Masters Degrees (Biochemistry)

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Intercellular communication between fibroblast phenotypes, macrophages and myoblasts during cellular migration.
(2022) Ramklowan, Dhamini Sanjay Hariduth.; Niesler, Carola Ulrike.
Abstract available in PDF.
Identifying small molecule binding ligands for the pup-ligase (PafA) of Mycobacterium tuberculosis.
(2022) Mthembu, Sandile Mehzi.; Hewer, Raymond.; Delport, Alexandre' Marie Chaplin.
The rapid emergence of resistant TB strains (Mycobacterium tuberculosis (Mtb)) renders traditional treatment options ineffective and necessitates the generation of novel anti-TB drugs that possess innovative modes of action. The pup-ligase (PafA) of Mtb that solely mediates protein proteasomal removal via the pupylation cascade has recently been identified as a suitable target for TB drug development. A novel approach would be to recruit proteolysis targeting chimeras (PROTACs) technology as an alternative anti-TB treatment option by developing PROTAC-like molecules capable of recruiting the pupylation cascade. Therefore, the identification of novel PafA small-molecule binding ligands is an essential first step to establish possible new TB therapies. To this effect, PafA recombinant expression was successfully optimised in E. coli cells at 20°C for 20 h, where a 50-kDa protein was observed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, the identity of the protein was confirmed via immunoblotting with anti-His antibodies and PafA subsequently purified via immobilized metal affinity chromatography (IMAC) to high purity. A thermal shift assay (TSA) of PafA against 48 small-molecule compounds from a chemical library pre-screened for non-specific inhibition activity was conducted. Seven Hit compounds were detected significantly binding PafA (P < 0.05), all inducing a > 5 °C increase of PafA melting temperature (Tm) upon binding. Future research on these novel PafA binding ligands will be to ascertain whether they possess inhibitor qualities. Additionally, they will be used in the synthesis of heterobifunctional molecules to create the first PROTAC-like molecules for targeted proteasomal degradation of essential Mtb proteins – a novel type of anti-TB drug.
Investigation of intravaginal practices as a factor associated with a high prevalence of genital human papillomavirus infection in adolescent girls.
(2021) Mntambo, Ntombenhle.; Gumbi, Pamela Phumelele.; Ngcapu, Sinaye.
Background: Human papillomavirus (HPV) is a common sexually transmitted infection (STI) in women which mainly infects the mucosal areas. Young women are disproportionately infected by HPV, and factors that may render adolescents or younger women more vulnerable to HPV acquisition than older women have not been fully elucidated. This study aimed to investigate the associations between the prevalence of HPV, the use of intravaginal products, the immune activation status of cervical T cells, and alterations of the composition and concentrations of antimicrobial peptides (AMPs) in vaginal fluid among adolescent females close to their sexual debut and older women in KwaZulu-Natal. Methodology: Genital specimens (cervical cytobrush and cervicovaginal swabs) were collected from 154 female participants aged 14-19 and 25-35 years. From cervicovaginal swabs, HPV genotyping was done using a deoxyribonucleic acid (DNA) Flow hybridization system. Flow cytometry was conducted from cervical cytobrush specimens (evaluating CD38+, HLA-DR+, andCCR5+ expressions) to assess T-cell immune activation status. Enzyme-linked Immunosorbent Assay (ELISA) kits were used to measure genital concentrations of human β- defensin (HBD-1, HBD-2), and psoriasin from cervicovaginal swabs. Statistical tests conducted were Robust Poisson regression models (RPRM), the Tukey multiple comparison adjustment, t-test, and Mann Whitney U test. 𝑃 values of <0.05 were statistically significant. Results: HPV prevalence was 85%, with high-risk genotypes being the most prevalent. The majority of the cohort was infected with multiple genotypes (76.62%). Genotypes associated with cancer and current Gardasil®9 HPV vaccine targets were more common (53.9%) than those associated with genital warts (14.9%). The risk of HPV in adolescent females was 15.9% higher than in adult females. The use of vaginal inserted products (VIPs) was associated with a 40% higher risk of contracting genotypes related to cancer (p=0.0503) compared to nonusers. The risk of HPV infection for adolescents using VIPs was 23% higher than that of adults using VIPs. However, these differences were not statistically significant at a 5% level of significance. Sexual debut after 18 years significantly reduced the risk of overall HPV infection (p=0.0040) and infection with genotypes associated with cancer (p=0.0024). When comparing HPV infected adolescents and adults, the proportion of activated CD4+ T cells was significantly higher in adolescents, particularly in CD4+ HLA-DR+ cells (p=0.0008). CD8+ T cells showed no difference. A significantly higher concentration of HBD-2 was observed in HPV+ adults compared to HPV- adults (p=0.0215) and HPV+ adolescents (p=0.0189). Conclusion: The overall HPV prevalence is higher than the previously reported prevalence in KwaZulu-Natal province. In addition, we demonstrate that the use of VIPs may be associated with some HPV infection risk, particularly in adolescent females. This finding suggests that young women should be warned about the potential risk of using VIPs. We also confirm that the age-phase, delay in sexual debut, and the number of sexual life partners have significant associations with HPV genotypes linked to cancer, highlighting the importance and the urgency of vaccinating young girls with Gardasil®9 HPV vaccine. Adolescents with HPV have significantly higher levels of activated CD4+ T cells in their cervical mucosa, suggesting the presence of reactive activated cells that lack efficiency in the clearing of HPV infection in this age group. HPV infection upregulates HBD-2 levels during HPV infection, notably significantly higher in adult females than in adolescents. This investigation has generated new insights into the risk factors for HPV acquisition in young women. These findings need to be confirmed further by larger cohort size studies, essential for HPV prevention.
