Electronic Theses and Dissertations (PhDs)

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Browsing Electronic Theses and Dissertations (PhDs) by SDG "SDG-3: Good health and well-being"

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    Biophysical studies of metal chelate binding by HSA: Towards an understanding of metallodrug transport
    (University of the Witwatersrand, Johannesburg, 2023) Sookai, Sheldon; Munro, Orde
    Human serum albumin (HSA) is the most abundant blood protein, transporting many exogenous compounds including clinically deployed and investigational drugs that are generally organic in nature. HSA may largely influence the pharmacokinetics and pharmacodynamics of these drugs. Therefore, studying their interactions with HSA is vital in progressing drug development. In this thesis we present work on the synthesis and characterisation of five Schiff base bis(pyrrolide-imine) ligands that were metalated with either Au(III) (Chapters 2 and 3) or Pt(II) (Chapters 4 and 5). One of the ligands H2L1 was further metalated with Ni(II) and Pd(II) (Chapter 6). In Chapters 2 and 3 focus on a patented class of anti-cancer bis(pyrrolide-imine) Au(III) Schiff base chelates. Three Au(III) chelates were synthesized in Chapter 2 and underwent National Cancer Institute (NCI)-60 cytotoxic screening. Among them, AuL1 and AuL3 underwent full-five dose testing and recorded GI50 values of 7.3 µM and 11.5 µM, and IC50 values of 15.7 µM and 30.9 µM, respectively. AuL1 was tested further and found to be an interfacial poison of topoisomerase II at 0.5–5 µM and a catalytic inhibitor at 50 µM. In Chapter 3, two chiral tetradentate cyclohexane-1,2-diamine-bridged bis(pyrrole-imine) Au(III) complexes were reported, both of which were found to be cytotoxic in the NCI-60 screen. The chiral Au(III) chelates had a different mode of action compared to AuL1. Hierarchical cluster analysis suggest that their mode of action is similar to that of taxol. All five Au(III) chelates bound to HSA with moderate affinity (104–105 M–1) and minimally perturbed the structure of the protein. This highlights the potential for the Au(III) complexes to be transported by the HSA-mediated pathway. Chapters 4 and 5 focused on the synthesis of novel and previously reported Pt(II) Schiff base chelates to spectroscopically and computationally study their interaction with HSA and elucidate if the chelates could act as theranostic agents. It was found that switching the linking bis(imine) carbon linkage altered the binding affinity of the complex. However, the Pt(II) ion ensured that all three Pt(II) chelates preferred binding to Sudlow’s site II of HSA. The data was corroborated by molecular docking simulations and ONIOM calculations. Only 2 was found to be cytotoxic when irradiated with UV light but was found to act as a photosensitizer rather than a theranostic agent. Chapter 6 investigated the influence of d8 metal ions (Ni(II), Pd(II) and Pt(II) within the same ligand scaffold (H2PrPyrr) binding to HAS, which was investigated by steady state fluorescence quenching. The affinity constants, Ka, ranged from -3.5 -103 M−1 to-1- 106 M–1 at 37 C, following the order Pd(PrPyrr) > Pt(PrPyrr) > Ni(PrPyrr) >H2PrPyrr. The Pd(II) chelate was prone to hydrolysis and had a unique binding mode which we attribute to the unusually high binding affinity. The complexes uptake is enthalpically driven, hinging mainly on London dispersion forces. In summation, twelve metal complexes were successfully synthesized, of which 11 bound to HSA with a moderate binding affinity. The Au(III) chelates preferred Sudlow’s site I, while the Pt(II) chelates preferred Sudlow’s site II. Overall, the metal complexes bound fully intact to HSA.
