Recent Developments in the Field of Quinolone Antibacterial Agents

Recently, a number of novel quinolones with potent activity against topoisomerase H and eukaryotic cells have been described. Many of these compounds contain aromatic substituents in their C-7 ring positions. To determine whether pyrimido[1,6-albenzimidazoles, a class of drugs modeled on quinolones, also display activity toward eukaryotic systems, the effects of Ro 46-7864 and Ro 47-3359 on Drosophila melanogaster topoisomerase II and Kc cells were characterized. While the former drug contains an aliphatic group (4-N-methylpiperazine) at the ring position equivalent to C-7 in quinolones, the latter compound contains an aromatic substituent (2,6-dimethylpyridine). Both pyrimido [1,6-

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A pyrimido[1,6-a]benzimidazole that enhances DNA cleavage mediated by eukaryotic topoisomerase II: a novel class of topoisomerase II-targeted drugs with cytotoxic potential

Corbett

Guerry

Pflieger

et al. 1993

“…1), one of the most potent members of this drug family currently in clinical use, is tricyclic in nature (3,38). Unlike its bicyclic counterparts, the methyl-substituted 1-8 oxazine bridge of ofloxacin possesses a chiral center.…”

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Novel 1-8-bridged chiral quinolones with activity against topoisomerase II: stereospecificity of the eukaryotic enzyme

Froelich-Ammon

McGuirk

Gootz

et al. 1993

A series of novel C-7 quinolyl-substituted enantiomers of ofloxacin were used to determine the stereospecificity of topoisomerase II for the C-il methyl group in tricyclic quinolones. In all cases, the S isomer was the most active compound against the eukaryotic enzyme. It was -2.2-fold more potent than the R isomer at inhibiting the overall catalytic activity of topoisomerase II (as monitored by DNA relaxation assays). A markedly greater difference in quinolone activity was observed in enzyme-mediated DNA cleavage reactions.While the S enantiomer stimulated nucleic acid breakage -3.5-fold, the R compound did not enhance and, in fact, decreased initial DNA cleavage levels by -50%o. The activity of the racemic mixture more closely resembled that of the R enantiomer. In competition experiments, the DNA cleavage-enhancing effects of the S isomer were attenuated by the R compound. Taken together, these latter results indicate that the R enantiomer is an antagonist of S isomer-promoted topoisomerase II-mediated DNA cleavage. Finally, the cytotoxic potential of quinolyl-substituted ofloxacin analogs correlated with the ability to stimulate enzyme-mediated DNA cleavage. Thus, stereochemistry appears to be a governing factor for the potential development of tricyclic quinolones as topoisomerase II-targeted drugs with antineoplastic activity.Quinolone-based drugs are a widely used class of oral antibiotics (11,37,42). The primary physiological target of these important pharmacological agents is DNA gyrase, a prokaryotic type II topoisomerase (11,27,36,42). Quinolones cause bacterial death by converting DNA gyrase into a cellular poison (18). This is accomplished by stabilizing covalent enzyme-cleaved DNA complexes (6,11,35) which are naturally occurring intermediates in the catalytic cycle of all type II topoisomerases (26,27).While most medically relevant quinolones have a bicyclic parent ring structure, ofloxacin (see Fig. 1), one of the most potent members of this drug family currently in clinical use, is tricyclic in nature (3,38). Unlike its bicyclic counterparts, the methyl-substituted 1-8 oxazine bridge of ofloxacin possesses a chiral center. This center, which is located at the C-il position, is indicated in Fig. 1. Although the clinical formulation of ofloxacin is a racemic mixture, enantiomerically pure R and S stereoisomers have been characterized (5,7,10,12,15,21). In all cases, the S isomer was substantially more active (.10-fold) than its R antipode at inhibiting the DNA supercoiling reaction of gyrase or preventing the growth of bacterial cells. The activity of the racemic ofloxacin generally was one-half that of the pure S isomer (5,7,10,12,15,21). Thus, it appears that the R enantiomer interacts poorly (at best) with DNA gyrase.The effects of ofloxacin enantiomers on the catalytic activity of eukaryotic topoisomerase II also have been examined (12). As determined by the inhibition of enzymecatalyzed DNA relaxation, an approximately twofold stereospecificity for the S configuration over the R configuratio...

“…The quinolones, a major group of DNA gyrase inhibitors, interrupt the DNA gyrase-mediated DNA cleavage and resealing cycle at the cleavage step (2,3,23). Addition of a protein denaturant, such as sodium dodecyl sulfate (SDS), to reaction mixtures containing linear, relaxed, or supercoiled DNA in the presence of quinolones releases double-stranded cleaved DNA (26,27).…”

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Effect of pyrimido[1,6-a]benzimidazoles, quinolones, and Ca2+ on the DNA gyrase-mediated cleavage reaction

Gmünder

Kuratli

Keck

1995

. Pyrimido[1,6-a]benzimidazoles have been found to represent a new class of potent DNA gyrase inhibitors which also act at the A subunit. To determine alterations in the DNA sequence specificity of DNA gyrase for cleavage sites in the presence of inhibitors of both classes or in the presence of Ca 2؉ , we used DNA restriction fragments of 164, 85, and 71 bp from the pBR322 plasmid as model substrates. Each contained, at a different position, the 20-bp pBR322 sequence around position 990, where DNA gyrase preferentially cleaves in the presence of quinolones. Our results show that pyrimido[1,6-a]benzimidazoles have a mode of action similar to that of quinolones; they inhibit the resealing step and influence the DNA sequence specificity of DNA gyrase in the same way. Differences between inhibitors of both classes could be observed only in the preferences of DNA gyrase for these cleavage sites. The 20-bp sequence appeared to have some properties that induced DNA gyrase to cleave all three DNA fragments in the presence of inhibitors within this sequence, whereas cleavage in the presence of Ca 2؉ was in addition dependent on the length of the DNA fragments.The prokaryotic DNA gyrase (EC 5.99.1.3) belongs to the class of topoisomerase II proteins and is able to catalyze the ATP-dependent negative supercoiling of double-stranded closed circular DNA (23). The active enzyme is a tetramer consisting of two A subunits and two B subunits. It is thought that in the initial stage of the supercoiling process the enzyme binds to double-stranded DNA and that about 120 bp is wrapped around the tetrameric protein in a single positive supercoil. There have been several proposals for the existence of specific sites for the interaction of DNA gyrase with DNA. In Escherichia coli and Salmonella typhimurium, there is a family of repetitive extragenic palindromic sequences, and there is evidence that DNA gyrase binds preferentially to these sites (30). It has been proposed that the par locus in the pSC101 plasmid represents a DNA gyrase binding site (29), and in bacteriophage Mu a sequence that is required for efficient replicative transposition is proposed as a strong DNA gyrase binding site (20). After binding, DNA gyrase cleaves each strand at sites separated by 4 bp and transiently forms a covalent phosphotyrosine bond between the 5Ј-phosphate groups of the cleaved DNA and Tyr-122 of the A subunits (8, 28). A segment of DNA is translocated through the break and presumably through at least part of the protein complex. There are data indicating that DNA gyrase binding to DNA is sufficiently stable to allow processive supercoiling before the enzyme dissociates from the DNA (16). At some point in the reaction, an ATP molecule binds to each B subunit and is hydrolyzed. Binding of ATP promotes a conformational change of the tetramer, and it is thought that this change brings the DNA segment to be translocated close to the doublestranded DNA break (21). The hydrolysis of ATP is required for further catalytic cycles.For the DNA supercoiling ...