Biochemical and structural characterization of ClpK from Klebsiella pneumoniae.
(2022) Motiwala, Tehrim .; Khoza, Thandeka.
Abstract available in PDF.
Enzymatic characterisation of a CATL-like protease from Trypanosoma brucei brucei and small-subunit rRNA sequence based phylogenetic analysis of freshwater fish trypanosomes.
(2021) Mthethwa, Bongumusa Comfort.; Coetzer, Theresa Helen Taillefer.; Willows-Munro, Sandi.
Abstract available in PDF.
Identification and analysis of Cryptosporidium Glutathione Transferase.
(2021) Mfeka, Sizamile Mbalenhle.; Khoza., Thandeka Ntokozo.
Cryptosporidiosis, caused by Cryptosporidium spp. is a gastrointestinal disease which gives rise to severe life threatening complications in immunecompromised patients. The disease causing parasite has a proficient defense system against xenobiotic compounds and substances that renders the only drug designed to treat the gastroenteritis infection inefficient in immune compromised patients. This defense system includes a phase II enzyme called Glutathione Transferase (GST) which detoxifies a wide range of oxidant based substrates. The overexpression of this protein in multi drug resistant cases and its presence in multiple stages of the parasites life cycle highlights the parasites dependence and utilization of the GST protein thus making it a suitable therapeutic target. This study was then set out to determine characteristic features of Cryptosporidium GSTs in comparison to well studied GSTs using molecular biology and bioinformatics tools. A genome wide search was performed across multiple protein databases to mine the Cryptosporidium GST. The 15 Cryptosporidium spp. found to possess full length proteins were compared amongst themselves within the species and against other species using phylogenetic analyses. This led to the discovery of three novel classes of Cryptosporidium GST based on amino acid sequence identity. The classes were named Gamma, Psi and Vega GSTs. The GSTs varied in amino acid length, and secondary structure characteristics determined through homology modeling. In comparison to preexisting GSTs, the Psi and Vega class GSTs did not have the typical active site Tyr7 found in most cytosolic GST, furthermore the Vega class GST also did not have the typical thioredoxin like fold conserved in the N-terminal region of all GSTs. The Gamma class GSTs were found to most resemble pre existing GSTs consisting of the typical thioredoxin fold and the active site Tyr7 and thus selected for expression and purification studies. pET, pCOLD1 and pCOLDTF vectors were used to determine a suitable vector to facilitate the expression of a soluble gamma class GST in Escherichia coli. pCOLDTF which utilizes cold shock proteins at low temperatures and a chaperone called trigger factor assisted in the recombinant expression of the gamma class GST resulting in a protein with the monomer size of ~50 kDa, which is double that of existing GSTs. This is owed to by the N-terminal and C-terminal extensions that the protein possesses. The protein was purified to homogeneity using affinity chromatography and size exclusion chromatography. The resulting protein was found to be dimeric under native conditions.
Recombinant cathepsin L-like cysteine proteases for species-specific diagnosis of animal African trypanosomiasis.
(2019) Mbhele, Ziphezinhle Elaine.; Coetzer, Theresa Helen Taillefer.
