Grepafloxacin, a Dimethyl Derivative of Ciprofloxacin, Acts Preferentially through Gyrase in Streptococcus pneumoniae : Role of the C-5 Group in Target Specificity

Abstract

Grepafloxacin, a 5-methyl-7-piperazinyl-3-methyl analogue of ciprofloxacin, was used to obtain stepwiseselected mutants of Streptococcus pneumoniae 7785. Analysis of the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes in these mutants revealed that gyrA mutations preceded those in parC. Given that ciprofloxacin (5-H,7-piperazinyl) and AM-1121 (5-H,7-piperazinyl-3-methyl) both act through topoisomerase IV, we conclude that the 5-methyl group of grepafloxacin favors gyrase in S. pneumoniae.In recent years, fluoroquinolones such as levofloxacin, grepafloxacin, gatifloxacin, moxifloxacin, and sparfloxacin have been introduced for the treatment of pneumonia and other diseases due to Streptococcus pneumoniae, a major gram-positive pathogen (2, 26). By interfering with DNA gyrase and topoisomerase IV, two essential enzymes that function through a double-stranded DNA break (3,7,8,13,31), these agents are also effective against S. pneumoniae isolates that exhibit decreased susceptibility to penicillins and macrolides (29).The precise mode of action of a quinolone against S. pneumoniae depends on its molecular structure (20,23). One group of quinolones, whose archetype is ciprofloxacin and which includes levofloxacin, norfloxacin, pefloxacin, and trovafloxacin, selects first-step mutants altered in the quinolone resistancedetermining region (QRDR) of parC or parE, indicating that topoisomerase IV is the intracellular target (5,9,11,16,18,24,28). A second group includes sparfloxacin and NSFQ-105 and selects first-step gyrA QRDR mutants, implying that gyrase is the preferred target (1,5,20,24). Clinafloxacin, gatifloxacin, gemifloxacin, and moxifloxacin select gyrA mutants at a rather low frequency (6, 10, 21) and seem to act through both enzymes, so they are called dual-targeting agents. Efforts to understand drug targeting have indicated a role for the C-7 and C-8 substituents (1, 6), but little is known about the effects of C-5 substitution. We realized that grepafloxacin, a simple 5-methyl-7-piperazinyl-3Ј-methyl derivative of ciprofloxacin (Fig. 1), could be informative. Previous work has shown that gyrA or parC mutations in S. pneumoniae raised the MIC of grepafloxacin some two-to fourfold, but no definite target assignment was made (30). Here we examine grepafloxacin action in S. pneumoniae by using defined mutants, by characterizing stepwise-selected mutants, and by using recombinant enzymes.(Part of this study was presented at the 39th InterscienceConference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., 26 to 29 September 1999.) Approximately 5 ϫ 10 9 CFU of susceptible isolate 7785 (18) was spread on brain heart infusion plates containing 10% horse blood and grepafloxacin at concentrations of 0.5 and 1 g/ml, i.e., one and two times the MIC for the parent strain (Fig. 2). Mutants appeared after 48 h of aerobic incubation at 37°C. Mutants 1G1 to 1G15 and 1G16 to 1G25 from the two independent drug challenges were studied further. Second-step mutants 2G1 to 2G12 were...

Mycobacterium tuberculosis DNA Gyrase: Interaction with Quinolones and Correlation with Antimycobacterial Drug Activity

“…Most of the classical fluoroquinolones developed for their activity against gram-negative bacteria (norfloxacin, pefloxacin, enoxacin, fleroxacin, ofloxacin, temafloxacin, and tosufloxacin) had moderate IC 50 s except for levofloxacin and ciprofloxacin, which had low IC 50 s against M. tuberculosis gyrase. Contrary to its effects against pneumococci, the presence of a group at C-5 (27) or a substituent in the 7-piperazinyl ring (1) does not seem to improve gyrase affinity. Moreover, the presence of a naphthyridone core (N-8) in gemifloxacin, which has the lowest MIC against gram-positive bacteria, seems unfavorable for a tight interaction with M. tuberculosis gyrase.…”

Topoisomerase Targeting with and Resistance to Gemifloxacin in Staphylococcus aureus

“…Stepwise accumulation of chromosomal mutations leads to quinolone resistance, with the first mutation usually occurring in the primary or more sensitive enzyme target (23). Quinolone structure affects the target preference of quinolones (1,34). In Staphylococcus aureus, topoisomerase IV has been shown to be the primary target for most quinolones, although recently nadifloxacin and sparfloxacin have been suggested to target gyrase primarily (46), and garenoxacin (BMS-284756) (26) and other nonfluorinated quinolones (41) appear to target both enzymes similarly.…”

Reactive metabolite of trovafloxacin activates inflammasomes: Implications for trovafloxacin‐induced liver injury

“…It is known to have a minimal potential for causing hepatic injury (Adikwu & Deo, 2012; Fish & North, 2001). Grepafloxacin, which has a structure identical to that of ciprofloxacin except for the addition of a methyl group at C‐5 and at the C‐3 position of the 7‐piperazinyl ring, had been shown to have high levels of bactericidal activity in the treatment of pneumococcal meningitis (Morris et al, 2002; Pfister et al, 2003). However, although grepafloxacin was withdrawn globally from use in 1999 due to severe cardiovascular events (Mandell & Tillotson, 2002), it had not been reported to cause other side effects, especially in the liver.…”