List of Titles

A novel, improved throughput bioassay for determining the delative speed of antimalarial drug action using fluorescent vitality probes

- Laming, Dustin

Authors: Laming, Dustin
Date: 2020
Subjects: Plasmodium falciparum , Malaria -- Treatment -- Africa , Antimalarials , Malaria vaccine
Language: English
Type: text , Thesis , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/139902 , vital:37810
Description: Malaria is one of the most prevalent diseases in Africa and Plasmodium falciparum is widely accepted as the most virulent of the malaria parasite species, with a fatality rate of 15 – 20 % of reported cases of infection. While various treatments have been accepted into early stage clinical trials, there has been little progress towards a proven vaccine. Pending a long-term solution, endemic countries rely heavily on the development of innovative drugs that are not only efficacious but are also quick acting. Traditional methods of evaluating antimalarial killing speeds via morphological assessments are inherently flawed by tedious, subjective interpretations of the heterogenous parasite morphology encountered in routine parasite culture conditions. This has led to the introduction of alternative assay formats to determine how rapidly compounds act on parasites in vitro: a parasite reduction ratio (PRR) assay that measures the recovery of parasite cultures from drug exposure; determining the shift in IC50 values of compounds when dose-response assays are carried out for different time periods; a bioluminescence relative rate of kill (BRRoK) assay that compares the extent to which compounds reduce firefly luciferase activity in transgenic parasites. Recent whole cell in vitro screening efforts have resulted in the generation of chemically diverse compound libraries such as the Medicines for Malaria Venture’s Pathogen Box, which houses 125 novel compounds with in vitro antiplasmodial activity. Assessing the relative killing speeds of these compounds would aid prioritizing fast-acting compounds that can be exploited as starting points for further development. This study aimed to develop a bioassay using the calcein-acetoxymethyl and propidium iodide fluorescent vitality probes, which would allow the relative speed of drug action on Plasmodium falciparum malaria parasites to be assessed and ranked in relation to each other using a quantitative, improved throughput approach. Initially applied to human (HeLa) cells, the assay was used to compare the relative speeds of action of 3 potential anti-cancer compounds by fluorescence microscopy. Subsequently adapted to P. falciparum, the assay was able to rank the relative speeds of action of standard antimalarials by fluorescence microscopy and two flow cytometry formats. Application of a multiwell flow cytometer increased throughput and enabled the assessment of experimental compounds, which included a set of artemisinin analogs and 125 antimalarial compounds in the MMV Pathogen Box. The latter culminated in the identification of five rapidly parasiticidal compounds in relation to the other compounds in the library, which may act as benchmark references for future studies and form the basis of the next generation of fast acting antimalarials that could be used to combat modern, resistant malaria.
Full Text:

Quick View

Application of machine learning, molecular modelling and structural data mining against antiretroviral drug resistance in HIV-1

