Electronic Theses and Dissertations (PhDs)

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    In Silico Exploration of Endocannabinoid Receptor–CB1 and CB2–Interactions Comparing Cannabidiol and Cannabidiol Diacetate: A Comprehensive Computational Study
    (University of the Witwatersrand, Johannesburg, 2024-09) Soobben, Marushka; Achilonu, Ikechukwu Anthony; Sayed, Yasien
    In the rapidly evolving field of cannabinoid research, acetylated phytocannabinoids such as cannabidiol diacetate (CBDDA) have shown prominence due to its enhanced effects compared to its natural counterpart, cannabidiol (CBD). Despite the growing popularity in the consumption of acetylated phytocannabinoids, in-depth research on its pharmacological impact, especially on CB1 and CB2 receptors, remains scarce. With rising reports of adverse reactions to acetylated phytocannabinoids, a molecular understanding of their interaction with endocannabinoid receptors (CBRs) is imperative. This study aimed to fill this knowledge gap by analysing receptor interactions of CBDDA in comparison with receptor interactions of CBD. The study showed that CBDDA forms stronger interactions with CBRs than CBD. Recognised for its heightened potency, the potential of CBDDA as a biopharmaceutical product was examined. CBR interactions with known endocannabinoids, agonists and inverse agonists validated the computational models used to determine the difference in conformational dynamics upon ligand binding. In this work, bioinformatics, molecular docking, and molecular dynamics (MD) simulations were used to determine the structural differences of CBRs when bound to CBD/CBDDA. Simulations in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and water environment successfully mimicked physiological conditions. Subsequent high-throughput virtual screening (HTVS) was conducted using CBDDA as a reference where ligands 142730975 and 21568811 were identified as the top scoring hits for CB1 and CB2 receptors, respectively. The identification of these ligands via HTVS highlights the therapeutic potential of targeting CBRs and the biopharmaceutical potential of CBDDA. This study elucidates the specific interactions of CBD and CBDDA with CB1 and CB2 receptors, laying a foundation for assessing the safety and efficacy of acetylated phytocannabinoids. Overall, the differential interaction of CBDDA compared to CBD with CBRs suggests that acetylation changes the conformational dynamics of CBRs thereby potentially affecting signalling. The identification of ligands 142730975 and 21568811 as strong interactors with the receptors may provide valuable leads for the development of new cannabinoid-based therapies.
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    Genome sequencing of the Southern Ground Hornbill (Bucorvus leadbeateri)
    (University of the Witwatersrand, Johannesburg, 2024-10) Patel, Jasmin Bharatkumar; De Maayer, Pieter; Mollett, Jean
    The Southern Ground Hornbill (SGH – Bucorvus leadbeateri) is one of the largest hornbill species worldwide, known for its complex social structures and breeding behaviours. These birds, endemic to Africa, have been of great concern due to their declining populations and disappearance from historic ranges. Despite being the focus of numerous conservation efforts, with research forming an integral part of these initiatives, there is a lack of knowledge regarding the molecular biology aspects of this bird species. This study bridges the gap by presenting the first whole genome sequence of the SGH. The SGH genome was further explored using comparative genomics, genetic variant, and selection analysis, providing deeper insights into the evolution and adaptation of this species. Chapter 1 comprehensively reviews pertinent literature on various aspects of avian evolution, including the role genomics has played in elucidating how these species have adapted and evolved. Furthermore, the current body of knowledge on the SGH is explored. In Chapter 2 the entire genome sequence of the SGH was sequenced using Illumina short-read and Pacific BioSciences long-read datasets. Subsequently, the performance of various assembly approaches was evaluated to attain a high-quality assembly of the SGH. This was coupled with parameter optimisation and reference-based refinement to improve the SGH draft genome assembly. The final draft genome assembly was structurally annotated, providing insight into the genetic blueprint underpinning the SGH. Chapter 3 presents the comparative genomic analysis of the SGH with the genomes of available hornbill species from the genera Bucorvus (Bucorvus abyssinicus and SGH) and Buceros (Buceros bicornis and Buceros rhinoceros subsp. silvestris). This included analysis of the pangenome of the hornbill species, functional characterisation of the core and genus-specific elements of the pan-genome and analysis of orthogroups with evidence of paralogy. In Chapter 4, a species-level comparative genomic analysis of the SGH and the Abyssinian Ground Hornbill (AGH) was performed. Here differences in the species-specific proteome of the two species were analysed and the functional implications of these differences on the adaptation and survival of these species were evaluated. Furthermore, genetic variations between the SGH and AGH were identified and selection analysis of key protein-coding genes with high-impact variants was undertaken. This provided insight into the genetic diversity between the SGH and AGH. Finally, the implications of the study on the understanding of the genetic basis underlying the evolution and adaptation of the SGH is discussed and the future perspective of large-scale population genetic studies is provided.
