Molecular analysis of human immuno-deficiency virus-1 (South African subtype C) protease drug resistance mutations emerging on Darunavir therapy.

Masters Degree. University of KwaZulu-Natal, Durban.Human immunodeficiency virus as the causative agent of acquired immune deficiency syndrome remains a serious infectious disease and the leading cause of deaths worldwide. According to UNAIDS, approximately 37 million individuals are living with HIV/AIDS and 770,000 AIDS related deaths. HIV-1 subtype C strain is responsible for approximately 70 % of individuals living with HIV. Even with this staggering statistic, not many studies have been conducted on this subtype. Currently, there exist no treatment that completely eradicates the virus from an infected individual. Although, three enzymes required by the virus to undergo intracellular replication have been targeted to delay the progression of the disease, these enzymes include; reverse transcriptase crucial for completion of the initial stages of HIV replication, integrase essential for the integration of pro-viral DNA into the host chromosomal DNA and finally the enzyme for which this study will focus only is protease which is vital for the development and assembly of infectious viral progeny. The HIV aspartyl protease plays a major role in the life cycle of the virus and has long been a target in antiviral therapy. This advancements in the knowledge of HIV biology, pathogenesis and pharmacology has led to unprecedented efforts to interpret basic findings in the development of novel antiviral drug therapies. Nonetheless, the emergence of drug resistant mutations has hampered the efficacy of HIV-1 protease inhibition therapy. These mutations reduce the binding affinity of inhibitors while maintaining viable catalytic activity and affinity for the natural substrate. In HIV-1 protease, mutations at the following positions V32I, I50V,154M, and I84V are associated with subtle structural changes that confer resistance to protease inhibitors especially darunavir. These mutations located at or adjacent to the active site cavity, compromise drug susceptibility due to weak Van der Waals interaction and binding site distortion resulting in treatment failure. In this study we analysed the functional effects of these mutations on the HIV-1 South African subtype C protease. To understand how these mutations influence drug susceptibility in HIV1 CSA protease, the mutations were introduced by site directed mutagenesis and confirmed by DNA sequencing. Over-expression and purification of wild-type and mutant protease. Followed by enzyme kinetics, inhibition (Ki) and thermodynamics studies carried out against six clinically approved drugs. Significant difference was not observed in the substrate affinity of the variant protease compared to the wildtype C-SA protease with a Michaelis constant (Km) values of 104 and 124 µM and turnover number (Kcat) of approximately 2.2 and 0.2 s-1 for variant and wildtype protease respectively. The six clinically approved drugs used in this study demonstrated reduced binding affinities and weaker inhibition towards the variant protease in comparison to the wild-type HIV-1 protease. Atazanavir, amprenavir, darunavir and saquinavir exhibited the weakest inhibition towards the variant protease with Ki ratio values of 163, 232, 465 and 247 respectively. Thermodynamic data showed less favourable Gibbs free binding energy in selected protease inhibitors towards the variant protease, largely due to decreased binding entropy. Vitality values for the variant protease against the selected protease inhibitors, confirm the impact of these mutations on the HIV-1 CSA protease. In the presence of these drug resistant mutations V32I, I50V,154M, and I84V the efficacy of the selected protease inhibitors used in this study is significantly reduced. Future studies would involve crystallization and structure determination. This will give an in-depth understanding on the structural interaction of the variant protease towards the protease inhibitors

Crystallographic Analysis and Molecular Modeling Studies of HIV-1 Protease and Drug Resistant Mutants

