug control. Armed with this knowledge, strategies to limit the expansion of HIVDR are proposed

In vitro selection and characterisation of human immunodeficiency virus type-1 subtype C integrase strand transfer inhibitor resistant mutants

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy in Medicine Johannesburg 2015The currently approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG) effectively halt HIV-1 replication but their use is limited by their low genetic resistance barrier and cross resistance. For instance, integrase amino acids N155 and Q148 represent genetic pathways selected by both drugs and are associated with considerable cross resistance to both RAL and EVG. Dolutegravir (DTG) is a second generation drug manufactured to exhibit a more robust resistance profile than RAL and EVG, and retains activity against RAL and EVG resistant isolates. Most research on drug resistance patterns have been carried out with emphasis on HIV-1 subtype B and inadequately assessed in HIV-1 subtype C. Thus, the aim of this study was to establish the drug resistance mutation profiles of HIV-1 subtype C primary virus isolates that evolve/emerge under selective pressure of the INSTIs RAL, EVG and DTG, and evaluate their impact on strand transfer. In vitro selection experiments were carried out using six primary virus isolates (three wild-type, FV, and three reverse transcriptase drug resistant, MR, viruses) grown in peripheral blood mononuclear cells in the presence of increasing concentrations of RAL, EVG and DTG, and monitored to beyond virus break-through. Viral RNA was extracted from various time points and the pol region was RT-PCR amplified and sequenced using conventional Sanger-based sequencing and next generation sequencing (Illumina MiSeq). HIV-1 subtype C FV6 wild-type and mutant recombinant integrase (generated by site-directed mutagenesis) were expressed, purified and used in strand transfer assays and surface plasmon resonance (SPR) experiments to establish the binding affinities of IN-DNA. Wild-type FV primary viruses were successfully grown in the presence of increasing concentrations of RAL, EVG and DTG, up to 266 nM, 66 nM and 32 nM, respectively. Drug resistant MR viruses were successfully grown in the presence of increasing concentrations of RAL, EVG and DTG, up to 266 nM, 16 nM and 8 nM, respectively. Sequence analysis on both platforms revealed the presence of the previously described drug resistance mutations T66IK, E92Q, F121Y, Q148R, N155H and R263K in some viruses, and additionally H114L was detected. RAL was observed to select for substitutions Q148R and N155H/H114L in isolates FV6 and MR69, respectively. EVG selected F121Y, T66I/R263K, T66K and T66I in FV3, FV6, MR69, MR81, and MR89, respectively. DTG selected substitutions E92Q and M50I in FV3 and MR81, respectively. In silico data exhibited changes in hydrophilicity, hydrophobicity and side chain changes as well as changes in polarity, and all substitutions displayed acceptable minimisation energies and distances between the atoms. Seven IN mutants were expressed and purified, and thereafter tested for efficiency in strand transfer. All mutant FV6T66I, FV6E92Q, FV6H114L, FV6F121Y, FV6Q148R, FV6N155H and FV6R263K IN enzymes demonstrated an overall loss in strand transfer capacity of 37.1%, 21.5%, 66.1%, 63.2%, 60.2%, 30.5% and 3.4%, respectively. This is the first report on loss of strand transfer activity associated with H114L. The loss in strand transfer capacity in all the mutants was not reflected by their overall binding affinities to donor DNA, as determined by surface plasmon resonance, likely attributed to the role of different residues associated with DNA and drug binding in the IN quaternary structure. In conclusion, this is the first report describing IN drug selection experiments using primary HIV-1 subtype C isolates, and a detailed genotypic and biochemical characterisation of the associated mutations

