Thank you for all your support and encouragement and for being an overall great mentor. Tim Sterling: Thank you for all your support and encouragement over the years. Tim Sterling: Thank you for allowing me to train with Amondrea Blackman at the BSL3 facilities to test my compounds against clinical isolates of Mycobacterium tuberculosis.
To Pan Chan: Thank you for providing Neisseria gonorrhoeae gyrase and topoisomerase IV and Eshcerischia coli gyrase. Thank you also for sending the lab geopotidazine and allowing me to investigate the interactions of geopotidazine with various bacterial type II topoisomerases. Also, thank you for being my football game buddy and going to one season of the Vanderbilt football games (sometimes while completely freezing).
To my church family in Georgia and Tennessee, thank you for all the prayers and encouragement. Thank you for allowing me to play piano and organ and continue to use my God-given talent of music.
Cell death
When an appropriate level of splicing complexes is maintained, topological problems are resolved and the cell can grow normally. If levels of fission complexes decrease, slow growth rates and mitotic failure can cause cell death. Conversely, if the levels of cleavage complexes are too high, these breaks can block essential nucleic acid functions and induce the SOS response, generating mutations and leading to cell death.
Compounds (such as fluoroquinolones) that increase the level of gyrase or topoisomerase IV cleavage complexes act as topoisomerase "poisons" by converting the proteins into cellular toxins that have the potential to fragment the genome. These compounds are referred to as gyrase or topoisomerase IV "poisons" because they are said to poison these proteins and convert them into cellular toxins that have the potential to fragment the genome. Compounds also act on the other side of the balance and can inhibit overall catalytic, leading to slow growth rates, mitotic failure and cell death.
Normal cell growth
Role of the water-metal ion bridge in mediating fluoroquinolone resistance and gyrase/topoisomerase IV interactions. It will also describe the first study of the activity of an NBTI against topoisomerase IV using N. In each of the reactions, the enzymes were incubated in the presence or absence of the compounds listed above.
In experiments performed in the absence of drug, MgCl2 in the cleavage buffer was replaced with 6 mM CaCl2 to increase baseline levels of DNA cleavage. Levels of gepotidacin-induced single-stranded or moxifloxacin-induced double-stranded DNA cleavage were set to 100% at time zero, as was enzyme-mediated DNA cleavage in the absence of drug. 77 In each of the reactions, the enzymes were incubated in the presence or absence of the MGIs, NBTIs or moxifloxacin.
The left panel shows the quantification of single-stranded (SS, closed circles) and double-stranded (DS, open circles) DNA breaks produced by GSK000 (red), GSK325 (blue) or GSK126 (black) in the presence of M The right panel shows the effects of GSK000 on gyrase-mediated DNA cleavage in the presence (red) or absence (black) of ATP (1 mM). Representative sites cleaved more frequently in the presence of the MGI (red arrows) or the fluoroquinolone (green arrows) are indicated.
The left and right sides of the gel show reactions processed without or after K+-SDS precipitation of the DNA cleavage complexes.
SDS K + -SDS
This latter divalent metal ion can replace Mg2+ in the active site of type II topoisomerases.129 Although most properties of the DNA cleavage and ligation reactions remain unchanged, significantly higher levels of enzyme-mediated double-strand breaks are generated in the presence of Ca2+. 65, 129 In the case of M. As seen in Figure 15, GSK000 induced high levels of single-stranded DNA breaks in the presence of Ca 2+ . However, it is probably due to a distortion of the DNA in the active site of the enzyme, similar to that generated in the presence of NBTIs.62.
The gel (top) shows DNA cleavage products after incubation of gyrase with increasing concentrations of GSK000 in the presence of Ca2+. Based on these studies, it does not seem possible that both drugs interact simultaneously in the same ternary complex. NBTIs are believed to capture the enzyme in the CRsym conformation, which can accommodate only one linkage in the DNA.
