Faculty Scholarship

Fluoroquinolones form drug-topoisomerase-DNA complexes that rapidly block transcription and replication. Crystallographic and biochemical studies showthat quinolone binding involves a water/metalion bridge between the quinolone C3-C4 keto-acid and amino acids in helix-4 of the target proteins, GyrA(gyrase) and ParC (topoisomerase IV). A recent cross-linking study revealed a second drug-binding modeinwhichtheotherendofthequinolone, the C7 ring system, interacts with GyrA. We report that addition of a dinitrophenyl (DNP) moiety to the C7 end of ciprofloxacin (Cip-DNP) reduced protection due to resistance substitutions in Escherichia coli GyrA helix-4, consistent with the existence of a second drug-binding mode not evident in X-ray structures of drug-topoisomerase-DNA complexes. Several other C7 aryl fluoroquinolones behaved in a similar manner with particular GyrA mutants. Treatment of E. coli cultures with Cip-DNP selectively enriched an uncommon variant, GyrA-A119E, a change that may impede binding of the dinitrophenyl group at or near the GyrA-GyrA interface. Collectively the data support the existence of a secondary quinolone-binding mode in which the quinolone C7 ring system interacts with GyrA; the data also identify C7 aryl derivatives as a new way to obtain fluoroquinolones that overcome existing GyrA-mediated quinolone resistance.

 DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have thefluoroquinolone C-7ring systemfacing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modifiedchloroacetylderivativeofciprofloxacin(CipAcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys466 gyrase as evidenced by resistance to reversal by both EDTAandthermaltreatments.Surprisingly,cross-linking was also readily seen with complexes formed by mutant GyrAG81Cgyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinoloneresistant GyrA-G81C variant of Escherichia coli and its Mycobacteriumsmegmatisequivalent(GyrA-G89C).Cip-AcClmediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinoloneC-7ringandbothGyrAandGyrB.BecausetheGyrAGly81andGyrB-Glu466residuesarefarapart(17Å)inthecrystal structureofcleavedcomplexes,twomodesofquinolonebinding mustexist. The presence of two binding modesraises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNAtopoisomerases.