Gyrase and Topoisomerase IV: Recycling Old Targets for New Antibacterials to Combat Fluoroquinolone Resistance

Abstract

Beyond their requisite functions in many critical DNA processes, the bacterial type II topoisomerases, gyrase and topoisomerase IV, are the targets of fluoroquinolone antibacterials. These drugs act by stabilizing gyrase/topoisomerase IV-generated DNA strand breaks and by robbing the cell of the catalytic activities of these essential enzymes. Since their clinical approval in the mid-1980s, fluoroquinolones have been used to treat a broad spectrum of infectious diseases and are listed among the five "highest priority" critically important antimicrobial classes by the World Health Organization. Unfortunately, the widespread use of fluoroquinolones has been accompanied by a rise in target-mediated resistance caused by specific mutations in gyrase and topoisomerase IV, which has curtailed the medical efficacy of this drug class. As a result, efforts are underway to identify novel antibacterials that target the bacterial type II topoisomerases. Several new classes of gyrase/topoisomerase IV-targeted antibacterials have emerged, including novel bacterial topoisomerase inhibitors, Mycobacterium tuberculosis gyrase inhibitors, triazaacenaphthylenes, spiropyrimidinetriones, and thiophenes. Phase III clinical trials that utilized two members of these classes, gepotidacin (triazaacenaphthylene) and zoliflodacin (spiropyrimidinetrione), have been completed with positive outcomes, underscoring the potential of these compounds to become the first new classes of antibacterials introduced into the clinic in decades. Because gyrase and topoisomerase IV are validated targets for established and emerging antibacterials, this review will describe the catalytic mechanism and cellular activities of the bacterial type II topoisomerases, their interactions with fluoroquinolones, the mechanism of target-mediated fluoroquinolone resistance, and the actions of novel antibacterials against wild-type and fluoroquinolone-resistant gyrase and topoisomerase IV.

Keywords: fluoroquinolone resistance; gyrase; novel bacterial topoisomerase inhibitors; spiropyrimidinetriones; topoisomerase IV; triazaacenaphthylenes.

Figure 1

DNA strand passage activities of gyrase and topoisomerase IV. Gyrase primarily modulates the superhelical state of the bacterial genome by relaxing (i.e., removing) positive supercoils and generating negative supercoils in relaxed DNA. Topoisomerase IV primarily removes tangles (catenanes) and knots from the genetic material but can also relax positive and negative DNA supercoils.

Figure 2

Catalytic cycle of bacterial type II topoisomerases. The double-stranded DNA passage reaction can be separated into discrete steps: 1) Capturing two segments of DNA, the gate, or G-segment (green), and the transport, or T-segment (yellow); 2) Bending the G-segment to assess DNA sites for cleavability; 3) Cleaving both strands of the G-segment; 4) Binding 2 molecules of ATP, which triggers N-gate dimerization, DNA gate opening, and T-segment strand passage through the DNA gate. The rate of the DNA passage step is increased if one of the two ATP molecules is hydrolyzed; 5) Closing the DNA gate and ligating the G-segment; 6) Hydrolyzing the second ATP molecule and releasing the T-segment through the C-gate; 7) Initiating enzyme turnover.

Figure 3

Activities of gyrase and topoisomerase IV during DNA replication and transcription. The movement of replication forks and transcription complexes along the double helix generates topological stress ahead of and behind these systems. The positive supercoils formed in front of DNA tracking machineries pose a physical barrier to progressing complexes. Gyrase (right) uses a DNA wrapping mechanism to rapidly remove these positive supercoils. The precatenanes (two intertwined partially replicated DNA duplexes) that trail replication complexes are untangled by topoisomerase IV (using a canonical strand passage mechanism) prior to cell division. The negative supercoils that accumulate behind transcription complexes are most likely removed by the ω protein, a type I topoisomerase.

Figure 4

Cellular death induced by gyrase/topoisomerase IV-targeted antibacterials. Under normal cellular conditions, DNA cleavage complexes generated by gyrase and topoisomerase IV are short-lived and readily reversible, leading to normal cellular growth (middle). However, topoisomerase poisons and inhibitors shift the balance between DNA cleavage and ligation. Topoisomerase poisons increase the level of enzyme-mediated DNA breaks by stabilizing cleavage complexes. In response to DNA damage, cells initiate the SOS response. If overwhelmed by DNA damage, bacteria can undergo mutagenesis or cell death (right). Conversely, topoisomerase inhibitors prevent gyrase and topoisomerase IV from completing their catalytic cycles. This robs the cell of essential enzyme functions. Inhibition of gyrase can stall DNA replication and transcription, which impedes bacterial growth, while inhibition of topoisomerase IV can lead to catastrophic cell division and death.

Figure 5

Fluoroquinolone structures. Nalidixic acid is a first-generation quinolone and the founding member of the drug class. It was approved to treat urinary tract infections but was eventually removed from the clinic due to poor pharmacodynamics. The addition of a fluorine atom at the C6 position spurred a new wave of fluoroquinolone drug development. The second-generation drugs, including norfloxacin, ciprofloxacin, and levofloxacin, had better pharmacodynamics and pharmacokinetics than their quinolone precursors and displayed activity against Gram-positive and Gram-negative pathogens. Subsequent third- (sparfloxacin) and fourth-generation (moxifloxacin and delafloxacin) drugs further improved pharmacokinetics/pharmacodynamics and extended antibacterial coverage to atypical bacteria (such as M. tuberculosis).

Figure 6

Schematic of the water–metal-ion bridge that mediates interactions between fluoroquinolones and bacterial type II topoisomerases. For simplicity, only interactions with the protein (and not the DNA) are shown. A noncatalytic divalent metal ion (orange, Mg²⁺) forms an octahedral coordination sphere (green dashed lines) between four water molecules (green) and the C3/C4 keto-acid of ciprofloxacin (black, shown as a representative fluoroquinolone). Two of the water molecules form hydrogen bonds (blue dashed lines) with the serine side chain hydroxyl group (blue), and one water molecule forms hydrogen bonds (red dashed lines) with the aspartic acid or glutamic acid side chain carboxyl group (red).

Figure 7

Novel bacterial topoisomerase inhibitor (NBTI), M. tuberculosis gyrase inhibitor (MGI), and triazaacenaphthylene structures. Members of these classes possess a left-hand substituent (LHS, orange), a central linker (green), a basic nitrogen (blue), and a right-hand substituent (RHS, red). While the LHS, central linker, and RHS are amenable to alteration, the basic nitrogen is critical for antibacterial activity. As an example, the structure of GSK299423 (top), an early piperidine-linked NBTI, is shown. GSK000 (middle) displays enhanced activity against M. tuberculosis gyrase and is a founding member of the MGI class. Gepotidacin (bottom) is a first-in-class triazaacenaphthylene antibacterial. Phase III trials that assessed the treatment of urinary tract infections and uncomplicated urogenital gonorrhea with gepotidacin were successfully concluded with positive outcomes.^,

Figure 8

Spiropyrimidinetrione (SPT) structures. The SPT class derives its name from the spiropyrimidinetrione group (yellow) that forms critical contacts with gyrase and topoisomerase IV and is essential for antibacterial activity. Optimization of an early progenitor of this class, QPT-1 (left), led to the synthesis of zoliflodacin (middle). Phase III trials for the treatment of uncomplicated gonorrhea with zoliflodacin were successfully concluded with positive outcomes in late 2023.^, Current drug development efforts are focused on synthesizing related SPTs with high activity against M. tuberculosis such as H3D-005722 (right).

Figure 9

Structures of allosteric gyrase/topoisomerase inhibitors and poisons. Thiophenes (top, defined as compound 1 in Chan et al.) defined a class of “allosteric” poisons that bind outside of the gyrase/topoisomerase IV active site. Recently, new allosteric inhibitors, including a biphenyl-based allosteric inhibitor (bottom, designated as compound 2 in Orritt et al.) and the antibiotic evybactin (right), have been described that bind to the type II enzymes in the same pocket as members of the thiophene class.