List of Titles

Computational search for nature-derived dual-action inhibitors of HIV-1 reverse transcriptase and integrase: a potential strategy to mitigate drug resistance progression

- Mwiinga, Luyando

Authors: Mwiinga, Luyando
Date: 2024-10-11
Subjects: HIV (Viruses) , Reverse transcriptase , Antiretroviral agents , RDKit , Drug resistance , Docking
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/463930 , vital:76458
Description: Human immunodeficiency virus Type 1 (HIV-1) is a devastating viral infection affecting millions worldwide and presents significant challenges in treatment and management. In 2022, approximately 39 million people were living with HIV with Sub-Saharan Africa having two thirds of these infections. Devastatingly, there were approximately 300 000 HIV/AIDS related deaths in Sub-Saharan Africa alone in 2022 alone. Antiretroviral therapy (ART) which is fundamental for HIV treatment, comprises of a combination of drugs such as nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTs), protease inhibitors (PIs) and integrase strand transfer inhibitors (INSTIs). However, although 28.7 million people out of the estimated 38.4 million people living with HIV in 2021 were receiving ART, the emergence of drug-resistant strains further complicates treatment efforts, highlighting the need for novel therapeutic approaches. This study aimed to address the challenges raised by drug resistance and significant side effects by identifying potential dual inhibitors against HIV-1 Reverse Transcriptase (RT) and Integrase (IN) using in silico techniques. RT RNase H and IN were chosen as targets for their shared dependency on Mg2+ ions within their active sites, which are crucial for catalytic activity. The selection of dual inhibitors was motivated by the fact that the virus would need to replicate at two points simultaneously to develop resistance, making it less likely. The objectives of this study included the creation of a natural derivative compound library using RDKit with the aid of SciFinder, utilizing (-)-epigallocatechin-3-O-gallate (EGCG), because of its dual inhibitory effects against RT and IN, as indicated by a study conducted by Sanna et al. 2019. The natural derivatives were chosen to take advantage of their chemical diversity and to explore potential novel therapeutic options for combating HIV drug resistance. The compound library created comprised of 125 203 compounds. Then docking studies were conducted to assess proteinligand binding. After the correlation of the RT and IN docking studies, 288 compounds were filtered to have potential dual inhibitory activity. Then quantitative estimation of druggability (QED) analysis identified three compounds with superior properties compared to EGCG and FDAapproved drug raltegravir (RAL). Molecular docking simulations revealed interactions between the inhibitors and the key active site residues of RT and IN, along with the chelation of at least one 3 Mg2+, suggesting the potential for enzymatic disruption. Furthermore, molecular dynamic (MD) simulations were then conducted to assess protein-ligand system behavior, through RMSD and RMSF analysis. The RMSD analysis uncovered instability in the IN-Sci30703 complex, leading to its exclusion as a potential dual action inhibitor. RMSF analysis for IN showed that all the inhibitors had the ability to limit the flexibility of the catalytic loop which is essential for catalytic activity. Therefore, further in vitro studies are required to evaluate the effectiveness of the remaining two EGCG derivatives (Sci33211 and Sci48919) in inhibiting RT and IN through the chelation of at least one Mg2+ ion to determine if they have superior dual inhibitory effects compared to EGCG. This study adds to the ongoing efforts to develop effective strategies against HIV-1 drug resistance and emphasizes the importance of continued research in this field. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
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Date Issued: 2024-10-11

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Evaluating baculovirus mixtures against false codling moth Thaumatotibia leucotreta Meyrick. (Lepidoptera: Tortricidae)

