published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

Non-nucleoside reverse transcriptase inhibitor (NNRTI) is one of the key components of antiretroviral drug regimen against human immunodeficiency virus type-1 (HIV-1) replication. However, the low genetic barriers to drug-resistance or cross-resistance, side effects, as well as the unaffordable cost of NNRTIs compromise their clinical usage. Therefore, to develop novel NNRTIs with potent antiviral activity against HIV-1 becomes a major concern in the treatment and prevention of HIV/AIDS. (+)-Calanolide A, which is a natural product initially extracted from the tropical rainforest tree Calophyllum lanigerum, was identified as an attractive NNRTI against HIV-1 despite virus strains containing drug-resistant K103N/Y181C mutations. In this study, a chemical library was constructed based on the three chiral carbon centers of (+)-Calanolide A. After screening the activity against HIVNL4-3 wild-type and several NNRTI-resistant pseudoviruses, a small molecule 10-chloromethyl-11- demethyl-12-oxo-calanolide A (F18) was identified as novel NNRTI with promising anti-HIV efficacy. Further studies were performed to investigate the antiviral breadth, drug resistance profile and underlying mechanism of the action of F18. F18 consistently displayed a potent activity against primary HIV-1 isolates including various subtypes of M group, CRF01_AE, and laboratory-adapted drug-resistant viruses in PBMC based assay. Moreover, F18 displayed distinct profiles against 17 NNRTI-resistant pseudoviruses, with an excellent potency especially against one of the most prevalent strains with the Y181C mutation (EC50=1.0nM) in cell line based assay, which was in stark contrast from the extensively used NNRTIs nevirapine and efavirenz. F18-resistant viruses were induced by in vitro serial passages, and mutation L100I was appeared to be the dominant contributor to F18-resistance, further suggesting a binding motif different from nevirapine and efavirenz. The efficacy of F18 was non-antagonistic when used in combination with other antiretrovirals against both wild-type and drug-resistant viruses in infected PBMCs. Interestingly, F18 displayed a highly synergistic antiviral effect with nevirapine against nevirapine-resistant virus (Y181C). Furthermore, in silico docking analysis suggested that F18 may bind to the HIV-1 reverse transcriptase in a way different to other NNRTIs. For the potential as an anti-HIV-1 microbicide, F18 also showed the stable and rapid release, as well as the sustained antiviral activity against HIV-1 wild-type virus in a formulation temperature-sensitive acidic gel. In summary, this study presents F18 as a new potential drug for clinical use and also underlies new mechanism-based design for future NNRTI. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

The human mitochondrial DNA (mtDNA) genome must be faithfully maintained by the mitochondrial DNA replication machinery. Deficiencies in mtDNA maintenance result in the accumulation of mutations and deletions, which have been associated with a number of neuromuscular degenerative disorders including, mtDNA depletion syndrome, Alpers syndrome, progressive external opthalmoplegia (PEO), and sensory ataxic neuropathy, dysarthria, and opthalmoparesis (SANDO). The mtDNA replication machinery is comprised of a nuclearly-encoded DNA polymerase gamma (Pol γ), single-stranded DNA binding protein (mtSSB), and a hexameric mtDNA helicase. In this work, we employed quantitative pre-steady state kinetic techniques to establish the mechanisms responsible for the replication of the human mitochondrial DNA by Pol γ and explored the effects of point mutations that are observed in heritable diseases. With our biochemical characterization of mutants of Pol γ, we have shown unique characteristics that would lead to profound physiological consequences over time. Additionally, we have made significant progress towards reconstitution of the mitochondrial DNA replisome by monitoring DNA polymerization that is dependent on helicase unwinding of double stranded DNA. Overall, this work provides a better understanding of the mechanism of mtDNA replication and has important implications toward understanding the role of mitochondrial DNA replication in mitochondrial disease, ageing and cancer. In addition to the work on the mtDNA replisome, we have applied pre-steady state kinetic techniques to better understand the mechanism of RNA-dependent DNA polymerization by HIV reverse transcriptase (HIV-RT). This enzyme is responsible for the replication of the viral genome in HIV and is a common target for anti-HIV drugs. We have characterized the role of enzyme conformational changes in the kinetics of incorporation of correct nucleotide and the Nucleotide Reverse Transcriptase Inhibitor (NRTI) AZT by wild-type enzyme, as well as a mutant with clinical resistance to AZT. This work provides a better understanding of the complete mechanism of RNA-dependent DNA polymerization, the changes in the mechanism in the presence of inhibitor and the development of resistance to this nucleoside analog; and thereby this work contributes to the long-term goal of designing more effective drugs that can possibly deter resistance and be used successfully for treatment of HIV. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

No description available.