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    Immunomodulation of the innate immune system: The role of vitamin D in the context of monocytes and macrophages
    (University of the Witwatersrand, Johannesburg, 2024-07) Mol, Bronwyn Ashleigh; Gentle, Nikki; Meyer, Vanessa
    Macrophages are widely distributed cells of the innate immune system with essential roles in homeostasis and disease. Despite concerted efforts, several aspects of macrophage origin, biology, and functionality remain poorly understood. To gain a deeper understanding of these cells, a physiologically relevant, but practical model is required. In vitro, macrophages are principally generated from primary monocytes and monocyte-like cell lines through a natural process referred to as monocyte-to-macrophage differentiation. Monocyte-like cell lines have several practical advantages over the use of primary monocytes with the most commonly employed monocyte-like cell lines being THP-1 and U937 cells. Despite their frequent use, no standardised protocol is employed in the differentiation of monocyte-like cell lines to macrophages. Naturally, this results in large discrepancies and a lack of comparability between studies. Furthermore, many of these protocols are not physiologically relevant and produce macrophages that are not responsive to downstream stimuli. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the biologically active form of vitamin D3, is a recognised immunomodulator that shows pronounced genomic and non-genomic effects in immune cells. It is also reported as an inducer of monocyte-to-macrophage differentiation, though heavily debated, and a potential macrophage polarisation agent. Despite this, there is relatively little information concerning the role of 1,25(OH)2D3 in monocyte-to-macrophage differentiation and macrophage biology. This study aimed to develop a more physiologically relevant differentiation protocol for the monocyte-like THP-1 and U937 cell lines. This model was then used to investigate the role of 1,25(OH)2D3 in monocyte-to-macrophage differentiation and macrophage biology. Assessment of morphological features and the macrophage markers, CD11b and CD14, indicated that in both THP-1 and U937 cells, differentiation induced using a combination of 5 nM of phorbol 12-myristate 13-acetate (PMA) and 10 nM 1,25(OH)2D3 over 96 hours produced the most mature macrophages. It was observed that 1,25(OH)2D3 alone was not capable of inducing differentiation, yet when combined with PMA, greatly enhanced macrophage features. THP-1 cells are the most widely employed monocyte-like cell line, and are proposed to be the most reflective of primary monocytes. In this study these cells were shown to be more responsive to the effects of 1,25(OH)2D3 than their U937 counterparts. As such, RNA-sequencing was used to explore the efficacy of the proposed differentiation protocols and the influence of 1,25(OH)2D3 on macrophage biology in THP-1 cells. Differential gene expression analysis confirmed that the most effective differentiation protocol was the combination of 5 nM PMA with 10 nM 1,25(OH)2D3 when considering macrophage associated features including transcription factor usage, adhesion, phagocytosis, and cytokine and cytokine receptor expression. This protocol also produced THP-1-derived macrophages that showed increased expression of genes considered to be primary macrophage markers. These results also suggested that THP-1 cells differentiated with neither PMA nor PMA with 1,25(OH)2D3 were likely to represent fully polarised macrophages. 1,25(OH)2D3 treatment of THP-1 monocytes and THP-1-derived macrophages produced distinct gene expression profiles with considerably less overlap than expected. Though 1,25(OH)2D3 treatment often affected similar biological processes in both cell types, the genes within these processes found to be differentially expressed in each cell line were often distinct. For example, in THP-1- derived macrophages, but not THP-1 monocytes, 1,25(OH)2D3 treatment resulted in the increased expression of genes encoding numerous antibacterial peptides, several small GTPases and their regulators. Additionally, several type I interferon response related proteins showed decreased expression, while expression of cytokines and cytokine receptors was variable. This, taken together with the morphological work, indicates two potential roles for 1,25(OH)2D3 in macrophages. Firstly, a protective role as it suggests the potential to prime an antibacterial response, while still balancing inflammatory responses and protecting against autoinflammation induced by aberrant type I interferon response. Secondly, a potential role in determining the morphological features, clearly demonstrated through microscopy, and further suggested by the differential expression of a variety of small GTPases and their regulators.
