Insights: elucidation of squalene monooxygenase inhibitors for lowering cholesterol in cardiovascular diseases
- Authors: Leoma, Mofeli Benedict
- Date: 2024-04-04
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/434861 , vital:73111
- Description: Statins have been used to lower high cholesterol levels in the past few decades. However, several studies have shown that some people taking statins experience side effects over time, especially elderly patients, women of childbirth possibility, and children. Several studies have shown that the majority of people with underlying cardiovascular complications caused by high cholesterol are at a greater risk of fatality due to COVID-19, regardless of age and sex. The literature suggests that antimycotic squalene monooxygenase inhibitors, terbinafine and its derivatives, and anticholesterolemic squalene monooxygenase (SM) inhibitors could be another option and a safer remedy for lowering cholesterol in mammals. Molecular docking calculations, molecular dynamics (MD) simulations, molecular mechanics generalized born surface area (MM-GBSA) calculations, quantum mechanics/molecular mechanics calculations (QM/MM), and density functional theory (DFT) calculations were used in this study. An early stage in drug discovery, in which small molecular hits from high- throughput screening (HTS) are evaluated and undergo limited optimization to identify promising lead compounds, is referred to as lead generation. To address the first step of lead generation, the number of compounds to be tested was narrowed down, and the hit compounds that could be taken for further tests were obtained. Thus, the molecular docking technique was taken advantage of, which assisted us in identifying the antimycotic ligand SDZ 18, which had a good binding affinity of about -8,4 kcal mol−1. Another widely employed strategy, the molecular mechanics-generalized born surface area (MM-GBSA), was used to investigate the binding free energies of the protein-ligand complexes to validate the binding affinities obtained from molecular docking. Despite the excellent docking results, it must be emphasized that the stability of the ligand in the binding pocket must be investigated. To address this, the protein-ligand complexes were then taken through molecular dynamics for 100 ns simulations calculations which showed that the inhibitors stayed in the binding pocket with the RMSD values below 3.5 Å for most systems. This provided insight into a realistic model because the docked complexes were placed in conditions closer to the physiological environment at 300 K and 1.01325 bar, and in an explicitly solvated dynamic environment. Density functional theory (DFT) at the B3LPY level of theory using the standard 6-31G(d,p) basis set was used to assess the reactivity and other properties of the SM inhibitors. ONIOM calculations were performed to explain what was happening at the microscopic level by calculating the total energy of the complex. The aim of this project was to efficiently uncover the non-physical aspects of SM inhibitors with the help of computational techniques to identify new drugs that can lower high cholesterol levels. From a theoretical perspective, the results obtained from docking indicated that the antimycotic ligands SDZ SBA 586 18 and TNSA 84 (trisnor-squalene alcohol ) have good binding affinities, and the MM-GBSA method provided free energy calculations. MD results indicated that the stability of the ligand in the binding pocket was achieved during the 100 ns simulations. The HOMO-LUMO energy gaps obtained from DFT calculations provided information on the reactivity of the ligands. Other insights into the protein-ligand complexes were obtained from a hybrid ONIOM QM/MM study. , Thesis (MSc) -- Faculty of Science, Chemistry, 2024
- Full Text:
- Date Issued: 2024-04-04
- Authors: Leoma, Mofeli Benedict
- Date: 2024-04-04
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/434861 , vital:73111
- Description: Statins have been used to lower high cholesterol levels in the past few decades. However, several studies have shown that some people taking statins experience side effects over time, especially elderly patients, women of childbirth possibility, and children. Several studies have shown that the majority of people with underlying cardiovascular complications caused by high cholesterol are at a greater risk of fatality due to COVID-19, regardless of age and sex. The literature suggests that antimycotic squalene monooxygenase inhibitors, terbinafine and its derivatives, and anticholesterolemic squalene monooxygenase (SM) inhibitors could be another option and a safer remedy for lowering cholesterol in mammals. Molecular docking calculations, molecular dynamics (MD) simulations, molecular mechanics generalized born surface area (MM-GBSA) calculations, quantum mechanics/molecular mechanics calculations (QM/MM), and density functional theory (DFT) calculations were used in this study. An early stage in drug discovery, in which small molecular hits from high- throughput screening (HTS) are evaluated and undergo limited optimization to identify promising lead compounds, is referred to as lead generation. To address the first step of lead generation, the number of compounds to be tested was narrowed down, and the hit compounds that could be taken for further tests were obtained. Thus, the molecular docking technique was taken advantage of, which assisted us in identifying the antimycotic ligand SDZ 18, which had a good binding affinity of about -8,4 kcal mol−1. Another widely employed strategy, the molecular mechanics-generalized born surface area (MM-GBSA), was used to investigate the binding free energies of the protein-ligand complexes to validate the binding affinities obtained from molecular docking. Despite the excellent docking results, it must