Bacterial resistance is eroding the clinical utility of existing antibiotics necessitating the discovery of new agents. Bacterial type II topoisomerase is a clinically validated, highly effective, and proven drug target. This target is amenable to inhibition by diverse classes of inhibitors with alternative and distinct binding sites to quinolone antibiotics, thus enabling the development of agents that lack cross-resistance to quinolones. Described here are novel bacterial topoisomerase inhibitors (NBTIs), which are a new class of gyrase and topo IV inhibitors and consist of three distinct structural moieties. The substitution of the linker moiety led to discovery of potent broad-spectrum NBTIs with reduced off-target activity (hERG IC 50 > 18 μM) and improved physical properties. AM8191 is bactericidal and selectively inhibits DNA synthesis and Staphylococcus aureus gyrase (IC 50 = 1.02 μM) and topo IV (IC 50 = 10.4 μM). AM8191 showed parenteral and oral efficacy (ED 50 ) at less than 2.5 mg/kg doses in a S. aureus murine infection model. A cocrystal structure of AM8191 bound to S. aureus DNA-gyrase showed binding interactions similar to that reported for GSK299423, displaying a key contact of Asp83 with the basic amine at position-7 of the linker.

The antibacterial activities and target inhibition of 15 quinolones against grlA and gyrA mutant strains were studied. The strains were obtained from wild-type Staphylococcus aureus MS5935 by selection with norfloxacin and nadifloxacin, respectively. The antibacterial activities of most quinolones against both mutant strains were lower than those against the wild-type strain. The ratios of MICs for the gyrA mutant strain to those for the grlA mutant strain (MIC ratio) varied from 0.125 to 4. The ratios of 50% inhibitory concentrations (IC 50 s) of quinolones against topoisomerase IV to those against DNA gyrase (IC 50 ratios) also varied, from 0.177 to 5.52. A significant correlation between the MIC ratios and the IC 50 ratios was observed (r ‫؍‬ 0.919; P < 0.001). These results suggest that the antibacterial activities of quinolones against the wild-type strain are involved not only in topoisomerase IV inhibition but also in DNA gyrase inhibition and that the target preference in the wild-type strain can be anticipated by the MIC ratios. Based on the MIC ratios, the quinolones were classified into three categories. Type I quinolones (norfloxacin, enoxacin, fleroxacin, ciprofloxacin, lomefloxacin, trovafloxacin, grepafloxacin, ofloxacin, and levofloxacin) had MIC ratios of <1, type II quinolones (sparfloxacin and nadifloxacin) had MIC ratios of >1, and type III quinolones (gatifloxacin, pazufloxacin, moxifloxacin, and clinafloxacin) had MIC ratios of 1. Type I and type II quinolones seem to prefer topoisomerase IV and DNA gyrase, respectively. Type III quinolones seem to target both enzymes at nearly the same level in bacterial cells (a phenomenon known as the dual-targeting property), and their IC 50 ratios were approximately 2.Quinolone antibacterial agents have potent activities against gram-positive and -negative bacteria, and they are currently used for the therapeutic treatment of various bacterial infections. Antibacterial activities of quinolones are involved in their inhibitory activities for DNA gyrase and topoisomerase IV (1,6,14). Both enzymes are members of the type II topoisomerase family that controls bacterial DNA topology by passing a DNA double helix through another, using a transient double-strand break. DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA and is involved in DNA replication, recombination, and transcription. Topoisomerase IV is also involved in supporting DNA replication, and the primary function of this enzyme seems to be the decatenation of multiply linked daughter chromosomes during the terminal stages of DNA replication.It has been proposed that the susceptibility of bacteria to quinolones is determined primarily by which one of the two target enzymes is more sensitive to quinolones (4,6,19). It has been reported that the primary target of many quinolones seems to be topoisomerase IV in Staphylococcus aureus (2-4, 6, 9, 19) and that the primary target in Streptococcus pneumoniae varies among the quinolones (5, 12, 13). Recently, some quinolones were reported to...

Bacterial resistance to antibiotics continues to pose serious challenges as the discovery rate for new antibiotics fades. Kibdelomycin is one of the rare, novel, natural product antibiotics discovered recently that inhibits the bacterial DNA synthesis enzymes gyrase and topoisomerase IV. It is a broad-spectrum, Gram-positive antibiotic without cross-resistance to known gyrase inhibitors, including clinically effective quinolones. To understand its mechanism of action, binding mode, and lack of cross-resistance, we have co-crystallized kibdelomycin and novobiocin with the N-terminal domains of Staphylococcus aureus gyrase B (24 kDa) and topo IV (ParE, 24 and 43 kDa). Kibdelomycin shows a unique "dual-arm", U-shaped binding mode in both crystal structures. The pyrrolamide moiety in the lower part of kibdelomycin penetrates deeply into the ATP-binding site pocket, whereas the isopropyl-tetramic acid and sugar moiety of the upper part thoroughly engage in polar interactions with a surface patch of the protein. The isoproramic acid (1,3-dioxopyrrolidine) and a tetrahydropyran acetate group (Sugar A) make polar contact with a surface area consisting of helix α4 and the flexible loop connecting helices α3 and α4. The two arms are connected together by a rigid decalin linker that makes van del Waals contacts with the protein backbone. This "dual-arm", U-shaped, multicontact binding mode of kibdelomycin is unique and distinctively different from binding modes of other known gyrase inhibitors (e.g., coumarins and quinolones), which explains its lack of cross-resistance and low frequency of resistance. The crystal structures reported in this paper should enable design and discovery of analogues with better properties and antibacterial spectrum.