- Sheik Amamuddy, Olivier Serge André

Authors: Sheik Amamuddy, Olivier Serge André
Date: 2020
Subjects: Machine learning , Molecules -- Models , Data mining , Neural networks (Computer science) , Antiretroviral agents , Protease inhibitors , Drug resistance , Multidrug resistance , Molecular dynamics , Renin-angiotensin system , HIV (Viruses) -- South Africa , HIV (Viruses) -- Social aspects -- South Africa , South African Natural Compounds Database
Language: English
Type: text , Thesis , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/115964 , vital:34282
Description: Millions are affected with the Human Immunodeficiency Virus (HIV) world wide, even though the death toll is on the decline. Antiretrovirals (ARVs), more specifically protease inhibitors have shown tremendous success since their introduction into therapy since the mid 1990’s by slowing down progression to the Acquired Immune Deficiency Syndrome (AIDS). However, Drug Resistance Mutations (DRMs) are constantly selected for due to viral adaptation, making drugs less effective over time. The current challenge is to manage the infection optimally with a limited set of drugs, with differing associated levels of toxicities in the face of a virus that (1) exists as a quasispecies, (2) may transmit acquired DRMs to drug-naive individuals and (3) that can manifest class-wide resistance due to similarities in design. The presence of latent reservoirs, unawareness of infection status, education and various socio-economic factors make the problem even more complex. Adequate timing and choice of drug prescription together with treatment adherence are very important as drug toxicities, drug failure and sub-optimal treatment regimens leave room for further development of drug resistance. While CD4 cell count and the determination of viral load from patients in resource-limited settings are very helpful to track how well a patient’s immune system is able to keep the virus in check, they can be lengthy in determining whether an ARV is effective. Phenosense assay kits answer this problem using viruses engineered to contain the patient sequences and evaluating their growth in the presence of different ARVs, but this can be expensive and too involved for routine checks. As a cheaper and faster alternative, genotypic assays provide similar information from HIV pol sequences obtained from blood samples, inferring ARV efficacy on the basis of drug resistance mutation patterns. However, these are inherently complex and the various methods of in silico prediction, such as Geno2pheno, REGA and Stanford HIVdb do not always agree in every case, even though this gap decreases as the list of resistance mutations is updated. A major gap in HIV treatment is that the information used for predicting drug resistance is mainly computed from data containing an overwhelming majority of B subtype HIV, when these only comprise about 12% of the worldwide HIV infections. In addition to growing evidence that drug resistance is subtype-related, it is intuitive to hypothesize that as subtyping is a phylogenetic classification, the more divergent a subtype is from the strains used in training prediction models, the less their resistance profiles would correlate. For the aforementioned reasons, we used a multi-faceted approach to attack the virus in multiple ways. This research aimed to (1) improve resistance prediction methods by focusing solely on the available subtype, (2) mine structural information pertaining to resistance in order to find any exploitable weak points and increase knowledge of the mechanistic processes of drug resistance in HIV protease. Finally, (3) we screen for protease inhibitors amongst a database of natural compounds [the South African natural compound database (SANCDB)] to find molecules or molecular properties usable to come up with improved inhibition against the drug target. In this work, structural information was mined using the Anisotropic Network Model, Dynamics Cross-Correlation, Perturbation Response Scanning, residue contact network analysis and the radius of gyration. These methods failed to give any resistance-associated patterns in terms of natural movement, internal correlated motions, residue perturbation response, relational behaviour and global compaction respectively. Applications of drug docking, homology-modelling and energy minimization for generating features suitable for machine-learning were not very promising, and rather suggest that the value of binding energies by themselves from Vina may not be very reliable quantitatively. All these failures lead to a refinement that resulted in a highly sensitive statistically-guided network construction and analysis, which leads to key findings in the early dynamics associated with resistance across all PI drugs. The latter experiment unravelled a conserved lateral expansion motion occurring at the flap elbows, and an associated contraction that drives the base of the dimerization domain towards the catalytic site’s floor in the case of drug resistance. Interestingly, we found that despite the conserved movement, bond angles were degenerate. Alongside, 16 Artificial Neural Network models were optimised for HIV proteases and reverse transcriptase inhibitors, with performances on par with Stanford HIVdb. Finally, we prioritised 9 compounds with potential protease inhibitory activity using virtual screening and molecular dynamics (MD) to additionally suggest a promising modification to one of the compounds. This yielded another molecule inhibiting equally well both opened and closed receptor target conformations, whereby each of the compounds had been selected against an array of multi-drug-resistant receptor variants. While a main hurdle was a lack of non-B subtype data, our findings, especially from the statistically-guided network analysis, may extrapolate to a certain extent to them as the level of conservation was very high within subtype B, despite all the present variations. This network construction method lays down a sensitive approach for analysing a pair of alternate phenotypes for which complex patterns prevail, given a sufficient number of experimental units. During the course of research a weighted contact mapping tool was developed to compare renin-angiotensinogen variants and packaged as part of the MD-TASK tool suite. Finally the functionality, compatibility and performance of the MODE-TASK tool were evaluated and confirmed for both Python2.7.x and Python3.x, for the analysis of normals modes from single protein structures and essential modes from MD trajectories. These techniques and tools collectively add onto the conventional means of MD analysis.
Full Text:

Quick View

Computer aided approaches against Human African Trypanosomiasis

- Kimuda, Magambo Phillip

Authors: Kimuda, Magambo Phillip
Date: 2020
Subjects: African trypanosomiasis , African trypanosomiasis -- Chemotherapy , Genomics , Macrophage migration inhibitory factor , Trypanosoma brucei , Pteridines , Tetrahydrofolate dehydrogenase , Adenylic acid , Molecular dynamics , Principal components analysis , Bioinformatics , Single nucleotide polymorphisms , Single Nucleotide Variants , Candidate Gene Association Study (CGAS)
Language: English
Type: Thesis , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/142542 , vital:38089
Description: The thesis presented here is divided into two parts under a common theme that is the use of computer based tools, genomics, and in vitro experiments to develop innovative ways of tackling Human African Trypanosomiasis (HAT). Part I of this thesis focused on the human host genetic determinants while Part II focused on the discovery of novel chemotherapeutics against the parasite. Part I is further sub-divided into two parts: The first involves a Candidate Gene Association Study (CGAS) on an African population to identify genetic determinants associated with disease and/or susceptibility to HAT. The second involves studying the effects of missense Single Nucleotide Variants (SNVs) on protein structure, dynamics, and function using Macrophage Migration Inhibitory Factor (MIF) as a case study. Part II is also sub-divided into two parts: The first involves a computer based rational drug discovery of potential inhibitors against the Trypanosoma the folate pathway; particularly by targeting Trypanosoma brucei Pteridine Reductase (TbPTR1) which is an enzyme used by trypanosomes to overcome T. brucei Dihydrofolate Reductase (TbDHFR) inhibition. Lastly the derivation of CHARMM force-field parameters that can be used to accurately model the geometry and dynamics of the T. brucei Phosphodiesterase B1 enzyme (TbrPDEB1) bimetallic active site center. The derived parameters were then used in MD simulations to characterise protein-ligand residue interactions that are important in TbrPDEB1 inhibition with the goal of targeting the cyclic Adenosine Monophosphate (cAMP) signalling pathway. In the CGAS we were unable to detect any genetic associations in the Ugandan cohort analysed that passed correction for multiple testing in spite of the study being sufficiently powered. Additionally, our study found no association of the Apo lipoprotein 1 (APOL1) G2 allele association with protection against acute HAT that has been previously reported. Future investigations for example, Genome Wide Association Studies using larger samples sizes (>3000 cases and controls) are required. Macrophage migration inhibitory factor (MIF) is a cytokine that is important in both innate and adaptive immunity that has been shown to play a role in T. brucei pathogenicity using murine models. A total of 27 missense SNVs were modelled using homology modelling to create MIF protein mutants that were investigated using in silico effect prediction tools, molecular dynamics (MD), Principal Component Analysis (PCA), and Dynamic Residue Network (DRN) analysis. Our results demonstrate that mutations P2Q, I5M, P16Q, L23F, T24S, T31I, Y37H, H41P, M48V, P44L, G52C, S54R, I65M, I68T, S75F, N106S, and T113S caused significant conformational changes. Further, DRN analysis showed that residues P2, T31, Y37, G52, I65, I68, S75, N106, and T113S are part of a similar local residue interaction network with functional significance. These results show how polymorphisms such as missense SNVs can affect protein conformation, dynamics, and function. Trypanosomes are auxotrophic for folates and pterins but require them for survival. They scavenge them from their hosts. PTR1 is a multifunctional enzyme that is unique to trypanosomatids that reduces both pterins and folates. In the presence of DHFR inhibitors, PTR1 is over-expressed thus providing an escape from the effects of DHFR inhibition. Both TbPTR1 and TbDHFR are pharmacologically and genetically validated drug targets. In this study 5742 compounds were screened using molecular docking, and 13 promising binding modes were further analysed using MD simulations. The trajectories were analysed using RMSD, Rg, RMSF, PCA, Essential Dynamics Analysis (EDA), Molecular Mechanics Poisson–Boltzmann surface area (MM-PBSA) binding free energy calculations, and DRN analysis. The computational screening approach allowed us to identify five of the compounds, named RUBi004, RUBi007, RUBi014, RUBi016 and RUBi018 that exhibited antitrypanosomal growth activities against trypanosomes in culture with IC50 values of 12.5 ± 4.8 μM, 32.4 ± 4.2 μM, 5.9 ± 1.4 μM, 28.2 ± 3.3 μM, and 9.7 ± 2.1 μM, respectively. Further when used in combination with WR99210 a known TbDHFR inhibitor RUBi004, RUBi007, RUBi014 and RUBi018 showed antagonism while RUBi016 showed an additive effect. These results indicate that the four compounds might be competing with TbDHFR while RUBi016 might be more specific for TbPTR1. These compounds provide scaffolds that can be further optimised to improve their potency and specificity. Lastly, using a systematic approach we derived CHARMM force-field parameters to accurately describe the TbrPDEB1 bi-metal catalytic center. For dynamics, we employed mixed bonded and non-bonded approach. We optimised the structure using a two-layer QM/MM ONIOM (B3LYP/6-31(g): UFF). The TbrPDEB1 bi-metallic center bonds, angles, and dihedrals were parameterized by fitting the energy profiles from Potential Energy Surface (PES) scans to the CHARMM potential energy function. The parameters were validated by means of MD simulations and analysed using RMSD, Rg, RMSF, hydrogen bonding, bond/angle/dihedral evaluations, EDA, PCA, and DRN analysis. The force-field parameters were able to accurately reproduce the geometry and dynamics of the TbrPDEB1 bi-metal catalytic center during MD simulations. Molecular docking was used to identify 6 potential hits, that inhibited trypanosome growth in vitro. The derived force-field parameters were used to simulate the 6 protein-ligand complexes with the aim of elucidating crucial protein-ligand residue interactions. Using the most potent ligand RUBi022 that had an IC50 of 14.96 μM we were able to identify key residue interactions that can be of use in in silico prediction of potential TbrPDEB1 inhibitors. Overall we demonstrate how bioinformatics tools can complement current disease eradication strategies. Future work will focus on identifying variants identified in Genome Wide Association Studies and partnering with wet labs to carry out further enzyme-ligand activity relationship studies, structure determination or characterisation of appropriate protein-ligand complexes by crystallography, and site specific mutation studies
Full Text:

Quick View

Cyclooxygenase-1 as an anti-stroke target: potential inhibitor identification and non-synonymous single nucleotide polymorphism analysis

- Muronzi, Tendai

Authors: Muronzi, Tendai
Date: 2020
Subjects: Cerebrovascular disease , Cerebrovascular disease -- Treatment , Cerebrovascular disease -- Chemotherapy , Cyclooxygenases , High throughput screening (Drug development) , Drug development , Molecular dynamics , South African Natural Compounds Database , ZINC database
Language: English
Type: Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/143404 , vital:38243
Description: Stroke is the third leading cause of death worldwide, with 87% of cases being ischemic stroke. The two primary therapeutic strategies to reduce post-ischemic brain damage are cellular and vascular approaches. The vascular strategy aims to rapidly re-open obstructed blood vessels, while the cellular approach aims to interfere with the signalling pathways that facilitate neuron damage and death. Unfortunately, popular vascular treatments have adverse side effects, necessitating the need for alternative chemotherapeutics. In this study, cyclooxygenase-1 (COX-1), which plays a significant role in the post- ischemic neuroinflammation and neuronal death, was targeted for identification of novel drug compounds and to assess the effect of nsSNPs on its structure and function. In a drug discovery part, ligands from the South African Natural Compounds Database (SANCDB-https://sancdb.rubi.ru.ac.za/) and ZINC database (http://zinc15.docking.org/) were used for high-throughput virtual screening (HVTS) against COX-1. Additionally, five nsSNPs were being investigated to assess their impact on protein structure and function. Three of these SNPs were in the COX-1 dimer interface. Molecular docking and molecular dynamics simulations revealed asymmetric nature of the protein. Several ligands, peculiar to each monomer, exhibited favourable binding energies in the respective active sites. SNP analysis indicated effects on inter-monomer interactions and protein stability.
Full Text:

Quick View

Evaluation of an NADPH-dependent assay for inhibition screening of Salmonella enterica DOXP Reguctoisomerase for identification of novel drug hit compounds

- Ngcongco, Khanyisile

Authors: Ngcongco, Khanyisile
Date: 2020
Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/167132 , vital:41440
Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
Full Text:

Quick View

Exploring the structural integrity of a picornavirus capsid

- Upfold, Nicole Sarah

Authors: Upfold, Nicole Sarah
Date: 2020
Subjects: Picornaviruses , Immunoglobulins , Capsids (Virology) , Viruses Morphology , RNA viruses
Language: English
Type: Doctoral theses , text
Identifier: http://hdl.handle.net/10962/131837 , vital:36758 , DOI https://doi.org/10.21504/10962/131837
Description: Picornaviruses are a diverse family of small RNA viruses that cause a broad range of human and veterinary diseases. Despite decades of research into the molecular biology of these pathogens, no antivirals and few vaccines are commercially available for the treatment and prevention of picornavirus infections. The capsids of these non-enveloped viruses are involved in many important aspects of the picornavirus lifecycle, such as cell attachment and entry, uncoating, and protection of the viral RNA. Although the structures of many picornavirus capsids have been solved, a broader understanding of the molecular determinants that are required for structural integrity and stability is imperative for an improved understanding of the basic biology of these viruses, and for designing effective control strategies. Collectively, this thesis aims to elucidate the molecular determinants of structural stability and integrity in the Theiler’s murine encephalomyelitis virus capsid (TMEV). To study the TMEV GDVII capsid using biochemical techniques, neutralising polyclonal antibodies were generated against GDVII particles. The antibodies recognised linear epitopes in the C-terminus of the VP1 protein, but not those present in VP2 or VP3. The VP1 C-terminal residues were mapped to a loop above the putative receptor binding pit on the capsid surface, which prompted an investigation into the potential binding site of the TMEV co-receptor, heparan sulphate. Molecular docking revealed that heparan interacts with residues of the receptor binding pocket, as well as residues of the adjoining VP1 C-terminal loop. These findings suggest that the antibodies neutralise virus infection by preventing attachment of the virus to the co-receptor and possibly the unknown primary receptor. Few studies have identified the specific residues and interactions at subunit interfaces that significantly contribute to picornavirus capsid stability, assembly, and function. A novel in-silico screen was developed for the prediction of hotspot residues at protein-protein interfaces of a virus capsid. This screen can be applied to elucidate the residues that contribute significantly to the intraprotomer, interprotomer and interpentamer interfaces of any picornavirus capsid, on condition that the structure of the virus is available. The screen was applied to TMEV GDVII resulting in the identification of hotspots, several of which correspond to residues that are known to be important for aspects of the virus lifecycle, such as those that contribute to pH stability or form part of receptor binding sites. This observation suggests that residues involved in specific capsid functions may also play a role in capsid stability. Many of the residues identified as hotspots in TMEV corresponded to those required for assembly, uncoating, and virus growth in representative picornaviruses from various genera, suggesting that the residues that regulate capsid stability may be somewhat conserved across the family. Hotspots identified at the interpentamer interfaces of TMEV were individually substituted to alanine to further explore their importance to the TMEV lifecycle. All the amino acid substitutions prevented completion of the virus lifecycle as no CPE was observed following transfection of susceptible cells. Immunofluorescence experiments demonstrated that virus protein synthesis and RNA replication were not inhibited by substitution of the hotspot residues, but that infectivity was severely impeded. This confirmed that the residues were required for some aspect of the virus lifecycle, such as capsid assembly, or were critical for maintaining the conformational stability of the TMEV particles. Virus capsids become unstable and are prone to dissociation under certain conditions such as extreme pH and non-physiological temperatures. The thermostability of TMEV was explored by selecting GDVII virions with improved thermal tolerance through serial passage and heat exposure. Thermostable virions that could tolerate temperatures above 57 °C had reduced infective titres compared to the wild type TMEV suggesting that the virus adapted to thermal stress at the expense of viral fitness. Sequencing the capsid encoding regions of the mutant virions revealed a pair of amino acid substitutions that were present in all mutants. Additional substitutions that were unique to viruses selected at different temperatures were also identified. Most of the substitutions were located within the intraprotomer interfaces of the virus, unlike previous studies on enteroviruses where mutations were mostly localised to the receptor binding pocket. This thesis provides the first analysis of the structural determinants of TMEV capsid stability. The generation of tools to further explore the capsid structures of TMEV and other picornaviruses provides an opportunity for future studies which may contribute to the development of novel control strategies against this important family of viruses. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
Full Text: false

Quick View

Mechanism of action of non-synonymous single nucleotide variations associated with α-carbonic anhydrases II, IV and VIII