- Tole, Siviwe

Authors: Tole, Siviwe
Date: 2024-10-11
Subjects: False codling moth Biological control , Baculoviruses , Integrated pest management , Natural pesticides , Granulovirus
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/463996 , vital:76464
Description: False codling moth (FCM), Thaumatotibia leucotreta, is an important pest of citrus, stone fruit, avocados, peppers, and other important agricultural crops in southern Africa. Baculovirus-based biopesticides are components in an integrated pest management (IPM) programme to manage the pest in the field. Cryptogran™ and Cryptex™ which are CrleGV-SA based-biopesticides have been effective in the control of T. leucotreta for the past 15 years. Recently, CrpeNPV-based Multimax™ and Codlmax™ have been commercialised to control T. leucotreta and other important agricultural pests. Despite these viruses being relatively host-specific and safe to humans and animals in comparison to chemical insecticides, their application is hindered by their slow speed of kill, sensitivity to UV light, and the potential for insect resistance. Research investigating the effects of mixed baculoviral interactions against target pests has been a growing field of interest due to their potential to overcome such shortcomings. Previous studies using a combination of CrleGV-SA and CrpeNPV against T. leucotreta observed a reduction in lethal concentration in laboratory bioassays, indicating that such mixtures may have the potential for application in the field. This has led to the motivation to investigate further interactions between CrleGV-SA in combination with CrpeNPV, CpGV-M, and HearNPV-Au to understand better how these viruses interact and to determine whether synergistic, additive, or antagonistic interactions can occur against T. leucotreta. The outcome of these interactions will inform researchers and farmers about best practices concerning these viruses should they be combined against T. leucotreta in the future. Prior to performing mixed baculovirus infections in laboratory bioassays, oligonucleotides targeting unique regions in the viral genomes of CrleGV-SA, CrpeNPV, CpGV-M, and HearNPV-Au were designed using Primer-BLAST. The specificity of these oligonucleotides was further tested in silico using Geneious R11 software (11.1.5). The stocks of CrpeNPV, CpGV-M, and HearNPV-Au were purified using crude OB extraction from diseased C. peltastica, C. pomonella, and H. armigera larval cadavers provided by River Bioscience (Pty) Ltd (Gqeberha, South Africa). The stock of CrleGV-SA was purified using crude OB extraction from infected T. leucotreta cadavers. Subsequently, the unique oligonucleotides were used in PCR assays to detect if the samples contained the baculoviruses of interest. Amplicons of the expected sizes were generated indicating the presence of CrleGV-SA, CrpeNPV, CpGV-M, and HearNPV-Au in each of the samples. The OBs were counted using darkfield microscopy and a counting chamber before the single and mixed infections were initiated against T. leucotreta neonate larvae. Surface-dose biological assays were used to evaluate the relative virulence in terms of lethal concentration of CrleGV-SA, CrpeNPV, and CpGV-M, alone against T. leucotreta. After 7 days, the dose mortality data was analysed using “drc” in R studio and the LC50 and LC90 were compared amongst each virus. The CrleGV-SA treatment was estimated to be the most virulent in comparison to CrpeNPV and CpGV-M. A dose discriminate assay confirmed that HearNPV does not cause mortality in T. leucotreta. Similarly, the relative virulence in terms of lethal concentration of CrleGV-SA in various ratios in combination with CrpeNPV, CpGV-M, and HearNPV-Au was determined using 7-day surface dose biological assays. The CrleGV/CrpeNPV was the most virulent mixture with lower LC50 and LC90 values measured in comparison to CrleGV/CpGV and CrleGV/HearNPV, respectively. The Tammes Bakuniak graphic method confirmed the CrleGV/CrpeNPV, CrleGV/CpGV, and CrleGV/HearNPV mixtures to be antagonistic against T. leucotreta neonate larvae in terms of lethal concentration. The last aspect of the study was to determine the probable cause of larval death. A modified CTAB protocol was used to extract genomic DNA from neonate-sized T. leucotreta cadavers collected in single and mixed infection assays. The gDNA served as templates in PCR assays using the unique oligonucleotides. In single infections, the presence of CrleGV-SA in CrpeNPV and HearNPV inoculated larvae was observed. The results suggest possible covert infections of CrleGV-SA in the T. leucotreta colony which may be caused by virus infection or an unknown stress factor. The results from the mixed infections showed the presence of each virus in all replicates except for the CrleGV/CpGV and CrleGV/HearNPV mixtures. In the CrleGV/CpGV mixture, only CrleGV-SA was present in the last replicate, suggesting a possible competition for host resources. In the CrleGV/HearNPV mixture, only CrleGV-SA was detected in all 3 replicates, suggesting that HearNPV did not have any effect and the larvae died of the CrleGV-SA infection. This is the first study to report mixtures of CrleGV-SA in combination with CpGV-M and HearNPV-Au against T. leucotreta neonate larvae. Despite the antagonistic interactions observed in the evaluated mixtures, this study has laid a foundation to further investigate how these viruses interact in dual infections for the improved control of T. leucotreta. This may be done by evaluating different ratios and combinations of baculoviruses to those used in this study. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
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Date Issued: 2024-10-11