African trypanosomiasis is a disease caused by protozoan parasites i.e. Trypanosoma spp. in livestock and humans and affects 37 sub-Saharan countries. Animal African trypanosomiasis (AAT) is known as nagana and African human trypanosomiasis (HAT) as sleeping sickness. Trypanosoma congolense, T. vivax and T. brucei brucei cause AAT which is an economic burden and hampers agricultural development in Africa. The parasite escapes the host’s immune response by switching the genes coding for the variable surface glycoproteins, resulting in new variable antigen types. This has made it unlikely to develop a vaccine against the disease and therefore many studies now focus on non-variant trypanosome antigens as potential diagnostic and drug targets. Trypanosomal cysteine proteases, such as the cathepsins B and L, have been identified and validated as potential diagnostic targets. They are expressed throughout the parasite life cycle and are essential for the survival of the parasite. The mammalian host produces an antibody response against trypanosomal cysteine proteases which do not affect the survival of the parasite, however, the antibodies are believed to play a role in trypanotolerance by neutralising the effects of the enzyme. Antigen-based ELISA is a good tool for accurate diagnosis of AAT, but also relies on good antibodies. The overall aim of this study was to produce antibodies against the cathepsin-L-like protease of T. b. brucei (TbbCATL) which can be used for specific diagnosis of T. b. brucei infections. This included polyclonal antibodies as well as single chain variable fragments (scFvs) using phage display. The protease, TbbCATL, was recombinantly expressed in E. coli for the first time as a 61 kDa protease (including the GST tag) using the pGEX-4T expression vector. The homologues from T. congolense (TcoCATL) (29 kDa) and T. vivax (TviCATL) (28 kDa and 32 kDa) were also recombinantly expressed using the P. pastoris yeast expression system and were shown to hydrolyse the Z-Phe-Arg-AMC substrate and to be inhibited by E-64. Antibodies against whole TbbCATL were produced in chickens and together with anti-TcoCATL peptide antibodies and anti-TviCATL antibodies, were evaluated in a western blot to determine possible cross-reactivity. Whereas the anti-TbbCATL antibodies were specific for TbbCATL, the anti-TcoCATL peptide and anti-TviCATL antibodies cross-reacted with TbbCATL. The production of scFvs was optimised using TviCATL as the panning antigen against the Nkuku® phage display library. The TviCATL-specific phages were enriched after the fourth round of panning and a total of seven clones gave high signals when analysed by monospecific ELISA. Future work will include recombinant expression and purification of the selected TviCATL-specific scFvs for testing in diagnostic assays as well as panning with the TbbCATL antigen. This study laid the groundwork for evaluating TbbCATL as a diagnostic target for AAT
Molecular cloning, recombinant expression and characterisation of serine and cysteine protease inhibitors from Trichinella zimbabwensis.
(2019) Maseko, Thando Glory.; Coetzer, Theresa Helen Taillefer.
Trichinellosis is a disease caused by parasitic helminths of the genus Trichinella. Infection occurs by ingestion of meat contaminated with infective Trichinella larvae. Cases of Trichinella zimbabwensis, a non-encapsulating species of Trichinella infecting mammals and reptiles, have been reported in Ethiopia, Mozambique and South Africa. The parasite life cycle alternates between the enteric and skeletal muscle phases of infection. Trichinella species release various excretory-secretory products that enable successful parasitism. These include cysteine protease inhibitors, cystatins and serine protease inhibitors. To illustrate, cystatins have roles in cellular invasion and immune evasion while serpins inhibit blood coagulation, resist host protease damage and interfere with host immunoregulatory signals. The potential roles of endogenous parasite cysteine and serine protease inhibitors in T. zimbabwensis make these inhibitors attractive targets for the development of novel antiparasitic interventions. The genes encoding a cysteine protease inhibitor, cystatin B, and a Kazal-type serine protease inhibitor, SPINK4, were identified in the T. zimbabwensis genome. Following the synthesis of cDNA from nematode extracted mRNA, the respective genes were amplified and cloned into E. coli expression vectors. The recombinantly expressed proteins, rTzcystatin B and rTzSPINK4, were purified using immobilised metal affinity chromatography, their inhibitory activity evaluated and antibodies were produced in chickens. The antibodies were used for the detection of the recombinant proteins on western blots and ELISA. Recombinant Tzcystatin B inhibited the activity of the catalytic domain of the cathepsin L-like peptidase from Trypanosoma congolense (TcoCATL), the homologues from T. vivax (TviCATL) and Theileria parva (ThpCATL) as well as cathepsin B from T. zimbabwensis (TzCATB). Conversely, rTzSPINK4 was unable to inhibit either host serine proteases, chymotrypsin or trypsin. Future studies will be aimed at exploring the effect of the T. zimbabwensis protease inhibitors on host proteases involved in antigen processing. This may indicate a possible role for the nematode protease inhibitors in host immunoregulation.
The stabilisation of the cysteine protease of Carica papaya (papain) and the catalytic domain of the Cathepsin L-like cysteine protease of Trypanosoma congolense (TcoCATL)
(2019) Chetty, Ryan.; Hewer, Raymond.; Coetzer, Theresa Helen Taillefer.
