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Antimicrob. Agents Chemother. doi:10.1128/AAC.00158-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Heteroaryl Isothiazolones in Staphylococcus aureus

Achillion Pharmaceuticals, New Haven, CT 06511

^* To whom correspondence should be addressed. Email: mpucci{at}achillion.com.

	Abstract

Heteroaryl isothiazolones (HITZs) are antibacterial agents that display excellent in vitro activity against Staphylococcus aureus. We recently identified a series of these compounds that show potent bactericidal activities against methicillin-resistant S. aureus (MRSA). We report here the results of in vitro resistance studies that reveal potential underlying mechanisms of action. HITZs selected gyrA mutations exclusively in first-step mutants of wild type S. aureus indicating that, in contrast to most quinolones, DNA gyrase is the primary target. The compounds displayed low mutation frequencies (10^-9 - 10^-10) at concentrations close to the MICs and maintained low MICs ( 0.016 µg/ml) against mutants with single mutations in either gyrA or grlA(parC). These data suggested that HITZs possess significant inhibitory activities against target enzymes, DNA gyrase and topoisomerase IV. This dual target inhibition was supported by low IC₅₀ values against topoisomerase IV as measured in a decatenation activity assay and against DNA gyrase as measured in a supercoiling activity assay. Good antibacterial activities ( 1 µg/ml) against staphylococcal gyrA-grlA double mutants as well as low frequencies (10^-9 - 10^-10) of selection of still higher-level mutants, also suggested that HITZs remained active against mutant enzymes. We further demonstrated that HITZs exhibit good inhibition of both S. aureus mutant enzymes and, thus, continue to possess a novel dual-targeting mode of action against these mutant strains. In step-wise acquisition of mutations, HITZs selected Quinolone Resistance Determining Region (QRDR) mutations gyrA Ser84Leu, grlA Ser80Phe, grlA Ala116Val and gyrA Glu88Lys sequentially, suggesting that these are key amino acids involved in the binding of HITZs to topoisomerases. The overall profile of these compounds suggests the potential utility of HITZs in combating infections caused by S. aureus, including multi-drug resistant MRSA.