Structural dynamic investigation of the mutation-induced resistance mechanisms of Mycobacterium tuberculosis DNA-directed RNA polymerase against Rifampicin
- Authors: Monama, Mokgerwa Zacharia
- Date: 2024-10-11
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Molecular dynamics Simulation methods
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/466849 , vital:76792 , DOI https://doi.org/10.21504/10962/466849
- Description: Emerging resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease, continue to plague mankind and reduce the efficacies of antitubercular therapies that have been an effective defence against TB for decades. More specifically, mutations located in the β subunit of the multisubunit Mtb RNA replicative machinery, RNA polymerase (RNAP), have been well established as the reason behind resistance to the first-line antitubercular drug rifampicin (RIF), which has resulted in therapeutic failure in several clinical cases. Additionally, elusive details pertaining to the underlying mechanisms associated with RIF resistance due to the presence of Mtb-RNAP-β mutations, have resulted in setbacks in the development of novel and effective drugs that might be able to curb the ongoing threat. Hence, in this investigation, we attempted to resolve the involved Mtb-RNAP structural events at the molecular level to discern potentially important details regarding the nine clinically relevant Mtb-RNAP-β missense mutations under investigation. Hence, for the first time, we conducted an in-silico RIF resistance investigation using the Mtb-RNAP complex. To accomplish the set-out task, we first employed the use of more traditional post-MD analytical approaches such as root mean square deviation, root mean square fluctuation, radius of gyration, center of mass distance analyses, hydrogen bond occupancies, and binding free energy calculations, to conduct a global analysis of the mutated Mtb-RNAP proteins referencing RIF efficacy. Our findings revealed that the mutations may have a perturbation effect resulting in the disruption of essential structural dynamics attributed to the protein’s catalytic functions. This was for instance observed for the βfork loop 2 domain, the β’zinc-binding domain, the β’ trigger loop domain, and the β’jaw domain, which happen to be in line with previously reported experiments detailing changes in RNAP processivity. Complementarily, some of the mutations more specifically perturbed the RIF binding pocket (RIF-BP) which observably led to the reorientation of RIF from the native or active orientation needed to obstruct the processive addition of nucleoside triphosphates to the growing RNA transcript. The mutation-induced repositioning from the active RIF orientation was also reflected through the loss of essential interactions between RIF and the RIF-BP along with the loss of binding affinities captured for a majority of the mutant proteins. In conjunction with traditional analytical approaches, we further employed computational alanine scanning, weighted contact map analyses, and dynamic residue network (DRN) analyses, a novel approach that delineates residue-residue communication pathways through several metrics, to further elucidate how a set of clinically relevant mutations affect Mtb-RNAP function. With that, we were able to observe several key changes in residue importance and interactions that may be instrumental in bringing about RIF resistance and the compensatory conformational changes we observed among the mt systems through global analysis. Furthermore, we identified persistent hubs that may be particularly important in maintaining transcriptional activities in the presence and absence of the investigated mutations and RIF that could serve as potential resistance markers for future therapeutic investigations. We believe these findings will significantly aid future efforts in the discovery of new treatment options with the potential to overcome antitubercular resistance. , Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Bioinformatics, 2024
- Full Text:
- Date Issued: 2024-10-11
- Authors: Monama, Mokgerwa Zacharia
- Date: 2024-10-11
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Molecular dynamics Simulation methods
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/466849 , vital:76792 , DOI https://doi.org/10.21504/10962/466849