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    Investigating 2-hydroxypropyl-β-cyclodextrin (HPβCD) as a novel therapeutic agent for breast cancer
    (University of the Witwatersrand, Johannesburg, 2019) Saha, Sourav Taru; Kaur, Mandeep
    Cancer cells have an increased need for cholesterol, which is required for cell membrane integrity. Cholesterol accumulation has been described in various malignancies including breast cancer. Cholesterol has also been known to be the precursor of estrogen and vitamin D, both of which play a key role in the histology of breast cancer. Elevated cholesterol levels have been linked to breast cancer therefore depleting cholesterol levels in cancer cells can be a viable strategy for treatment. 2-hydroxypropyl-β-cyclodextrin (HPβCD) is a cholesterol depleting compound which is a cyclic amylose oligomer composed of glucose units. It solubilizes cholesterol and is proven to be toxicologically benign in humans. This led us to hypothesise that it might deplete cholesterol from cancer cells and may prove to be a clinically useful compound. Our work provides experimental evidences to support this hypothesis. We identified the potency of HPβCD in vitro against two breast cancer cell lines: MCF7 (Estrogen positive, ER+), MDA-MB-231 [Triple negative breast cancer (TNBC)], and compared the results against two normal cell lines: MRC-5 (Normal Human Lung Fibroblasts) and HEK-293 (Human embryonic kidney) using cytotoxic, apoptosis and cholesterol based assays. HPβCD treatment reduced intracellular cholesterol resulting in significant breast cancer cell growth inhibition through apoptosis. The results hold true for both ER+ and TNBC. We have also tested HPβCD in vivo in MF-1 mice xenograft model and obtained 73.9%, 94% and 100% reduction in tumour size for late, intermediate and early stage TNBC. These data suggest that HPβCD can prevent cholesterol accumulation in breast cancer cells and is a promising anti- cancer agent
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    Insights into silver(I) phosphine complexes in targeting cell death and metastatic mechanisms in malignant cell lines
    (University of the Witwatersrand, Johannesburg, 2023-09) Roberts, Kim Elli; Engelbrecht, Zelinda; Cronjé, Marianne J.
