Masters Degrees (Medical Science)

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Drug susceptibility testing of second and third line anti-tuberculosis drugs used in the management of extensively drug resistant tuberculosis.
(2013) Moodley, Salona.; Moodley, Prashini.
Drug resistant tuberculosis is a major contributor to South Africa’s quadruple burden of disease. Management of this infection in a highly HIV endemic area is a constant challenge. There is a paucity of new anti-tuberculosis agents in the developmental and clinical trial phases to address the problem of extensively-drug resistant tuberculosis (XDR-TB). In an attempt to affect a cure in patients with XDR-TB, it has become necessary to re-introduce previously used anti-tuberculosis drugs, as well as antimicrobial agents designed for treatment of non-tuberculosis infections. Whilst these drugs may have previously been tested and shown efficacy in drug susceptible tuberculosis, their activity in XDR TB strains was not tested before introduction for management of XDR-TB in KwaZulu-Natal, South Africa. Drug susceptibility testing (DST) plays an integral role in the diagnosis and treatment options for tuberculosis. It is able to decrease the burden and spread of resistant tuberculosis. However DSTs methods for second line anti –TB drugs (SLDs) and third line anti-TB drugs (TLDs) have not been standardised. Critical concentrations of these anti-TB drugs remain unknown or vary within and between settings thus further hampering the control of TB.
Genital tract immune activation, inflammation and sexually transmitted infections in CAPRISA 008 trial participants.
(2016) Mhlungu, Sanele Nobleman.; Liebenberg, Lenine Julie.; Ngcapu, Sinaye.
Abstract available in PDF file.
Antimicrobial susceptibility testing of four 5-nitroimidazoles against trichomonas vaginalis.
(2016) Mtshali, Andile.; Joubert, Bronwyn C.
Abstract available in PDF file.
Molecular characterization of resistance and virulence in Methicillin Resistant Staphylococcus Aureus (MRSA) from the private sector in KwaZulu-Natal, South Africa.
(2016) Amoako, Daniel Gyamfi.; Esaack, Sabiha Yasuf; Bester, Linda Antionette.
Abstract available in PDF file.
Computational studies of pentacycloundecane peptide based HIV-1 protease inhibitors.
(2013) Chibi, Buyisile.; Soliman, Mahmoud Elsayed Soliman.; Kruger, Hendrik Gerhardus.; Govender, Thavendran.; Maguire, Glenn Eamonn Mitchel.
Abstract available in PDF.
Identification of anti-cancer agents using integrated computational tools.
(2015) Mbatha, Sbongile Happyness.; Soliman, Mahmoud Elsayed Soliman.
Cancer is a heterogeneous disease that is responsible for various molecular changes and pathological entities that play vital roles in its response to treatment, survival, and growth. Better understanding into the critical pathways and molecular events involved in cancer has enabled the identification of novel targets and development of anti-cancer therapies. In this study, two enzymes that have been shown to be involved in different stages of cancer were used, namely cathepsin B and Hsp90. The main aim of this study was to employ integrated in-silico approaches to study inhibitory routes of these enzymes to develop novel anti-cancer agents. Cathepsin B is the most well studied of the cathepsin family as a potential therapeutic target to treat cancer. To accomplish practical aim 1 of this study, the Michael-acceptor type compounds obtained from a chemical database that irreversibly inhibit cathepsin B were investigated. Validation was carried out using compounds with experimentally determined anti-cathepsin B activity. Four novel compounds exhibited better covalent binding affinity when compared against the experimentally determined prototypes. Molecular dynamics simulations were performed to ensure the stability of the docked complexes and to allow further analysis. Per-residue interaction decomposition analysis provided deeper insight into the interaction themes with active site residues. It was found that polar and hydrophobic interactions had the highest contribution towards drug binding. Recent experimental studies have documented FDA-approved protease inhibitors involvement in anti-cancer activity, however, there was limited understanding to the mode of inhibition. To accomplish practical aim 2 of this study, the mode of inhibition of protease inhibitors against Hsp90 cysteine protein was investigated. The lack of an X-ray crystal structure of human Hsp90 prompted the creation of its homology model for subsequent simulations. Two possible binding sites, C-terminal and N-terminal domains, were identified and considered in this study. Molecular docking followed by molecular dynamic simulations and post-dynamic analyses were performed to elaborate on the binding mechanism and relative binding affinities of nine FDA-approved HIV-1 protease inhibitors against human Hsp90. Our findings from thermodynamics calculations revealed that these inhibitors were more likely to bind to the N-terminal domain (~ 54.7 -83.03 kcal/mol) when compared to C-terminal domain. This appears to be the first account of a detailed computational investigation aimed to understand the binding mechanism of HIV protease inhibitors binding to Hsp90. Information gained from this study should also provide a significant route map towards the design and optimisation of potential derivatives of protease inhibitors to treat breast cancer. The results obtained will serve as a powerful tool in the drug design and development process. However, further experimental investigations will be useful to improve our computational findings.