Gatifloxacin (8-methoxy, 7-piperazinyl-3-methyl) at the MIC selected mutant strains that possessed gyrA mutations at a low frequency (3.7 ؋ 10 ؊9 ) from wild-type strain Streptococcus pneumoniae IID553. AM-1147 (8-methoxy, 7-piperazinyl-3-H) at the MIC or higher concentrations selected no mutant strains. On the other hand, the respective 8-H counterparts of these two compounds, AM-1121 (8-H, 7-piperazinyl-3-methyl) and ciprofloxacin (8-H, 7-piperazinyl-3-H), at one and two times the MIC selected mutant strains that possessed parC mutations at a high frequency (>2.4 ؋ 10 ؊6 ). The MIC of AM-1147 increased for the gyrA mutant strains but not for the parC mutant strains compared with that for the wild-type strain. These results suggest that fluoroquinolones that harbor 8-methoxy groups select mutant strains less frequently and prefer DNA gyrase, as distinct from their 8-H counterparts. The in vitro activities of gatifloxacin and AM-1147 are twofold higher against the wild-type strain, eight-and twofold higher against the first-step parC and gyrA mutant strains, respectively, and two-to eightfold higher against the second-step gyrA and parC double mutant strains than those of their 8-H counterparts. These results indicate that the 8-methoxy group contributes to enhancement of antibacterial activity against target-altered mutant strains as well as the wild-type strain. It is hypothesized that the 8-methoxy group of gatifloxacin increases the level of target inhibition, especially against DNA gyrase, so that it is nearly the same as that for topoisomerase IV inhibition in the bacterial cell, leading to potent antibacterial activity and a low level of resistance selectivity.

bThe mechanism of quinolone resistance in Mycoplasma genitalium remains poorly understood due to difficulties with in vitro culture, especially of clinical isolates. In this study, to confirm the association between mutations in topoisomerases and antimicrobial susceptibilities to quinolones, ciprofloxacin-resistant mutant strains were selected using the cultivable type strain ATCC 33530. Sequence analysis revealed that the mutant strains harbored mutations in topoisomerase IV: Gly81Cys in ParC, Pro261Thr in ParC, or Asn466Lys in ParE. The MICs of all quinolones tested against the mutant strains were 2-to 16-fold higher than those against the wild-type strain. No cross-resistance was observed with macrolides or tetracyclines. We determined the inhibitory activities of quinolones against DNA gyrase and topoisomerase IV in order to investigate the correlation between antimicrobial susceptibility and inhibitory activity against the target enzymes, considered the primary targets of quinolones. Furthermore, using enzymatic analysis, we confirmed that Gly81Cys in the ParC quinolone resistance-determining region (QRDR) contributed to quinolone resistance. This is the first study to isolate quinolone-resistant mutant strains of M. genitalium harboring substitutions in the parC or parE gene in vitro and to measure the inhibitory activities against the purified topoisomerases of M. genitalium. Mycoplasma genitalium was first isolated in urethral cultures from men with nongonococcal urethritis (NGU) in 1981 (1). M. genitalium is an important cause of NGU in men and has been shown to be associated with cervicitis, endometritis, salpingitis, and pelvic inflammatory diseases in women (2-5).M. genitalium has been reported to be susceptible to macrolides and is highly susceptible to azithromycin. The current guidelines recommend an azithromycin regimen for the treatment of NGU (6, 7). On the other hand, macrolide-resistant clinical strains of M. genitalium have emerged due to selection by the azithromycin regimen, and these clinical isolates with elevated azithromycin MICs have been shown to harbor an A2058G or A2059G substitution in the 23S rRNA gene (8).Some quinolones, such as moxifloxacin, have potent activity against M. genitalium, and treatment with moxifloxacin is considered an effective second-line treatment for persistent or recurrent NGU (9). The antibacterial activities of quinolones are due to their inhibitory activities against type II topoisomerases, DNA gyrase (composed of two GyrA and two GyrB subunits), and topoisomerase IV (composed of two ParC and two ParE subunits). It has been reported that mutations in the quinolone resistance-determining regions (QRDR) of the genes encoding DNA gyrase and/or topoisomerase IV contribute to quinolone resistance in various bacterial species, including other mycoplasmas (10-13). We recently detected the amino acid substitution Ser83Asn, Asp87Tyr, or Asp87Val in the ParC QRDR of M. genitalium DNAs in urine specimens from men with NGU (14-16).Due to the difficulties of isolatin...