Faculty of Science (ETDs)
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Item Investigating 2-hydroxypropyl-β-cyclodextrin (HPβCD) as a novel therapeutic agent for breast cancer(University of the Witwatersrand, Johannesburg, 2019) Saha, Sourav Taru; Kaur, MandeepCancer 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 agentItem Characterising the Role of Cholesterol in Hypoxia-induced Epithelial- Mesenchymal Transition in Breast Cancer(University of the Witwatersrand, Johannesburg, 2022) Abdulla, Naaziyah; Kaur, MandeepThe cellular epithelial-mesenchymal transition (EMT) process is a complex labyrinth dependent on subversion of critical cellular signalling pathways, which crosstalk extensively to confer cancer cells with characteristics that mediate metastasis. Based on the pleotropic role of cholesterol in the cell, it is not surprising that cancer cells have evolved several mechanisms to facilitate cholesterol dyshomeostasis. In addition to meeting the increased metabolic demands of cancer cells, deregulated cholesterol metabolism also facilitates increased cellular cholesterol availability which is crucial to regulating the activity of protein intermediates in EMT-related signalling pathways. Despite evidence indicating that cholesterol directly regulates signalling pathways related to EMT, no publication to date has attempted to address the effect of EMT induction on cellular cholesterol levels in cancer. To shed light on the dynamics of cholesterol in the relationship between hypoxia and EMT, cholesterol content in MCF-7 cells pre- and post-hypoxia induced EMT was assessed. This dissertation presents findings indicating increased levels of free cholesterol, cholesteryl esters as well as lipid raft cholesterol in MCF-7 cells following hypoxia-induced EMT. Interestingly, MCF-7 cells post- EMT induction displayed increased sensitivity to treatment with cholesterol targeting agents and presented with reversion to an epithelial state as evidenced by the increased expression of epithelial markers, decreased expression of mesenchymal markers and also reduced invasive potential. Importantly, treatment with cholesterol targeting agents is also seen to abrogate the drug resistant potential following hypoxia-induced EMT. Based on these observations, it is proposed that targeting cellular cholesterol could be a promising area to invest in the search for novel therapeutics effective in combatting cancer metastasisItem 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.Item Antibacterial activity and susceptibility testing of bacterial isolates from nematodes (Cruznema spp.)(University of the Witwatersrand, Johannesburg, 2023-09) Mothapo, Maletjema Magdeline; Lephoto, Tiisetso E.Nematodes are unsegmented worms found in different niches associated with a diverse range of bacteria. Various types of nematodes exist including those that are parasitic to insects, known as entomopathogenic nematodes (EPNs). EPNS of genera Steinernema, Heterorhabditis and Oscheuis are symbiotically associated with Xenorhabdus, Photorhabdus and Serratia, respectively. The symbiotic bacteria of EPNs have been reported to produce a broad spectrum of antimicrobial compounds active against human pathogens. The aim of this study was to isolate and identify nematodes and their associated bacteria from soil samples collected from a vegetative farm in Lesotho and study their antimicrobial activity against four species of pathogenic bacteria (E. coli, S. aureus, E. faecalis and P. aeruginosa). An uncharacterized species of Cruznema was isolated and named Cruznema NTM-2021 (GenBank 18S rDNA accession number: OQ408141). Based on the BLASTN search incorporating the phylogenetic analysis of the 16S rDNA region, three genera of bacteria were identified as Alcaligenes sp., Enterobacter sp. and Elizabethkingia sp. The study revealed that all three bacterial isolates were pathogenic to Tenebrio molitor. Symbiosis tests, using lipid agar method demonstrated the ability of the host nematodes to develop and reproduce in the presence of their associated bacteria. Bacterial supernatants of Alcaligenes sp. and Enterobacter sp. showed some inhibitory activity against Escherichia coli and Enterococcus faecalis, by disk diffusion method. Staphylococcus aureus and Pseudomonas aeruginosa were the most resistant bacteria