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Immobilisation of an Aspergillus niger derived endo-1,4-β-mannanase, Man26A, for the production of prebiotic mannooligosaccharides from soybean meal

- Anderson, Amy Sage

Authors: Anderson, Amy Sage
Date: 2024-10-11
Subjects: Aspergillus niger , Soybean meal , Mannosidases , Oligosaccharides , Immobilized microorganisms
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/463897 , vital:76455
Description: This study investigated the potential for antibiotic alternatives in the form of prebiotics produced from the enzymatic breakdown of soybean meal (SBM). This study first investigated the immobilisation of an endo-1,4-β-mannanase derived from Aspergillus niger on glutaraldehyde-activated chitosan nanoparticles (CTS) and glutaraldehyde-activated chitosan-coated magnetic Fe3O4 nanoparticles (MAGS-CTS) - which could be effectively used to hydrolyse the galactomannan contained in SBM in a recyclable manner. The mannooligosaccharides (MOS) produced from the enzymatic digestion of SBM were then analysed for their prebiotic and antimicrobial capabilities to determine whether the strategy employed was capable of promoting and inhibiting probiotic and pathogenic growth, respectively. An Aspergillus niger endo-1,4-β-mannanase, Man26A, was confirmed by FTIR (Fourier-transform infrared spectroscopy) and XRD (X-ray powder diffraction) to be immobilised onto CTS and MAGS-CTS by covalent bonding. The immobilisation (%) and activity yields (%) were 81.14% and 35.45%, and 55.75% and 21.17%, respectively. The biochemical properties (pH and temperature optima, and temperature stability) of both the free CTS and MAGS-CTS immobilised Man26A enzymes were evaluated, with the pH optima shifting to a lower pH range after immobilisation (pH 2.0 – 3.0 vs. 5.0), while the temperature optima and stabilities remaining unchanged (at 60°C). CTS and free enzymes exhibited identical thermal stabilities, maintaining 100% activity for the first 6 hours at 55°C, while MAGS-CTS showed an immediate drop in relative activity after the first 30 minutes of incubation. Recyclability analysis revealed that CTS could be effectively reused for six reaction cycles, while the MAGS-CTS immobilised enzyme could only be used once. Both enzymes could be efficiently stored at 4ºC, showing a relative residual activity of 73% after 120 hours of storage. Substrate kinetic analysis showed that the free enzyme had the highest catalytic capabilities in hydrolysing locust bean gum (LBG), with the CTS immobilised enzyme was the most efficient in hydrolysing SBM, the insoluble, complex substrate. Sugar residues produced from the hydrolysis of LBG illustrated the effective breakdown of galactomannan to mannobiose (M2), mannotriose (M3), mannotetrose (M4), and mannohexose (M6). SBM-produced sugars analysed via TLC and HPLC indicated that the MOS residues were most probably glucose, galactose, and galactomannans (GM2 and GM3). The SBM-produced sugars were then evaluated for their prebiotic effect, illustrating their successful utilisation as a carbon source by probiotic bacteria; Streptococcus thermophilus, Bacillus subtilis and Lactobacillus bulgaricus. Evaluation of the antimicrobial activities of the SBM-produced sugars digested by probiotics suggested that their metabolites had the potential to be used as an antibiotic alternative. This study therefore illustrated that an endo-1,4-β-mannanase derived from Aspergillus niger could be immobilised successfully, for use in a recyclable reaction to produce MOS products. This study also described the successful use of SBM-sugars as a prebiotic, indicating a successful alternative to antibiotic growth promoters (AGP) by illustrating their positive effect on inhibiting growth of pathogenic bacterial species. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
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Date Issued: 2024-10-11