- Description: Emerging resistant strains of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) disease, continue to plague mankind and reduce the efficacies of antitubercular therapies that have been an effective defence against TB for decades. More specifically, mutations located in the β subunit of the multisubunit Mtb RNA replicative machinery, RNA polymerase (RNAP), have been well established as the reason behind resistance to the first-line antitubercular drug rifampicin (RIF), which has resulted in therapeutic failure in several clinical cases. Additionally, elusive details pertaining to the underlying mechanisms associated with RIF resistance due to the presence of Mtb-RNAP-β mutations, have resulted in setbacks in the development of novel and effective drugs that might be able to curb the ongoing threat. Hence, in this investigation, we attempted to resolve the involved Mtb-RNAP structural events at the molecular level to discern potentially important details regarding the nine clinically relevant Mtb-RNAP-β missense mutations under investigation. Hence, for the first time, we conducted an in-silico RIF resistance investigation using the Mtb-RNAP complex. To accomplish the set-out task, we first employed the use of more traditional post-MD analytical approaches such as root mean square deviation, root mean square fluctuation, radius of gyration, center of mass distance analyses, hydrogen bond occupancies, and binding free energy calculations, to conduct a global analysis of the mutated Mtb-RNAP proteins referencing RIF efficacy. Our findings revealed that the mutations may have a perturbation effect resulting in the disruption of essential structural dynamics attributed to the protein’s catalytic functions. This was for instance observed for the βfork loop 2 domain, the β’zinc-binding domain, the β’ trigger loop domain, and the β’jaw domain, which happen to be in line with previously reported experiments detailing changes in RNAP processivity. Complementarily, some of the mutations more specifically perturbed the RIF binding pocket (RIF-BP) which observably led to the reorientation of RIF from the native or active orientation needed to obstruct the processive addition of nucleoside triphosphates to the growing RNA transcript. The mutation-induced repositioning from the active RIF orientation was also reflected through the loss of essential interactions between RIF and the RIF-BP along with the loss of binding affinities captured for a majority of the mutant proteins. In conjunction with traditional analytical approaches, we further employed computational alanine scanning, weighted contact map analyses, and dynamic residue network (DRN) analyses, a novel approach that delineates residue-residue communication pathways through several metrics, to further elucidate how a set of clinically relevant mutations affect Mtb-RNAP function. With that, we were able to observe several key changes in residue importance and interactions that may be instrumental in bringing about RIF resistance and the compensatory conformational changes we observed among the mt systems through global analysis. Furthermore, we identified persistent hubs that may be particularly important in maintaining transcriptional activities in the presence and absence of the investigated mutations and RIF that could serve as potential resistance markers for future therapeutic investigations. We believe these findings will significantly aid future efforts in the discovery of new treatment options with the potential to overcome antitubercular resistance. , Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Bioinformatics, 2024
- Full Text:
- Date Issued: 2024-10-11
Quinolone-Pyrazinamide Derivatives: synthesis, characterisation, in silico ADME analysis and in vitro biological evaluation against Mycobacterium tuberculosis
- Authors: Rukweza, Kudakwashe Gerald
- Date: 2023-10-13
- Subjects: Quinolone antibacterial agents , Mycobacterium tuberculosis , Antitubercular agents , Tuberculosis Chemotherapy , Drug resistance , Moxifloxacin , Isoniazid
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/390901 , vital:68596
- Description: Tuberculosis is one of the leading causes of death worldwide caused by an infectious species, Mycobacterium tuberculosis (Mtb). Some of the factors that contribute to the prevalence of this disease include the complexity of diagnosis, prolonged period of therapy, side effects associated with current TB drugs, the prevalence of resistance against the current treatment options and a high incidence of co-infection with HIV/AIDS. Thus, there is a need for new alternative drugs to provide safer and shorter treatment therapy options that are not susceptible to the development of drug resistance. In this project, we focus our attention on the quinolone pharmacophore. Quinolones are currently used as alternative options in the treatment of resistant strains of Mtb. Previous work pertaining to quinolone-isoniazid hybrid compounds showed promising in vitro activity against the H37Rv strain of Mtb and served as the inspiration to pursue this project. The journey commenced with the synthesis of quinolone-pyrazinamide hybrid compounds (Figure 3.1). These compounds were synthesised, through the attachment of the quinolone and the pyrazinamide entity through a hydrazine linker. The synthesised compounds were purified, and their structural identity confirmed using