Molecular Basis for the Differential Quinolone Susceptibility of Mycobacterial DNA Gyrase

Abstract

b DNA gyrase is a type II topoisomerase that catalyzes the introduction of negative supercoils in the genomes of eubacteria. Fluoroquinolones (FQs), successful as drugs clinically, target the enzyme to trap the gyrase-DNA complex, leading to the accumulation of double-strand breaks in the genome. Mycobacteria are less susceptible to commonly used FQs. However, an 8-methoxysubstituted FQ, moxifloxacin (MFX), is a potent antimycobacterial, and a higher susceptibility of mycobacterial gyrase to MFX has been demonstrated. Although several models explain the mechanism of FQ action and gyrase-DNA-FQ interaction, the basis for the differential susceptibility of mycobacterial gyrase to various FQs is not understood. We have addressed the basis of the differential susceptibility of the gyrase and revisited the mode of action of FQs. We demonstrate that FQs bind both Escherichia coli and Mycobacterium tuberculosis gyrases in the absence of DNA and that the addition of DNA enhances the drug binding. The FQs bind primarily to the GyrA subunit of mycobacterial gyrase, while in E. coli holoenzyme is the target. The binding of MFX to GyrA of M. tuberculosis correlates with its effectiveness as a better inhibitor of the enzyme and its efficacy in cell killing. The DNA gyrase is an essential enzyme responsible for the maintenance of DNA topology in eubacteria. The enzyme catalyzes ATP-dependent negative supercoiling of relaxed circular DNA (1). It is composed of two GyrA and two GyrB subunits resulting in a heterotetrameric holoenzyme. Negative supercoiling by DNA gyrase involves a series of sequential events. The enzyme cleaves duplex DNA through which another segment of the same DNA molecule is transferred. The cleaved DNA is resealed followed by the release of substrate DNA, completing one round of DNA supercoiling (2-4). Owing to its indispensability for bacterial survival and its absence in mammals, gyrase has been a much sought-after drug target, culminating in the characterization of a number of inhibitors with diverse mechanisms of action. Among them, coumarins (5) and cyclothialidines (6) inhibit ATPase activity, and quinolones (7), CcdB (8), and microcin B17 (9) arrest the gyrase-DNA covalent complex. Several other compounds inhibit the enzyme activity (10-15). Among all of these molecules, fluorine substituted quinolones (FQs) act as bactericidal agents by poisoning the gyrase-DNA complex. Arrest of gyrase-DNA covalent complex leads to the accumulation of double-strand DNA breaks, triggering the activation of SOS pathways, depletion of reducing equivalents, and generation of reactive oxygen intermediates causing cell death (16,17). Due to this potent bactericidal effect, FQs are used for the treatment of a wide range of bacterial infections (18).Among all bacterial infections, tuberculosis (TB) continues to pose a major global challenge. The treatment of TB includes a combination of several drugs. FQs are now used to treat infection caused by rifampin-resistant tubercle bacilli (19). Although DNA gyrase from many path...

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

“…Moxifloxacin is a well-known type II topoisomerase poison which targets mycobacterial DNA gyrase (40,41). The experiments described above indicated that imipramine poisons the topoI reaction.…”

Topoisomerase Inhibitors: Fluoroquinolone Mechanisms of Action and Resistance

“…In GyrA, a noncatalytic Mg 2þ ion coordinated with four water molecules appears as a bridge for hydrogen bonding between quinolone and Ser83 and Asp87 (Wohlkonig et al 2010), the two most commonly mutated amino acids in quinolone-resistant mutants. Although quinolones can bind to mycobacterial gyrase in the absence of DNA (Kumar et al 2014), in E. coli, the gyrase -DNA complex shows increases in the amount and specificity of quinolone binding relative to gyrase alone (Shen et al 1989;Willmott and Maxwell 1993).…”

Boldine-Derived Alkaloids Inhibit the Activity of DNA Topoisomerase I and Growth of Mycobacterium tuberculosis

“…Both compounds inhibited S. pneumoniae TopoI activity in vitro at concentrations equivalent to those necessary to inhibit bacterial growth (∼10 μM) without affecting human cell viability ( García et al, 2011 ). M. tuberculosis possess two DNA topoisomerases: one type II enzyme, DNA gyrase, which is targeted by fluoroquinolone antibiotics ( Kumar et al, 2014 ) and one type I enzyme, topoisomerase I (MtbTopoI), which is encoded by Rv3646c ( topA ) gene. This gene has been shown to be essential for M. tuberculosis growth ( Kumar et al, 2014 ).…”