be emphasized that the stability of the ligand in the binding pocket must be investigated. To address this, the protein-ligand complexes were then taken through molecular dynamics for 100 ns simulations calculations which showed that the inhibitors stayed in the binding pocket with the RMSD values below 3.5 Å for most systems. This provided insight into a realistic model because the docked complexes were placed in conditions closer to the physiological environment at 300 K and 1.01325 bar, and in an explicitly solvated dynamic environment. Density functional theory (DFT) at the B3LPY level of theory using the standard 6-31G(d,p) basis set was used to assess the reactivity and other properties of the SM inhibitors. ONIOM calculations were performed to explain what was happening at the microscopic level by calculating the total energy of the complex. The aim of this project was to efficiently uncover the non-physical aspects of SM inhibitors with the help of computational techniques to identify new drugs that can lower high cholesterol levels. From a theoretical perspective, the results obtained from docking indicated that the antimycotic ligands SDZ SBA 586 18 and TNSA 84 (trisnor-squalene alcohol ) have good binding affinities, and the MM-GBSA method provided free energy calculations. MD results indicated that the stability of the ligand in the binding pocket was achieved during the 100 ns simulations. The HOMO-LUMO energy gaps obtained from DFT calculations provided information on the reactivity of the ligands. Other insights into the protein-ligand complexes were obtained from a hybrid ONIOM QM/MM study. , Thesis (MSc) -- Faculty of Science, Chemistry, 2024
- Full Text:
- Date Issued: 2024-04-04
Characterization of Trypanosoma brucei Sti1 and its interactions with Trypanosoma brucei Hsp83 and human Hsp90
- Authors: Jamabo, Miebaka
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422629 , vital:71963 , DOI 10.21504/10962/422629
- Description: Neglected tropical diseases continue to pose global concern due to their impact on health and socio-economic status of developing countries in sub-Saharan Africa. African trypanosomiasis is one of the neglected tropical diseases caused by the kinetoplastid flagellate parasite Trypanosoma brucei (T. brucei). The disease is fatal if untreated and the toolbox to combat the disease has been plagued with many difficulties such as drug resistance, toxic chemotherapeutics, and cumbersome drug delivery processes. In recent years, the disease has received attention from organizations such as the Drugs for Neglected Diseases initiative (DNDi) in partnership with WHO as well as academia and industry to provide alternatives to the existing drugs as part of a targeted approach to eliminate human African trypanosomiasis by 2030. The life cycle of the T. brucei parasite requires that it transitions between a cold-blooded vector (the tsetse fly) and a human host. To survive this extreme environmental change and maintain its infectious cycle, the parasite has evolved an arsenal of tools which include a strong immune evasion technique and a robust molecular chaperone system. Heat shock protein 90 (Hsp90) is one of the most abundant eukaryotic molecular chaperones that has been extensively studied in many organisms. It is indispensable for maintaining proteostasis in some organisms and its inhibition is currently being explored as a drug target for cancer and other parasitic diseases. In T. brucei, cytosolic Hsp90 is specifically referred to as Hsp83 due to variations in the sizes amongst different orthologues. Hsp90 is present in high levels in all stages of the T. brucei cell cycle both constitutively and on exposure to stress. To function in the cell, Hsp90 is dependent on co-chaperones, one of which can be found in most organisms, namely, the stress-inducible protein 1 (Sti1). The Hsp90-Sti1 interaction was shown to be crucial for growth in the intracellular kinetoplastid parasite, Leishmania donovani. However, this partnership has not been explored in the extracellular parasite T. brucei. To analyse the interaction of Hsp90 with Sti1 in T. brucei, this study combined in silico, in vitro and in vivo tools. In silico analyses of the Hsp90 complement in T. brucei revealed the presence of twelve putative Hsp90 genes, ten of which code for the cytosolic protein and are arranged in tandem in a head to tail fashion on the same chromosome. One gene each was found for the mitochondrial and ER paralogues of Hsp90, similar to all other species analysed. Eight putative co-chaperones specific to T. brucei were also discovered: six tetratricopeptide repeat domain (TPR) containing co-chaperones and two non-TPR containing co-chaperones. Structural and evolutionary analysis also confirmed that the domains were conserved across the species analysed. T. brucei Sti1 (TbSti1), T. brucei cytosolic Hsp90 (TbHsp83) and human cytosolic Hsp90 (hHsp90) were heterologously overproduced in E. coli and purified using nickel affinity chromatography. With specific antibodies, the expression and localization of the proteins were confirmed. TbSti1 showed strong affinity to the Hsp90s in the nanomolar range, with higher affinity for hHsp90 compared to TbHsp83. TbHsp83 and hHsp90 showed typical chaperone properties by suppressing the aggregation of thermolabile substrate MDH at equimolar concentrations and both chaperones had potent ATP hydrolysis activity. TbSti1, on the other hand, showed no MDH suppression activity and did not affect the ATP hydrolysis activity of TbHsp83 or hHsp90. Ex-vivo experiments using HeLa CRISPR Hop knockout (KO) human cell lines transfected with pcDNA3.1(+)HA-TbSti1 revealed TbSti1 also localized to the cytoplasm. The transfected cells showed a distinct fibroblast-like morphology which was different from the circular morphology seen in the Hop KO untransfected and wild type untransfected cells. Finally, co-immunoprecipitation studies revealed that TbSti1 co-immunoprecipitated with hHsp90. These results show the first characterization of the TbHsp83-TbSti1 partnership in T. brucei. The strong association between both proteins suggests a functional role for this partnership in T. brucei and could provide an updated context for understanding Trypanosome brucei biology. , Thesis (PhD) -- Faculty of Science, Biotechnology and Innovation Centre, 2023
