Identification of selective novel hits against Mycobacterium tuberculosis KasA potential allosteric sites using bioinformatics approaches
- Authors: Hare, Fadzayi Faith
- Date: 2022-10-14
- Subjects: Tuberculosis , Docking , Molecules Models , Virtual screening , Multidrug-resistant tuberculosis , Fatty acids Synthesis , Drugs Design
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362842 , vital:65367
- Description: Tuberculosis (TB) is a global health threat that has led to approximately 1.5 million deaths annually. According to the World Health Organization (WHO), TB is among the top ten deadly diseases and is the leading cause of death due to a single infectious agent. The main challenge in the effective treatment and control of TB is the ongoing emergence of resistant strains of Mycobacterium tuberculosis (Mtb) which lead to multi-drug resistant (MDR) and extensive-drug resistant (XDR) TB. Hence, the identification and characterization of novel drug targets and drugs that modulate the activity of the pathogen are an urgent priority. The current situation even necessitates the reengineering or repurposing of drugs in order to achieve effective control. The β-ketoacyl-acyl carrier protein synthase I (KasA) of Mycobacterium tuberculosis is an essential enzyme in the mycobacterial fatty acid synthesis (FAS-II) pathway and is believed to be a promising target for drug discovery in TB. It is one of the five main proteins of the FAS-II pathway and catalyzes a key condensation reaction in the synthesis of meromycolate chains, the precursors of mycolic acids involved in cell wall formation. Although this protein has been extensively studied, little research has been devoted to the allosteric inhibition of potential drug compounds. The main aim of this research was to identify the allosteric sites on the protein that could be involved in the inhibition of substrate binding activities and novel drug compounds that bind to these sites by use of in-silico approaches. The bioinformatics approaches used in this study were divided into four main objectives namely identification of KasA homolog sequences, sequence analysis and protein characterization, allosteric site search and lastly virtual screening of DrugBank compounds via molecular docking. Fifteen homolog sequences were identified from the BLASTP analysis and were derived from bacteria, fungi and mammals. In order to discover important residues and regions within the KasA proteins, sequence alignment, motif analysis and phylogenetic studies were performed using Mtb KasA as a reference. Sequence alignment revealed conserved residues in all KasA proteins that have functional importance such as the catalytic triad residues (Cys171, His311 and His345). Motif analysis identified 18 highly conserved motifs within the KasA proteins with structural and functional roles. In addition, motifs unique to the Mtb KasA protein were also identified and explored for inhibitor drug design purposes. Phylogenetic analysis of the homolog sequences showed a distinct clustering of prokaryotes and eukaryotes. A distinctive clustering was also observed for species belonging to the same genus. Since the mechanism of action of most drugs involves the active site, allosteric site search was conducted on Mtb KasA and the human homolog protein using a combination of pocket detection algorithms with the aim of identifying sites that could be utilized in allosteric modulator drug discovery. This was followed by the virtual screening of 2089 FDA approved DrugBank compounds against the entire protein surfaces of Mtb KasA and Hsmt KasA, performed via molecular docking using AutoDock Vina. Screening of the compounds was based on the binding energies, with more focus on identifying ligands that bound exclusively to the acyl-binding tunnel of Mtb KasA. This reduced the data set to 27 promising drug compounds with a relatively high binding affinity for Mtb KasA, however, further experiments need to be performed to validate this result. Among these compounds were DB08889, DB06755, DB09270, DB11226, DB00392, DB12278, DB08936, DB00781, DB13720 and DB00392, which displayed relatively low binding energies for Mtb KasA when compared to the human homolog protein. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Hare, Fadzayi Faith
- Date: 2022-10-14
- Subjects: Tuberculosis , Docking , Molecules Models , Virtual screening , Multidrug-resistant tuberculosis , Fatty acids Synthesis , Drugs Design
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362842 , vital:65367
- Description: Tuberculosis (TB) is a global health threat that has led to approximately 1.5 million deaths annually. According to the World Health Organization (WHO), TB is among the top ten deadly diseases and is the leading cause of death due to a single infectious agent. The main challenge in the effective treatment and control of TB is the ongoing emergence of resistant strains of Mycobacterium tuberculosis (Mtb) which lead to multi-drug resistant (MDR) and extensive-drug resistant (XDR) TB. Hence, the identification and characterization of novel drug targets and drugs that modulate the activity of the pathogen are an urgent priority. The current situation even necessitates the reengineering or repurposing of drugs in order to achieve effective control. The β-ketoacyl-acyl carrier protein synthase I (KasA) of Mycobacterium tuberculosis is an essential enzyme in the mycobacterial fatty acid synthesis (FAS-II) pathway and is believed to be a promising target for drug discovery in TB. It is one of the five main proteins of the FAS-II pathway and catalyzes a key condensation reaction in the synthesis of meromycolate chains, the precursors of mycolic acids involved in cell wall formation. Although this protein has been extensively studied, little research has been devoted to the allosteric inhibition of potential drug compounds. The main aim of this research was to identify the allosteric sites on the protein that could be involved in