- Sanyanga, Taremekedzwa Allan

Authors: Sanyanga, Taremekedzwa Allan
Date: 2020
Subjects: Carbonic anhydrase , Carbonic anhydrase -- Therapeutic use , Nucleotides
Language: English
Type: text , Thesis , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/167346 , vital:41470
Description: The carbonic anhydrase (CA) group of enzymes are Zinc (Zn2+) metalloproteins responsible for the reversible hydration of CO2 to bicarbonate (BCT or HCO− 3 ) and protons (H+) for the facilitation of acid-base balance and homeostasis within the body. Across all organisms, a minimum of six CA families exist, including, α (alpha), β (beta), γ (gamma), δ (delta), η (eta) and ζ (zeta). Some organisms can have more than one family, with exception to humans that contain the α family solely. The α-CA family comprises of 16 isoforms (CA-I to CA-XV) including the CA-VIII, CA-X and CA-XI acatalytic isoforms. Of the catalytic isoforms, CA-II and CA-IV possess one of the fastest rates of reaction, and any disturbances to the function of these enzymes results in CA deficiencies and undesirable phenotypes. CA-II deficiencies result in osteopetrosis with renal tubular acidosis and cerebral calcification, whereas CA-IV deficiencies result in retinitis pigmentosa 17 (RP17). Phenotypic effects generally manifest as a result of poor protein folding and function due to the presence of non-synonymous single nucleotide variations (nsSNVs). Even within the acatalytic isoforms such as CA-VIII that llosterically regulates the affinity of inositol triphosphate (IP3) for the IP3 receptor type 1 (ITPR1) and regulates calcium (Ca2+) signalling, the presence of SNVs also causes phenotypes cerebellar ataxia, mental retardation, and dysequilibrium syndrome 3 (CAMRQ3). Currently the majority of research into the CAs is focused on the inhibition of these proteins to achieve therapeutic effects in patients via the control of HCO− production or reabsorption as observed in glaucoma and diuretic medications. Little research has therefore been devoted into the identification of stabilising or activating compound that could rescue protein function in the case of deficiencies. The main aim of this research was to identify and characterise the effects of nsSNVs on the structure and function of CA-II, CA-IV and CA-VIII to set a foundation for rare disease studies into the CA group of proteins. Combined bioinformatics approaches divided into four main objectives were implemented. These included variant identification, sequence analysis and protein characterisation, force field (FF) parameter generation, molecular dynamics (MD) simulation and dynamic residue network analysis (DRN). Six variants for each of the CA-II, CA-IV and CA-VIII proteins with pathogenic annotations were identified from the HUMA and Ensembl databases. These included the pathogenic variants K18E, K18Q, H107Y, P236H, P236R and N252D for CA-II. CA-IV included the pathogenic R69H, R219C and R219S, and benign N86K, N177K and V234I variants. CA-VIII included pathogenic S100A, S100P, G162R and R237Q, and benign S100L and E109D variants. CA-II has been more extensively studied than CA-IV and CA-VIII, therefore residues essential to its function and stability are known. To discover important residues and regions within the CA-IV and CA-VIII proteins sequence and motif analysis was performed across the α-CA family, using CA-II as a reference. Sequence analysis identified multiple conserved residues between the two acatalytic CA-II and CA-IV, and the acatalytic CA-VIII isoforms that were proposed to be essential for protein stability. With exception to the benign N86K CA-IV variant, none of the other pathogenic or benign CA-II, CA-IV and CA-VIII SNVs were located at functionally or structurally important residues. Motif analysis identified 11 conserved and important motifs within the α-CA family. Several of the identified variants were located on these motifs including K18E, K18Q, H107Y and N252D (CA-II); N86K, R219C, R219S and V234I (CA-IV); and E109D, G162R and R237Q (CA-VIII). As there were no x-ray crystal structures of the variant proteins, homology modelling was performed to calculate the protein structures for characterisation. In CA-VIII, the substitution of Ser for Pro at position 100 (variant S100P) resulted in destruction of the β-sheet that the SNV was located on. Little is known about the mechanism of interaction between CA-VIII and ITPR1, and residues involved. SiteMap and CPORT were used to identify binding site amino for CA-VIII and results identified 38 potential residues. Traditional FFs are incapable of performing MD simulations of metalloproteins. The AMBER ff14SB FF was extended and Zn2+ FF parameters calculated to add support for metalloprotein MD simulations. In the protein, Zn2+ was noted to have a charge less than +1. Variant effects on protein structure were then investigated using MD simulations. Root mean square deviation (RMSD) and radius of gyration (Rg) results indicated subtle SNV effects to the variant global structure in CA-II and CA-IV. However, with regards to CA-VIII RMSD analysis highlighted that variant presence was associated with increases to the structural rigidity of the protein. Principal component analysis (PCA) in conjunction with free energy analysis was performed to observe variant effects on protein conformational sampling in 3D space. The binding of BCT to CA-II induced greater protein conformational sampling and was associated with higher free energy. In CA-IV and CA-VIII PCA analysis revealed key differences in the mechanism of action of pathogenic and benign SNVs. In CA-IV, wild-type (WT) and benign variant protein structures clustered into single low energy well hinting at the presence of more stable structures. Pathogenic variants were associated with higher free energy and proteins sampled more conformations without settling into a low energy well. PCA analysis of CA-VIII indicated the opposite to CA-IV. Pathogenic variants were clustered into low energy wells, while the WT and benign variants showed greater conformational sampling. Dynamic cross correlation (DCC) analysis was performed using the MD-TASK suite to determine variant effects on residue movement. CA-II WT protein revealed that BCT and CO2 were associated with anti-correlated and correlated residue movement, highlighting at opposite mechanisms. In CA-IV and CA-VIII variant presence resulted in a change to residue correlation compared to the WT proteins. DRN analysis was performed to investigate SNV effects of residue accessibility and communication. Results demonstrated that SNVs are associated with allosteric effects on the CA protein structures, and effects are located on the stability assisting residues of the aromatic clusters and the active site of the proteins. CA-II studies discovered that Glu117 is the most important residue for communication, and variant presence results in a decrease to the usage of the residue. This effect was greatest in the CA-II H107Y SNV, and suggests that variants could have an effect on Zn2+ dissociation from the active site. Decreases to the usage of Zn2+ coordinating residues were also noted. Where this occurred, compensatory increases to the usage of other primary and secondary coordination residues were observed, that could possibly assist with the maintenance of Zn2+ within the active site. The CA-IV variants R69H and R219C highlighted potentially similar pathogenic mechanisms, whereas N86K and N177K hinted at potentially similar benign mechanisms. Within CA-VIII, variant presence was associated with changes to the accessibility of the N-terminal binding site residues. The benign CA-VIII variants highlighted possible compensatory mechanisms, whereby as one group of N-terminal residues loses accessibility, there was an increase to the accessibility of other binding site residues to possibly balance the effect. Catalytically, the proton shuttle residue His64 in CA-II was found to occupy a novel conformation named the “faux in” that brought the imidazole group even closer to the Zn2+ compared to the “in” conformation. Overall, compared to traditional MD simulations the incorporation of DRN allowed more detailed investigations into the variant mechanisms of action. This highlights the importance of network analysis in the study of the effects of missense mutations on the structure and function of proteins. Investigations of diseases at the molecular level is essential in the identification of disease pathogenesis and assists with the development of specifically tailored and better treatment options especially in the cases of genetically associated rare diseases.
Full Text:

Quick View

Molecular simulations of potential agents and targets of Alzheimer’s disease

- Carlisle, Tanya

Authors: Carlisle, Tanya
Date: 2020
Subjects: Alzheimer's disease -- Treatment , Alzheimer's disease -- Molecular aspects , Amyloid beta-protein precurs , Amyloid beta-protein
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/140025 , vital:37825
Description: The World Alzheimer Report statedin 2016 that approximately 46.8 million people were living with dementia and this figure is expected to triple by 2050. Alzheimer’s Disease was discovered to be a precursor to dementia in 1976 and since then efforts to understand Alzheimer’s have been prioritized. To date, there are very few effective forms of treatment for Alzheimer’s, many are known to offer only mild calming of the symptoms and have side effects such as diarrhea, nausea, loss of appetite and sleep disturbances. This has been due to lack of understanding on how Alzheimer’s is caused. With the two main hallmarks of the disease now being more understood it has opened the doorway into the discovery of new treatments for this disease. This study focuses on the hallmark involving the aggregation of the β-amyloid protein to form plaques surrounding the neurons of the brain. Copper, Zinc and Iron have also been found in high concentrations in and surrounding these plaques. This study focused on the screening of the South African Natural Compound database (SANCDB) to discover hits that have potential destabilizing action against the Beta-amyloid aggregate. If one of these compounds could prove to have destabilizing action on the aggregate it could open the doorway to new potential forms of treatment. Over 700 SANCDB compounds were docked, and the top hits were taken to molecular dynamics to further study the interactions of the compounds and the aggregate. However, the hits identified had strong binding to the aggregate causing it to become stable instead of the desired effect of destabilizing the structure. This information, however, does not rule out the possibility of these compounds preventing the formation of the aggregates. Further, interactions of copper with β-amyloid and copper were determined by solubilizing the aggregate and introducing copper ions in a dynamics simulation. Possible interactions between copper and the methionine residues were visualised.
Full Text:

Quick View

Orchid mycorrhizal and endophytic fungal diversity of three co-occurring terrestrial orchids in the large African genus Disa (Orchidaceae)