A retrospective analysis of induction of labour at a regional hospital in KwaZulu-Natal, South Africa.
(2012) Malende, Brenden.; Kambaran, A.
Abstract available in PDF file.
Benign uterine conditions: correlating MRI and US findings: retrospective analysis of 33 patients.
(2016) Ramaema, Dibuseng Paulina.
Abstract not available.
Defining current facial fracture patterns in a quaternary institution following high-velocity blunt trauma.
(2016) Magagula, Senzwesihle Clive.
Background: In the early 20th century, René Le Fort studied facial fractures resulting from blunt trauma and devised a classification system still in common use today. This classification, however, was based on low-velocity trauma. In modern practice, in a quaternary-level referral hospital, patients are often admitted following high-velocity injuries that mostly result from motor vehicle collisions. Objectives: A retrospective study to define facial bone fractures occurring subsequent to highvelocity trauma. Method: A retrospective study comprising the review of CT scans of 52 patients with highvelocity facial fractures was performed between April 2007 and March 2013. Injuries were classified using the Le Fort classification system. Deviations from the true Le Fort types, which are often depicted in the literature as occurring bilaterally and symmetrically, were documented; these included unilaterality, occurrence of several Le Fort fractures on one side of the face, occurrence of several Le Fort fractures on different levels and on different sides of the face, and occurrence of other fractures in addition to Le Fort fractures. Results: Of the 52 cases, 12 (23%) had Le Fort injuries, with true Le Fort fractures occurring in only 1, and 11 deviating from the classic description. Nine patients had Le Fort fractures and additional fractures. Mandibular and zygomatic bone fractures were found to be common associations with Le Fort injuries, occurring in 58% and 33% of the cases respectively. Conclusion: Fractures occurring in modern practice often deviate from the traditional Le Fort classification. Precise recognition of these deviations and recognition of additional associated fractures is pivotal in their management, assisting the surgeon in determining the treatment plan, such as the surgical approach and the order in which to fix the various fractured components.
Seroprevalence and viral quantification of Kaposi Sarcoma-associated Herpes Virus (KSHV) in a Human Immunodeficiency Virus (HIV) infected adult South African cohort.
(2017) Singh, Shoohana.; Mosam, Anisa.; Shaik, Fahmida.; Uldrick, Thomas S.; Naidoo, Kogieleum.
Background Kaposi sarcoma-associated herpes virus (KSHV), also known as human herpes virus 8 (HHV8), is aetiologically implicated in Kaposi’s sarcoma (KS). Although HIV associated KS has increased in incidence and is a public health problem in South Africa, serological studies of KSHV have not been extensively documented in this population. This cross-sectional study investigates the seroprevalence and viral load of KSHV in an adult South African cohort. Method Cross-sectional data of 140 participants attending an urban research HIV counseling and testing (HCT) clinic site in Durban, KwaZulu-Natal, between July and October 2013 was analyzed. Detection of antibodies against latent (Orf73) and lytic (K8.1) KSHV antigens was performed on 70 HIV-seropositive and 70 HIV-seronegative participants. Subjects reactive to either antigen were considered KSHV seropositive and analyzed for salivary KSHV DNA, which was quantified using primers for the K6 gene region. Results The demographic characteristics of the two groups were similar, with 36% males (median age, 35yrs.) and 64% females (med. age, 34yrs.) in the HIV-positive group, and 31% males (med. age, 36.5yrs.) and 69% females (med. age, 36.5yrs.) in the HIV-negative group. Of 70 HIV-positive participants, 100% were black Africans, as was 97% of the HIV-negative group, with the remaining 3% being Indian/Asian and Mixed race. Only 24% of HIV-positive patients were on Anti-retro viral treatment. Fifty-four percent of all participants tested positive for KSHV, with 33% reactive to lytic K8.1, 37% to latent Orf73 and 21% to both. Of those HIV-positive, 50% were seropositive for K8.1 and 46% for Orf73. In those HIV-negative, 16% were seropositive for K8.1 and 29% for Orf73. The HIV-positive group demonstrated a significantly higher percentage KSHV seropositivity (70% vs. 37%, p=0.0001). Amongst the KSHV seropositive participants, KSHV DNA was detected in 41 % HIV-positive and 23% HIV-negative participants. Conclusion KSHV seroprevalence was high in South African adults attending an urban HCT clinic. HIV positive status was associated with a higher KSHV seropositivity and a greater KSHV salivary shedding. HIV positive individuals should be tested for KSHV infection and those found infected, be monitored aggressively for development of KS.