the inhibition of substrate binding activities and novel drug compounds that bind to these sites by use of in-silico approaches. The bioinformatics approaches used in this study were divided into four main objectives namely identification of KasA homolog sequences, sequence analysis and protein characterization, allosteric site search and lastly virtual screening of DrugBank compounds via molecular docking. Fifteen homolog sequences were identified from the BLASTP analysis and were derived from bacteria, fungi and mammals. In order to discover important residues and regions within the KasA proteins, sequence alignment, motif analysis and phylogenetic studies were performed using Mtb KasA as a reference. Sequence alignment revealed conserved residues in all KasA proteins that have functional importance such as the catalytic triad residues (Cys171, His311 and His345). Motif analysis identified 18 highly conserved motifs within the KasA proteins with structural and functional roles. In addition, motifs unique to the Mtb KasA protein were also identified and explored for inhibitor drug design purposes. Phylogenetic analysis of the homolog sequences showed a distinct clustering of prokaryotes and eukaryotes. A distinctive clustering was also observed for species belonging to the same genus. Since the mechanism of action of most drugs involves the active site, allosteric site search was conducted on Mtb KasA and the human homolog protein using a combination of pocket detection algorithms with the aim of identifying sites that could be utilized in allosteric modulator drug discovery. This was followed by the virtual screening of 2089 FDA approved DrugBank compounds against the entire protein surfaces of Mtb KasA and Hsmt KasA, performed via molecular docking using AutoDock Vina. Screening of the compounds was based on the binding energies, with more focus on identifying ligands that bound exclusively to the acyl-binding tunnel of Mtb KasA. This reduced the data set to 27 promising drug compounds with a relatively high binding affinity for Mtb KasA, however, further experiments need to be performed to validate this result. Among these compounds were DB08889, DB06755, DB09270, DB11226, DB00392, DB12278, DB08936, DB00781, DB13720 and DB00392, which displayed relatively low binding energies for Mtb KasA when compared to the human homolog protein. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
Evaluating metabolism-induced toxicity using a non-hepatic cell line
- Authors: Weyers, Carli
- Date: 2018
- Subjects: Cytochrome P-450 , Drugs Metabolism , Drugs Design
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/61950 , vital:28087
- Description: The drug discovery pipeline is a complicated process taking roughly 15 years to complete, costing in excess of $1 billion per new chemical entity. It has been estimated that for every 100, 000 promising hit or lead compounds, only one will make it onto the market due to numerous drug candidates being discarded because of many complications. One such complication is metabolism-induced toxicity. Accordingly, an early understanding of the metabolism of any new chemical entity is becoming an integral part of the pipeline. In order to explore this, various methods have been developed including in silico and in vitro techniques. One such method involves performing cell viability assays on human liver cancer cell lines, which overexpress specific metabolic cytochrome P450 enzymes. If a toxic metabolite is produced it would result in reduced cell viability of the transformed cell line in comparison to a control. Since the liver is the primary site of metabolism in the human body, we were curious as to the extent to which background metabolism may play a role in the degree to which toxic metabolites would be produced in these cell lines. The aim of this project, therefore, was to establish if a non-hepatic cell-based system which overexpresses CYP3A4 could be used to detect the metabolism and any subsequent toxicity of compounds which have been reported to be substrates of the CYP450 enzyme. The HEK293 cell line was stably transfected with a plasmid vector for human CYP3A4 to create a model overexpression system for our metabolism studies. The activity of the enzyme was confirmed using the substrate, 7-benzyloxy-4-trifluoromethyl-coumarin. Subsequently, cytotoxicity testing was done on four known pharmaceuticals reported to generate toxic metabolites in hepatic cell-based assays. In silico metabolic predictions on the four known compounds were performed and compared to the results of published literature. Finally, the metabolism of one compound was studied using a combination of high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) in order to detect predicted metabolites. We observed no change in cellular toxicity nor did we detect the formation of metabolites, even though the overexpressed CYP3A4 enzyme was active. The results suggest that caution should be taken when interpreting the results of cell-based metabolism studies, and background metabolism may play a significant role in the data. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Date Issued: 2018
- Authors: Weyers, Carli
- Date: 2018
- Subjects: Cytochrome P-450 , Drugs Metabolism , Drugs Design
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/61950 , vital:28087