- Khambule, Nondumiso Venessia

Authors: Khambule, Nondumiso Venessia
Date: 2020
Subjects: Orchids South Africa , Mycorrhizal fungi South Africa , Endomycorrhizas South Africa , Endophytes , Orchids Roots
Language: English
Type: Doctoral theses , text
Identifier: http://hdl.handle.net/10962/163341 , vital:41030
Description: Orchids (in the family Orchidaceous) are one of the richest plant families and approximately 500 species are found in South Africa. A number of orchid species are found on disturbed areas and many of the terrestrial species grow in poor soils with low mineral nutrient availability. Most orchid species are thought to be associated with mycorrhizal fungi for germination and mycorrhiza provides nutrients for the survival of adult plants. The aim of this study was to select Orchidaceous plant species and to isolate, identify and characterize the orchid endophytes and assess these isolates for potential antimicrobial and enzymatic activities Isa is the largest genus in South Africa and three Disa species co-occurring in a small geographical area were selected. These included Disa bracteata, D. cornuta and D. polygonoides which span three sections of the genus. Roots were stained to confirm the mycorrhizal status of the Disa species. Mycorrhizal pelotons structures were microscopically observed inside root cells. The presence of pelotons is indictive of mycorrhizal fungal interactions within the orchid roots and areas associated with the site of nutrient exchange between plant and fungus. The presence of pelotons, however, does not give n indication of the fungal species involved. The endophytes were successfully isolated in pure cultures on potato dextrose agar (PDA). All slow growing isolates were selected, and further molecular identification undertaken; DNA was extracted, and PCR amplified using internal transcribed spacer (ITS1F and ITS4) fungal primers. The amplified products were then sequenced and analysed by comparison to sequences in the GenBank database. Trichoderma, Penicillium, Metapochonia, Talaromyces, Oidiodendron Neopestalotiopsis, and Chaetomium were identified from these sequences. The presence of other fungal root endophytes was suspected despite the rigorous surface sterilization procedure used. The primers used to amplify the ITS region are the universal barcoding primers which are specific to fungi. ITS1F is one of the primers designed to amplify a broad range of fungi. DNA was extracted from orchid roots and amplicons were cloned into a pGEMT plasmid vector. Individual clones were sequenced and aligned with Mega software and compared to sequences in the GenBank and UNITE database. Based on percentage sequence identity, unidentified Tulasnella species, Tullasnela colaspora, and various Ascomycota endophytes were identified as contributing to the endophytic root fungal diversity of the selected Disa species. The Disa species investigated in this study were associated with several soil endophytes. D. bracteata, D. polygonoides were collected from the same site along the road verge which is regarded as being disturbed. Based on both culture – dependent and independent techniques employed Oidiodendron was found associated with both species. Antimicrobial activity was determined using a well diffusion method using extracts from the isolated fungi against the bacterial isolates Bacillus cereus, Staphylococcus aureus, Escherichia coli and Pseudomonas puptida. Most of the isolated fungi showed at least one potential inhibition effect against one of the bacterial isolates. The extracts that showed potential antimicrobial activity could be further screened to determine the compounds produced as secondary metabolites using techniques such as LC-MS Enzymatic activities of protease, cellulose and amylase were determined using solid media amended with milk protein, carboxymethylcellulose (CMC) and starch. The majority of fungal isolates tested positive with amylase and cellulose with only a few fungal isolates testing positive for protease activity. Broth cultures containing CMC and starch were shown to enhance biomass production in approximately 40 % of the fungal isolates. Degradation of the substrates is required in order to provide carbon to the fungus under test in order to optimize fungal growth as well as to gain insight into their ecological role. Enzyme activity was evident particularly when cellulose and starch were provided as substrates. All the fungal isolates tested grew on the amended medium, with 40% of the isolates preferring to utilize CMC and/or starch, indicating the ability of these fungi to utilize various resources for carbon acquisitions. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
Full Text:

Quick View

Prediction of mass spectra for natural products using an ab initio approach

- Novokoza, Yolanda

Authors: Novokoza, Yolanda
Date: 2020
Subjects: Molecular dynamics , Molecular dynamics -- Computer simulation , Mass spectroscopy , Electron impact ionization
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/167166 , vital:41443
Description: Mass spectrometry (MS) is a technique that measures the fragmentation of molecules, dependent on the molecule’s chemical composition and structure, by first introducing a charge on the molecules. The instrument records the mass to charge ratio, but the energy from the ionization process causes the molecule to fragment. The resultant mass spectrum is highly indicative of not only the molecule analyzed, but also its chemical composition. MS is used in research and industry for both routine and research purposes. One such way to ionize molecules for MS is by bombarding the molecule with electrons which is the basis of electron impact mass spectrometry (EIMS). Although EIMS is widely used, prediction of electron impact mass spectra from first principles is a challenging problem due to a need to accurately determine the probability of different fragmentation pathways of a molecule. Ab initio molecular dynamics based methods are able to explore in an automatic fashion the energetically available fragmentation paths thus give reaction mechanisms in an unbiased way. The mass spectra of five molecules have been explored in work-flows leading to the prediction of mass spectra. These molecules include three natural products alpha-hispanolol, PFB oxime derivative and boronolide (for which experimental mass spectra were not available) and two compounds from the NIST database (for which experimental mass spectra were available). For each of these systems many random conformations were generated using the RDKit library. To all conformations random velocities were applied to each atom. Ab initio molecular dynamics was performed on each conformer, using these initial random velocities using CP2K software, at DFTB+ level at a variety of highly raised temperatures (to accelerate the formation of fragments) Fragmentation was monitored by iterating through all bonds, and identifying bond breakages during dynamics. Graph theoretical packages were used then to track distinct fragments generated. For each of these fragments, charges were determined from Mulliken analysis for all atoms on the fragment from the QM calculations and sum of atomic spin densities per fragment was also plotted. The fragment with the greatest charge (corresponding to the formation of a cation fragment) was taken for plotting on the mass spectrum. Finally, from the mass of the fragment and its elemental composition, the isotopic distribution for the fragment was determined, and this distribution was included by addition in to the mass spectrum. For all trajectories, the sum of all isotopic distributions determined the final mass spectrum.
Full Text:

Showing items 1 - 20 of 26