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Search for acrylonitrile-based inhibitors of SAR-Cov-19 main and papain-like proteases through covalent docking and high-throughput virtual screening

- Ntantiso, Yamkela

Authors: Ntantiso, Yamkela
Date: 2024-10-11
Subjects: Uncatalogued
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/463941 , vital:76459
Description: The sudden outbreak of SARS-CoV-2 formerly known as the 2019 novel coronavirus (2019-nCoV) quickly turned into a pandemic of coronavirus disease 2019 (COVID-19), the scale of which has never been seen before. High infection rates and mortality from COVID-19 placed pressure on global health services, and this has been to the detriment of the global economy. However, treatment options for COVID-19 are still very limited; hence, it is now as important as ever that researchers explore searching for new compounds with pharmacokinetic properties that inhibit the two COVID proteases - the main protease (Mpro) and the papain-like protease (PLpro). The main protease is a cysteine protease; as such, it is susceptible to permanent inhibition by reactive species (warheads) that may covalently bind to cysteine residues. One such class of compounds is acrylonitriles, in which the reactive acrylonitrile is reactive towards cysteine through a Michael addition reaction. The resulting covalent interaction is permanent and inactivates the cysteine residue and hence the protease within the context of the COVID-19 life-cycle. In this context, this study seeks to utilize computational-based approaches to identify acrylonitrile-based inhibitors of coronavirus drug targets. To do this, the ZINC database has been screened for compounds containing acrylonitrile functionality, due to its known nature as a warhead that binds to cysteine residues. Pharmacokinetic properties are computed to evaluate the viability of identified inhibitors, and covalent and non-covalent molecular docking approaches to the Mpro enzyme crystal structure have also been used to assess the identified systems. To gather more information and evaluate the most promising systems, a subset of the most promising compounds have been subjected to molecular dynamics simulation (for both covalently bound and non-covalently bound systems). , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
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Date Issued: 2024-10-11

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The expression and evaluation of CrpeNPV gp37 as a formulation additive for enhanced infectivity with CrleGV-SA and improved Thaumatotibia leucotreta control