HIV-1 protease (PR) is an effective target protein for drugs in anti-retroviral therapy (ART). Using PR inhibitors (PIs) in clinical therapy successfully reduces mortality of HIV infected patients. However, drug resistant variants are selected in AIDS patients because of the fast evolution of the viral genome. Structural, kinetic and MD simulations of PR variants with or without substrate or PIs were used to better understand the molecular basis of drug resistance. Information obtained from these extensive studies will benefit the design of more effective inhibitor in ART. Amprenavir (APV) inhibition of PRWT, and single mutants of PRV32I, PRI50V, PRI54M, PRI54V, PRI84V and PRL90M were studied and X-ray crystal structures of PR variants complexes with APV were solved at resolutions of 1.02-1.85 Å to identify structural alterations. Crystal structures of PRWT, PRV32I and PRI47V were solved at resolutions of 1.20-1.40 Å. Reaction intermediates were captured in the substrate binding cavity, which represent three consecutive steps in the catalytic reaction of HIV PR. HIV-1 PR20 variant is a multi-drug resistant variant from a clinical isolate and it is of utility to investigate the mechanisms of resistance. The crystal structures of PR20 with inactivating mutation D25N have been determined at 1.45-1.75 Å resolution, and three distinct flap conformations, open, twisted and tucked, were observed. These studies help understand molecular basis of drug resistance and provide clues for design of inhibitors to combat multi-drug resistant PR. The evaluation of electrostatic force in MD simulations is the computationally intensive work, which is of order theta(N2) with integration of all atom pairs. AMMP invokes Amortized FMM in summation of electrostatic force, which reduced work load to theta(N). A hybrid, CPU and GPU, parallel implementation of Amortized FMM was developed and improves the elapsed time of MD simulation 20 fold faster than CPU based parallelization

Biochemical characterization of highly mutated South African HIV-1 subtype C protease.

Doctoral Degree. University of KwaZulu-Natal, Durban.Understanding the underlying molecular mechanism of HIV-1 protease (PR) inhibition by HIV-1 protease inhibitors (PIs) is essential to gain mechanistic insight into the evolution of resistance to HIV-1 PIs. HIV-1 PIs have improved patient care management, but the accumulation of drug resistance mutations in the HIV-1 PR gene diminishes their inhibitory capacity. The current study investigated the kinetic and structural characteristics of highly mutated South African HIV-1 subtype C PR from clinical isolates obtained from individuals failing a lopinavir (LPV) inclusive regimen at the point of switch to darunavir (DRV) based therapy. In this study, enzyme activity and inhibition assays were used to determine the biochemical fitness of HIV-1 PR variants and the inhibitory constants of HIV-1 PIs for drug-resistant HIV-1 subtype C proteases. The mechanistic insight into the impact of the accumulated drug resistance mutations on the HIV-1 PR structure and its interaction with LPV and DRV was obtained using fluorescence spectroscopy and molecular dynamic simulation. The study showed that the unfavorable binding landscape caused by the accumulation of drug-resistance mutations resulting from LPV associated drug pressure would shape the outcome of DRV-based therapy after a switch in the treatment regimen. This is related to the distortion of the HIV-1 PR structure associated with increased solvent exposure and instability of the HIV-1 PR dimer caused by these mutations leading to a shorter lifetime of the enzyme-inhibitor complex. Analysis of the binding kinetics of LPV and DRV with the HIV-1 PR variants showed that the drug resistance mutations caused an imbalance between the association and dissociation rate constants favoring a fast dissociation rate. The latter resulted in a reduced inhibitor residence time. Our findings showed that LPV had a longer residence time than DRV when bound to the HIV-1 PR variants; this shows LPV can be a suitable platform for developing newer HIV-1 PIs with a longer residence time. However, the enzyme inhibition mechanism shows both LPV and DRV act via a two-step tight-binding mixed inhibition mechanism, suggesting the existence of a second binding site on HIV-1 PR for these inhibitors. The information provided in this thesis adds to existing knowledge about HIV-1 PI drug resistance and for the design of novel HIV-1 PIs with the potential to evade drug resistance mutations