There is an increasing number of patients failing second line highly active antiretroviral therapy (AZT, DDI and LPV/r) in South Africa, where HIV-1 subtype C predominates. Mutations at gag cleavage sites (CS) have been found to correlate with resistance mutations in protease (PR). Therefore, it is important to collect data on subtype C protease and gag sequences from patients as these mutations may affect the efficacy of protease inhibitor (PI) containing drug regimens. In this study, 30 subtype-C infected second-line failures were genotyped using the ViroSeqTM resistance genotyping kit and the gag region from these isolates were then characterised. These sequences were then compared to 30 HIV-1 subtype C infected first-line failures (PI-naïve) and subtype B, C and group M naïve sequences that were downloaded from the Los Alamos Sequence Database. Amino acid diversity at the CS was measured using Mega version 4.0. To investigate the effect of CS mutations on replication capacity, a mutation was introduced by site-directed mutagenesis (Stratagene’s QuikChange Site-Directed Mutagenesis kit). Of the 30 second-line failures that we genotyped, only 16 had resistance mutations in PR and 23 in gag. The most frequent major PI mutations were: I54V/L, M46I, V82A, and I84V and in gag CS were V390L/I and A431V. Interestingly the A431V mutation significantly correlated with protease mutations M46I/L, I54V and V82A. The virus carrying the A431V mutation in vitro was found to have a lower replication capacity compared to the wild type. These findings emphasize the need for further investigation of gag mutations and their contribution to the evolution of HIV resistance to PIs. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2010.

No description available.

The mitochondrial small-subunit ribosomal RNA (mt. SSU rRNA = rns) gene appears to be a reservoir for a number of group I and II introns along with the intron- encoded proteins (IEPs) such as homing endonucleases (HEases) and reverse transcriptases. The key objective for this thesis was to examine the rns gene among different groups of ophiostomatoid fungi for the presence of introns and IEPs. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent loses. Some of the novel findings of this work were the discovery of a twintron complex inserted at position S1247 within the rns gene, here a group IIA1 intron invaded the ORF embedded within a group IC2 intron. Another new element was discovered within strains of Ophiostoma minus where a group II introns has inserted at the rns position S379; the mS379 intron represents the first mitochondrial group II intron that has an RT-ORF encoded outside Domain IV and it is the first intron reported to at position S379. The rns gene of O. minus WIN(M)371 was found to be interrupted with a group IC2 intron at position mS569 and a group IIB1 intron at position mS952 and they both encode double motif LAGLIDADG HEases referred as I-OmiI and I-OmiII respectively. These IEPs were examined in more detail to evaluate if these proteins represent functional HEases. To express I-OmiI and I-OmiII in Escherichia. coli, a codon-optimized versions of I-OmiI and I-OmiII sequences were synthesized based on differences between the fungal mitochondrial and bacterial genetic code. The optimized I-OmiI and I-OmiII sequences were cloned in the pET200/D TOPO expression vector system and transformed into E. coli BL21 (DE3). These two proteins were biochemically characterized and the results showed that: both I-OmiI and I-OmiII are functional HEases. Detailed data for I-OmiII showed that this endonuclease cleaves the target site two nucleotides upstream of the intron insertion site generating 4 nucleotide 3’overhangs.

Age-related macular degeneration (AMD) is a principal cause of blindness in the United States and other industrialized nations. An estimated 10 million Americans are afflicted with AMD, which is comparable in scope to the 12 million living with cancer, or the 5 million with Alzheimer’s disease. The prevalence of AMD steadily increases with age, affecting 2% of the population at age 40, and one in four people by age 80. For reasons that are not fully understood, AMD is more common in lightly-pigmented and female populations. Treatment of AMD is largely an unmet need: There are no FDA approved therapies except for a small percentage of individuals with end-stage disease. This dissertation investigates the mechanisms of AMD pathogenesis and offers insight into novel therapeutic strategies for this disease.