- Full Text:
- Date Issued: 2023-03-31
- Authors: Jamabo, Miebaka
- Date: 2023-03-31
- Subjects: Uncatalogued
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422629 , vital:71963 , DOI 10.21504/10962/422629
- Description: Neglected tropical diseases continue to pose global concern due to their impact on health and socio-economic status of developing countries in sub-Saharan Africa. African trypanosomiasis is one of the neglected tropical diseases caused by the kinetoplastid flagellate parasite Trypanosoma brucei (T. brucei). The disease is fatal if untreated and the toolbox to combat the disease has been plagued with many difficulties such as drug resistance, toxic chemotherapeutics, and cumbersome drug delivery processes. In recent years, the disease has received attention from organizations such as the Drugs for Neglected Diseases initiative (DNDi) in partnership with WHO as well as academia and industry to provide alternatives to the existing drugs as part of a targeted approach to eliminate human African trypanosomiasis by 2030. The life cycle of the T. brucei parasite requires that it transitions between a cold-blooded vector (the tsetse fly) and a human host. To survive this extreme environmental change and maintain its infectious cycle, the parasite has evolved an arsenal of tools which include a strong immune evasion technique and a robust molecular chaperone system. Heat shock protein 90 (Hsp90) is one of the most abundant eukaryotic molecular chaperones that has been extensively studied in many organisms. It is indispensable for maintaining proteostasis in some organisms and its inhibition is currently being explored as a drug target for cancer and other parasitic diseases. In T. brucei, cytosolic Hsp90 is specifically referred to as Hsp83 due to variations in the sizes amongst different orthologues. Hsp90 is present in high levels in all stages of the T. brucei cell cycle both constitutively and on exposure to stress. To function in the cell, Hsp90 is dependent on co-chaperones, one of which can be found in most organisms, namely, the stress-inducible protein 1 (Sti1). The Hsp90-Sti1 interaction was shown to be crucial for growth in the intracellular kinetoplastid parasite, Leishmania donovani. However, this partnership has not been explored in the extracellular parasite T. brucei. To analyse the interaction of Hsp90 with Sti1 in T. brucei, this study combined in silico, in vitro and in vivo tools. In silico analyses of the Hsp90 complement in T. brucei revealed the presence of twelve putative Hsp90 genes, ten of which code for the cytosolic protein and are arranged in tandem in a head to tail fashion on the same chromosome. One gene each was found for the mitochondrial and ER paralogues of Hsp90, similar to all other species analysed. Eight putative co-chaperones specific to T. brucei were also discovered: six tetratricopeptide repeat domain (TPR) containing co-chaperones and two non-TPR containing co-chaperones. Structural and evolutionary analysis also confirmed that the domains were conserved across the species analysed. T. brucei Sti1 (TbSti1), T. brucei cytosolic Hsp90 (TbHsp83) and human cytosolic Hsp90 (hHsp90) were heterologously overproduced in E. coli and purified using nickel affinity chromatography. With specific antibodies, the expression and localization of the proteins were confirmed. TbSti1 showed strong affinity to the Hsp90s in the nanomolar range, with higher affinity for hHsp90 compared to TbHsp83. TbHsp83 and hHsp90 showed typical chaperone properties by suppressing the aggregation of thermolabile substrate MDH at equimolar concentrations and both chaperones had potent ATP hydrolysis activity. TbSti1, on the other hand, showed no MDH suppression activity and did not affect the ATP hydrolysis activity of TbHsp83 or hHsp90. Ex-vivo experiments using HeLa CRISPR Hop knockout (KO) human cell lines transfected with pcDNA3.1(+)HA-TbSti1 revealed TbSti1 also localized to the cytoplasm. The transfected cells showed a distinct fibroblast-like morphology which was different from the circular morphology seen in the Hop KO untransfected and wild type untransfected cells. Finally, co-immunoprecipitation studies revealed that TbSti1 co-immunoprecipitated with hHsp90. These results show the first characterization of the TbHsp83-TbSti1 partnership in T. brucei. The strong association between both proteins suggests a functional role for this partnership in T. brucei and could provide an updated context for understanding Trypanosome brucei biology. , Thesis (PhD) -- Faculty of Science, Biotechnology and Innovation Centre, 2023
- Full Text:
- Date Issued: 2023-03-31
Ethics matter: A critical realist account of research ethics for social science and humanities researchers
- Authors: Dlova, Lisolethu
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192440 , vital:45226
- Description: Thesis (MEd) -- Faculty of Education, Centre for Higher Education Research, Teaching and Learning, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Dlova, Lisolethu
- Date: 2021-10-29
- Subjects: Uncatalogued
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/192440 , vital:45226
- Description: Thesis (MEd) -- Faculty of Education, Centre for Higher Education Research, Teaching and Learning, 2021
- Full Text:
- Date Issued: 2021-10-29
- «
- ‹
- 1
- ›
- »