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    Design and synthesis of chronic wound healing collagen peptide mimics
    (University of the Witwatersrand, Johannesburg, 2024) Lesotho, Ntlama Francis
    The South African wound care management market is expecting a compound annual growth rate (CAGR) of 6.75%. The numbers are expected to further increase because South Africa has the highest number (4.6 million) of people living with diabetes in Africa. Annually approximately 2% of patients with diabetes develop diabetic foot ulcers and hence chronic wounds. Many chronic wound patients must deal with the financial burden, as many current wound treatment options are expensive, ineffective, and inconvenient. Intervention in the form of synthetic collagen mimetic peptides has been limited due to cytotoxicity and susceptibility to protease degradation. These challenges have, for an ardent time affected the clinical and commercial development of synthetic wound healing peptides. The aim of the current study is to develop novel wound healing peptides by derivatizing bioactive peptides into selective and protease stable peptidomimetics. All the synthesized peptides are meant to mimic the function of collagen type I. Thus, the designed peptides comprise of the retro- integrin binding type I collagen motif, -GFOGER-, the DGD tripeptide for attraction of growth factors, the retro- tripeptides Thr-Thr-Lys (TTK), Gly-His-Lys (GHK), Gln-Pro-Arg (QPR) and Glu-Glu-Met (EEM) to stimulate collagen production. The importance of collagen is evidenced by the fact that it features in all four stages of wound healing. This therefore means, its inclusion in any biomaterial meant to curb chronicity in wound healing is indispensable. With this approach, the biomaterial would overcome the challenge of excess matrix metalloproteinases (MMPs), which degrade both viable and nonviable collagen used in the wound healing process. It would further provide a collagen-based wound scaffold that compensates for the loss of collagen required for proper tissue regeneration. The applications of collagens in wound healing are immense. Due to its material properties, and apparent effectiveness, collagen has the potential to be utilized as an unprecedented treatment protocol for chronic, slow-healing wounds. Sixteen palmitate and adamantane collagen mimetic peptides were designed and successfully synthesized using the solid-phase peptide synthesis strategy. Eight of the sixteen peptidescomprise of lipophilic moieties (adamantane and palmitic acid) for improved membrane permeability and different collagen inducing retro-tripeptides namely, TTK, GHK, QPR and EEM (retro-DGD-GG-GFOGER-GG-TTK-Adamantane (NL010)/palmitate (NL009), retro-DGD- GG-GFOGER-GG-GHK-Adamantane/palmitate, retro-DGD-GG-GFOGER-GG-QPR- Adamantane/palmitate and retro-DGD-GG-GFOGER-GG-EEM-Adamantane/palmitate). Another eight are control peptides without the retro-tripeptides (retro-DGRGOF- Adamantane/palmitate, retro-GOP-GFOGER-GOP-Adamantane/palmitate, retro-GG- GFOGER-GG-Adamantane/palmitate and retro-DGD-GG-GFOGER-GG-Adamantane (NL008)/palmitate). The tertiary structure and secondary features (folding patterns) of the peptides were determined using the Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD). From NMR experiments, medium-range couplings were detected for NL010 and NL009, suggesting a possibility of alpha helices. Temperature 1H NMR experiment for the peptide DGRGOF- Adamantane proved the presence of cis and trans geometric isomers. CD experiments revealed that NL009 mainly has α-helix while NL010 mainly consists of a parallel conformation. Synthesis of adamantane and palmitate peptides with enhanced integrin binding was accomplished by incorporation of para-fluorophenylalanine in place of phenylalanine in the peptide retro-GG-GFOGER-GG-Adamantane/palmitate. The peptides were obtained in low yields but with increased hydrophobicity. Structural features for the improvement of the stability of the peptides against protease degradation were accomplished by the synthesis of peptoids and N-methylated peptides. The peptoids were synthesized in low yields but with increased hydrophobicity. The efficacy of NL009 and NL010 in wound healing was tested both in vitro and in vivo. In the former, the efficiency of both NL009 and NL010 in inducing migration of cells in a scratch wound was accentuated by hyaluronic acid. In in vivo studies, NL010 performed better than NL009. However, NL010 was outperformed by a comparator, Puramatrix® The peptides have the ability to induce migration of cells and therefore have an ability to create an environment needed for proper wound healing. The peptides could be used in place of native collagen and bring about proper healing of wounds
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    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, Manoko
    The 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.