to supernatants of the three isolates. This study also showed that the Alcaligenes, Enterobacter, and Elizabethkingia species isolated from Cruznema NTM-2021 were resistant to ampicillin, amoxicillin, cefuroxime/sodium, vancomycin and cephalothin but susceptible to gentamicin.Item Detecting and Understanding COVID-19 Misclassifications: A Deep Learning and Explainable AI Approach(University of the Witwatersrand, Johannesburg, 2023-08) Mandindi, Nkcubeko Umzubongile Siphamandla; Vadapalli, Hima BinduInterstitial Lung Disease (IDL) is a catch-all term for over 200 chronic lung diseases. These diseases are distinguished by lung tissue inflammation (Pulmonary fibrosis). They are histologically heterogeneous dis eases with inconsistent microscopic appearances, but they have clinical manifestations similar to other lung disorders. The similarities in symptoms of these diseases make differential diagnosis difficult and may lead to COVID-19 misdiagnosis with various types of IDLs. Be cause the turnaround time is shorter and more sensitive for diagnosis, imaging technology has been mentioned as a critical detection method in combating the prevalence of COVID-19. The aim of this research is to investigate existing deep learning architectures for the aforementioned task, as well as incorporate evaluation modules to determine where and why misclassification occurred. In this study, three widely used deep learning architectures, ResNet-50, VGG-19, and CoroNet, were evaluated for detecting COVID-19 from other IDLs (bacterial pneumonia, nor mal (healthy), viral pneumonia, and tuberculosis). The baseline results demonstrate the effectivities of Coronet having a classification performance of 84.02% for accuracy, specificity of 89.87%, a sensitivity of 70.97%. Recall 84.12%, and F1 score of 0.84. The results further emphasize the effectiveness of transfer learning using pre-trained domain-specific architectures, resulting in fewer learnable parameters. The proposed work used Integrated Gradients (IG), an Explainable AI technique that uses saliency maps to observe pixel feature importances, to understand mis classifications. This refers to visually prominent features in input im ages that were used by the model to make predictions. As a result, the proposed work envisions future research directions for improved classi fication through misclassification understanding.Item HIV-1 subtype C protease: enzyme kinetics, thermodynamics, and X-ray crystal structure(University of the Witwatersrand, Johannesburg, 2023-09) Dlamini, Nozinhle Precious; Sayed, YasienHuman immunodeficiency virus (HIV), a precursor for AIDS is still one of the most devastating pandemics in history. In 2021 alone there were 650 000 deaths associated with the virus and the number of people living with the infection was recorded to be 38.4 million globally. Sub-Saharan Africa suffers the most burden of the virus with approximately 8.3 million people living with virus, HIV-1 subtype C is the main driver of the disease in South Africa and accounts for 46% of global infections. Even with these alarming statistics this subtype is not the main focus point for the majority of HIV-1 research which mainly focuses on subtype B though it only accounts for 12% of infections globally. There is no vaccine or cure against HIV; however, great strides have been made in suppressing the virus. Viral suppression drugs have been developed to target different stages of viral replication such as those targeting the three important enzymes (protease, reverse transcriptase and integrase). In this study the focus will be on HIV-1 subtype C protease. This is a homodimeric aspartyl protease with 99 amino acids in each monomer. It plays a crucial role in the replication cycle of HIV-1 by producing mature infectious virions through cleavage of the Gag and Gag-Pol polyproteins. The subtype C protease differs from subtype B protease in that it has eight naturally occurring polymorphisms which are substitution mutations, some occurring in different regions of the protease with some in the fulcrum (T12S, I15V and L19I), others in the hinge region (M36I and R41K), with H69K and L89M found in the loops and I93L in the α helix. In this study, structural and functional characterisation of HIV-1 subtype C protease was carried out. The secondary structure was