In the absence of MGI, moxifloxacin readily induced enzyme-mediated DNA double-strand breaks (~36% in the presence of 100 µM drug), but also induced ~12% single-strand breaks (peak at 25 µM) (Figure 17 , left panel). Modeling studies suggest that moxifloxacin and GSK000 cannot interact simultaneously in the same drug-enzyme-DNA ternary complex. In the right panel, gyrase was saturated with 10 µM GSK000 followed by a subsequent titration of 0-200 µM moxifloxacin.
Changes in the level of double- and single-strand breaks were monitored (Figure 17, right panel) to determine whether there was competition or additivity between the two compounds. As the concentration of moxifloxacin increased, levels of double-strand breaks increased, albeit to a lesser extent than observed in the absence of the MGI (similar changes in DNA cleavage were observed when gyrase was incubated with moxifloxacin before the addition of GSK000). To distinguish between these latter possibilities, I monitored the single-strand breaks generated in the presence of both drugs (Figure 17, right panel).
In contrast to double-strand breaks, single-strand breaks decreased in the presence of moxifloxacin. If both drugs were present in the ternary complex, levels of single-strand breaks would be expected to rise, because both drugs cause enzyme-mediated single-strand DNA cleavage. This finding confirms that the competition between moxifloxacin and GSK000 seen in Figure 17 was due to the replacement of the MGI by the fluoroquinolone in the active site of gyrase.
GSK000
GSK126
Therefore, the ability of GSK126 to induce enzyme-mediated single-strand breaks was examined for each of the type II enzymes discussed above. No increase in DNA DS breaks was observed for any of the bacterial type II enzymes tested. Graph shows the effects of GSK126 on gyrase-mediated enhancement of single-stranded (SS, closed circles) or double-stranded (DS, open circles) in the presence of ATP (1.5 mM).
The graph shows the effects of GSK126 on the enhancement of topoisomerase IV-mediated single-stranded (SS, closed circles) or double-stranded (DS, open circles) DNA breaks in the presence of ATP (1.5 mM). Like GSK126, all breaks created in the presence of gepotidacin were single-stranded. Gepotidacin potently enhanced gyrase-mediated DNA cleavage in the nanomolar range [EC50 (concentration required to induce 50% maximal DNA cleavage)≈ 130 nM] and generated single-stranded DNA breaks in more than 30% of the original substrate at low micromolar concentrations.
The right panel shows the effects of moxifloxacin on single- and double-stranded DNA gyrase cleavage of negatively (black) and positively (green) supercoiled DNA. As reported for other types of fluoroquinolone and MGI gyrases, gepotidacin induced approximately 2-fold lower levels of single-strand breaks in the presence of S. Therefore, the effects of gepotidacin on gyrase-induced DNA cleavage were performed in the presence of 1.5 mM ATP, to determine whether the high-energy cofactor affects the compound's ability to generate single vs.
While the compound is more potent in the presence of ATP (EC50 ≈ 40 nM compared to EC50 ≈ 130 nM in the absence of ATP), no double-stranded DNA cleavage was observed. The stability of these single-stranded DNA cleavage complexes was similar to or slightly greater than that of the double-spliced counterparts generated in the presence of moxifloxacin. In contrast, in the absence of the NBTI or fluoroquinolone the lifetime of single or double strands.
The increase in gyrase-mediated single-stranded (SS, closed circles) or double-stranded (DS, open circles) DNA breaks generated by gyrase in the presence of 1.5 mM ATP is shown. The stability of ternary gyrase-drug-DNA cleavage complexes was monitored by loss of DNA single-strand breaks in the presence of 5 μM gepotidacin (blue) or double-strand DNA breaks in the presence of 25 μM moxifloxacin ( red), or loss of single- (open circle, white) or double-stranded DNA cleavage in the absence of the drug (closed circle, black). In contrast to fluoroquinolones, only a single molecule of gepotidacin binds to the active site of S.
First, I investigated the ability of GSK126 to enhance DNA cleavage by a wide range of bacterial type II topoisomerases. I first investigated the ability of gepotidacin to enhance DNA cleavage by bacterial type II topoisomerases from S.
