1997;388:903C906. reported. INTRODUCTION The quinolones are broad-spectrum antibacterial agents that are receiving increasing attention as resistance develops to other compounds. Unfortunately, the quinolones are also losing their utility due to bacterial resistance, which creates a sense of urgency to develop new, more effective derivatives. As a result, biochemical insights continue to emerge, and we can now begin to discuss crystal structures of drug-target-DNA complexes. Our understanding of intracellular quinolone action is also deepening. For example, evidence is accumulating that lethal action is due to chromosome fragmentation and the resulting surge in reactive oxygen species (ROS). While finding new quinolone derivatives has continued along conventional lines that seek low MIC, that effort is expanding to include identification of compounds having good activity with mutants resistant to existing compounds. We expect studies with fluoroquinolone resistance to eventually lead regulatory agencies to add anti-mutant Ciproxifan maleate properties to the evaluation of new compounds. These and other developments make an update of quinolone action and resistance timely. We use the term quinolone in a generic sense that refers loosely to a class of inhibitors that includes naphthyridones, quinolones, quinazolines, isothiazoloquinolones, and related agents. These compounds have as their targets two essential bacterial enzymes, DNA gyrase (topoisomerase II) [1] and DNA topoisomerase IV [2]. The two enzymes, each of which contains 4 subunits (2 GyrA or ParC and 2 GyrB or ParE), act by passing one region of duplex DNA through another [3-6]; during that process, the quinolones form Rabbit polyclonal to AMIGO2 complexes with enzyme and DNA [1, 7]. The DNA moiety in the complex is broken, as revealed by detection of fragmented DNA following addition of protease, ionic detergent (sodium dodecyl sulfate, SDS), or both to quinolone-containing reaction mixtures or lysates from quinolone-treated bacterial cells [1, 7, 8]. The complexes are called cleaved or cleavable to indicate the presence of broken DNA that is covalently attached to the enzyme at the 5 ends. Chromosomal DNA remains supercoiled when obtained from cells treated with quinolones at bacteriostatic concentrations, provided that the complexes are kept intact by omission of protein denaturants from cell lysis procedures [8]. The presence of supercoils indicates that the DNA breaks in the complexes are constrained in a way that prevents the rotation of DNA ends that would otherwise relax supercoils. However, when cells are treated with lethal drug concentrations, the supercoils are absent, indicating release of the DNA ends from the complexes. That release is expected to fragment chromosomes. The hallmark of quinolone action is formation of cleaved complexes. and [20]. It also underlies use of the mutant selection window hypothesis as a framework for suppressing the emergence of resistance (the hypothesis maintains that resistant mutant subpopulations are selectively enriched and amplified when drug concentrations fall in a range above the MIC for the susceptible population and below the MIC of the least susceptible mutant subpopulation, a value called the MPC). The selection window can be used to formulate dosing regimens, to choose compounds for therapy, Ciproxifan maleate and to design new agents. Below we turn first to biochemical studies of cleaved complex formation. Knowledge gained from crystal structures is moving us toward an atomic description of the complexes, with current data appearing to require a two-step model. An underlying assumption of structural studies is that the type II topoisomerases have very similar structures; consequently, conclusions drawn with one enzyme are often applied to others. While this assumption is generally sound, the enzymes differ; in the second section we discuss the C-terminal domains of the GyrA and ParC proteins, regions where major differences between gyrase and topoisomerase IV appear. We.Biochim. observed with static agar plate assays, dynamic in vitro systems, and experimental infection of rabbits. The gap between MIC and MPC can be narrowed by compound design that should restrict the emergence of resistance. Resistance is likely to become increasingly important, since three types of plasmid-borne resistance have been reported. INTRODUCTION The quinolones are broad-spectrum antibacterial agents that are receiving increasing attention as resistance develops to other compounds. Unfortunately, the quinolones are also losing their utility due to bacterial resistance, which creates a sense of urgency to develop new, more Ciproxifan maleate effective derivatives. As a result, biochemical insights continue to emerge, and we can now begin to discuss crystal structures of drug-target-DNA complexes. Our understanding of intracellular quinolone action is also deepening. For example, evidence is accumulating that lethal action is due to chromosome fragmentation and the resulting surge in reactive oxygen species (ROS). While finding new quinolone derivatives has continued along conventional lines that seek low MIC, that effort is expanding to include identification of compounds having good activity with mutants resistant to existing compounds. We expect studies with fluoroquinolone resistance to eventually lead regulatory agencies to add anti-mutant properties to the evaluation of new compounds. These and other developments make an update of quinolone action and resistance timely. We use the term quinolone in a generic sense that refers loosely to a class of inhibitors that includes naphthyridones, quinolones, quinazolines, isothiazoloquinolones, and related agents. These compounds have as their targets two essential bacterial enzymes, DNA gyrase (topoisomerase II) [1] and DNA topoisomerase IV [2]. The two enzymes, each of which contains 4 subunits (2 GyrA or ParC and 2 GyrB or ParE), act by passing one region of duplex DNA through another [3-6]; during that process, the quinolones form complexes with enzyme and DNA [1, 7]. The DNA moiety in the complex is broken, as revealed by detection of fragmented DNA following addition of protease, ionic detergent (sodium dodecyl sulfate, SDS), or both to quinolone-containing reaction mixtures or lysates from quinolone-treated bacterial cells [1, 7, 8]. The complexes are called cleaved or cleavable to indicate the presence of broken DNA that is covalently attached to the enzyme at the 5 ends. Chromosomal DNA remains supercoiled when obtained from cells treated with quinolones at bacteriostatic concentrations, provided that the complexes are kept intact by omission of protein denaturants from cell lysis procedures [8]. The presence of supercoils indicates that the DNA breaks in the complexes are Ciproxifan maleate constrained in a way that Ciproxifan maleate prevents the rotation of DNA ends that would otherwise relax supercoils. However, when cells are treated with lethal drug concentrations, the supercoils are absent, indicating release of the DNA ends from the complexes. That release is expected to fragment chromosomes. The hallmark of quinolone action is formation of cleaved complexes. and [20]. It also underlies use of the mutant selection window hypothesis as a framework for suppressing the emergence of resistance (the hypothesis maintains that resistant mutant subpopulations are selectively enriched and amplified when drug concentrations fall in a range above the MIC for the susceptible population and below the MIC of the least susceptible mutant subpopulation, a value called the MPC). The selection window can be used to formulate dosing regimens, to choose compounds for therapy, and to design new agents. Below we turn first to biochemical studies of cleaved complex formation. Knowledge gained from crystal structures is moving us toward an atomic description of the complexes, with current data appearing to need a two-step model. An root assumption of structural research is that the sort II topoisomerases possess very similar buildings; therefore, conclusions drawn with one enzyme tend to be put on others. While this assumption is normally audio, the enzymes differ; in the next section we discuss the C-terminal domains from the GyrA and ParC protein, regions where main distinctions between gyrase and topoisomerase IV show up. We then change to biological implications of cleaved complicated development: inhibition of DNA replication, chromo-some fragmentation, and.