common spectroscopic techniques including 1H and 13C NMR, infra-red (IR) and mass spectrometry. In vitro biological assays were performed by testing for the activity against the H37RvMA strain of Mtb. The bioassays were performed in triplicates to ensure the accuracy of the results. Moxifloxacin and isoniazid were tested as control compounds. Finally, the resultant compounds were profiled in silico for physicochemical and ADMET properties using open access software SwissADME. All the synthesised compounds 3.8a-f showed no activity against H37RvMA. In most cases, the resulting compounds showed minimal to no activity (MICs ≥ 57.3 μM) in all three media. During the in vitro studies, the compounds showed significant precipitation in the media over time suggesting poor aqueous solubility. The SwissADME analysis of these compounds indicated poor solubility in aqueous media, which is likely linked to their molecular size and complexity. Despite poor aqueous solubility, compounds 3.8a-f showed acceptable physicochemical properties and ADME parameters. No PAINs (Pan-assay interference compounds) were observed. Minimal to no interaction with CYP enzymes were predicted. Most of the compounds were compatible with the Lipinski’s rules of five. , Thesis (MSc) -- Faculty of Science, Pharmacy, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Rukweza, Kudakwashe Gerald
- Date: 2023-10-13
- Subjects: Quinolone antibacterial agents , Mycobacterium tuberculosis , Antitubercular agents , Tuberculosis Chemotherapy , Drug resistance , Moxifloxacin , Isoniazid
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/390901 , vital:68596
- Description: Tuberculosis is one of the leading causes of death worldwide caused by an infectious species, Mycobacterium tuberculosis (Mtb). Some of the factors that contribute to the prevalence of this disease include the complexity of diagnosis, prolonged period of therapy, side effects associated with current TB drugs, the prevalence of resistance against the current treatment options and a high incidence of co-infection with HIV/AIDS. Thus, there is a need for new alternative drugs to provide safer and shorter treatment therapy options that are not susceptible to the development of drug resistance. In this project, we focus our attention on the quinolone pharmacophore. Quinolones are currently used as alternative options in the treatment of resistant strains of Mtb. Previous work pertaining to quinolone-isoniazid hybrid compounds showed promising in vitro activity against the H37Rv strain of Mtb and served as the inspiration to pursue this project. The journey commenced with the synthesis of quinolone-pyrazinamide hybrid compounds (Figure 3.1). These compounds were synthesised, through the attachment of the quinolone and the pyrazinamide entity through a hydrazine linker. The synthesised compounds were purified, and their structural identity confirmed using common spectroscopic techniques including 1H and 13C NMR, infra-red (IR) and mass spectrometry. In vitro biological assays were performed by testing for the activity against the H37RvMA strain of Mtb. The bioassays were performed in triplicates to ensure the accuracy of the results. Moxifloxacin and isoniazid were tested as control compounds. Finally, the resultant compounds were profiled in silico for physicochemical and ADMET properties using open access software SwissADME. All the synthesised compounds 3.8a-f showed no activity against H37RvMA. In most cases, the resulting compounds showed minimal to no activity (MICs ≥ 57.3 μM) in all three media. During the in vitro studies, the compounds showed significant precipitation in the media over time suggesting poor aqueous solubility. The SwissADME analysis of these compounds indicated poor solubility in aqueous media, which is likely linked to their molecular size and complexity. Despite poor aqueous solubility, compounds 3.8a-f showed acceptable physicochemical properties and ADME parameters. No PAINs (Pan-assay interference compounds) were observed. Minimal to no interaction with CYP enzymes were predicted. Most of the compounds were compatible with the Lipinski’s rules of five. , Thesis (MSc) -- Faculty of Science, Pharmacy, 2023
- Full Text:
- Date Issued: 2023-10-13
Understanding the underlying resistance mechanism of Mycobacterium tuberculosis against Rifampicin by analyzing mutant DNA - directed RNA polymerase proteins via bioinformatics approaches
- Authors: Monama, Mokgerwa Zacharia
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167508 , vital:41487
- Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
- Full Text:
- Date Issued: 2020
- Authors: Monama, Mokgerwa Zacharia
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167508 , vital:41487
- Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
- Full Text:
- Date Issued: 2020
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