Mechanism of Action of Mycobacterium tuberculosis Gyrase Inhibitors: A Novel Class of Gyrase Poisons

Abstract

Tuberculosis is one of the leading causes of morbidity worldwide, and the incidences of drug resistance and intolerance are prevalent. Thus, there is a desperate need for the development of new antitubercular drugs. Mycobacterium tuberculosis gyrase inhibitors (MGIs) are napthyridone/aminopiperidine-based drugs that display activity against M. tuberculosis cells and tuberculosis in mouse models [Blanco, D., et al. (2015) Antimicrob. Agents Chemother. 59, 1868-1875]. Genetic and mutagenesis studies suggest that gyrase, which is the target for fluoroquinolone antibacterials, is also the target for MGIs. However, little is known regarding the interaction of these drugs with the bacterial type II enzyme. Therefore, we examined the effects of two MGIs, GSK000 and GSK325, on M. tuberculosis gyrase. MGIs greatly enhanced DNA cleavage mediated by the bacterial enzyme. In contrast to fluoroquinolones (which induce primarily double-stranded breaks), MGIs induced only single-stranded DNA breaks under a variety of conditions. MGIs work by stabilizing covalent gyrase-cleaved DNA complexes and appear to suppress the ability of the enzyme to induce double-stranded breaks. The drugs displayed little activity against type II topoisomerases from several other bacterial species, suggesting that these drugs display specificity for M. tuberculosis gyrase. Furthermore, MGIs maintained activity against M. tuberuclosis gyrase enzymes that contained the three most common fluoroquinolone resistance mutations seen in the clinic and displayed no activity against human topoisomerase IIα. These findings suggest that MGIs have potential as antitubercular drugs, especially in the case of fluoroquinolone-resistant disease.

Keywords: Mycobacterium tuberculosis gyrase inhibitors; fluoroquinolones; gyrase; single-stranded DNA cleavage; tuberculosis.

Figure 1.

Structures of selected compounds that alter the activity of gyrase. GSK000 and GSK325 are Mycobacterium tuberculosis Gyrase Inhibitors (MGIs); GSK 126 is a Novel Bacterial Topoisomerase Inhibitor (NBTI); and moxifloxacin is a fluoroquinolone antibacterial.

Figure 2.

MGIs induce single-stranded DNA breaks mediated by M. tuberculosis gyrase. The left panel shows the quantification of single-stranded (SS, closed circles) and double-stranded (DS, open circles) DNA breaks induced by GSK000 (red), GSK325 (blue), or GSK126 (black) in the presence of M. tuberculosis gyrase. Error bars represent the standard deviation (SD) of four independent experiments. The top right gel shows DNA cleavage products produced by gyrase that was incubated with increasing concentrations of GSK000. The bottom right gel shows DNA products following cleavage reactions containing 10 μM GSK000 (000), GSK325 (325), or GSK126 (126), or 20 μM moxifloxacin (Moxi) in the presence of gyrase or 200 μM GSK000, GSK325, or GSK126 in the absence of enzyme. Negatively supercoiled (SC) and linear (Lin) DNA controls are shown along with a reaction that contained gyrase, but no drug (Gyr). The mobilities of negatively supercoiled DNA [(−)SC], nicked circular DNA (Nick), and linear DNA (Lin) are indicated. Gels are representative of at least four independent experiments.

Figure 3.

DNA cleavage induced by GSK000 is mediated by M. tuberculosis gyrase. The bar graph shows results from reactions that contained negatively supercoiled DNA in the presence of M. tuberculosis gyrase (Gyr, black bar), GSK000 (200 μM) in the absence of gyrase (GSK000), or complete reaction mixtures containing 10 μM GSK000 and gyrase that were stopped with SDS prior to the addition of EDTA (blue bar), and reactions that were treated with EDTA (green bar) or NaCl (yellow bar) prior to SDS. Error bars represent the SD of at least 4 independent experiments.

Figure 4.

GSK000 enhances only single-stranded DNA breaks mediated by M. tuberculosis gyrase. The panel on the left shows the enhancement of gyrase-mediated single-stranded (SS, closed circles) or double-stranded (DS, open circles) DNA breaks generated by gyrase over time in reactions that contained 10 μM (red) or 200 μM (black) GSK000. The right panel shows the effects of GSK000 on gyrase-mediated DNA cleavage in the presence (red) or absence (black) of ATP (1 mM). Error bars represent the SD of at least 3 independent experiments.

Figure 5.

Effects of GSK000 and moxifloxacin on ligation and persistence of cleavage complexes mediated by M. tuberculosis gyrase. The rate of gyrase-mediated DNA ligation (left) and the stability of ternary gyrase–drug–DNA cleavage complexes (right) were monitored by the loss of single-stranded DNA breaks in the presence of 10 μM GSK000 (red) or the loss of double-stranded DNA cleavage in the absence of drug (black) or in the presence of 50 μM moxifloxacin (blue). Levels of DNA cleavage at time 0 (42% single-stranded breaks in the presence of GSK000 and 36% double-stranded breaks in the presence of moxifloxacin) were set to 1 to allow direct comparison. Error bars represent the SD of at least 3 independent experiments.

Figure 6.

