Characterization of compounds inhibiting recombinant M. tuberculosis topoisomerase I expressed in M. smegmatis
Department
Biochemistry
Faculty Advisor
Yuk-Ching
Start Date
1-10-2020 1:00 PM
End Date
1-10-2020 2:00 PM
Abstract
Topoisomerase I is an enzyme involved in the maintenance of genomic stability within an organism by mainly interacting with DNA to relieve topological strain. This serves as an essential target for inhibition as a means of preventing bacterial growth, and that was the goal of this study. More specifically, this project aimed to study the bactericidal activity of differing strains of Mycobacterium smegmatis: WT, M+, and Mnol. The project compared two different compounds, NSC 65858 and NSC 76027, as they expressed inhibitory mechanisms on topoisomerase I through various phases. The first phase employed the use of a MIC assay where both the M+ and Mnol strains showed similar values of growth inhibition. The values of these compounds shown to decrease growth successfully were around 25 micromolar concentrations for M+ and 12.5 micromolar concentrations for the Mnol strain. Because these compounds both showed sensitivity with the enzyme of interest, the study could continue to phase two: a bacterial survival assay. This phase involved studying the growth of the M. smegmatis over a 24-hour period at the concentrations of 50 and 100 micromolar concentrations, and these showed similar values when compared to each other. However, in comparison to a non-treated sample, they showed much less growth than when exposed to the compound. To ensure this inhibition was due to inhibition of the enzyme, the third phase comprised of a relaxation assay with NSC 76027 to ensure the mechanism behind the decreased growth was in fact due to topoisomerase I inhibition.
File Type
Event
Characterization of compounds inhibiting recombinant M. tuberculosis topoisomerase I expressed in M. smegmatis
Topoisomerase I is an enzyme involved in the maintenance of genomic stability within an organism by mainly interacting with DNA to relieve topological strain. This serves as an essential target for inhibition as a means of preventing bacterial growth, and that was the goal of this study. More specifically, this project aimed to study the bactericidal activity of differing strains of Mycobacterium smegmatis: WT, M+, and Mnol. The project compared two different compounds, NSC 65858 and NSC 76027, as they expressed inhibitory mechanisms on topoisomerase I through various phases. The first phase employed the use of a MIC assay where both the M+ and Mnol strains showed similar values of growth inhibition. The values of these compounds shown to decrease growth successfully were around 25 micromolar concentrations for M+ and 12.5 micromolar concentrations for the Mnol strain. Because these compounds both showed sensitivity with the enzyme of interest, the study could continue to phase two: a bacterial survival assay. This phase involved studying the growth of the M. smegmatis over a 24-hour period at the concentrations of 50 and 100 micromolar concentrations, and these showed similar values when compared to each other. However, in comparison to a non-treated sample, they showed much less growth than when exposed to the compound. To ensure this inhibition was due to inhibition of the enzyme, the third phase comprised of a relaxation assay with NSC 76027 to ensure the mechanism behind the decreased growth was in fact due to topoisomerase I inhibition.