- Description: The drug discovery pipeline is a complicated process taking roughly 15 years to complete, costing in excess of $1 billion per new chemical entity. It has been estimated that for every 100, 000 promising hit or lead compounds, only one will make it onto the market due to numerous drug candidates being discarded because of many complications. One such complication is metabolism-induced toxicity. Accordingly, an early understanding of the metabolism of any new chemical entity is becoming an integral part of the pipeline. In order to explore this, various methods have been developed including in silico and in vitro techniques. One such method involves performing cell viability assays on human liver cancer cell lines, which overexpress specific metabolic cytochrome P450 enzymes. If a toxic metabolite is produced it would result in reduced cell viability of the transformed cell line in comparison to a control. Since the liver is the primary site of metabolism in the human body, we were curious as to the extent to which background metabolism may play a role in the degree to which toxic metabolites would be produced in these cell lines. The aim of this project, therefore, was to establish if a non-hepatic cell-based system which overexpresses CYP3A4 could be used to detect the metabolism and any subsequent toxicity of compounds which have been reported to be substrates of the CYP450 enzyme. The HEK293 cell line was stably transfected with a plasmid vector for human CYP3A4 to create a model overexpression system for our metabolism studies. The activity of the enzyme was confirmed using the substrate, 7-benzyloxy-4-trifluoromethyl-coumarin. Subsequently, cytotoxicity testing was done on four known pharmaceuticals reported to generate toxic metabolites in hepatic cell-based assays. In silico metabolic predictions on the four known compounds were performed and compared to the results of published literature. Finally, the metabolism of one compound was studied using a combination of high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) in order to detect predicted metabolites. We observed no change in cellular toxicity nor did we detect the formation of metabolites, even though the overexpressed CYP3A4 enzyme was active. The results suggest that caution should be taken when interpreting the results of cell-based metabolism studies, and background metabolism may play a significant role in the data. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Date Issued: 2018
Formulation development, manufacture and evaluation of hydralazine hydrochloride microspheres
- Kangausaru, Shakemore Tinashe
- Authors: Kangausaru, Shakemore Tinashe
- Date: 2017
- Subjects: Hydralazine , Microspheres , Drugs Controlled release , Drugs Design , Drug development , Hypertension Chemotherapy
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59220 , vital:27482
- Description: Despite improvements in its detection and treatment since the 1970s, hypertension is the most common and important risk factor for cardiovascular diseases. Hypertension is a chronic condition often underdiagnosed and/or inadequately treated in Sub-Saharan Africa. Recent survey results illustrate that the condition continues to contribute significantly to mortality and morbidity in adults and that it is poorly controlled in clinical practice. Hydralazine (HYD) is used either alone or in combination for the management of chronic hypertension, chronic cardiac failure and hypertensive crises. Due to its short plasma half-life of between 2 to 4 hours, HYD is normally administered two to four times daily, therefore making it a potential candidate for inclusion in sustained release formulations. The formulation of sustained release microsphere dosage forms may be useful to improve patient adherence and to achieve predictable and optimised therapeutic plasma concentrations. A stability indicating reversed-phase high performance liquid chromatography (RP-HPLC) method for the quantitation of HYD in pharmaceutical dosage forms was developed and optimised using a Central Composite Design (CCD) approach. UV/Vis detection method was selected as HYD contains an ultraviolet light-absorbing chromophore. The method was validated with respect to linearity and range, limits of quantitation (LOQ) and detection (LOD), accuracy, precision, sensitivity, selectivity and specificity as per International Conference on Harmonisation (ICH) guidelines. The method was applied to commercially available HYD tablets. No interfering peaks were observed from excipients used in the commercially available tablets. Preformulation studies were conducted to ensure the manufacture of high quality, stable sustained release HYD microspheres. The results revealed that there was an interaction between HYD and Carbopol® 971P, therefore Carbopol® polymers were not included during formulation studies. HYD was found to be compatible with Methocel® K100LV, Eudragit® RS PO and Avicel® 101 and HYD formulations were developed and optimised using these excipients. An oil-in-oil (o/o) solvent evaporation technique was selected for the manufacture of HYD microspheres due to its simplicity and to avoid exposure of HYD to moisture that could have been encountered if a water-in-oil (w/o) manufacturing process was used. The selection of o/o solvent evaporation technique was also based on the hydrophilicity of HYD and the polymers selected. Different grades of Methocel® and Eudragit® were selected to evaluate their effect on encapsulation efficiency (EE), in vitro release and microparticle shape and morphology. The best combination of these polymers which resulted in the desired EE, in vitro release, microparticle shape and size were then selected for formulation optimisation. A numerical optimisation approach was used to predict a formulation composition that would produce minimal HYD release initially and maximum HYD release after 12 hours of dissolution testing. The release kinetics of HYD from the manufactured microspheres were established by fitting in vitro release data to several mathematical models. The in vitro release data for the optimised formulations was best described using Higuchi model. The short-term stability of the optimised formulations was established by undertaking stability studies at 4°C, 25 °C/60 % RH and 40 °C/75 % RH. The results revealed that there was no significant change in appearance and physicochemical properties of the microspheres over a period of one month. However, long-term stability studies would be required to determine the shelf-life of the formulations. In addition, a gas chromatographic (GC) method was selected for determining residual amounts of acetone and n-hexane in the optimised formulations. GC methods were developed and optimised by evaluation of process parameters. System suitability testing was performed with respect to resolution, theoretical number of plates and selectivity. Method validation was performed with respect to linearity, range, inter- and intra-day precision, retention time (Rt) precision, limit of quantitation (LOQ) and detection (LOD). A solvent extraction method was used to analyse residual solvents in the optimised formulations. The drying process was sufficient in evaporating acetone and n-hexane from the optimised formulations. Solvent evaporation technique has been successfully used in the manufacture of HYD microspheres. The microspheres have potential for further development, scale up formulation studies and long-term stability studies. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2017
- Full Text:
- Date Issued: 2017
- Authors: Kangausaru, Shakemore Tinashe
- Date: 2017
- Subjects: Hydralazine , Microspheres , Drugs Controlled release , Drugs Design , Drug development , Hypertension Chemotherapy
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59220 , vital:27482
- Description: Despite improvements in its detection and treatment since the 1970s, hypertension is the most common and important risk factor for cardiovascular diseases. Hypertension is a chronic condition often underdiagnosed and/or inadequately treated in Sub-Saharan Africa. Recent survey results illustrate that the condition continues to contribute significantly to mortality and morbidity in adults and that it is poorly controlled in clinical practice. Hydralazine (HYD) is used either alone or in combination for the management of chronic hypertension, chronic cardiac failure and hypertensive crises. Due to its short plasma half-life of between 2 to 4 hours, HYD is normally administered two to four times daily, therefore making it a potential candidate for inclusion in sustained release formulations. The formulation of sustained release microsphere dosage forms may be useful to improve patient adherence and to achieve predictable and optimised therapeutic plasma concentrations. A stability indicating reversed-phase high performance liquid chromatography (RP-HPLC) method for the quantitation of HYD in pharmaceutical dosage forms was developed and optimised using a Central Composite Design (CCD) approach. UV/Vis detection method was selected as HYD contains an ultraviolet light-absorbing chromophore. The method was validated with respect to linearity and range, limits of quantitation (LOQ) and detection (LOD), accuracy, precision, sensitivity, selectivity and specificity as per International Conference on Harmonisation (ICH) guidelines. The method was applied to commercially available HYD tablets. No interfering peaks were observed from excipients used in the commercially available tablets. Preformulation studies were conducted to ensure the manufacture of high quality, stable sustained release HYD microspheres. The results revealed that there was an interaction between HYD and Carbopol® 971P, therefore Carbopol® polymers were not included during formulation studies. HYD was found to be compatible with Methocel® K100LV, Eudragit® RS PO and Avicel® 101 and HYD formulations were developed and optimised using these excipients. An oil-in-oil (o/o) solvent evaporation technique was selected for the manufacture of HYD microspheres due to its simplicity and to avoid exposure of HYD to moisture that could have been encountered if a water-in-oil (w/o) manufacturing process was used. The selection of o/o solvent evaporation technique was also based on the hydrophilicity of HYD and the polymers selected. Different grades of Methocel® and Eudragit® were selected to evaluate their effect on encapsulation efficiency (EE), in vitro release and microparticle shape and morphology. The best combination of these polymers which resulted in the desired EE, in vitro release, microparticle shape and size were then selected for formulation optimisation. A numerical optimisation approach was used to predict a formulation composition that would produce minimal HYD release initially and maximum HYD release after 12 hours of dissolution testing. The release kinetics of HYD from the manufactured microspheres were established by fitting in vitro release data to several mathematical models. The in vitro release data for the optimised formulations was best described using Higuchi model. The short-term stability of the optimised formulations was established by undertaking stability studies at 4°C, 25 °C/60 % RH and 40 °C/75 % RH. The results revealed that there was no significant change in appearance and physicochemical properties of the microspheres over a period of one month. However, long-term stability studies would be required to determine the shelf-life of the formulations. In addition, a gas chromatographic (GC) method was selected for determining residual amounts of acetone and n-hexane in the optimised formulations. GC methods were developed and optimised by evaluation of process parameters. System suitability testing was performed with respect to resolution, theoretical number of plates and selectivity. Method validation was performed with respect to linearity, range, inter- and intra-day precision, retention time (Rt) precision, limit of quantitation (LOQ) and detection (LOD). A solvent extraction method was used to analyse residual solvents in the optimised formulations. The drying process was sufficient in evaporating acetone and n-hexane from the optimised formulations. Solvent evaporation technique has been successfully used in the manufacture of HYD microspheres. The microspheres have potential for further development, scale up formulation studies and long-term stability studies. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2017
- Full Text:
- Date Issued: 2017
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