characterised using far-UV CD, which is a technique that measures the difference in left and right circularly polarised light. The subtype C protease was estimated to be predominantly β-sheeted, with spectra showing a maximum at 195 nm and a minimum between 215-225 nm. Tertiary structure characterisation of protease was performed using fluorescence spectroscopy. The maximum emission at 347 nm close to that of water (350 nm), demonstrated that the tertiary conformation of the HIV-1 protease was conserved, and that the tryptophan residues within the protease are solvent exposed. SE-HPLC was used to characterise the quaternary structure of the protease and the homodimeric size was determined to be approximately 22 kDa. Steady-state enzyme kinetics to assess the catalytic activity of the subtype C protease was performed using a fluorogenic substrate. The activity of the enzyme was confirmed, with the specific activity of 24.22±1.72 µmol. min-1.mg-1 and the binding of the substrate to the HIV-1 protease was demonstrated by the KM value of 79.546±6.491 µM. This correlates to literature indicating that the substrate was weakly bound and that a high substrate concentration will be required to reach the maximum velocity (Vmax), and Vmax was determined to be 0.036±0.003 µmol. min-1. Enzyme kinetics was coupled with displacement isothermal titration calorimetry for determination of thermodynamics parameters using second generation PIs (atazanavir, darunavir and lopinavir). Thermodynamic studies indicated that the HIV-1 protease has a high affinity for LPV (Kd = 1 nM), compared to ATV (Kd = 18.57nM) and DRV (Kd = 42.26 nM) and binding reactions were all spontaneous with ΔG values(ATV = -43.39 kJ/mol, DRV = -41.39 kJ/mol and LPV = -50.51 kJ/mol). The values also indicated that LPV complexed with HIV-1 is more a stable complex. Also, all the binding reactions were exothermic as indicated by the negative ΔH values of ATV = -45.54 kJ/mol, DRV = -55.62 kJ/mol and LPV = -54.71 kJ/mol. The entropy of all the reactions were determined to be unfavourable with the -T∆S of DRV = 14.23 kJ/mol followed by LPV: 4.2 kJ/mol and ATV: 2.15 kJ/mol. Overall this suggested that all the binding reactions were enthalpically driven. Furthermore, the three-dimensional structure of the HIV-1 subtype C protease was elucidated using X-ray crystallography. The three-dimensional structure the HIV-1 CSA (PDB ID: 8CI7) was solved at a 2.4 Å resolution which is better than the 2.7 Å (PDB ID: 3U71) initially solved in our lab. The high-resolution three-dimensional structure of the protease will provide precise information about the arrangement of atoms within the protease molecule, thus enabling the design and development of protease inhibitors that will be specific for the subtype C protease. This study emphasised the significance of investigating subtype C protease in the context of enzyme kinetics, thermodynamics and detailed X-ray crystallography.Item Synthesis of carbon nanodots-peptide conjugates decorated with germanium for bioimaging(University of the Witwatersrand, Johannesburg, 2023-10) Machumele, Khanani Peggy; Makatini, Maya Mellisa; Maubane-Nkadimkeng, ManokoThe World Health Organization Global Cancer Observatory estimates that cancer caused 9.96 million deaths worldwide in 2020, making early detection crucial for diagnosis and treatment. Accurate identification of cancer plays a crucial role in the diagnosis and treatment process. It allows for customized and efficient therapies, minimizes unnecessary procedures and adverse effects, and improves the prognostic insights for patients and healthcare providers alike. The challenges in diagnosis include overdiagnosis, false positives/negative outcomes, and limited sensitivity. Advanced technologies are needed for better imaging accuracy and minimizing harm. This study aims to fabricate carbon dot-peptide conjugates to enhance bio-imaging capacity and selectivity. The peptides used are derived from the GKPILFF cell-penetrating peptide sequence and the RLRLRIGRR peptide, which is selective to cancerous cells. The Carbon dots were used to provide the photoluminescent properties required for bio-imaging of cancerous cells. Carbon dots (CDs) were synthesized using iso-ascorbic acid as the source of carbon using a microwave-assisted method. The nitrogen and germanium-modified