F1-2022. Dual Targeting of Gyrase and Topoisomerase IV by ACH-702 and Mutation Analysis in Staphylococcus aureus
Session: Poster Session: New Topoisomerase Inhibitors
Monday, October 27, 2008: 12:00 AM
Room: Hall C
Background: ACH-702 is an isothiazoloquinolone (ITQ) with potent antibacterial activity against Staphylococcus aureus. Selection of resistant mutants and the target genes involved were investigated. Methods: Standard in vitro methods were used to analyze resistance and antibacterial mechanisms of action. These included isolation of mutants on solid medium containing compound and biochemical enzyme assays to calculate enzyme inhibition. Results: First-step mutants of S. aureus ATCC 29213 (parent MIC = 0.004 µg/ml) were present exclusively in the DNA gyrase gene, gyrA, but not in the topoisomerase IV gene, grlA, with low frequencies of resistance (10-9 - 10-10). Low MIC values, ≤0.016 μg/ml, against mutants with single mutations in either gyrA or grlA suggested that ITQs possessed significant inhibitory activities against both target enzymes. This dual target inhibition was supported by low μM IC50 values against both topoisomerase IV (~1 μM), measured in a decatenation activity assay, and DNA gyrase (~1-10 μM), measured in a supercoiling activity assay. Retention of good MICs (≤1 μg/ml) against staphylococcal gyrA-grlA double mutants, as well as low frequencies of higher-level resistance (10-9 - 10-10), indicated that ITQs remained active against both mutant enzymes. This was supported by demonstration of in vitro inhibition of both mutant enzymes with IC50 values about 10-fold higher than against wild type enzyme. Macromolecular synthesis inhibition assays confirmed that ACH-702 inhibits DNA synthesis in S. aureus. Conclusions: Isothiazoloquinolones display good antibacterial activities against both laboratory mutants and clinical MRSA strains with multiple mutations in topoisomerases. Low spontaneous resistance frequencies and an apparent dual-targeting mode of action with exceptional activity against DNA gyrase suggest the potential utility of ITQs in combating infections caused by S. aureus, including multi-drug resistant MRSA.
Barton Bradbury, PhD1, Jane Thanassi1, Jijun Cheng, PhD1, Michael Pucci, PhD2, Milind Deshpande, PhD1, Steven Podos, PhD2 and  J. A. Thanassi,
Achillion Pharmaceuticals Role(s): Employee, Received: Salary., (1)Achillion Pharmaceuticals, (2)Achillion Pharmaceuticals, New Haven, CT

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