Although the functional target of quinolone antibacterials such as nalidixic acid and norfloxacin has been identified as the enzyme DNA gyrase, the direct binding site of the drug is the DNA molecule [Shen, L. L., & Pernet, A. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 307-311]. As described in this paper, binding specificity and cooperativity of quinolones to DNA were further investigated with the use of a variety of DNA species of different structures and different base compositions. Results show that the drug binding specificity is controlled and determined largely by the DNA structure. The drug binds weakly and demonstrates no base preference when DNA strands are paired. The drug binds with much greater affinity when the strands are separated, and consequently, binding preference emerges: it binds better to poly(G) and poly(dG) over their counterparts including poly(dI). The results suggest that the drug binds to unpaired bases via hydrogen bonding and not via ring stacking with DNA bases. The weak binding to relaxed double-stranded DNA and the stronger binding to single-stranded DNA are both nonspecific as they do not demonstrate binding saturation and cooperativity. The specific type of binding, initially demonstrated in our previous publication with the supercoiled DNA and more recently with complex formed between linear DNA and DNA gyrase [Shen, L. L., Kohlbrenner, W. E., Weigl, D., & Baranowski, J. (1989) J. Biol. Chem. (in press)], occurs near the drug's supercoiling inhibition concentration. As shown in this paper, binding saturation curves of this type are highly cooperative (with Hill constant greater than 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Two novel series of 2-pyridones were synthesized by transposition of the nitrogen of 4-quinolones to the bridgehead position. This subtle interchange of the nitrogen atom with a carbon atom yielded two novel heterocyclic nuclei, pyrido[1,2-alpha]pyrimidine and quinolizine, which had not previously been evaluated as antibacterial agents and were found to be potent inhibitors of DNA gyrase. Quinolizines with a methyl group at the 9-position such as (S)-45a (ABT-719) demonstrate exceptional broad spectrum antibacterial activity. Most notably, they are active against resistant bacteria such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant strains of enterococci, and ciprofloxacin-resistant organisms. In addition, 2-pyridones also possess favorable physiochemical and pharmacokinetic properties. These 2-pyridones were synthesized from the commercially available starting materials by 10-17 linear transformations. The structure of an adduct yielded by this sequence, (S)-45a (ABT-719), was determined by X-ray crystallographic analysis.

(11,19,20,36,60DNA break to form a gate allowing subsequent DNA strand passing. Effective topoisomerase inhibitors stabilize the intermediate, termed the cleavage complex, and this stabilization in the cell triggers an unknown process causing cell death (7,30). The stabilization of the cleavage complex by topoisomerase inhibitors thus converts these enzymes into cellular poisons (28). The strategy of discovering therapeutic agents of this class is, therefore, plausible and easily justifiable because of the fact that the enzyme need not be proven to be essential to the viability of that particular cell. In principle, any cell which contains high levels of a topoisomerase is vulnerable to killing by topoisomerase inhibitors capable of stabilizing the complex.Candida albicans andAspeigilus niger are two important, life-threatening systemic fungal pathogens in individuals with impaired immunity, such as transplant recipients, patients with AIDS, and patients with cancer undergoing chemotherapy. More effective and safe antifungal agents are urgently needed for the treatment of these increasingly important opportunistic infections. Our results showed that both types of topoisomerases exist in abundance in these pathogenic fungi, indicating that the enzymes are potential targets for the discovery of fungicidal agents. By using isolated C. albicans DNA topoisomerase II and DNA breakage assays, we have further found that the responses of the fungal and the mammalian topoisomerase II to topoisomerase poisons differ in magnitude, suggesting that the discovery of specific inhibitors safe for the host is feasible. MATERIAL1 AND METHODSChemicals and reaents. Agarose (type II), Tris, EDTA, dimethyl sulfoxide, and amiloride were from Sigma. EcoRI

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