Proteases play an intricate role in the numerous functions of a living organism. Proteases are responsible for the cleavage of proteins into smaller fragments by catalysing the hydrolysis of peptide bonds. The class of cysteine proteases have a cysteine thiol group in their active site have been found in lower and higher organisms. They have been investigated as promising drug targets for various diseases due to their fundamental functions in catabolism and protein processing. The thermal stability of a protease is a key characteristic feature that is largely dependent on its amino acid sequence and composition and quantified through the determination of its melting temperature (TM). Papain is the most well characterised cysteine protease and is commonly used as a model for other cysteine proteases. Congopain is the major cysteine protease of Trypanosoma congolense which has been identified as the main causative agent of trypanosomiasis in livestock. The thermal stability for papain and congopain were investigated in this study via the thermal shift assay. Papain was purchased and the catalytic domain of congopain was expressed using the Pichia pastoris yeast expression system. The thermal stability of the proteases were determined under neutral pH conditions and the effect of pH and ligand binding were evaluated to determine if the proteases could be further stabilised. The most stable forms of papain and the catalytic domain of congopain in its monomeric form was observed at pH 5.0 with 50 μM chymostatin. The thermal stability of both cysteine proteases was successfully evaluated via the thermal shift assay and conditions to further stabilise papain and the catalytic domain of congopain were determined. The thermal shift assay has been proven to be a reliable technique in identifying factors which increase the stability of a protein. More specifically, the technique serves as a simple and primary diagnostic tool to screen potential inhibitors of a protein and detect changes in the TM of a protein.
Transferrin receptor-mediated gene delivery using functionalised gold nanoparticles.
(2018) Padayachee, Jananee.; Singh, Moganavelli.
Gene therapy strategies have shown their potential in treating numerous central nervous system (CNS) disorders, including highly aggressive brain cancers. Gold nanoparticles (AuNPs) are popular vectors for gene delivery, due to their low toxicity, and ease of synthesis and functionalisation. However, the in vivo efficacy of these vectors is dependent on their ability to cross the blood-brain barrier (BBB), a specialised capillary network preventing the movement of compounds into the CNS. Passage across the BBB is often facilitated through targeting of the transferrin (Tf) receptor, leading to uptake by receptor-mediated transcytosis. This study aimed to develop untargeted and Tf-targeted functionalised AuNP (FAuNP) vectors and assess their potential as gene delivery vectors. AuNPs were prepared through citrate reduction and functionalised with chitosan (CS) and poly(ethylene) glycol 2000 (PEG2000) in two weight ratios [2% and 5% (ww⁄)] to produce untargeted FAuNPs. The holo-transferrin protein was conjugated to both PEGylated and unPEGylated FAuNPS to produce the Tf-targeted FAuNPs (TfAuNPs). The physicochemical characteristics of FAuNPs were evaluated using UV spectroscopy, Fourier-transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). TEM revealed AuNP to be spherical and relatively monodisperse. FAuNPs displayed hydrodynamic diameters ranging from 94.7 – 196.4 nm with good colloidal stability, as evidenced by NTA. Binding studies viz. band shift and ethidium bromide intercalation assays showed that all FAuNPs were able to fully complex and efficiently condense pCMV-luc plasmid DNA, with PEGylated and targeted FAuNPs being capable of partially protecting DNA from nuclease degradation, as determined in nuclease protection assays. In vitro studies were conducted in the HEK293, Caco-2, and the Tf receptor-positive HeLa cell lines. Cytotoxicity was assessed using the MTT cytotoxicity assay, which revealed FAuNPs to be relatively non-toxic to HeLa and HEK293 cells. Notably, TfAuNPs displayed low cytotoxicities, and generally exhibited increased cell viabilities compared to the untargeted FAuNPs. The luciferase gene reporter assay was conducted to assess the transfection efficiency of the FAuNPs. Transfection levels were highest in Caco-2 cells, with PEGylated FAuNPs observed to produce reduced transfection compared to the unPEGylated FAuNPs. TfAuNPs displayed favourable transfection in HeLa cells; with the competition binding assays confirming receptor-mediated uptake for AuCSTf and AuCSTf-5% PEG FAuNPs only, suggesting that a grafting density of the 2% (ww⁄) PEG interfered with receptor binding. These Tf-targeted FAuNPs show the potential to be utilised as vectors for brain delivery; however further optimisation and investigations in an in vivo system are required.
Expression and evaluation of a 297-amino acid fragment of the blood stage protein PfC0760c from Plasmodium falciparum.
(2019) Baig, Zainab.; Goldring, James Philip Dean.
Abstract available in pdf.
Evaluating the effect of nitric oxide on myoblast proliferation, migration and differentiation.
(2018) Sibisi, Ntethelelo Charles.; Niesler, Carola Ulrike.; Myburgh, Kathryn Helen.