carbon dots (Iso-N-Ge-CDs) demonstrated the highest photoluminescent properties compared to the unmodified CDs (Iso-CDs) and those with either N (Iso-N-CDs) or Ge (Iso-Ge-CDs). Photoluminescence emissions of longer wavelengths suitable for cell imaging were observed for the CDs, and the Iso-N-Ge-CDs demonstrated excitation-dependent emission wavelength behavior, pH sensitivity, and Fe3+ sensitivity. The 13 peptides derived from the peptide accelerating sequence GKPILFF and the cancer-selective peptide RLRLRIGRR were successfully synthesized. The peptides were characterized using Liquid Chromatography Mass Spectrometry (LCMS) and purified using preparative High-Pressure Liquid Chromatography (prep-HPLC). The secondary structure of the L-GKPILFF penetration acceleration peptide sequence (Pas) adopted a helical secondary structure. The D-GKPILFF derivative was found to adopt a random coil structure. These were confirmed using Nuclear Magnetic Resonance (NMR) techniques such as Total Correlation Spectroscopy (TOCSY) and Rotating Frame Overhauser Enhancement Spectroscopy (ROESY) NMR. The CDs-peptide conjugates were successfully synthesized, and the confirmation of conjugation involved multiple methods, including UV-Vis and PL techniques. To the best of our knowledge, the thesis incorporates the first study to demonstrate long-range interactions through ROESY NMR. The NMR analysis indicated that the helical structure of the peptide could be affected after conjugation, leading to notable peak shifts. Since the helical structure is crucial for the peptide's bioactivity and stability enhancement, NMR spectra with fewer structural changes in the peptide region may improve its biological properties. The research contained valuable information for scientists aiming to design and characterize Carbon dot-peptide conjugates with enhanced permeability and selectivity that can effectively deliver materials into cytosolic space.Item The political geography of hemp Cannabis in South Africa: A development critique(University of the Witwatersrand, Johannesburg, 2023-06) Moore, Wendell; Mnwana, Sonwabile; Wafer, AlexThe discourse on Cannabis that is generally understood in the public domain, revolves mostly around its recreational uses and more recently its medicinal potential. The plants industrial usages and overall worth to society are often framed in relation to these more popular parts of Cannabis. The study focuses its attention on hemp Cannabis to tell a different story but acknowledges that all the uses of Cannabis are interlinked. Using an extensive desktop analysis, Cannabis webinars and conferences, as well as autoethnography, the thesis shows that there is in fact more to the plant than what first meets the eye. It especially illuminates two important dimensions of Cannabis that are often overlooked when discussing the plant. Firstly, by its nature Cannabis is political, which is deeply embedded in how the plant is understood in the geographic diaspora of the Global South. Secondly, it shows that any serious social scientific analysis of contemporary Cannabis development must think through how the ways of knowing, produced during prohibition, will become a part of the plant’s future. This thesis therefore argues for an appraisal and maintenance of the historical resistances of Cannabis used prior to legalization, if its development in the legal era is to be successful. To be sure, such innovative and alternative models of practical development should become a standard part of the geographies of all work economies. Moreover, making use of these forms of resistance is not about Cannabis gaining the approval from ‘the market’. In fact, the skills learnt while labouring for Cannabis, when it was prohibited, has become such a permanent feature of its development that liberal democratic consent is not an option. Therefore, the thesis proposes that Cannabis development delink from the prohibition narratives inscribed onto the plant. It continues by showing that the current medicalisation of Cannabis only updates gatekeeping models. Hence, an agrarian alternative was suggested that consolidates all the uses of Cannabis without undermining any one part of the plant. Lastly, the thesis documents the possibilities of gaining access into the industry for black people, the poor working