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    Synthesis, characterization and investigation of the mode of action in the anticancer activity of novel platinum complexes
    (University of the Witwatersrand, Johannesburg, 2024) Peega, Tebogo; Harmse, Leonie; Kotzé, Izak. A.
    Cancer remains a global health concern, causing approximately 10 million deaths in 2020. Lung cancer, accounting for 18% of cancer-related deaths, and colorectal cancer, contributing 9.4%, are major contributors to this alarming statistic, emphasizing the urgent need for innovative and effective treatment options. Despite the success of platinum-based drugs such as cisplatin, carboplatin, and oxaliplatin, their limitations and severe adverse effects necessitate the exploration of alternative chemotherapeutic agents. This research project focused on synthesizing and characterizing square planar platinum(II) complexes bearing variations of two bidentate coordinating ligands; disubstituted acylthiourea and diimine ligands, each possessing unique physical and chemical properties. A series of cationic [Pt(diimine)(Ln-κO,S)]Cl complexes were successfully synthesized and characterized using nuclear magnetic resonance spectroscopy, infrared spectroscopy, mass spectrometry, and elemental analysis. The anticancer activity of these complexes was evaluated against two lung cancer cell lines, A549 and H1975, and a colorectal cancer cell line, HT-29. In vitro cytotoxicity studies included the determination of IC50 values of active complexes and assessing their cell death mechanisms through multiple biochemical marker assays. These included annexin-V binding, caspase-3/7 and caspase-8 activity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and immunofluorescence for the expression of key proteins involved in the DNA damage response and oxidative stress response, such as p21 and haemoxygenase-1 (HO-1). A proteome array was employed to investigate the effects on apoptosis-associated proteins. The results indicated that these platinum complexes were more cytotoxic than cisplatin with IC50 values ranging between 0.68 μM and 2.28 μM. Further investigation showed that the platinum complexes induced cell stress, chromatin condensation, nuclear fragmentation, increased phosphatidylserine (PS) on the outer cell membranes and activated caspase-3/7. Platinum complexes induced intrinsic apoptosis in cancer cells, as evidenced by the loss of mitochondrial membrane potential and the absence of caspase-8 activity. Elevated ROS levels, increased HO-1 expression and increased expression of p21 suggested oxidative stress and DNA damage as the trigger source for intrinsic apoptotic cell death. The active complexes downregulated pro-survival proteins (IGFs) in lung cancer cells and anti-apoptotic proteins (survivin and HSP70) and upregulated pro-apoptotic proteins (p21, TRAIL R2), across the three cancer cell lines, indicating potential dual activation of apoptotic pathways. DNA binding studies indicated groove binding and intercalation as the mode of interaction with DNA. The findings highlight the potential of these platinum complexes as promising candidates for further development as cancer therapeutics.
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    The Design, Synthesis and Structure-Activity Relationship of Antitubercular Lassomycin Derivatives
    (University of the Witwatersrand, Johannesburg, 2023) Ngqinayo, Ntombizanele; Makatini, Maya
    Tuberculosis (TB) is a potentially fatal infectious disease caused by Mycobacterium tuberculosis (Mtb) and is a global health risk responsible for over 1.5 million deaths worldwide annually. Tuberculosis is treated with a combinatory regimen of approved first- line drugs such as rifampicin and isoniazid as well as second-line anti-TB drugs such as fluoroquinolones, most of which use similar mechanisms to cause cell death. The formation of multidrug resistance (MDR) TB strains, biofilms, and dormant persister cells (non- replicating cells) are some factors that prolong TB treatment and hence the need for developing novel antitubercular agents with a different mode of action. Furthermore, the emergence of multidrug resistance TB poses a challenge in controlling and eradicating tuberculosis. Lassomycin is a novel antimicrobial peptide (AMP) that has garnered much interest across various research groups due to its ability to effectively target and kill Mtb, including MDR strains and latent TB, with a potency that is similar to that of rifampicin. Lassomycin is highly basic and targets the highly acidic N-terminal domain (NTD) of the caseinolytic enzyme that forms part of the caseinolytic protease crucial for Mtb cell