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|Title:||Characterization of the novel 4-chloro-1-piperidin-1ylmethyl-1H-indole-2,3-dione compound (Raja 42) for its antibacterial activity against Escherichia coli, Clostridium difficile, Staphylococcus aureus and Helicobacter pylori|
|Keywords:||Novel drug discovery;antibiotic resistance;Escherichia coli,;γ-lactam antibiotics;reactive oxygen species;bacterial genomics;metabolism;Clostridium difficile|
|Abstract:||According to the World Health Organization (WHO), drug-resistant bacteria are prevalent in 83.3% of the regions where WHO conducts surveillance. Furthermore, the number of antibiotic resistant bacterial strains increases every year, necessitating the development of new classes of antibacterial agents. Toward developing a novel class of antibacterial agents, we have created a chemical library using chloroquine as the basic scaffold. We screened our chemical library of 211 compounds to identify antibacterial activity. Twenty-seven were effective on drug-sensitive E. coli strains as well as on those resistant to ampicillin, kanamycin or NDM-1. In addition, they were also effective against Staphylococcus aureus and methicillin-resistant S. aureus. Since all of them contain an isatin moiety, they are classified as the γ-lactam class of antibiotics. Although similarities can be seen in the spectrum of activities of γ-lactam-based and β-lactam-based antibiotics, there are marked differences in the activity against antibiotic resistant bacterial strains. One of the new compounds, Raja 42 (4-chloro-1-piperidin-1ylmethyl-1H-indole-2,3-dione), displayed a lowered MIC value and, therefore, was chosen for further studies. In addition to its excellent activity against E. coli, Raja 42 is also notably effective against Helicobacter pylori and Clostridium difficile isolates from patients. I set out to unravel the molecular mechanism by which Raja 42 exhibits its antibacterial effects. Data from cellular and fluorescent microscopic assays showed that bacteria were killed rapidly in the presence of Raja 42. A time-kill and membrane depolarization assays confirmed the rapid cell killing by Raja 42, suggesting that the mode of killing by the compound is likely due to the disruption of bacterial cell membrane. To further investigate this possibility, I carried out protein 2-D gel electrophoresis in an attempt to identify proteins involved in the Raja 42-mediated cell killing. In the process, those proteins differentially expressed in response to Raja 42 were isolated and their identities were determined by peptide fingerprinting using mass spectrometry. The resultant data revealed that several proteins involved in the reactive oxygen species (ROS) pathway are upregulated in the Raja 42-treated samples. In parallel, ten clones resistant to Raja 42 were generated, and their nucleotide sequences were determined. A 27 bp deletion upstream of the promoter region of ghrA, a necessary catalytic converter of glyoxylate to glycolate in the glyoxylate shunt pathway, was found to be present in all of the Raja 42-resistant clones. This data suggests that the ablation of ghrA is directly related to the Raja 42 resistant phenotype. To determine the quantitative gene expression of bacteria in response to Raja 42 treatment, QPCR analysis was carried out. To solidify the mechanism of Raja 42 further, rescue experiments were performed to determine the importance of ghrA. Taken all the data together, Raja 42 appears to kill bacteria by upregulating the level of cellular ROS through rapidly redirecting the metabolic pathways.|
|Appears in Collections:||Biomolecular Sciences - Doctoral Theses|
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|PhD Thesis-A Fong, Final & corrected version,2020,12,21.pdf||4.96 MB||Adobe PDF|
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