Fig 1.
Chemical structures of novel DNA gyrase inhibitors and reference compounds.
Fig 2.
Inhibition of macromolecule synthesis in E. coli.
(A) Dose-dependent, selective inhibition of DNA synthesis by MRL-770 in E. coli JL553. (B) Dose-dependent, selective inhibition of DNA synthesis by ciprofloxacin. (C) Schematic representation of the E. coli GyrB mutations conferring resistance to MRL-770 series compounds. For each mutant listed in the first column a black-filled cell identifies the mutation site and amino acid substitution (top row). GYR106-108 mutants were isolated following reselection for higher-level resistance to MRL-423. Red cells designate second site amino acid location and substitution acquired in the reselected mutants.
Table 1.
Antibacterial activity of novel gyrase inhibitors.
Table 2.
Antibacterial susceptibility of E. coli gyrB mutants.
Fig 3.
Dose-dependent inhibition of E. coli (A) and P. aeruginosa (B) DNA gyrase by MRL-423 and MRL-1082 respectively. (C) Dose-dependent stabilization of cleavage complex formation in E. coli DNA gyrase by MRL-423. Relaxed, closed circular substrate (rel.), linear (lin.), and super-coiled (sc.) DNA species are indicated to the left of each gel image.
Fig 4.
Time-dependent bactericidal growth inhibition by MRL-1082.
E. coli HS151 was treated with ciprofloxacin (MIC = 0.00195 μg/mL), novobiocin (MIC = 2 μg/mL) or MRL-1082 (MIC = 0.0625 μg/mL) at the indicated concentrations. The ciprofloxacin treated culture was below the lower limit of quantitation (LOQ = 20 CFU/mL) at the 4 hour time point and the 4-8XMIC MRL-1082-treated cultures were below the LOQ after 8 hours. Isolates from the 2XMIC MRL-1082 treated culture at 24 hours were not resistant to the compound (MIC = 0.0625 μg/mL) suggesting that MRL-1082 had either deteriorated or precipitated to a level below the MIC.
Fig 5.
E. coli DNA gyrase GyrB (F513L) is resistant to inhibition by MRL-1082.
(A) Agarose gel showing dose-dependent inhibition of wild-type E. coli DNA gyrase and resistance of DNA gyrase containing GyrB (F513L). (B) Quantitation of supercoiled product (sc). The 0.1 μM compound concentration served as the 100% control value.
Table 3.
Effect of a plasmid-based fluoroquinolone resistance determinant and gyrase regulatory factor YacG on activity of novel gyrase inhibitor.
Fig 6.
Mapping of the MRL-770/423/1082 resistance mutations onto a model of E. coli DNA gyrase (GyrBA fusion dimer) suggests a novel inhibitor interaction domain.
Amino acids in the GyrB domain (light blue) where MRL-770/MRL-423 resistant primary mutations reside are rendered in stick form. The GyrA domains of monomers 1 and 2 are colored in light green and green respectively, S83 and N87 of GyrA monomer 1 are shown in stick form. The two ciprofloxacin molecules are displayed in CPK and carbon atoms colored in yellow. Nicked DNA is shown in orange and Mn+2 ions are in purple.