Effects of GSK000 and moxifloxacin on the sites of DNA cleavage generated by M. tuberculosis gyrase. An autoradiogram of a polyacrylamide gel is shown. Reaction mixtures contained DNA with no enzyme (DNA), enzyme in the absence of drug (Gyr), or enzyme in the presence of 100 μM GSK000 (GSK000) or 100 μM moxifloxacin (Moxi). The left- and right-hand sides of the gel show reactions processed without or following K⁺-SDS precipitation of DNA cleavage complexes. Red arrows indicate representative strong sites where GSK000 induced greater levels of DNA cleavage than did moxifloxacin, green arrows indicate representative strong sites where moxifloxacin induced greater levels of cleavage than did GSK000, and blue arrows indicate representative sites where GSK000 and moxifloxacin induced similar levels of cleavage. The autoradiogram is representative of at least 4 independent experiments.

Figure 7.

GSK000 suppresses double-stranded DNA breaks generated by M. tuberculosis gyrase. The gel (top) shows DNA cleavage products following incubation of gyrase with increasing concentrations of GSK000 in the presence of Ca²⁺. Negatively supercoiled (SC) and linear controls (Lin) are shown. The gel is representative of at least 4 independent experiments. The graph quantifies the effects of GSK000 on M. tuberculosis gyrase-mediated single-stranded (SS, closed circles) and double-stranded (DS, open circles) DNA cleavage. Error bars represent the SD of at least 4 independent experiments.

Figure 8.

Modeling studies suggest that moxifloxacin and GSK000 cannot interact simultaneously in the same drug-enzyme-DNA ternary complex. A. View down the two-fold axis of a crystal structure of a ternary complex formed with M. tuberculosis gyrase, DNA, and moxifloxacin. Gyrase subunits are shown in cartoon representation, in blue/cyan (GyrA) or red/dark red (GyrB). The catalytic tyrosine (Tyr 129) which has cleaved the DNA is shown in stick representation. Moxifloxacin (orange carbons) and DNA (green carbons) are also shown in stick representation. B. Model of a complex of GSK000 (yellow carbons) with M. tuberculosis gyrase and uncleaved DNA that was based on the crystal structure of GSK299423 with Staphylococcus aureus gyrase. C. Superimposition of A and B require that base-pairs overlap with the compounds.

Figure 9.

The actions of GSK000 and moxifloxacin on gyrase-mediated DNA cleavage are mutually exclusive. In the left panel, enhancement of M. tuberculosis gyrase-mediated single-stranded (SS, closed circles) and double-stranded (DS, open circles) DNA cleavage is shown in the presence of moxifloxacin alone (blue). In the right panel, gyrase was saturated with 10 μM GSK000 followed by a subsequent titration of 0–200 μM moxifloxacin. Error bars represent the SD of at least 3 independent experiments.

Figure 10.

GSK000 maintains lower levels of gyrase-mediated single-stranded DNA breaks on positively supercoiled DNA. The effects of GSK000 on the enhancement of gyrase-mediated single-stranded cleavage of negatively (closed circles) and positively (open circles) supercoiled DNA is shown. Error bars represent the SD of at least 3 independent experiments.

Figure 11.

Inhibition of gyrase catalyzed reactions by GSK000 and moxifloxacin. The effects of GSK000 (left panels) and moxifloxacin (right panels) on the supercoiling of relaxed DNA (top panels) and the relaxation of positively supercoiled DNA (bottom panels) are shown. The positions of relaxed (Rel), negatively supercoiled [(−)SC], and positively supercoiled [(+)SC] DNA are indicated. Gels are representative of at least four independent experiments.

Figure 12.

GSK000 acts preferentially against M. tuberculosis gyrase. The effects of GSK000 on single-stranded DNA cleavage mediated by M. tuberculosis gyrase (Mt gyr, red), B. anthracis gyrase (Ba gyr, purple) and topoisomerase IV (Ba TIV, blue), N. gonorrhoeae gyrase (Ng gyr, green) and topoisomerase IV (Ng TIV, yellow), and E. coli gyrase (Ec gyr, orange) and topoisomerase IV (Ec TIV, black) are shown. Error bars represent the SD of at least 3 independent experiments.

Figure 13.

MGIs/NBTI maintain activity against M. tuberculosis gyrase containing the most common mutations associated with clinical fluoroquinolone resistance. Effects of the MGIs GSK000 (left panel), GSK325 (middle panel), and the NBTI GSK126 (right panel) on wild-type (red) M. tuberculosis gyrase and gyrase containing the fluoroquinolone resistance mutations at GyrA^A90V (blue), GyrA^D94G (purple), or GyrA^D94H (green) are shown. Single-stranded (SS) and double-stranded (DS) DNA breaks are denoted by closed and open circles respectively. Error bars represent the SD of at least 3 independent experiments.

Figure 14.

The actions of GSK000 and moxifloxacin on DNA cleavage mediated by GyrA^D94G. The mutant fluoroquinolone-resistant gyrase was saturated with 10 μM GSK000 followed by a subsequent titration of 0–200 μM moxifloxacin. Error bars represent the SD of at least 3 independent experiments.

Figure 15.

MGIs/NBTI do not enhance DNA cleavage mediated by human topoisomerase IIα. The left and right panels show the effects of the MGIs GSK000 (red) and GSK325 (blue) and the NBTI GSK126 (black) on single-stranded (closed circles) and double-stranded (open circles) DNA cleavage mediated by the human enzyme. The effects of etoposide (green), a widely prescribed anti-cancer drug, on topoisomerase IIα are shown as a positive control. Error bars represent the SD of at least 3 independent experiments.