Case series of subtotal exenteration with buccal mucosal graft for orbital squamous cell carcinoma.
(2016) Surajballi, Sharisha.; Kruse, Carl-Heinz.
The aim of the study was to look for a safe alternative to a disfiguring total orbital exenteration for orbital squamous cell carcinoma, so that a standard hospital issue inexpensive stock ocular prosthesis can be fitted with improved aesthetic results, rather than an expensive custom made prosthesis for the patient’s own cost. The subjects and methods involved a retrospective case review which was performed of patients from St Aidan’s Missionary Hospital initially, which was later amalgamated into the McCords Provincial Eye Hospital, Durban, KwaZulu-Natal, South Africa. Ten consecutive patients who underwent an ‘extended’ lid-sparing subtotal exenteration with minimally preserved healthy conjunctiva and a buccal mucosal graft were identified over a 3 year period from 1 January 2011 to 31 December 2013. Patients’ clinical records were reviewed. Results included all of the ten patients having a good aesthetic outcome at 4 weeks and six months with a standard hospital issue stock ocular prosthesis. One patient had a repeat buccal mucosal graft after forniceal shortening. Three patients had local recurrences within one year but all recurrences were identified easily and total exenteration was successfully performed. The survival rate at 3 years was ninety percent as one patient was lost to follow-up. A subtotal orbital exenteration with minimally preserved healthy conjunctiva and a buccal mucosal graft is cost effective, safe and cosmetically acceptable with a standard ocular prosthesis.
Extrapulmonary tuberculosis at King Edward hospital : a descriptive retrospective study.
(2016) Gounden, Strinivasen.; Magula, Nombulelo Princess.
Background Globally, South Africa remains one of the top twenty high tuberculosis(TB) burden countries. In addition, South Africa has the highest burden of tuberculosis/Human Immunodeficiency virus (TB/HIV) coinfection in the world, with the province of KwaZulu-Natal representing the global epicenter of TB/HIV. With the scaling up of one of the world’s largest antiretroviral therapy programs, it was envisioned that the burden of tuberculosis would be reduced. While significant progress has been made to improve the diagnosis of pulmonary tuberculosis, the diagnosis of extrapulmonary TB(EPTB) remains a significant challenge in resource constrained settings. This study describes the profile of patients with EPTB at a tertiary hospital in a TB/HIV hyperendemic setting in Durban, South Africa. Methods A retrospective chart review was conducted, and included all adult patients diagnosed with EPTB at a tertiary hospital in Durban, South Africa, between 1 January 2016 and 31 March 2016. Data was extracted from the facility TB register, as well as patient clinical records. All data was analysed using SPSS software (SPSS 23.0, Armonk NY: IBM Corp). For all statistical comparisons, a 5% level of significance was used; correspondingly 95% confidence intervals were used to describe effect size. All data was assessed for normality, and non-parametric tests were used where necessary. Medians and interquartile ranges were used for data not amenable to parametric description. Pearson’s Chi-square test was utilised for comparison between subgroups. All p values were 2-tailed and considered significant below 0.05. Significant findings were analysed for association using Phi and Kramers V test for symmetric measures. Results There were 188 new cases of TB during the study period, with 80 patients diagnosed with EPTB. The mean age of patients was 34.73 years (SD ±9.44). Forty two (52.5%) patients were female, while 76(96%) were black African. The most common risk factor for EPTB was HIV co-infection (88.8%). The median CD4 cell count was 68 (IQR 32-165) cells/mm3. Pleural (36.3%), lymph node (28.7%) and abdominal(27.5%) involvement were the most common sites of extrapulmonary disease Eleven of the 80 patients (13.8%) presented with EPTB involving more than one anatomical system. Weight loss, fever, night sweats and cough were amongst the most common symptoms reported. Signs varied according to the site of infection. Non-specific symptoms were common. In the majority of cases, more than one diagnostic method was used to confirm the presence of TB in distant organs. Conclusion A high index of suspicion is required when assessing a patient with known risk factors for EPTB. Immunosuppression remains the most significant risk factor for the development of EPTB. In our setting, HIV co-infection remains the most common risk factor. Advancements in Xpert MTB/Rif and computer tomography have greatly assisted in rapidly diagnosing EPTB. Despite improved access to antiretroviral therapy over the past years, advanced HIV disease remains a significant challenge to eradicating TB.