Wound healing is the process of restoring tissue integrity in response to injury. This process involves four major phases namely; haemostasis, inflammation, regeneration and remodelling. These phases are regulated by various growth factors and cytokines that are released at the site of injury to facilitate wound repair. Cells involved in wound healing include neutrophils, macrophages, lymphocytes, fibroblasts and stem cells. Satellite cells are mesenchymal stem cells which facilitate skeletal muscle regeneration through a process known as myogenesis. These cells are quiescently located underneath the sarcolemma of the muscle fiber and are activated upon injury to enter the growth phase of cell cycle. They then proliferate and migrate to the injury site to differentiate and fuse with existing fibers to form multinucleated muscle cells.Growth factors and signalling molecules, such as hepatocyte growth factor (HGF) and nitric oxide (NO), induce satellite cell activation by altering the expression of transcription factors such as paired box transcription factor 7 (Pax7), myogenic regulatory factor 5 (Myf5), myogenic differentiation antigen (MyoD) and Myogenin. The role of NO in the subsequent process of myoblast proliferation, migration and differentiation is however unclear. The present study therefore evaluated the effect of nitric oxide on myoblast proliferation, migration and terminal differentiation. C2C12 myoblasts were cultured in standard growth media and subsequently plated for analysis in serum free media. Proliferation or differentiation was induced via the addition of either 2 ng/ml HGF or 2% horse serum respectively, while migration was stimulated using the standard in vitro wound healing assay. L-NAME (a NOS inhibitor; 100 µM and 200 µM) and SIN-1 (a NO donor; 10 µM or 25 µM) were utilized to modify NO levels in vitro, while NO levels were assessed using a nitric oxide colorimetric assay kit. Proliferation was assessed via cell counts, migration by assessing the percentage wound closure and differentiation determined by calculating myoblast alignment and subsequent fusion into multinucleated myotubes. There was no significant change in nitric oxide generated by myoblasts during proliferation and migration studies. However, NO levels increased significantly in response to differentiation, L-NAME significantly prevented this NO increase at day of differentiation. L-NAME also significantly decreased myoblast terminal differentiation by inhibiting myoblast alignment and fusion at day 5 of differentiation. L-NAME also significantly reduced the proliferative effect of HGF on myoblasts at 24 hours, and significantly reduced percentage wound closure at 16 hours post-injury. NO release by C2C12 myoblast was observed to increase in response to SIN-1 in a dose dependent manner. NO levels significantly increased from 0.58 nmol in a control up to 1.35 nmol and 1.9 nmol at 1 hour in response to 10 µM and 25 µM SIN-1 respectively. These levels increased until they reached 2.5 nmol in response to 25 µM SIN-1 at 16 hours. SIN-1 showed no significant effect on myoblast proliferation, however, it significantly promoted myoblast migration in a dose dependent manner by increasing the percentage wound closure to 42% and 45% at 7 hours for 10 µM and 25 µM respectively compared to 38% of the control. SIN-1 also significantly stimulated myoblast fusion with myofiber area of 26% as compared to 18.6% of the control at day 5 of differentiation. In conclusion, nitric oxide levels increase significantly during myoblast differentiation, but not during proliferation and migration. Despite this, inhibition of nitric oxide synthase significantly affects all these processes. In contrast NOS-independent elevation of NO (through incubation with SIN-1) significantly increased myoblast migration and fusion, but not proliferation. This suggests a central role for NO in regulating myogenesis; however, this role requires further investigation.
Mechanisms behind the anti-diabetic effects of caffeine in a Type 2 diabetes model of rats.
(2018) Reddy, Rebecca.; Islam, Mohammad Shahidul.
Caffeine has been used for many years and is one of the most extensively consumed active food ingredient throughout the world. Caffeine has been broadly studied in a variety of areas regarding human health and performance. Various reports have shown that consumption of coffee or caffeine containing drinks are associated with the reduction of type 2 diabetes related symptoms, promotes weight loss and acts as an antioxidant. However, the fundamental mechanisms have not been understood. Therefore, the main objective of this study was to investigate the mechanisms behind the anti-diabetic effects of caffeine. Various in vitro, ex vivo and in vivo models were used to achieve this objective. The results of this study showed that caffeine possessed strong antioxidant potential and was able to inhibit key enzymes linked to type 2 diabetes in vitro. The results of this study further demonstrate that caffeine can modulate T2D-induced oxidative stress in various organs in vivo. Caffeine was able to reduce small intestinal glucose absorption, increase muscle glucose uptake ex vivo, improve pancreatic β-cell function and stimulate insulin secretions in an animal model of type 2 diabetes. Pancreatic histopathology showed that caffeine ameliorated T2D-induced pancreatic β-cell destruction and their functions at the end of the study. Data of this study suggest that caffeine can be used an anti-diabetic supplement in anti-diabetic foods and food products, however, a safer effective dose still needs to be identified. Hence, further studies are warranted in experimental animals and humans to determine the most effective and safer dose of caffeine for achieving its maximum anti-diabetic effects.