Patterns of resistance development with integrase inhibitors in HIV

Raltegravir, the only integrase (IN) inhibitor approved for use in HIV therapy, has recently been licensed. Raltegravir inhibits HIV-1 replication by blocking the IN strand transfer reaction. More than 30 mutations have been associated with resistance to raltegravir and other IN strand transfer inhibitors (INSTIs). The majority of the mutations are located in the vicinity of the IN active site within the catalytic core domain which is also the binding pocket for INSTIs. High-level resistance to INSTIs primarily involves three independent mutations at residues Q148, N155, and Y143. The mutations significantly affect replication capacity of the virus and are often accompanied by other mutations that either improve replication fitness and/or increase resistance to the inhibitors. The pattern of development of INSTI resistance mutations has been extensively studied in vitro and in vivo. This has been augmented by cell-based phenotypic studies and investigation of the mechanisms of resistance using biochemical assays. The recent elucidation of the structure of the prototype foamy virus IN, which is closely related to HIV-1, in complex with INSTIs has greatly enhanced our understanding of the evolution and mechanisms of IN drug resistance

Novel therapeutic strategies targeting HIV integrase

Integration of the viral genome into host cell chromatin is a pivotal and unique step in the replication cycle of retroviruses, including HIV. Inhibiting HIV replication by specifically blocking the viral integrase enzyme that mediates this step is an obvious and attractive therapeutic strategy. After concerted efforts, the first viable integrase inhibitors were developed in the early 2000s, ultimately leading to the clinical licensure of the first integrase strand transfer inhibitor, raltegravir. Similarly structured compounds and derivative second generation integrase strand transfer inhibitors, such as elvitegravir and dolutegravir, are now in various stages of clinical development. Furthermore, other mechanisms aimed at the inhibition of viral integration are being explored in numerous preclinical studies, which include inhibition of 3' processing and chromatin targeting. The development of new clinically useful compounds will be aided by the characterization of the retroviral intasome crystal structure. This review considers the history of the clinical development of HIV integrase inhibitors, the development of antiviral drug resistance and the need for new antiviral compounds

Hiv Integrase Mechanisms Of Resistance To Raltegravir, Elvitegravir, And Dolutegravir

ABSTRACT HIV INTEGRASE MECHANISMS OF RESISTANCE TO RALTEGRAVIR, ELVITEGRAVIR, AND DOLUTEGRAVIR by KYLA ROSS December 2015 Advisor: Dr. Ladislau Kovari Major: Biochemistry and Molecular Biology Degree: Master of Science HIV-1 integrase (HIV-1 IN or IN) is a multimeric enzyme that integrates the HIV-1 genome into the chromosomes of infected CD4+ T-cells. Currently there are three FDA approved HIV-1 IN strand transfer inhibitors (INSTIs) used in clinical practice: raltegravir (RAL), elvitegravir (ELV), and dolutegravir (DTG). The [Q148H], [Q148H, G140S], [Q148R], [Q148R, G140A] and [N155H, E92Q] mutations decrease IN susceptibility to RAL and ELV and may result in therapeutic failure. As an indicator of protein flexibility, the root mean square deviation (RMSD) of each HIV-1 IN residue in the last 5 ns of a 40 ns molecular dynamics simulation was calculated for HIV-1 IN catalytic core domain as an apoprotein and in complex with RAL, ELV, and DTG to study how the mutations affect HIV-1 IN flexibility. In addition, we studied the relationship between HIV-1 IN flexibility and resistance. We found that the mutants reduced overall HIV-1 IN flexibility relative to the WT IN apoprotein. We also observed that the catalytic 140s loop in the HIV-1 IN-INSTI complexes were more flexible in mutants that displayed higher reported EC50 FC (fold change) values. To further investigate the mutations effect on the more complexed full length HIV-1 IN structure, we used molecular dynamics simulations to study the impact of the mutants on binary (IN-viral DNA complex) and ternary (IN-viral DNA- INSTI) IN flexibility. RMSD analyses revealed that that the mutants have a rigid structure relative to the WT IN. Furthermore, mutant IN showed transient changes in the secondary structure of the 140s loop compared to the WT. In addition to these reduced flexibility and structural changes, resistance mutations alter the binding mode of RAL, ELV, and DTG to IN and viral DNA. This study is the first to identify a structural basis of IN mechanism of resistance to INSTIs that develops under treatment pressure in HIV-1 IN