Evaluation of the left ventricular ejection fraction post right ventricular pacing at Inkosi Albert Luthuli Central Hospital (IALCH), Durban, KwaZulu-Natal (KZN).
(2016) Kasipersad, Sherlina.; Magula, Nombulelo Princess.
Since the implantation of the first artificial pacemaker in 1958, these devices have become the treatment of choice in bradycardias. Despite its widespread use, only a few studies have looked at the effects of single chamber right ventricular(RV) pacing on left ventricular(LV) function in patients with sinus node dysfunction or atrioventricular node dysfunction. In addition, these studies have produced conflicting results with no consensus reached. Furthermore, the limitation to these studies were the small sample sizes and the absence of sequential echocardiographic monitoring of LV function in each patient. To the best of the authors’ knowledge, no such studies have been conducted in South Africa. This study reviewed data collected from Inkosi Albert Luthuli Central Hospital (IALCH), which is a government hospital in Durban, KwaZulu-Natal (KZN), South Africa. The objective of the study was to evaluate the effects of RV pacing on LV function in a setting where the majority of patients requiring a permanent pacemaker receive single chamber RV apical pacing. The focus of this study was to assess the effect of RV pacing on LV function by assessing the ejection fraction(EF), on echocardiography, pre and post pacemaker insertion. A retrospective chart review of 465 patients managed at the IALCH pacemaker clinic from 2003 up to 2012 was undertaken. Adult patients 18 years and older with a documented EF at the time of insertion of a pacemaker were included in the study. Patients were excluded from the study if they had coronary artery disease (CAD), unrepaired valvular heart disease, atrial fibrillation or dual chamber pacemakers. After enforcing the exclusion criteria, 430 patients were excluded and only 35 patients were eligible for the study. LV dysfunction was pre-defined as a left ventricular ejection fraction (LVEF) of < 50%. This study showed that RV pacing did not have a statistically significant effect on LV function post pacemaker insertion, based on the assessment of EF. The study was limited by the low number of eligible patients as it was a retrospective study and obtaining data was difficult as most patients who require a pacemaker do not routinely have a baseline echocardiograph done prior to insertion of the pacemaker. Another limiting factor in the study was that EF was the only modality of LV function that was assessed. Moreover, evaluation of the EF on echocardiography is subjective and user dependent. International studies have shown that the site of RV pacing has an impact on the degree of LV dyssynchrony and function. This factor could not be assessed in the current study as the site of RV pacing was not documented and was not standardised. Pacing in the correct clinical context is a necessity and is lifesaving. Current literature shows that RV pacing is a safe, relatively simple, convenient procedure that is well tolerated and is effective. This study showed no deterioration in LV function in patients post RV pacemaker insertion, which is important as the RV remains the most common site of lead placement especially in the resource limited state sector. Some studies have reported that RV pacing is associated with LV dysfunction. However, since there is a paucity of level 1 evidence regarding this aspect of RV pacing, the need for prospective studies on the long-term effects of RV pacing on LV function is required. In addition, the impact of alternative pacing sites on LV function should be explored.
Frequency and phenotype of Immune cell subsets in different regions of the human cervix : implications for HIV susceptibility.
(2018) Pillay, Nishlin.; Ngcapu, Sinaye.
Abstract available in PDF file.