survival. Lassomycin has an unusual mode of action that causes Mtb cell death by disrupting the highly controlled and tightly regulated proteolysis by inhibiting proteolytic activities as well as increasing unfoldase activities. Thus, lassomycin shows great potential as a candidate for drug development. This study aimed to design lassomycin derivatives with improved stability and potency; and synthesize them using shorter and cost-effective synthetic routes. Peptide modifications includes (i) replacing the macrolactam ring in the peptide sequence with a disulfide bridge via a simpler ring-formation method resulting in an enlarged cyclic ring; (ii) replacing ‘difficult’ arginine residues with less basic lysine residues; (iii) forming cationic derivatives by increasing the number of basic lysine residues to enhance selectivity for the bacterial membrane; (iv) conjugating peptide derivatives to lipophilic molecules including palmitic acid and 1-adamantane carboxylic acid to improve bacterial cell penetration and binding; (v) conjugating the peptides to silver nanoparticles for improved drug delivery and antimicrobial effect; (vi) incorporating N-methylated residues to improve peptide stability; (vii) making non-polar peptide derivatives by replacing all basic amino acids with alanine to investigate the importance of the basic residues and study structure activity relationship (SAR) (viii) synthesizing linear derivatives in order to investigate the effect of the ring and (ix) shortening the peptide sequences to include only the cyclic ring or the tail sequence portions in order to shorten the synthetic route. Peptides were synthesized via the Fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis strategy (SPPS) and purified using a semi-preparative High-Performance Liquid Chromatogram (prep-HPLC). They were then analysed using High-Performance Liquid Chromatography Mass Spectrometry (HPLC-MS), Circular Dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Silver nanoparticles and the peptide conjugates were characterized using ultraviolet-visible (UV-Vis) spectrophotometry and transmission electron microscopy (TEM) imaging. Two-dimensional (2D) Nuclear magnetic resonance (NMR) spectroscopy, including [1H, 1H] COSY, [1H, 1H] TOCSY, [1H, 13C] HSQC, [1H, 1H] HMBC and [1H, 1H] ROESY were used to determine the structural conformation of Pep-2- NN, a lassomycin derivative that has activity against tuberculosis. Furthermore, the secondary structure of selected derivatives was examined using circular dichroism (CD) spectroscopy. Computational studies were utilized to determine the structure of the active lassomycin derivatives, Pep-2-NN and Pep-2-NNA. All the peptide derivatives were successfully synthesized, including non-polar, short-chained, and those conjugated to silver nanoparticles and lipophilic molecules. The disulfide bridge was successfully added to replace the lactam bridge of the parent lassomycin peptide by oxidising sidechain thiol groups of two cysteine residues inserted at appropriate positions in the sequences. All the peptides were purified to varying degrees of success, and their behaviour was analysed to investigate structure-activity relationships. The silver nanoparticles were successfully synthesized in-house and conjugated to Pep-2-NN. Transmission Electron Microscopy (TEM) imaging revealed that the silver nanoparticles have a spherical morphology at sizes that ranged between 7 nm and 9 nm whilst peptide conjugated nanoparticles were between 9 – 12 nm. Caseinolytic protease (ClpP1P2 or ClpP) assay studies revealed that the peptides display inhibiting and activating properties when screened against the protease, including lassomycin derivatives with shortened chains such as Ring-2-NNA-Ada, Ring-2-NN, and Tail-2-NN. The secondary structure of selected lassomycin derivatives was studied using circular dichroism (CD), revealing that the structures are comprised of anti-parallel beta- (β) sheets at slightly higher proportions followed by alpha- (α) helix and, to some extent, β-turn motif. Computational studies were conducted on selected derivatives to predict their secondary structure and revealed that the peptides form stable α-helical conformations. NMR revealed that Pep-2-NN formed a ‘knotted’ structure, where the tail sequence was threaded inside the cyclic ring with a curved loop, and certain residues in the ring acted as ‘steric plugs’ to prevent unthreading. In conclusion, the insertion of the disulfide bridge remains an effective alternative to the lactam bond found originally on lassomycin and can result in the formation of biologically active derivatives with the desired stable ‘lasso’ conformation.