Resistance to Integrase Inhibitors

Integrase (IN) is a clinically validated target for the treatment of human immunodeficiency virus infections and raltegravir exhibits remarkable clinical activity. The next most advanced IN inhibitor is elvitegravir. However, mutant viruses lead to treatment failure and mutations within the IN coding sequence appear to confer cross-resistance. The characterization of those mutations is critical for the development of second generation IN inhibitors to overcome resistance. This review focuses on IN resistance based on structural and biochemical data, and on the role of the IN flexible loop i.e., between residues G140-G149 in drug action and resistance

Clinical use of HIV integrase inhibitors : a systematic review and meta-analysis

Background: Optimal regimen choice of antiretroviral therapy is essential to achieve long-term clinical success. Integrase inhibitors have swiftly been adopted as part of current antiretroviral regimens. The purpose of this study was to review the evidence for integrase inhibitor use in clinical settings. Methods: MEDLINE and Web-of-Science were screened from April 2006 until November 2012, as were hand-searched scientific meeting proceedings. Multiple reviewers independently screened 1323 citations in duplicate to identify randomized controlled trials, nonrandomized controlled trials and cohort studies on integrase inhibitor use in clinical practice. Independent, duplicate data extraction and quality assessment were conducted. Results: 48 unique studies were included on the use of integrase inhibitors in antiretroviral therapy-naive patients and treatment-experienced patients with either virological failure or switching to integrase inhibitors while virologically suppressed. On the selected studies with comparable outcome measures and indication (n = 16), a meta-analysis was performed based on modified intention-to-treat (mITT), on-treatment (OT) and as-treated (AT) virological outcome data. In therapy-naive patients, favorable odds ratios (OR) for integrase inhibitor-based regimens were observed, (mITT OR 0.71, 95% CI 0.59-0.86). However, integrase inhibitors combined with protease inhibitors only did not result in a significant better virological outcome. Evidence further supported integrase inhibitor use following virological failure (mITT OR 0.27; 95% CI 0.11-0.66), but switching to integrase inhibitors from a high genetic barrier drug during successful treatment was not supported (mITT OR 1.43; 95% CI 0.89-2.31). Integrase inhibitor-based regimens result in similar immunological responses compared to other regimens. A low genetic barrier to drug-resistance development was observed for raltegravir and elvitegravir, but not for dolutegravir. Conclusion: In first-line therapy, integrase inhibitors are superior to other regimens. Integrase inhibitor use after virological failure is supported as well by the meta-analysis. Careful use is however warranted when replacing a high genetic barrier drug in treatment-experienced patients switching successful treatment

Prevalence of resistance mutations related to integrase inhibitor S/GSK1349572 in HIV-1 subtype B raltegravir-naive and -treated patients

Objectives To compare the frequency of previously in vitro-selected integrase mutations (T124A, T124A/S153F, S153Y, T124A/S153Y and L101I/T124A/S153Y) conferring resistance to S/GSK1349572 between HIV-1 subtype B integrase inhibitor (INI)-naive and raltegravir-treated patients. Methods Integrase sequences from 650 INI-naive patients and 84 raltegravir-treated patients were analysed. Results The T124A mutation alone and the combination T124A/L101I were more frequent in raltegravir-failing patients than in INI-naive patients (39.3% versus 24.5%, respectively, P = 0.005 for T124A and 20.2% versus 10.0%, respectively, P = 0.008 for T124A/L101I). The S153Y/F mutations were not detected in any integrase sequence (except for S153F alone, only detected in one INI-naive patient). Conclusions T124A and T124A/L101I, more frequent in raltegravir-treated patients, could have some effect on raltegravir response and their presence could play a role in the selection of other mutations conferring S/GSK1349572 resistance. The impact of raltegravir-mediated changes such as these on the virological response to S/GSK1349572 should be studied further