In vitro and in vivo evaluation of metal-chelating agents as novel metallo beta-lactamase inhibitors against carbapenem-resistant enterobacteriaceae.
(2018) Omolabi, Kehinde Foluke.; Baijnath, Sooraj.; Kruger, Hendrik Gerhardus.
Infectious diseases remain one of the leading causes of death worldwide, despite the discovery of new and improvements on existing antibiotics. Bacteria are constantly developing sophisticated mechanisms of resisting the effects of antibiotics, this in turn has increased their pathogenicity and virulence. Drugs belonging to the beta-lactam class of antibiotics are most commonly prescribed as they display a broad-spectrum activity against both gram-positive and gramnegative bacteria. Carbapenems which are a member of this class is regarded as the last line of defence against bacterial infections. Resistance to carbapenems is on the increase especially by bacterial strains that are capable of producing metallo-beta lactamase enzymes. Infections caused by carbapenem resistant Enterobacteriaceae are deadly especially those mediated by metallo beta-lactamases. Efforts are being made to synthesize compounds that can inhibit these enzymes. Thus far little progress has been made as a clinically available metallo beta-lactamase inhibitor has not yet emerged, hence the scourge of carbapenem resistant infections rages on. Therefore, the main aim of this study was to evaluate the in vitro and in vivo activities of metal chelating agents NO3PY and NOTA as potential metallo beta-lactamase inhibitors against carbapenem resistant Enterobacteriaceae. The metal-chelating agents used in this study were NOTA and NO3PY. In vitro analysis was performed to determine the minimum inhibitory concentrations by broth microdilution of meropenem alone and when co-administered with the chelators against resistant bacterial strains. The strains used in this study were Escherichia coli NDM-1, Klebsiella pneumoniae 449, Escherichia coli IMP-1 and Enterobacter cloacae NDM-1. Time kill kinetics was also evaluated at graded concentrations of MIC, 1*MIC, 2*MIC, 4*MIC, 8*MIC and 16*MIC. For the in vivo pharmacokinetics were determined using LC-MS/MS analysis. Forty-eight healthy male Balb/c mice were divided into two groups; meropenem+NO3PY group and meropenem+NOTA group. Both groups received intraperitoneal doses at 10 mg/kg of meropenem and the MBLIs. Thereafter, the in vivo efficacy of meropenem co-administered with NOTA (100 mg/kg each) in a murine thigh infection was determined. Both chelators were able to restore the efficacy of meropenem to a concentration as low as 0.06 µg/ml. The time kill kinetics also showed that both compounds were able to significantly extend the killing time of meropenem. In vivo pharmacokinetic analysis revealed that NO3PY may not xiv be a suitable candidate for in vivo efficacy study as the MBLI was not bioavailable in plasma at 10mg/kg. NOTA on the other hand was bioavailable at the same concentration as NO3PY. The former was able to potentiate the effect of meropenem in vivo in a murine thigh infection model. It was evident by a significant reduction of colony forming unit counts in groups treated with meropenem co-administered with NOTA when compared to infected controls Further preclinical work such as in vitro and in vivo cytotoxicity tests, post beta-lactamase inhibitor effects among others are recommended for NOTA to further ascertain its suitability as a potential clinical metallo beta-lactamase inhibitor.
A comparative chemistry of coa® herbal medicine and herbal extracts of Vernonia Amygdalina (Bitter leaf) and Persea Americana (Avocado).
(2018) Boadu, Akwasi; Nlooto, Manimbulu.