    Cancer is the leading cause of death worldwide, with 18.1 million new cases and 9.6 million deaths reported annually. Cisplatin, a popular chemotherapeutic drug, exhibits certain limitations in terms of selectivity and efficacy. This emphasizes the necessity for novel therapeutic approaches in addressing a variety of cancer types. Multiple studies have shown that silver-based compounds suppress cancer cell proliferation and induce apoptosis. Thirteen novel silver(I) mono-dentate phosphine complexes were investigated for their anticancer effects on seven different human malignant cell lines; A375 non-pigmented melanoma, A549 lung adenocarcinoma, HEP-G2 hepatocellular carcinoma, HT-29 colorectal adenocarcinoma, MCF-7 and MDA-MB-231 breast adenocarcinoma, and SNO oesophageal squamous cell carcinoma. Two non-malignant human cell lines, HEK-293 embryonic kidney cells and MRHF foreskin fibroblast cells, were used to assess the selectivity of the complexes. Cisplatin and the efficient silver(I) phosphine complexes were selected for dose-response experiments to determine IC50 concentrations for the respective cell lines. On the basis of these screening results (chapter two), five difficult-to-treat cancer cell lines, and their most efficient complexes were selected for further investigation. Various cellular characteristics were investigated in chapter three (A549, HEP-G2, HT-29); these included morphological changes, ATP levels, GAPDH levels, Ptd-L-Ser externalization, mitochondrial membrane potential, oxidative stress levels, and the activity of a metabolic enzyme, cytochrome P450 isoform CYP1B1. The antimetastatic activity of the selected complexes was assessed by evaluating their ability to impede the migration of A549 cells. The fourth chapter examines the anticancer effect of selected complexes on hormone-dependent (MCF-7) versus triple-negative (MDA-MB-231) breast cells. Changes in morphology, Ptd-L-Ser externalization, alterations in mitochondrial membrane potential, oxidative stress levels, cytochrome c release, and DNA damage were studied. Furthermore, in chapter five, molecular docking simulations were used to determine whether the most potent silver(I) phosphine complex across all cell lines bonds to estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β). Seven of the thirteen silver(I) phosphine complexes significantly reduced cell viability in malignant cell lines while being less toxic to non-malignant cells. Complex 4 best targeted all cancer types, with IC50 values ranging from 5.75 to 10.80 µM across malignant cell lines. In the malignant treated cells, morphological changes, reactive oxygen species production, mitochondrial membrane depolarization, and Ptd-L-Ser externalization were observed. Complexes 1 and 4 repressed cell migration in the A549 cells. The presence of damaged nuclei, metabolically inactive mitochondria and cytochrome c translocation from the mitochondria’ intermembrane to the cytosol in MCF-7 cells were observed. These findings suggest that complexes 2, 4 and 7 induced apoptotic cell death. Furthermore, in silico computational predictions suggested a promising interaction between complex 4, and ER-α and ER-β. Overall, this study demonstrates the potential of silver(I) phosphine complexes as anticancer agents, with promising effects on various cancer cell lines.
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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.
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    Evaluating the in vitro anti-metastatic effects of silver(I) phosphine complexes on malignant breast cancer cell lines
    (University of the Witwatersrand, Johannesburg, 2023-08) Ferreira, Mizan; Engelbrecht, Zelinda; Cronje, Marianne Jacqueline
    Breast cancer is the most diagnosed cancer type among females worldwide. Metastasis, the spread of cancer cells from the primary tumour and establishment of macroscopic secondary tumours, is regarded as the most dangerous characteristic of cancer cells as it is responsible for over 90% of cancer-related deaths. Globally there is a lack of drugs available to specifically target or prevent either the dissemination of cells from the primary tumour or the establishment of distant metastases. The purpose of this study was to ascertain whether a series of silver(I) phosphine complexes, which have previously been shown to display anti-cancer properties in vitro, are also effective as anti-metastatic compounds. The migration, invasion and adhesive abilities of two malignant breast cancer cell lines, MCF-7 and MDA-MB-231, in response to silver(I) phosphine treatment were evaluated. In addition, the colony-forming abilities of cells under both anchorage-dependent and -independent conditions were investigated. Furthermore, the effects of silver(I) phosphine treatment on the expression and activities of key metastatic proteins, matrix metalloproteinases (MMPs), were studied. Of the nine complexes evaluated, all of them showed the ability to reduce one or more metastatic steps namely cell migration, invasion through collagen towards a chemoattractant or adhesion to collagen. In addition, a selected number of complexes reduced the colony-forming abilities of MCF-7 and/or MDA-MB-231 cells in culture plates as well as in soft agar. Moreover, three of these complexes increased the in vitro invasion and colony formation of breast cancer cells. Further investigation into complexes showing anti-metastatic abilities revealed that, apart from one complex on MDA-MB-231 cells, anti-metastatic effects were not achieved through a reduction in MMP levels or activities. The findings presented here show the potential for silver(I) phosphine complexes to reduce the in vitro metastatic abilities of breast cancer cells, warranting further investigations into these complexes for their use as anti-metastatic drugs.
