Fluoroquinolone interactions with Mycobacterium tuberculosis gyrase: Enhancing drug activity against wild-type and resistant gyrase

Abstract

Mycobacterium tuberculosis is a significant source of global morbidity and mortality. Moxifloxacin and other fluoroquinolones are important therapeutic agents for the treatment of tuberculosis, particularly multidrug-resistant infections. To guide the development of new quinolone-based agents, it is critical to understand the basis of drug action against M. tuberculosis gyrase and how mutations in the enzyme cause resistance. Therefore, we characterized interactions of fluoroquinolones and related drugs with WT gyrase and enzymes carrying mutations at GyrA(A90) and GyrA(D94). M. tuberculosis gyrase lacks a conserved serine that anchors a water-metal ion bridge that is critical for quinolone interactions with other bacterial type II topoisomerases. Despite the fact that the serine is replaced by an alanine (i.e., GyrA(A90)) in M. tuberculosis gyrase, the bridge still forms and plays a functional role in mediating quinolone-gyrase interactions. Clinically relevant mutations at GyrA(A90) and GyrA(D94) cause quinolone resistance by disrupting the bridge-enzyme interaction, thereby decreasing drug affinity. Fluoroquinolone activity against WT and resistant enzymes is enhanced by the introduction of specific groups at the C7 and C8 positions. By dissecting fluoroquinolone-enzyme interactions, we determined that an 8-methyl-moxifloxacin derivative induces high levels of stable cleavage complexes with WT gyrase and two common resistant enzymes, GyrA(A90V) and GyrA(D94G). 8-Methyl-moxifloxacin was more potent than moxifloxacin against WT M. tuberculosis gyrase and displayed higher activity against the mutant enzymes than moxifloxacin did against WT gyrase. This chemical biology approach to defining drug-enzyme interactions has the potential to identify novel drugs with improved activity against tuberculosis.

Keywords: Mycobacterium tuberculosis; antibiotic resistance; complex stability; fluoroquinolones; gyrase.

Fig. 1.

Catalytic and DNA cleavage activities of WT and mutant M. tuberculosis gyrase in the absence of drugs. (Left) The ability of WT (black), GyrA^A90S (A90S, blue), GyrA^A90V (A90V, red), GyrA^D94H (D94H, green), and GyrA^D94G (D94G, yellow) to supercoil relaxed pBR322 plasmid DNA is shown. Gels are representative of three independent experiments. The positions of negatively supercoiled [(-)SC] and relaxed (Rel) DNA controls are indicated. (Right) The ability of the enzymes to cleave negatively supercoiled pBR322 plasmid DNA is shown. Error bars represent the SD of at least three independent experiments.

Fig. 2.

Drug-induced DNA cleavage mediated by WT and mutant M. tuberculosis gyrase. The ability of WT (black), GyrA^A90S (A90S, blue), GyrA^A90V (A90V, red), GyrA^D94H (D94H, green), and GyrA^D94G (D94G, yellow) to mediate DNA cleavage in the presence of the clinically used fluoroquinolones ciprofloxacin and moxifloxacin (Top Left and Top Right, respectively), the experimental quinazolinediones 8-methyl-3′-(AM)P-dione and 8-H-3′-(AM)P-dione (Middle Left and Bottom Left, respectively), and the experimental fluoroquinolones 8-methyl-3′-(AM)P-FQ and 8-H-3′-(AM)P-FQ (Middle Right and Bottom Right, respectively) is shown. Compound structures are shown in or above their respective panels. Error bars represent the SD of at least three independent experiments.

Fig. 3.

Effects of Mg²⁺ concentration on DNA cleavage mediated by WT and mutant M. tuberculosis gyrase. Results are shown for 10 µM ciprofloxacin (Left) and 10 µM 8-methyl-3′-(AM)P-dione (Right) with WT (black), GyrA^A90S (A90S, blue), and GyrA^A90V (A90V, red). DNA cleavage for each drug–enzyme pair was normalized to 100% at 6 mM Mg²⁺ to facilitate direct comparisons. Error bars represent the SD of at least three independent experiments.

Fig. 4.

Effects of Mn²⁺ on drug-induced DNA cleavage mediated by WT and mutant M. tuberculosis gyrase. Results are shown for cleavage mediated by WT (black), GyrA^A90S (A90S, blue), and GyrA^A90V (A90V, red) in the presence of ciprofloxacin. Assays included 2.5 mM Mn²⁺, the concentration that yielded maximal enzyme activity, instead of Mg²⁺. Error bars represent the SD of at least three independent experiments.

