Electronic Theses and Dissertations (PhDs)
Permanent URI for this collectionhttps://hdl.handle.net/10539/38017
Browse
12 results
Search Results
Item 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, YasienIn 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.Item 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, VanessaMacrophages 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.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 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 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.Item 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 JacquelineBreast 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.Item Investigating FOXP2 dynamics, stability, and DNA-binding capabilities(University of the Witwatersrand, Johannesburg, 2024) Perumal, Cardon; Fanucchi, SylviaAll 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 investigatedItem 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, AkeelPHARMACOPHORE 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 schistosomiasisItem A Phenotype Prediction Framework for Classifying Colorectal Cancer Patients’ Response to FOLFOX Treatment: An Integrated Approach(University of the Witwatersrand, Johannesburg, 2024) Mashatola, Lebohang; Kaur, MandeepColorectal cancer (CRC), characterised by its prevalence and heterogeneity, poses a significant challenge in understanding drug resistance, especially in the context of FOLFOX treatment. This study presents an innovative methodology that integrates diverse data analysis approaches to address the challenge of predicting the phenotype of CRC patients resistant or sensitive to FOLFOX. The initial analysis involved dierential and co-expression analyses, identifying pivotal hub genes crucial to drug resistance in CRC, regulating intricate molecular networks. Subsequent enrichment analysis revealed their significant roles in biological functions, particularly influencing DNA repair and nuclear division. To capture inherent topological characteristics within genetic expression data, a novel technique utilising topological data analysis (TDA) was employed. By applying persistence homology to generate persistence images, the Vietoris-Rips complex was constructed using the signed-topological overlap matrix, comprehensively capturing numerous topological features, including high-dimensional Betti-1 and Betti-2. This provided valuable insights into the structural patterns of gene expression between the hub genes. Furthermore, the integration of whole-slide images enhanced understanding of tissue anatomy, which is crucial for predicting cancer stages. Using a MobileNet architecture, a deep learning model classified cancer stages, contributing to a holistic understanding of colorectal tumor microenvironments. For predictive modelling of drug resistance, a multilayer perceptron applied topological summaries generated by TDA. The developed framework, GeTopology, exhibited remarkable performance metrics, achieving an overall 83% accuracy in predicting the FOLFOX response, demonstrating a 3% improvement over a previously published phenotype prediction framework (NSCLC ) that utilised similar data modes. Robust accuracies were consistently observed in independent datasets, classifying both cancer patients and healthy individuals. The results indicated an approximate 10% increase in model prediction accuracy compared to NSCLC, emphasising the potential clinical impact of this integrative approach. In conclusion, this study advances the understanding of drug resistance in CRC by proposing a novel approach that integrates topology with histopathological images, oering transformative insights into predictive modelling and precision medicineItem Identification and characterisation of the interaction between FOXP2 and the ligand binding domain of oestrogen receptor α(University of the Witwatersrand, Johannesburg, 2023-10) Lakhi, Aasiya Yakub; Fanucchi, SylviaForkhead box P2 (FOXP2) regulates the expression of various genes and is associated with language and speech, neural development and outgrowth, and cancer. As transcription factors rarely function in isolation, this study aims to investigate whether FOXP2 directly associates with oestrogen receptor α (ER1), a nuclear receptor responsible for sexual differentiation and cancer progression and outcome. The association between ER1 and FOXP2 was first identified in MCF-7 cells using co-immunoprecipitation. Thereafter, the interaction was characterised biophysically by overexpressing the FOXP2’s DNA-binding forkhead domain (FHD) and N-terminal region (NT), and ER1’s ligand-binding domain (LBD) in E. coli cells. Isothermal titration calorimetry and fluorescence anisotropy were used to investigate the thermodynamic parameters and regulation of interaction between FOXP2 FHD and ER1 LBD, respectively. Electrophoretic mobility shift assay was used to investigate the effect of the interaction on FOXP2’s DNA binding ability. Following the successful overexpression and purification of all three proteins, ER1 LBD was found to interact with FOXP2 FHD but not with FOXP2 NT. The affinity of the ER1 LBD for FOXP2 FHD increases with an increase in salt concentration. ITC shows a similar trend and reveals that the interaction is enthalpically favoured at lower salt concentrations but enthalpically opposed at higher salt concentrations. Additionally, the FOXP2-ER1 LBD interaction remains unaffected by the inclusion of oestrogen, but addition of FOXP2 cognate DNA results in inhibition of the formation of the complex. This research identifies a novel interaction between ER1 LBD and FOXP2 FHD and shows that the DNA simultaneously suggesting a probable role of this interaction in regulating the transcriptional pathway of FOXP2. This study serves as a foundation for further investigation into the interaction between FOXP2 and ER1 in different cell lines and its relevance in FOXP2-mediated outcomes in cancer and neurodevelopmental disorders.