Population dynamics in HIV-1 transmitted antiretroviral drug resistance

A dissertation submitted to Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Science in Medicine, Johannesburg, June 2018It is well known that antiretroviral (ARV) drug resistant variants of HIV-1 can be sexually transmitted. Several studies have shown that in resource-rich geographical locations as many as 15-20% of individuals are newly infected with HIV-1 containing at least one drug resistant mutation. In contract, resource limited geographical locations, such as Sub-Saharan Africa, have shown prevalences in the range of 5 to 10%. Since the ART rollout in these resource-limited locations are generally not well monitored with virological genotyping, the transmission of drug resistant HIV-1 is likely to increase, with significant clinical and public health consequences. HIV-1 transmission is characterised by the transmission of a single founder virus, or narrow spectrum of founder viruses, that develop into the viral quasispecie. It is unlikely that drug resistant virus will coexist with wild type (wt) virus, in the case of non-drug resistance transmission. However, initiating in ARV treatment, drug non-adherence may select of ARV drug resistance mutations and may subsequent lead to treatment failure. Drug resistant virus may be transmitted to a new host, as drug resistant mutations do not appear to hamper transmission efficiency of the mutated virus. Several studies have shown that transmitted drug resistance mutations (TDRMs) persist either as the dominant species or as minority variants, or revert to wild type over time, in the absence of drug pressure. It is generally acknowledged that many drug resistance mutations decrease the replicative capacity of HIV-1, and thus reversion confers a potential survival advantage. Because of the emergence of wild type variants from TDRM quasispecies requires evolution and back-mutation, the rate at which individual TDRMs become undetectable may vary substantially. Contradictory findings of persistence versus reversion of TDRMs have been reported, and may be attributed to the fact that minority variants are difficult to detect by conventional population based Sanger sequencing, and patient numbers studied are small. Consequently, individuals infected with HIV-1 harbouring TDRM have a higher chance of failing their first-line therapy. Understanding the population dynamics of transmitted drug resistant HIV-1 in the absence of drug pressure is essential for clinical management and public health strategies. The individuals identified with TDRMs from the IAVI-Early Infections Cohort (Protocol C) provides a unique research opportunity to address the aforementioned issue. This study describes III the evolutionary mechanisms of ARV drug resistant HIV-1 after transmission to a new host to provide insight into persistence and/or rates of reversion to wild type. TDRMs initially identified by Price et al. (2011) in the IAVI-Early Infections Cohort (Protocol C) using population-based Sanger sequencing (the current diagnostic gold standard), were confirmed in this study by newer ultra-deep next generation sequencing (NGS) technology on the Illumina Miseq platform. Longitudinal samples were made available for individuals in which transmitted drug resistance were identified, and we also sequenced using NGS on the Illumina Miseq platform. Additional minority variants (present at <20% of the sequenced viral population) were identified by NGS. This study found a large percentage of TDRMs to persist for a significant amount of time after transmission to a new, drug naïve host, in the longitudinal samples. The level of persistence, or rate of reversion of TDRMs, appear to be subject to the type of resistance (NRTI, NNRTI or PI), level of resistance the mutation confers, as well as the combination of mutations that are cotransmitted. Findings of this study highlight the importance of drug resistance screening prior to ART initiation, as well as the importance of the drug resistance screening assay sensitivity. As rates of transmitted drug resistance are increasing in developing countries of which the IAVI-Early Infections Cohort (Protocol C) are composed of, understanding the population dynamics of transmitted drug resistant HIV-1 in the absence of drug pressure is essential for clinical management, public health strategies and informing future vaccine design.XL201

A Comparative Insight into Amprenavir Resistance of Mutations V32I, G48V, I50V, I54V, and I84V in HIV‑1 Protease Based on Thermodynamic Integration and MM-PBSA Methods

Drug resistance of mutations V32I, G48V, I50V, I54V, and I84V in HIV-1 protease (PR) was found in clinical treatment of HIV patients with the drug amprenavir (APV). In order to elucidate the molecular mechanism of drug resistance associated with these mutations, the thermodynamic integration (TI) and molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) methods were applied to calculate binding free energies of APV to wild-type PR and these mutated PRs. The relative binding free energy differences from the TI calculations reveal that the decrease in van der Waals interactions of APV with mutated PRs relative to the wild-type PR mainly drives the drug resistance. This result is in good agreement with the previous experimental results and is also consistent with the results from MM-PBSA calculations. Analyses based on molecular dynamics trajectories show that these mutations can adjust the shape and conformation of the binding pocket, which provides main contributions to the decrease in the van der Waals interactions of APV with mutated PRs. The present study could provide important guidance for the design of new potent inhibitors that could alleviate drug resistance of PR due to mutations