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 of the underlying resistance mechanism of the Kat-G protein against isoniazid in Mycobacterium tuberculosis using bioinformatics approaches
- Authors: Barozi, Victor
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Isoniazid , Drug resistance in microorganisms , Proteins -- Microbiology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/146592 , vital:38540
- Description: Tuberculosis (TB) is a multi-organ infection caused by rod-shaped acid-fast Mycobacterium tuberculosis. The World Health Organization (WHO) ranks TB among the top 10 fatal infections and the leading the cause of death from a single infection. In 2017, TB was responsible for an estimated 1.3 million deaths among both the HIV negative and positive populations worldwide (WHO, 2018). Approximately 23% (roughly 1.7 billion) of the world’s population is estimated to have latent TB with a high risk of reverting to active TB infection. In 2017, an estimated 558,000 people developed drug resistant TB worldwide with 82% of the cases being multi-drug resistant TB (WHO, 2018). South Africa is ranked among the 30 high TB burdened countries with a TB incidence of 322,000 cases in 2017 accounting for 3% of the world’s TB cases. TB is curable and is clinically managed through a combination of intensive and continuation phases of first-line drugs (isoniazid, rifampicin, ethambutol, and pyrazinamide). Second-line drugs which include fluoroquinolones, injectable aminoglycoside and injectable polypeptides are used in cases of first line drug resistance. The third-line drugs include amoxicillin, clofazimine, linezolid and imipenem. These have variable but unproven efficacy to TB and are the last resort in cases of total drug resistance (Jilani et al., 2019). TB drug resistance to first-line drugs especially isoniazid in M. tuberculosis has been attributed to single nucleotide polymorphisms (SNPs) in the catalase peroxidase enzyme (katG), a protein important in the activation of the pro-drug isoniazid. The SNPs especially at position 315 of the katG enzyme are believed to reduce the sensitivity of the M. tuberculosis to isoniazid while still maintaining the enzyme’s catalytic activity - a mechanism not completely understood. KatG protein is important for protecting the bacteria from hydro peroxides and hydroxyl radicals present in an aerobic environment. This study focused on understanding the mechanism of isoniazid drug resistance in M. tuberculosis as a result of high confidence mutations in the katG through modelling the enzyme with its respective variants, performing MD simulations to explore the protein behaviour, calculating the dynamic residue network analysis (DRN) of the variants in respect to the wild type katG and finally performing alanine scanning. From the MD simulations, it was observed that the high confidence mutations i.e. S140R, S140N, G279D, G285D, S315T, S315I, S315R, S315N, G316D, S457I and G593D were not only reducing the backbone flexibility of the protein but also reducing the protein’s conformational variation and space. All the variant protein structures were observed to be more compact compared to the wild type. Residue fluctuation results indicated reduced residue flexibility across all variants in the loop region (position 26-110) responsible for katG dimerization. In addition, mutation S315T is believed to reduce the size of the active site access channel in the protein. From the DRN data, residues in the interface region between the N and C-terminal domains were observed to gain importance in the variants irrespective of the mutation location indicating an allosteric effect of the mutations on the interface region. Alanine scanning results established that residue Leucine at position 48 was not only important in the protein communication but also a destabilizing residue across all the variants. The study not only demonstrated change in the protein behaviour but also showed allosteric effect of the mutations in the katG protein.
- Full Text:
- Date Issued: 2020
- Authors: Barozi, Victor
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Isoniazid , Drug resistance in microorganisms , Proteins -- Microbiology
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/146592 , vital:38540
- Description: Tuberculosis (TB) is a multi-organ infection caused by rod-shaped acid-fast Mycobacterium tuberculosis. The World Health Organization (WHO) ranks TB among the top 10 fatal infections and the leading the cause of death from a single infection. In 2017, TB was responsible for an estimated 1.3 million deaths among both the HIV negative and positive populations worldwide (WHO, 2018). Approximately 23% (roughly 1.7 billion) of the world’s population is estimated to have latent TB with a high risk of reverting to active TB infection. In 2017, an estimated 558,000 people developed drug resistant TB worldwide with 82% of the cases being multi-drug resistant TB (WHO, 2018). South Africa is ranked among the 30 high TB burdened countries with a TB incidence of 322,000 cases in 2017 accounting for 3% of the world’s TB cases. TB is curable and is clinically managed through a combination of intensive and continuation phases of first-line drugs (isoniazid, rifampicin, ethambutol, and pyrazinamide). Second-line drugs which include fluoroquinolones, injectable aminoglycoside and injectable polypeptides are used in cases of first line drug resistance. The third-line drugs include amoxicillin, clofazimine, linezolid and imipenem. These have variable but unproven efficacy to TB and are the last resort in cases of total drug resistance (Jilani et al., 2019). TB drug resistance to first-line drugs especially isoniazid in M. tuberculosis has been attributed to single nucleotide polymorphisms (SNPs) in the catalase peroxidase enzyme (katG), a protein important in the activation of the pro-drug isoniazid. The SNPs especially at position 315 of the katG enzyme are believed to reduce the sensitivity of the M. tuberculosis to isoniazid while still maintaining the enzyme’s catalytic activity - a mechanism not completely understood. KatG protein is important for protecting the bacteria from hydro peroxides and hydroxyl radicals present in an aerobic environment. This study focused on understanding the mechanism of isoniazid drug resistance in M. tuberculosis as a result of high confidence mutations in the katG through modelling the enzyme with its respective variants, performing MD simulations to explore the protein behaviour, calculating the dynamic residue network analysis (DRN) of the variants in respect to the wild type katG and finally performing alanine scanning. From the MD simulations, it was observed that the high confidence mutations i.e. S140R, S140N, G279D, G285D, S315T, S315I, S315R, S315N, G316D, S457I and G593D were not only reducing the backbone flexibility of the protein but also reducing the protein’s conformational variation and space. All the variant protein structures were observed to be more compact compared to the wild type. Residue fluctuation results indicated reduced residue flexibility across all variants in the loop region (position 26-110) responsible for katG dimerization. In addition, mutation S315T is believed to reduce the size of the active site access channel in the protein. From the DRN data, residues in the interface region between the N and C-terminal domains were observed to gain importance in the variants irrespective of the mutation location indicating an allosteric effect of the mutations on the interface region. Alanine scanning results established that residue Leucine at position 48 was not only important in the protein communication but also a destabilizing residue across all the variants. The study not only demonstrated change in the protein behaviour but also showed allosteric effect of the mutations in the katG protein.
- Full Text:
- Date Issued: 2020
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