3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Structural and enzymatic studies of HIV-1 subtype C protease(2019) Shabangu, BonginkosiHuman immunodeficiency virus (HIV) is a retrovirus that infects T-lymphocytes in the human immune system causing a condition known as Acquired Immunodeficiency Syndrome (AIDS). At the moment, there is an estimated 1.2 million people dying annually from diseases related to AIDS and ~ 36.7 million people are currently infected with HIV across the globe. There are 10 subtypes found within the major group of HIV-1 and subtype C overwhelmingly drives the South African epidemic and accounts for more than 50% of the world infections. The continuous infection and maturation of HIV-1 is yielded by the three viral enzymes; namely, reverse transcriptase, integrase and protease. HIV-1 relies on the catalytic efficacy of the protease enzyme for cleaving precursor polyproteins to yield structural and functional proteins. When HIV-1 protease is inactivated, either by an inhibitor or mutagenically, the virion will remain non-infectious. Thus, the HIV-1 protease is an opportune drug target. It is an important variant in the study of the pathogenesis, treatment and prevention of HIV-1 infections. The study intended to characterise and evaluate the catalytic activity of HIV-1 South African subtype C protease which exhibits high variability compared to subtype B protease. For characterisation properties, far-UV circular dichroism, intrinsic fluorescence spectroscopy and size exclusion high-performance liquid chromatography were used to evaluate secondary, tertiary and quaternary structure, respectively. Secondary structure results indicated a trough at 216 nm which means the protein is predominantly β-sheeted. Using intrinsic tryptophan as a probe, the tertiary structure of protease revealed that the local structural environment had not been perturbed and it was indicated by fluorescence emission intensity peak at 355 nm. The results of the size exclusion-high performance liquid chromatography study revealed that the dimeric molecular size of the wild-type protease was 22 kDa. The proteolytic efficiency of the subtype C wild-type protease was evaluated following the hydrolysis of a fluorogenic substrate resembling the CA/p2 cleavage site in the gag-pol polyprotein precursors. The KM was determined as 42.07 μM, Vmax was 0.047 μmol.min-1, specific activity was 76.46 μmol/min/mg, kcat was 24.02 s-1 and kcat/KM was 0.084 s-1μM-1. In the presence of darunavir, the Ki value for protease was determined as 3.4 nM, for saquinavir it was 6.2 nM and for ritonavir it was 9.4 nM. The protease inhibitor GRD 110D had a Ki of 27 nM, showing high susceptibility compared to the other three FDA-approved HIV protease inhibitors (PIs). A molecular docking study was carried out to analyse and compare the binding mode of the FDA-approved HIV PIs. The docking results indicated that the protease recognition site is hydrophobic due to the two-flexible glycine-dense β-sheets and that subtype C protease (PDB code: 3U71) polymorphisms result in an altered flap-hinge region, thus making it less susceptible to inhibitors as compared to subtype B protease (PDB code: 2P3B). The results of the comparative binding mode analysis of all the FDA-approved drugs could be useful for the design of new potent inhibitors of HIV-1 protease.Item Prevalence of dyslipidaemia in HIV-infected children and adolescents treated with protease inhibitors(2018) Sipambo, NosisaBackground – HIV infection and antiretroviral therapy (ART) are associated with dyslipidaemia in children. Protease inhibitor (PI) based regimens, in particular, have shown the highest association. Methods – We conducted a retrospective study of children treated with either a first- or second-line lopinavir/ritonavir (LPV/r) regimen who had any lipid tests done from 2004 to 2015. Dyslipidaemia was defined as hypercholesterolaemia [total cholesterol ≥5.13mmol/l (≥200mg/dl)] and /or hypertriglyceridaemia [total triglycerides ≥1.69 mmol/l (≥150mg/dl)]. There were 4 cross sectional points of analysis in this study: ART start, LPV/r-start, 12 and 24 months after starting LPV/r. Demographic and clinical characteristics were compared using univariate and multivariate analyses to determine risk factors for dyslipidaemia at each time point using logistic regression to obtain odds ratios and 95% confidence intervals (95% CI). Results - Few children had lipids measured over the follow-up period, increasing from 7% (146/2145) at ART initiation to 24% (365/1522) after 24 months on LPV/r. The median age at ART-start was 1.6 (0.4; 4.4) increasing to 3.6 (2.6; 6.2) years by 24 months. The majority (51%) of the children had severe immune suppression at ART-start. The prevalence of dyslipidaemia at ART-start was 47%, decreasing to 36% at 24 months. Multivariate analysis at 12 months found that children less than 10 years of age with near suppressed viral loads (viral load < 4 logs) were more likely to have dyslipidaemia. At 24 months on LPV/r, ART duration greater than 60 months and high viral loads were protective factors. Conclusion – The high prevalence of dyslipidaemia in young children is concerning as lopinavir/ritonavir is the mainstay of ART in young children for the foreseeable future.Item Antiretroviral drug susceptibility of a hinge region variant of HIV-1 subtype C protease(2018) Zondagh, JakeSince their discovery, protease inhibitors continue to be an essential component of antiretroviral treatment for human immunodeficiency virus type 1 (HIV-1). However, the development of resistance to protease inhibitors remains one of the most significant challenges in the fight for sustained viral suppression in those infected with HIV-1. Studies show that specific mutations arising within the HIV-1 gag and protease genes can lead to the development of resistance. In this research, a South African HIV-1 subtype C Gag-protease variant (W1201i) was investigated. This variant was considered due to the presence of a mutation and insertion (N37T↑V), located within the hinge region of the protease enzyme. Moreover, the variant displayed the following polymorphisms: Q7K, I13V, G16E, M36T, D60E, Q61E, I62V and M89L. Genotyping of W1201i Gag revealed a previously unreported MSQAG insertion between the CA/p2 and p2/NC cleavage sites. Additionally, a mutation and insertion (I372L↑M), and multiple polymorphisms (S369N, S371N, I373M and G377S) were discovered within the p2/NC cleavage site. Single-cycle phenotypic assays were performed to determine the drug susceptibility and replication capacity of the variant. The results show that the mutations present in the N37T↑V protease conferred a replicative advantage and reduced susceptibility to lopinavir, atazanavir and darunavir. Interestingly, the mutations in W1201i Gag were found to modulate both replication capacity and protease inhibitor susceptibility. In silico studies were performed to understand the physical basis for the observed variations. Molecular dynamics simulations showed that the N37T↑V protease displayed altered dynamics around the hinge and flap region and highlighted the amino acids responsible for the observed fluctuations. Furthermore, induced fit docking experiments showed that the variant bound the iv protease inhibitors with fewer favourable chemical interactions than the wild-type protease. Collectively, these data elucidate the biophysical basis for the selection of hinge region mutations and insertions by the HI virus and show that protease, as well as Gag, needs to be evaluated during resistance testing.