class and women. What it argues is that ownership, just employment and leadership of Cannabis industries in South Africa must be repurposed so that ordinary people can also play a part in making sure the plants contemporary development is truly sustainable. Taken together, this is the critique of development that the political geography of hemp Cannabis in South Africa reveals.Item The synthesis of aryl benzamides as potential HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs)(University of the Witwatersrand, Johannesburg, 2023-07) Mohasoa, Likhopotso Cecilia; Zimuwandeyi, Memory; Bode, Moira L.Dihydro-alkoxybenzyloxopyrimidines are heteroaryl-containing compounds that have previously been shown to exhibit excellent activity against HIV-1 reverse transcriptase (RT) enzyme. In our own laboratory, 2-chloro-N-(6-(piperidin-1-yl)pyridin-2-yl)benzamide was identified as a compound with activity against wild-type HIV-1. Using these two structural types as a guide, as part of our ongoing studies to search for anti-HIV therapeutic agents that target the RT enzyme, a library of arylbenzamide compounds bearing a pyrimidine ring as a central core was synthesized. These compounds contained an oxygen linker to allow flexible rotation of the molecule in the RT active site, with the aim of achieving activity against wild-type and mutant HIV-1. As a starting point, in order to first identify a suitable synthetic method and then apply it for our target novel compounds, four different carboxylic acids and two classes of amines were tested. Amidation reactions were carried out on unsubstituted benzoic acid, 3-methoxybenzoic acid, 3-hydroxybenzoic acid, and 3-((2,6-dichloropyrimidin-4-yl)oxy)benzoic acid. In this last case, the 3-hydroxybenzoic acid moiety had already been linked to the pyrimidinyl core in order to test which order of reaction worked best: linking followed by amidation, or the reverse. Reaction of these benzoic acid derivatives with anilines and aminopyridines gave the resulting benzamides in 22-99% yields after optimization. When triethylamine was used as a base in amidation reactions involving 2-amino-3-bromopyridine, 2-amino 5-bromopyridine and 2-amino-5-methylpyridine, diacylation was favoured, while when pyridine was used, monoacylation predominated. The reactions to link benzoic acid derivatives to the pyrimidinyl core were carried out by displacement of chlorine on 2,4,5-trichloropyrimidine. The displacement of the first chloride was tested using three types of nucleophiles. The first nucleophile was methyl 3-hydroxybenzoate, effectively a protected benzoic acid, which afforded methyl 3-((2,6-dichloropyrimidin-4-yl)oxy)benzoate in 81% yield. Problems with subsequent hydrolysis of the ester made this route impractical. The second nucleophile was 3-hydroxybenzoic acid which provided 3-((2,6-dichloropyrimidin-4-yl)oxy)benzoic acid in 81% yield. The third nucleophile was N-(5-bromopyridin-2-yl)-3-hydroxybenzamide, where amidation had already been performed, which transformed into the desired compound N-(5-bromopyridin-2-yl)-3- ((2,6-dichloropyrimidin-4-yl)oxy)benzamide in 28%. The low yield obtained from reaction of the amidated nucleophile identified the most promising route to be linking of 3-hydroxybenzoic acid to 2,4,5-trichloropyrimidine first, followed by amidation. After the successful displacement of the first chlorine atom, two of the resulting analogues 3-((2,6-dichloropyrimidin-4-yl)oxy)-N-(p-tolyl)benzamide and N-(4-bromophenyl)-3-((2,6-dichloropyrimidin 4-yl)oxy)benzamide were functionalized with sulfur and nitrogen nucleophiles by displacement of a second chlorine atom. Ethanethiol proved to be highly nucleophilic, leading to pyrimidine C-O bond cleavage and sulfur disubstitution, while the nitrogen ucleophiles propylamine and piperidine afforded their corresponding derivatives in good yields without breaking the carbon-oxygen bond. The newly coupled propyl compound was further derivatized by means of hydrolysis with sodium hydroxide to yield the desired novel 3-((6-hydroxy-2-(propylamino)pyrimidin-4-yl)oxy)-N-(p tolyl)benzamide or 3-((6-oxo-2-(propylamino)-1,6-dihydropyrimidin-4-yl)oxy)-N-(p-tolyl)benzamide compound.Item 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 AnthonySchistosomiasis, 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.