Recombinant expression and initial characterisation of two Plasmodium falciparum copper binding proteins: Cox11 and Cox19.
(2018) Salman, Abdumalik Abdullahi.; Goldring, James Philip Dean.
Emerging drug resistance hinders the efforts to control malaria and so novel antimalarial drugs are required. Copper is essential for the survival of plasmodial parasites but the proteins involved in copper homeostasis are not well characterised. This study looked at plasmodial copper homeostasis by identifying and partially characterising two P. falciparum copper metallochaperones, Cox11 and Cox19. The Basic Local Alignment Search Tool (BLASTp) screen of the Plasmodium database (www.plasmodb.org) identified Cox11 and Cox19 gene orthologues in nine Plasmodium spp. The plasmodial Cox11 amino acid sequence contained a single N-terminus membrane-spanning region and three conserved cysteine residues, two of which are in a CFCF motif. These features are found in mammalian and yeast Cox11 amino acid sequences. The plasmodial Cox19 amino acid sequence has a domain containing a twin Cx9C motif, and a conserved Tyr-Leu dipeptide between the pair of cysteine of one Cx9C motif, similar to the amino acid sequences of human and yeast Cox19. The cloned and expressed recombinant MBP-PfCox11Ct and MBP-PfCox19 fusion proteins resolved on SDS-PAGE gels as ~62 kDa and ~66 kDa proteins respectively. Polyclonal IgY antibodies raised in chickens against rMBP-PfCox11Ct and rMBP-PfCox19 detected the native murine parasite, P. berghei, proteins on a western blot. Both recombinant proteins bound copper in the form of the cuprous ion in vitro and in vivo using the: bicinchoninic acid release, ascorbic acid oxidation, atomic absorption spectroscopy, and differential scanning fluorimetry assays. Three P. falciparum Cox11 mutants (two single- and a double-mutant) were engineered with site-directed mutagenesis, where an alanine replaced the corresponding cysteine residue and the mutant proteins were expressed as MBP fusion proteins. The two P. falciparum Cox11 cysteines, Cys155 and Cys157, in a CFCF motif were shown to be essential for the binding of copper in several assays. P. falciparum Cox11 and Cox19 bind copper in vitro and in an in vivo environment. Both rMBP-PfCox11Ct and rMBP-PfCox19 bound copper in an in vivo environment, enabling the growth of E. coli host cells expressing the proteins in the presence of toxic concentrations of copper. The localisation of the plasmodial Cox11 and Cox19 proteins suggested by proteomic data to be mitochondrial requires experimental confirmation. This study provides the foundation for further experiments to study P. falciparum Cox11 and Cox19 biochemistry and the evaluation of the two proteins as possible drug targets.
Recombinant expression of plasmodium falciparum histidine rich protein-2 (PfHRP-2) and characterisation of chicken anti-PfHRP-2 antibodies.
(2019) Shezi, Mlondi.; Goldring, James Philip Dean.
The diagnosis of malaria using rapid diagnostic tests (RDTs) targets three Plasmodial proteins namely, Plasmodium falciparum histidine rich protein-2 (PfHRP-2); lactate dehydrogenase (LDH) and aldolase. Early diagnosis is important for malaria control and accurate diagnosis guides treatment. The diagnosis of malaria prioritises the detection of P. falciparum (Pf), the species with the highest mortality. As a result, PfHRP-2-based RDTs are the most commonly used RDTs to detect P. falciparum infections. PfHRP-2 is a Plasmodium falciparum protein expressed during the blood stages of the parasite infection and is abundant in the blood of patients. The PfHRP-2 protein is stable in the blood, urine, saliva and cerebrospinal fluids of patients where it can be detected. Detection of PfHRP-2 in the field is problematic due to gene deletion, PfHRP-2 protein sequence variation, low heat tolerance of mammalian antiPfHRP-2 antibodies in RDTs, prozone effect, low quality assurance standards and low sensitivity of RDTs in some cases. False positive results caused by mammalian antibodies reacting with human rheumatoid factor also affect the antibody-based detection of malaria. This limits the diagnosis of P. falciparum malaria and undermines malaria control efforts. Optimising the performance of current PfHRP-2-based RDTs could prevent some of these problems and would strengthen malaria surveillance. Most malaria RDT problems associated with defective or cross-reactive antibodiescould be abated using a different species of antibodies. Chicken IgY antibodies are stable at room temperatures and do not react with human rheumatoid factor. To raise anti- PfHRP-2 chicken IgY antibodies, recombinant histidine rich protein-2 (rPfHRP-2) was expressed in E. coli BL21 (DE3) at 30°C overnight, induced with lactose. The recombinant protein was affinity purified and analysed on SDS-PAGE gels, resolving as a single protein band of 54 kDa. rPfHRP-2 induced high antibody titers after 13-weeks in immunised chickens. Purified IgY antibodies against rPfHRP-2 detected the 54 kDa band, a rPfHRP-2 dimer and trimer which were also detected by human anti-rPfHRP-2 antibodies. The thermal stability of anti-rPfHRP-2 IgY antibodies was assessed by exposure to temperatures resembling the field were RDTs are deployed. IgY antibodies stored at room temperatures for sixteen weeks were shown to be stable and still detected rPfHRP-2 in an ELISA after storage. To determine epitopes or peptides detected by the anti-rPfHRP-2 IgY antibodies, the recombinant protein was cleaved with trypsin and the fragments probed with IgY. Trypsin cleavage of rPfHRP-2 produced fewer fragments than predicted and the fragments resolved larger than their estimated sizes on SDS-PAGE gels. Thefragments were detected by the anti-rPfHRP- 2 IgY antibodies. Chicken IgY antibodies have potential for possible application in malaria diagnostics to detect Plasmodial proteins. The IgY used in this study was stable at room temperatures, and detected trypsin hydrolysed rPfHRP-2 fragments whose identity requires further investigation to better understand the fragment’s behavior and determine epitopes of anti-rPfHRP-2 IgY antibodies.