The aim of this study is to investigate the phytochemical compounds present in standard COA®, dichloromethane (DCM), ethanol (EtOH), hexane (HEX), and ethyl acetate (EtOAc) , extracts of COA® compared to leaf extracts of Vernonia amygdalina and Persea americana collected from Cape Coast (Ghana) and Durban (South Africa) by phytochemical screening techniques and gas chromatography-mass spectrometry (GC-MS) analysis. Findings from this study revealed that leaf extracts of P. americana and V. amygdalina have been used in many local African communities for management of various diseases. Ethnomedicinal use and pharmacological properties of leaf extracts of P. americana andV. amygdalina may justify polyherbal formulation involving the two plants in the treatment of diseases such as diabetes, hypertension and other diseases. Outcomes of the preliminary phytochemicals screening showed the presence of alkaloids, anthraquinones, saponins, flavonoids, tannins, terpenoids and glucosides in ethanolic leaf extracts and standard COA®. GC-MS study of standard COA®, COA® extract and leaf extracts of P. americana and V. amygdalina collected from Ghana and South Africa had 60y phytochemical compounds identified in the hexane extract of COA® extract, 47 in DCM, 37 in ethyl acetate, 18in ethanol and 11 in standard COA® herbal medicine. The identification of phytochemical compounds and pharmacological actions was based on the name of the chemical compound, retention time and molecular formula from GC-MS analysis. The major phytochemicals common to COA® extract and leaf extracts of P. americana and V. amygdalina were heneicosane, phytyl acetate, pyrene, octadecanoic acid, eicosane, 2-methyltetracosane, pentadecanoic acid, hexadecanamide, and octadecanamide. Most of these major phytochemicals are present in ethanolic extracts of both COA® and leaf extracts of P. americana and V. amygdalina Leaf extracts of P. americana and V. amygdalina collected from Ghana have more phytochemicals compared to that of South Africa. The finding, of this study, confirm the presence of P. americana and V. amygdalina leaf extracts in COA® herbal medicine. It also confirms the profound variations in phytochemicals of P. americana and V. amygdalina leaf extracts due to the effects of environmental factors and geographical locations.
Deoxynivalenol downregulates NRF2-induced cytoprotective response in human hepatocellular carcinoma (HepG2) cells.
(2017) Ndlovu, Siqiniseko Sinikiwe.; Chuturgoon, Anil Amichund.; Nagiah, Savania.
Deoxynivalenol (DON) is a mycotoxin produced by Fusarium species that commonly infect agricultural foods. DON exhibits multiple toxic effects in both animals and humans, binding to the A site of the 28S ribosome and inhibits peptidyl transferase and protein elongation. It induces cytotoxicity through oxidative stress and inhibition of protein synthesis. Liver cells possess the antioxidant signalling mediator - Nuclear erythroid-2-Related factor (NRF2) that is activated in response to oxidative stress. There is no sufficient work done to show if the HepG2cells have an ability to withstand the molecular modifications induced by DON. The aim of the study was to investigate the cytotoxicity of DON and its effect on the NRF2 antioxidant response in HepG2 cells. The MTT assay was used to determine a dose response of DON (72 hr) on cell viability and to generate an IC50 value to use in subsequent assays. The intracellular concentration of GSH and ATP was determined using Luminometry. Lipid peroxidation and membrane damage were assessed by TBARS and LDH cytotoxicity assays respectively. Protein expression of NRF2, phosphorylated (p-)NRF2, catalase (CAT), superoxide dismutase (SOD)2, and Sirtuin (Sirt)3 was quantified by Western Blotting. The mRNA expressions of GPx, CAT and SOD2 were quantified using qPCR. DON decreased cell viability in a dose-dependent manner with an IC50 value of 26.17 μM. DON caused a significant decrease in the intracellular GSH concentration (1.77-fold, p= 0.0005). There was a significant decrease in the intracellular ATP content (1.92-fold, p= 0.0002).The study shows an induced lipid peroxidation and membrane damage in HepG2 cells by DON, as there was a significant increase in extracellular levels of both MDA (1.89-fold, p=0.0020) and LDH (1.35-fold, p=0.0207). DON reduced total NRF2 expression (0.30-fold, p= 0.0017), however activated p-NRF2 was significantly up-regulated (3.54-fold, p= 0.0085). There was a downregulation in the NRF2 target antioxidant proteins: CAT (0.33-fold, p= 0.005) with a concomitant decrease in CAT mRNA levels (0.02-fold, p= 0.0003), SOD2 (0.02-fold, p= 0.0137), with a parallel trend in the levels of SOD2 mRNA (0.06-fold, p= 0.0020) by DON. This toxin also significantly decreased the mRNA expression of GPx levels (0.03-fold, p= 0.0006). The expression of a mitochondrial stress response Sirt3 was significantly decreased (0.14-fold, p= 0.0058). Taken together, the data shows that DON causes oxidative stress and downregulates the NRF2-induced cytoprotection in HepG2 cells. Keywords: Deoxynivalenol Antioxidant response NRF-2
Tissue distribution of an anti-TB drug, TBA-354 in rats via mass spectrometric investigations.
