Antibacterial small molecules targeting the conserved TOPRIM domain of DNA gyrase

<div><p>To combat the threat of antibiotic-resistant Gram-negative bacteria, novel agents that circumvent established resistance mechanisms are urgently needed. Our approach was to focus first on identifying bioactive small molecules followed by chemical lead prioritization and target identification. Within this annotated library of bioactives, we identified a small molecule with activity against efflux-deficient <i>Escherichia coli</i> and other sensitized Gram-negatives. Further studies suggested that this compound inhibited DNA replication and selection for resistance identified mutations in a subunit of <i>E</i>. <i>coli</i> DNA gyrase, a type II topoisomerase. Our initial compound demonstrated weak inhibition of DNA gyrase activity while optimized compounds demonstrated significantly improved inhibition of <i>E</i>. <i>coli</i> and <i>Pseudomonas aeruginosa</i> DNA gyrase and caused cleaved complex stabilization, a hallmark of certain bactericidal DNA gyrase inhibitors. Amino acid substitutions conferring resistance to this new class of DNA gyrase inhibitors reside exclusively in the TOPRIM domain of GyrB and are not associated with resistance to the fluoroquinolones, suggesting a novel binding site for a gyrase inhibitor.</p></div

Time-dependent bactericidal growth inhibition by MRL-1082.

<p><i>E</i>. <i>coli</i> 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.</p

Chemical structures of novel DNA gyrase inhibitors and reference compounds.

<p>Chemical structures of novel DNA gyrase inhibitors and reference compounds.</p

Antibacterial small molecules targeting the conserved TOPRIM domain of DNA gyrase - Fig 3

<p><b>Dose-dependent inhibition of <i>E</i>. <i>coli</i> (A) and <i>P</i>. <i>aeruginosa</i> (B) DNA gyrase by MRL-423 and MRL-1082 respectively</b>. (C) Dose-dependent stabilization of cleavage complex formation in <i>E</i>. <i>coli</i> 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.</p

Mapping of the MRL-770/423/1082 resistance mutations onto a model of <i>E</i>. <i>coli</i> DNA gyrase (GyrBA fusion dimer) suggests a novel inhibitor interaction domain.

<p>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.</p

E. coli DNA gyrase GyrB (F513L) is resistant to inhibition by MRL-1082.

<p>(A) Agarose gel showing dose-dependent inhibition of wild-type <i>E</i>. <i>coli</i> 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.</p

Antibacterial susceptibility of <i>E</i>. <i>coli gyrB</i> mutants.

<p>Antibacterial susceptibility of <i>E</i>. <i>coli gyrB</i> mutants.</p

Inhibition of macromolecule synthesis in <i>E</i>. <i>coli</i>.

<p>(A) Dose-dependent, selective inhibition of DNA synthesis by MRL-770 in <i>E</i>. <i>coli</i> JL553. (B) Dose-dependent, selective inhibition of DNA synthesis by ciprofloxacin. (C) Schematic representation of the <i>E</i>. <i>coli</i> 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.</p