Recombinant expression, purification, analysis and immunization of mice with plasmodium yoelii glyceraldehyde-3-phosphate dehydrogenase (rPyGAPDH) and lactate dehydrogenase (rPyLDH) and evaluation of protection against P. berghei challenge.
(2019) Khuzwayo, Sinothile Sementha.; Goldring, James Philip Dean.
The principal focus in malaria research is the development of an effective malaria vaccine which will help to prevent death and eliminate the disease. This study investigated two plasmodial glycolytic enzymes as possible malaria vaccine candidates. Plasmodium yoelii glyceraldehyde-3-phosphate (rPyGAPDH) and lactate dehydrogenase (rPyLDH) recombinantly as His-tagged fusion proteins were affinity purified using a cobalt affinity matrix. Ethanol has been reported to enhance expression of recombinant proteins in E. coli bacteria. Concentrations of 1%, 2% and 3% (v/v) ethanol were tested for enhancement of rPyLDH and rPyGAPDH expression in both lysogenic broth and terrific broth. Ethanol reduced the overall expression of proteins by inhibiting the growth of PyLDH and PyGAPDH E. coli host cells. The recombinant proteins were evaluated using a metal ion gel shift assay. Both rPyLDH and rPyGAPDH did not show a size shift on the gel following incubation with Cu2+, Co2+ or Ni2+. Both the reduced and non-reduced forms of rPyLDH and rPyGAPDH had similar migration on a 10%, 12.5% and 15% SDS-PAGE gels. Recombinant PyLDH and rPyGAPDH were prepared in Freund’s adjuvants and mice were immunized to evaluate immunogenicity. Both rPyGAPDH and rPyLDH were immunogenic in mice and produced high antibody titers. Mice were immunized with rPyLDH or rPyGAPDH and challenged with P. berghei infection. All PyLDH immunized mice developed high parasitemia and 1/5 of the PyGAPDH immunized mice maintained low parasitemia below 5% throughout the study. Further work includes repeating the immunization and challenge experiments.
Differential scanning fluorometry analysis of antibodies, P. falciparum proteins and assessing two copper-based assays to detect P. falciparum Histidine Rich Protein-2.
(2019) Sookai, Sheldon.; Goldring, James Philip Dean.
Abstract available in pdf.
The impact of aromatic ring count on the bioavailability of chemical compounds.
(2019) Mcoyi, Michael Phumlani.; Hewer, Raymond.
Aromatic rings are a negative contributing factor in the bioavailability of chemical compounds in drug discovery. Due to the inevitable effect the aromatic rings possess on the bioavailability of chemical compounds, methods to continuously evaluate their effect should be ceaseless. As such, this study aimed at investigating the impact of aromatic ring count on the bioavailability of chemical compounds by screening a small collection compound library comprising 13 compounds. The permeability of chemical compounds was evaluated using the PAMPA assay; the results further analysed through spectrophotometry. The PAMPA assay is an automated system and was used to allow for rapid screening of compounds and the screening was successful. Overall, only four chemical compounds showed poor permeability: ammonium bromide, gibberellic acid, salicylic acid, and PMSF. Osiris Property Explorer was used to screen all 13 compounds for cLogP, LogS, TPSA, Toxicity risks, drug-likeness and drug score. Most of these compounds produced suitable logP (< 5) and were therefore predicted to possess good absorption and permeability properties. Percentage protein binding was assessed separately; all chemical compounds were screened for percentage protein binding using Amicon Ultra-15 Centrifugal Filter method. The results indicated a broad range of protein binding for all chemical compounds tested (70.29 – 98.23%). Lipinski’s Rule of Five was applied to all chemical compounds and compounds were scored against the four rules. Most compounds adhered to all four rules with only two compounds violating one or two rules. A relationship between parameters: permeability, drug-score, and percentage protein binding, against the aromatic ring count of chemical compounds, was explored. Overall, no real relationship existed between any of these parameters and aromatic ring count, as was indicated by low correlation coefficients (R2) (< 0.95). Overall, this study resulted in the successful screening of 13 chemical compounds and the establishment of effective permeability, percentage protein binding assays for future studies.