(2016) Ntshangase, Sphamandla.; Govender, Thirumala.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.
Tuberculosis (TB) (TB) is one of the most well-recognized ancient human diseases in the history of mankind and it remains the major cause of human death amongst the transmittable diseases despite the use of antitubercular antibiotics. TB is caused by a pathogenic bacterium known as Mycobacterium tuberculosis (M.tb). The M.tb bacteria primary site of infection is the human lungs (resulting in pulmonary TB) but it can also affect other body parts (extrapulmonary TB) such as the bones, central nervous system (CNS), liver and many more others. Present-day TB research is focused on the development of more effective anti-TB drugs that can help shorten the treatment period. One of the major set-backs in TB drug development is to find the balance between the potential drug’s side effects and its activity. The present demonstrates the potential of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) techniques in the evaluation of the fundamental in-vivo pharmacokinetics and tissue distribution properties of a bicyclic nitroimidazole derivative, TBA-354. This drug is recognized to have an excellent activity against M.tb strains but also known to have mild signs of neurotoxicity. The use of MSI in this study shows the exact localization and accumulation of the drug in the brain, providing evidence as to why it showed certain neurotoxic signs during clinical trials. The study was conducted on healthy female Sprague-Dawley rats by administering 20 mg/kg of the drug, via an intraperitoneal (i.p.) route. After dosing the biological samples (plasma, lungs and brain) were collected at different time points for analysis. A validated LC-MS/MS method was used to quantify TBA-354 in rat plasma, lung and brain homogenate samples. LC-MS/MS cannot provide enough information regarding the drug localization and where it accumulates in the brain, therefore, MSI was then used to study the accumulation of the drug in different regions of the brain. As per LC-MS/MS results, the drug showed significant pharmacokinetic and distribution properties in the rat model with the highest levels in plasma compared to lung and brain. MALDI-MSI results showed that the drug was effectively able cross the blood-brain barrier (BBB) resulting in toxic accumulation in the neocortical regions of the brain. This study has proven the efficacy of MSI as a suitable analytical technique that can be used in future preclinical studies to evaluate the neurotoxicity of drugs targeting the brain, thus minimizing possible side effects.
In silico studies on Zika NS3 helicase: bedrock for antiviral drug design.
(2017) Oguntade, Sofiat Yetunde.; Soliman, Mahmoud Elsayed Soliman.
Zika virus is a re-emerging infectious disease, which was declared to be a public health emergency of international concern due to its various reported complications ranging from microcephaly in newborn to Guillain-Barré Syndrome (GBS). Because less attention has been paid to this virus over time, literature has been lacking regarding the structural and conformational features of its proteins particularly the NS3 helicase protein. This dissertation has addressed two major aspects of Zika NS3 helicase protein: (i) the binding interactions and (ii) structural dynamics and conformational changes of the protein. Investigations were carried out on the various Zika NS3 helicase ligand binding landscapes using 10 ligands via molecular docking, of which the best 3 were subjected to molecular dynamic simulations and several post dynamics analyses. Ivermectin, HMC-HO1α and lapachol emerged as the best 3 ligands. The result of the analysis showed that the binding of Ivermectin to ssRNA site and Lapachol and HMC-HO1α to the ATPase site induces a more compact protein structure, thus stabilizing residue fluctuations. The pharmacophoric characteristics found in Lapachol, HMC-HO1α and Ivermectin may be utilized in the design of a potent hybrid drug that can show efficient inhibition of a multitude of diseases including the detrimental co-infection of ZIKV, Dengue and Chikungunya. Also in this study, a detailed structural dynamic analysis was carried out on the structural flexibility of the NS3 helicase protein after NITD008 binding via molecular dynamics simulation and other posts dynamic analysis including the Principal Component Analysis (PCA) and the Dynamic Cross Correlation (DCC) analysis. Result revealed a prominent shift in the P-Loop found at the ATP site of the helicase. This loop and helical flexible regions give new insight into the dynamic structural features of ZIKV NS3 Helicase. The PCA and DCC analysis result revealed a significant structural flexibility of the NITD008-NS3 Helicase system compared to the rigid unbound form of the protein. Furthermore, the NITD008-NS3 Helicase complex stability was also ensured via a 130ns molecular dynamic simulation, this has proven NITD008 as an effective potential inhibitor of the NS3 helicase protein. This research is of immense importance to the discovery of a potent Zika inhibitor and in medicine as it has proven potential inhibitors with a good binding affinity towards Zika NS3 helicase. Also, this study hopes to fill in the gap of information that has been missing regarding molecular studies on Zika virus to some extent.