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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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    Investigating FOXP2 dynamics, stability, and DNA-binding capabilities
    (University of the Witwatersrand, Johannesburg, 2024) Perumal, Cardon; Fanucchi, Sylvia
    All forkhead box (FOX) transcription factors share a conserved DNA-binding domain called the forkhead domain. They regulate gene expression in different organisms and have widespread biological roles, ranging from embryogenesis to immune regulation. The FOXP subfamily, unlike other FOX transcription factors, have the capacity for dimerisation, an evolutionary fate yet to be fully understood. In particular, the FOXP2 forkhead domain (FHD) has been shown in vitro to form domain-swapped dimers. Additionally, the FOXP2 leucine zipper domain forms heterotypic associations with FOXP1, 2, and 4. These somewhat isolated structural characterisations of FOXP2 have informed the domain-specific functionality of the DNA-binding forkhead domain but the leucine zipper domain has been characterized to a lesser extent, although it is thought to be implicated in FOXP2 DNA-binding as well. Elucidating the structural and functional impact of the leucine zipper on FOXP2 DNA-binding remains challenging, as it is unclear how these motifs work together to achieve binding and whether complexation is a requirement for DNA-binding. Owing to this, the cooperative structural contributions of both the leucine zipper and forkhead domain and the effect of DNA on the structure and stability of these domains have not been considered. Consequently, the aim of this study was to gain a comprehensive understanding of the FOXP2 DNA-binding mechanism by comparing the structure, stability, and dynamics of both the leucine zipper domain and the forkhead domain in the presence and absence of DNA. Assessed here, for the first time, is the conformational dynamics of the FOXP2 leucine zipper domain and FHD flanking disordered regions using hydrogen-deuterium exchange mass spectrometry. The results confirm the binding of DNA to the FHD recognition helix as well as the change in dynamics of the interlinking loop region. Additionally, the FOXP2 folding mechanisms and stability for each domain was characterised, revealing a 2-state unfolding mechanism for the forkhead domain and a 3-state mechanism for the longer LeuZip variant. Fluorescence anisotropy studies revealed that the LeuZip variant bound DNA with a higher affinity than the forkhead domain. The findings of this study highlight the structural significance of the leucine zipper domain and unstructured regions and the functional cooperativity of the FOXP2 domains investigated
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    Computational modeling approaches to validate the druggability of the 26- and 28-kDa Schistosoma glutathione transferase enzymes using bromosulfophthalein as a benchmark ligand
    (University of the Witwatersrand, Johannesburg, 2024) Valli, Akeel
    PHARMACOPHORE MODELS are 3-D representations of the chemical and spatial features required for interaction with a drug target. These. models offer advantages in early-phase drug design by expediting screening experiments and enabling the sampling of highly specific chemical. space subsets, such as those containing quality drug-like candidates. The glutathione transferase enzyme of Schistosoma spp. (SGST) has been identified as an attractive drug target for the novel treatment of human schistosomiasis. We observed selective inhibition of SGST by bromosulfophthalein. Bromosulfophthalein was found to complex with SGST at a drug binding site in the target dimer interface, providing a suitable benchmark for the design of discriminative SGST pharmacophores. The aim of this research is to construct, deploy and evaluate pharmacophore models of the SGST drug binding site. The objectives are: to characterise the SGST drug binding site, to develop the pharmacophore models and finally to evaluate the drug-resolving ability of the models. We observed significant differences in the drug-binding character of SGST, compared to human glutathione transferase (hGST) counterparts, particularly that SGST supports binding of phenol and sulfonate moieties. Five- and four feature pharmacophores were developed for the respective 26- and 28 kDa SGST variants. Finally, the models demonstrated remarkable ability to retrieve candidates displaying drug-like qualities. In conclusion, we characterised and developed pharmacophore models of the drug binding domains from two major SGST variants. Assessment of drug-resolving power validates the capability of the models to sample drug-like chemicals. Altogether, these accomplishments enable efficient and reliable screening toward novel drug treatment for human schistosomiasis