Development of a novel biochemical assay for the identification of promiscuous compounds.
(2019) Ntombela, Nomfundo Praise.; Hewer, Raymond.
Promiscuous compounds, specifically aggregate-based inhibitors that result in false positives in biochemical activity assays present a serious and increasing interference to early-stage drug discovery processes. Although under-used, a number of purpose-specific assays have been developed to enable the identification of promiscuous inhibitors. To advance on these efforts, this study aimed to develop and optimise a novel thermal shift assay to concurrently identify both true and promiscuous inhibitors. In particular, stem bromelain selected as the model protein for this study was successfully isolated from the bromelain mixture through molecular exclusion chromatography and shown to be enzymatically active in the titrimetric assay (gelatin digestive unit/gm enzyme of 2024.36-2085.5). The radial diffusion assay predicted that the promiscuous compound Epigallocatechin gallate and the known aggregate based-promiscuous compound Congo Red activated the digestion of gelatin by stem bromelain. In the thermal shift assay, bromelain yielded a melting temperature of ~75-76 °C which then shifted by 9 °C in the presence of a true inhibitor E-64. A similar shift was surprisingly observed in the presence of Epigallocatechin gallate and both these compounds were similarly not affected by the presence of a detergent (0.004% sodium dodecyl sulfate). The protein was aggregated in the presence of Congo Red, however, the addition of the detergent effectively restored the protein to its original melting temperature. Both Epigallocatechin gallate and Congo Red demonstrated cytotoxicity. As a proof of concept, this study showed that in addition to identifying true inhibitors, the detergent based thermal shift assay can be successfully employed to identify promiscuous inhibitors and to determine different mechanisms.
The evaluation of novel metal -based compounds as potential BACE1 ligands.
(2018) Karrian, Jezelle.; Hewer, Raymond.
The proposed primary cause of Alzheimer’s disease (AD) is the cleavage of the Amyloid-β precursor protein by the β- Amyloid cleavage enzyme 1 (BACE1). As such it has been proposed that inhibiting this enzyme would help reduce the formation of Aβ plaques and slow down the progression of the disease. However, there is currently no treatment available that slows down the progression of AD and current drugs on the market only treat the symptoms of the disease. Metal-based compounds have been studied extensively for use as anticancer treatments. Success in the of these metal-based compounds have introduced new avenues in drug development and as such, there have been studies conducted on the introduction into metal-based compounds as potential AD drug candidates. In this study, 13 novel metal-based compounds were evaluated using five simple assessment techniques to determine potential BACE1 ligands. Molecular docking studies were able to predict that compound 12 was able to bind readily with BACE1 as it had a greater docking score (-4.630) which correlated with the thermal shift assay as the ΔTm of 9.1 ⁰C was compared to the other compounds. Compounds 7 and 13 were found to be aggregating compounds when results of the chymotrypsin assay were assessed. In addition, the lack of inhibition in the presence and absence of detergent in the chymotrypsin assay was able to determine specificity of these metal-based compounds to the BACE1 protein. Furthermore, the DNA cleavage assay determined that copper-containing compounds 9, 10 and 11 were able to cause scissions in supercoiled plasmid DNA. Theoretical predictions of the physiochemical properties were evaluated to determine probable CNS/oral drug candidates according to Lipinski’s rule of 5 and Veber’s rules. All results obtained in this study predicted most favourable results with seven compounds producing an RO5 score of 4 thus making them potential BACE1 ligands with probable CNS/oral drug candidate properties and with fewer toxic effects. Furthermore, the chymotrypsin assay revealed that compound 13 was an aggregator and that compound 7 had binding affinities to both BACE1 and chymotrypsin. Overall assessment of these compounds has revealed that the compounds with the most favourable properties and an oral and CNS drug candidate as well as a good BACE1 ligand was compound 6. In addition, the overall positive outcomes of the molecular docking and TSA indicate that metal-based compounds have great potential in the drug design and discovery of new drug candidates for the treatment of AD.

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