Electronic Theses and Dissertations (PhDs)

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Browsing Electronic Theses and Dissertations (PhDs) by Keyword "Computational modelling"

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    Elucidating the Structure-Function Relationships of Enterococcus faecium Nicotinate-Nucleotide Adenylyltransferase through X-Ray Crystallography, Computational Modelling and Binding Studies
    (University of the Witwatersrand, Johannesburg, 2024) Jeje, Olamide Adetomi; Pandian, Ramesh; Achilonu, Ikechukwu A.
    Nicotinate nucleotide adenylyltransferase (NNAT) is a vital enzyme at the heart of NAD biosynthesis, catalysing a crucial reaction that leads to the formation of pyridine dinucleotides. NAD+ is an essential coenzyme in numerous metabolic processes, DNA repair, and cellular signalling. Given its pivotal role, NNAT has emerged as a compelling drug target, particularly for its potential to disrupt the survival mechanisms of bacterial pathogens. By inhibiting NNAT, it is possible to undermine the metabolic integrity of these pathogens, making NNAT a promising focal point in the fight against bacterial infections and antibiotic resistance. However, understanding the structure-function relationship of Enterococcus faecium NNAT (EfNNAT) has remained elusive. Hence, this study aimed to address this gap bycharacterising EfNNAT and validating its potential as a druggable target. EfNNAT was overexpressed and purified using the Escherichia coli system and IMAC purification technique. Subsequently, biophysical characterisation was performed, followed by the determination of the three-dimensional structure in both apo and liganded forms using X-ray crystallography. High-throughput virtual screening, along with SP and XP docking, was conducted using a library of synthesizable flavonoids. Molecular dynamic simulation and fluorescence studies were employed to establish and validate the binding of identified inhibitors to EfNNAT. Successful expression and purification of EfNNAT yielded approximately 101 mg per 7.8 g of wet E. coli cells, with a purity exceeding 98%. High-resolution crystal structures of EfNNAT in native, adenine-bound, and NMN-bound forms were determined at 1.90 Å, 1.82 Å, and 1.84 Å, respectively. These structures provided insights into EfNNAT's substrate preference and revealed a potential allosteric site at the dimer interface of the NMN-bound structure. Virtual screening identified quercetin 3-O-beta-D-glucose- 7-O-beta-D-gentiobioside as the only potential inhibitor from the flavonoid library used. A 500 ns atomistic molecular dynamics simulation showed the compound interacted through hydrogen bonding and water bridges, albeit unstable within the receptor. ANS and mant-ATP fluorescence spectroscopy confirmed quercetin binding, while thermal shift assay revealed minimal impact of the inhibitor on the protein stability and structure. This study establishes a pipeline from expression and purification to structure solution and potential inhibitor identification for EfNNAT, validating its druggability. The mechanistic insights offer a foundation for advancing drug discovery efforts targeting EfNNAT and other bacterial NNAT enzymes.
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    The ligandin activity of Schistosoma 26-kDa and 28-kDa glutathione transferases towards 17β-Hydroxyandrost-4-ene-3-one from a biophysical perspective
    (University of the Witwatersrand, Johannesburg, 2023) Makumbe, Hattie Hope; Achilonu, Ikechukwu Anthony
    Schistosomiasis, caused by helminth worms, ranks second amongst parasitic diseases and accounts for over 220 million fatalities globally. Statistics show that in South Africa, schistosomiasis (bilharzia) has infected approximately 4 million individuals. Currently, there are parasite resistance challenges with the sole available remedy. The World Health Organisation (WHO) acknowledges the need for new effective drugs. The 26-kDa Schistosoma bovis/haematobium (Sbh26GST) and 28-kDa Schistosoma haematobium (Sh28GST) are parasite Glutathione S-transferases (GSTs) which consist of two identical subunits that perform a vital role in mitigating the adverse effects of harmful electrophilic substances within the parasite since the parasite is devoid of the neutralizing cytochrome P-450. This automatically renders these parasite GSTs as potential therapeutic targets for schistosomiasis. Testosterone, the major hormone responsible for sexual characteristics and growth in males, can be repurposed as a drug target against schistosomiasis. In this study, we examined the structural, stability and functional interactions between the parasite GSTs and testosterone. After confirmation of inhibition, IC50 experiments were performed. The enzymes were overexpressed in Escherichia coli (E.coli) and then purified through a single-step nickel ion-immobilized metal affinity chromatography (IMAC). Extrinsic fluorescence spectroscopy was also done to provide evidence for the binding of the recombinant GSTs with testosterone. The GST activity was measured by employing 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate. Additionally, we investigated if the enzyme activity was influenced by the presence of testosterone. To analyse the stability of the enzymes, a SYPRO Orange-based thermal shift assay was used in the presence and absence of testosterone. In addition to empirical investigations, computational modelling, molecular docking, and molecular dynamic simulations were used to provide complementary insights to show binding affinities, prediction of binding modes and stability of the GST-testosterone complex. The secondary structural composition was found to be predominantly alpha-helical. Insights into tertiary structure analysis revealed the presence of buried solvent exposed tryptophan residues. The findings from spectroscopy with 8-anilino-1-naphthalenesulfonate (ANS) indicated that both Human GST-mu and parasite GSTs bound to ANS. Enzyme kinetic studies show that testosterone is a potent inhibitor of the parasite GSTs, with a specific activity that decreases from 16 μmol min-1mg-1 to 0.03 μmol min-1mg-1 and IC50 in the nanomolar range of 20 µM for Sh28GST. Sbh26GST exhibited a specific activity that decreased from 20 μmol min-1mg-1 to 0.14 μmol min-1mg-1, and a testosterone IC50 of 23 µM. The thermal stability assay confirmed Sh28GST to be more stable than Sbh26GST, and this stability of Sh28GST intensified when the enzyme bound to testosterone and GSH. Steady state kinetics towards glutathione (GSH) revealed a Km of 4.2mM and 6.6 mM for Sh28GST and Sbh26GST respectively. The present study has practical implications for novel application of the enzymes to serve as a basis for future studies aimed at development of inhibitors with potential therapeutic benefits through rational drug design.