- Muleya, Naho

Authors: Muleya, Naho
Date: 2024-10-11
Subjects: Cryptophlebia leucotreta Biological control , False Codling Moth , Cryptophlebia leucotreta granulovirus , Cryptophlebia peltastica nucleopolyhedrovirus , Citrus Diseases and pests South Africa , Baculoviruses
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/463919 , vital:76457
Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a significant pest native to Africa, causing damage to citrus and posing a threat to the export of fresh citrus in South Africa. Classified as a phytosanitary risk by several South African export markets, this pest necessitates effective control measures. Baculoviruses emerge as promising biological control agents against T. leucotreta due to their inherent safety and eco-friendly characteristics. Among these, Cryptophlebia leucotreta Granulovirus (CrleGV-SA) and Cryptophlebia peltastica Nucleopolyhedrovirus (CrpeNPV) stand out, both causing larval mortality upon infecting T. leucotreta. CrleGV-SA has been formulated into the products Cryptogran™, CryptoMax™ and Cryptex®, while CrpeNPV has been formulated into the product Multimax™. Both viruses are used in integrated pest management programmes to reduce fruit damage in agricultural fields, with CrleGV-SA having been employed against T. leucotreta for nearly 20 years in South Africa. However, these control options are limited by factors such as virulence and the slow speed of kill. This limitation can be addressed by exploiting potential synergistic relationships between baculoviruses infecting the same host. Previous studies have demonstrated that the truncated CpGV gp37 can enhance the infectivity of NPVs on other lepidopteran pests, such as Spodoptera exigua (Hübner). Although the mechanism behind this phenomenon remains unclear, it presents an opportunity to enhance the effectiveness of baculovirus-based management strategies. Notably, the genome of CrpeNPV encodes gp37, while CrleGV-SA lacks this gene. The potential interaction between CrleGV-SA and CrpeNPV gp37 remains unexplored. Therefore, investigating whether they exhibit synergistic or antagonistic effects is essential for optimising baculovirus-based management of T. leucotreta. This study aims to express CrpeNPV gp37 in a bacterial system and then evaluate its effect on larval mortality when combined with CrleGV-SA in laboratory bioassays. The initial step involved extracting genomic DNA (gDNA) from occlusion bodies (OBs) of CrpeNPV. A modified Quick DNA Miniprep plus kit was utilised, which entailed pre-treatment with Na2CO3 followed by neutralisation with Tris-HCI before gDNA extraction using the kit. Subsequently, the concentration of the gDNA was estimated using a Nanodrop spectrophotometer. Oligonucleotides targeting the CrpeNPV gp37 gene were designed for PCR amplification, with the gDNA serving as a template. The gp37 amplicon was identified through agarose gel electrophoresis and then gel purified in preparation for cloning. Secondly, the purified PCR product was cloned into the intermediate vector pJET1.2/blunt and then subcloned into the bacterial expression vector pCA528 through DNA ligation. The construction of recombinant plasmids (pJET-gp37 and pCA-gp37) was conducted and verified using Colony PCR, plasmid extraction, restriction enzyme analysis, and Sanger sequencing. Thirdly, the recombinant protein (6×His-SUMO-gp37) was expressed and purified using Nickel affinity chromatography and analysed through SDS-PAGE and Western blot techniques. The expression of 6×His-SUMO-gp37 was carried out at both 25 °C and 18 °C. A time course induction study was conducted, inducing transformed cells for 0-, 3-, 5-, and 24-hours post induction (hpi). SDS-PAGE and Western blotting of samples collected at various time points revealed that 6×His-SUMO-gp37, approximately 42 kDa in size, was visible from 3 hpi, with maximal expression at 24 hpi. Solubility analysis of 6×His-SUMO-gp37 was performed at both temperatures, showing solubility at 18 °C but predominantly present in the insoluble fraction. The soluble protein was purified under native conditions, while the insoluble protein was purified under denaturing conditions. Despite being unable to elute 6×His-SUMO-gp37 under native conditions, successful elution was achieved under denaturing conditions, confirmed via Western blot analysis. No further experiments were conducted on the eluted 6×His-SUMO-gp37 under denaturing conditions. Lastly, a preliminary surface dose bioassay was conducted to evaluate the efficacy of pelleted bacteria expressing 6×His-SUMO-gp37 in combination with CrleGV-SA against T. leucotreta neonates. Two lethal concentration doses of CrleGV-SA were prepared: a low concentration (2.96×104 OBs/mL) capable of killing 40 % of the T. leucotreta population, and a high concentration (2.96×105 OBs/mL) capable of killing 90 % of the population. The target protein, 6×His-SUMO-gp37, and the control, pCA528, were obtained by lysing the cells, centrifuging the samples, and collecting the insoluble fractions in pellet form. These fractions were then resuspended in PBS and used as treatments in combination with the prepared CrleGV-SA concentration doses. The concentration of the pellets was estimated using a Nanodrop spectrophotometer by measuring the absorbance at 280 nm. The bioassay results revealed that the combination of 100 μg/mL of pelleted bacteria expressing 6×His-SUMO-gp37 with CrleGV-SA had no effect on T. leucotreta larval mortality compared to CrleGV-SA alone. A one-way ANOVA was performed to assess differences among the virus treatment groups, concluding that no statistically significant differences were observed among the groups. The experiments in this study provided valuable insights for future research, particularly in exploring the use of a protein-virus combination as a novel method for pest control. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
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Date Issued: 2024-10-11

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