Fig. 5.

Ability of ciprofloxacin to compete out DNA cleavage induced by 10 µM 8-methyl-3′-(AM)P-dione with mutant M. tuberculosis gyrase. Results are shown for GyrA^A90V (A90V, red) and GyrA^D94G (D94G, yellow). Both drugs were added to reaction mixtures simultaneously. The low level of cleavage seen in the presence of ciprofloxacin alone was used as a baseline and was subtracted from DNA scission observed in the presence of both drugs. The level of DNA cleavage observed in the presence of the quinazolinedione alone was set to 1.0 to facilitate direct comparisons. Error bars represent the SD of at least three independent experiments.

Fig. 6.

Effects of ciprofloxacin- and moxifloxacin-based fluoroquinolones on DNA cleavage mediated by WT and mutant M. tuberculosis gyrase and human topoisomerase IIα. The ability of WT (Top Left), GyrA^A90V (A90V, Top Right), and GyrA^D94G (D94G, Bottom Left) gyrase and human topoisomerase IIα (hTIIα, Bottom Right) to cleave DNA in the presence of fluoroquinolones containing a C8-H (black), -methyl (blue), or -methoxy (red) group and a C7 piperazinyl (closed symbols) or diazabicyclononyl (open symbols) group is shown. For human topoisomerase IIα, DNA cleavage levels induced by the enzyme in the presence of CP-115,955 or etoposide (dashed lines) are shown for comparison. These latter data are from Aldred et al. (32). The ciprofloxacin and moxifloxacin fluoroquinolone cores are shown at the top. Error bars represent the SD of at least three independent experiments.

Fig. S1.

Effects of moxifloxacin (Top) and 8-methyl-moxi (Bottom) on DNA cleavage mediated by WT (Left) and GyrA^D94G mutant gyrase (Right). The positions of nicked (single-stranded breaks), linear (double-stranded breaks), and the negatively supercoiled (SC) substrate are indicated. Ethidium bromide-stained agarose gels are shown and are representative of three independent experiments.

Fig. 7.

Ability of fluoroquinolones with a C8-H to compete out DNA cleavage induced by 50 µM 8-methoxy-cipro or 8-methyl-moxi with mutant M. tuberculosis gyrase. Results are shown for GyrA^A90V (A90V, black) and GyrA^D94G (D94G, blue). Ciprofloxacin (Cipro, closed symbols) or 8-H-moxi (open symbols) and 8-methoxy-cipro (Left) or 8-methyl-moxi (Right) were added to reaction mixtures simultaneously. The low level of cleavage seen in the presence of the C8-H fluoroquinolones alone was used as a baseline and was subtracted from the DNA scission observed in the presence of both drugs. The level of DNA cleavage observed in the presence of 8-methoxy-cipro or 8-methyl-moxi alone was set to 1.0 to facilitate direct comparisons. Error bars represent the SD of at least three independent experiments.

Fig. S2.

Ability of 50 or 100 µM ciprofloxacin (Cipro) or 50 or 100 µM 8-H-moxi to compete out DNA cleavage induced by 50 µM 8-methoxy-cipro with mutant GyrA^D94G Mycobacterium tuberculosis gyrase. Cipro or 8-H-moxi and 8-methoxy-cipro were added to reaction mixtures simultaneously. The low level of cleavage seen in the presence of ciprofloxacin or 8-H-moxi alone is shown at right. Ethidium bromide-stained agarose gels are shown and are representative of three independent experiments.

Fig. 8.

Effects of fluoroquinolones on the persistence of cleavage complexes formed by WT and mutant M. tuberculosis gyrase. The stability of ternary enzyme–drug–DNA cleavage complexes formed with WT (Left), GyrA^A90V (A90V, Middle), and GyrA^D94G (D94G, Right) was determined in the presence of 100 µM ciprofloxacin (Cipro, black), 50 µM moxifloxacin (Moxi, blue), 50 µM 8-methoxy-cipro (red), or 10 µM 8-methyl-moxi (yellow). The data table (Inset, Middle) lists the t_1/2 of DNA cleavage complexes formed with each drug–enzyme combination. Initial DNA cleavage-religation reactions were allowed to come to equilibrium and were then diluted 20-fold with reaction buffer. Levels of DNA cleavage at time 0 were set to 100%. Error bars represent the SD of at least three independent experiments.