Computational studies of mycobacterium tuberculosis L, d-transpeptidase2.
(2018) Ntombela, Thandokuhle.; Honarparvar, Bahareh.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.
Tuberculosis (TB) is still one of the most highly elusive lethal transmittable diseases to eradicate and persist to be a major threat to public health due to emergence of drug resistance. Drug-resistant is steadily increasing worldwide, therefore, there is an urgent need for development of improved efficacious antibiotics and novel drug targets to successfully contain the disease. Peptidoglycan layer (PG) is the major attribute in bacterial cell envelope and is essential for protection and growth in all bacterial species including Mycobacterium tuberculosis(Mtb). The biosynthesis pathway for PG is extremely intricate and involves numerous interconnected metabolites such as N-acetylmuramic(NAM) acid and N-acetylglucosamine(NAG), that are required during transpeptidation. These two sugar molecules are linked together by a β (1-4) glycosidic bond and the NAM attaches 3-5 amino acid peptide stems. Consequently, the peptidoglycan strands are cross linked by transpeptidases, namely D, D- and L, D-transpeptidases, forming crucial 4→3 and 3→3 cross-linkages respectively. Both D, D- and L, D-transpeptidases need to be inhibited concomitantly to eradicate the bacterium. L, D-transpeptidase 2 (LdtMt2) is one of the paralogs that is essential for Mtb growth, cell morphology and virulence during the chronic stage of the disease. This paralog has major influence in drug resistance and persistence of tuberculosis. The traditional β-lactam family of antibiotics have been reported to be effective against Mtb following the inactivation of β-lactamases (BlaC) known to rapidly hydrolyze the core β-lactam ring. The classic penicillins inhibit D, D-transpeptidases, while L, D transpeptidases are blocked by carbapenems. Despite several studies in this field, to the best of our knowledge, limited attention has been paid to the inhibition mechanism of LdtMt2 using carbapenem derivatives. In this regard, we need to explore reliable alternative strategies that are most cost-effective in terms of investigating the interactions of FDA approved carbapenems against Mtb L, D-Transpeptidases and study the role of explicit water molecule confined in the active site. As a result, computational chemistry has provided the possibility to sightsee and investigate this problem with relatively cost effective computational techniques. In this thesis, we applied a hybrid quantum mechanics and molecular mechanics techniques (QM:MM), Our own N-layered Integrated molecular Orbital and Molecular Mechanics (ONIOM) approach, to investigate the binding interaction energies of carbapenems (biapenem, imipenem, meropenem and tebipenem) against L, D-transpeptidase 2. Furthermore, the role of explicit water molecule confined in the active site was also explored using the same hybrid method to ascertain the nature of binding interaction energies of carbapenems against LdtMt2. In all the investigated carbapenem─LdtMt2 complexes, the carbapenems and the catalytic active site residues of LdtMt2 (Cys205, His187, Ser188, His203 and Asn207) were treated at QM (B3LYP/6-31+G(d)) level of theory whereas the remaining part of the complexes were treated at MM level (AMBER force field). The explicit water molecules near the carbapenems were considered and treated at QM as well. The obtained findings of Gibbs free energy (G), enthalpy (H) and entropy (S) for all studied complexes showed that the carbapenems exhibit reasonable binding interactions towards LdtMt2. This can be attributed by the structural dissimilarities of the carbapenems side chain which significantly induce conformational changes in the LdtMt2. In comparison, the binding free energy calculations of the model system with explicit water molecule yielded significant binding interaction energies. The QTAIM and NBO results confirmed the nature of binding free energies that the topological properties of atoms in molecules and the delocalization of electrons are from a bonding to antibonding orbitals in hydrogen bond interactions and this has enhanced the stability of carbapenem―LdtMt2 complexes. We believe that molecular insight of the carbapenems binding to LdtMt2 and the role of explicit solvent will enable us to understand the inhibition mechanisms.

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