The C7-aminomethylpyrrolidine group rescues the activity of a thio-fluoroquinolone.

A Mg2+-water bridge between the C-3, C-4 diketo moiety of fluoroquinolones and the conserved amino acid residues in the GyrA/ParC subunit is critical for the binding of a fluoroquinolone to a topoisomerase-DNA covalent complex. The fluoroquinolone UING-5-249 (249) can bind to the GyrB subunit through its C7-aminomethylpyrrolidine group. This interaction is responsible for enhanced activities of 249 against the wild type and quinolone-resistant mutant topoisomerases. To further evaluate the effects of the 249-GyrB interaction on fluoroquinolone activity, we examined the activities of decarboxy- and thio-249 against DNA gyrase and conducted docking studies using the structure of a gyrase-ciprofloxacin-DNA ternary complex. We found that the 249-GyrB interaction rescued the activity of thio-249 but not that of decarboxy-249. A C7-group that binds more strongly to the GyrB subunit may allow for modifications at the C-4 position, leading to a novel compound that is active against the wild type and quinolone-resistant pathogens.

Novel N-1 substituted fluoroquinolones inhibit human topoisomerase I activity and exhibit anti-proliferative activity.

Fluoroquinolone-class agents selectively target the bacterial type IIA topoisomerases DNA gyrase and topoisomerase IV, with a few exceptions that target eukaryotic type IIA topoisomerases. Fluoroquinolones bind and stabilize type IIA topoisomerase-DNA covalent complexes that contain a double-strand break. This unique mode of action is referred to as 'topoisomerase poisoning'. We discovered that two novel fluoroquinolones having aryl functionality at the N-1 position, UITT-3-217 (217) and UITT-3-227 (227), could inhibit the catalytic activity of human topoisomerase II without stabilizing topoisomerase-DNA complexes, i.e., without poisoning it. Surprisingly, these compounds are more effective in inhibiting the catalytic activities of human and bacterial topoisomerase I. The National Cancer Institute's 60 human tumor cell lines screen revealed significant anti-proliferative activities with 217 and 227 against the majority of 60 cancer cell lines. A proof of concept in vivo efficacy study using an HT-29 xenograft model of human colorectal cancer showed that 217 could inhibit the proliferation of human colorectal cancer cells to a degree comparable to fluorouracil in mice. Although 227 also exhibited anti-proliferative activity, it was not as effective as 217 in this xenograft model. These novel fluoroquinolones may serve as promising lead compounds for the development of new anticancer drugs.

Design, synthesis, and evaluation of novel N-1 fluoroquinolone derivatives: Probing for binding contact with the active site tyrosine of gyrase.

Structural studies of topoisomerase-fluoroquinolone-DNA ternary complexes revealed a cavity between the quinolone N-1 position and the active site tyrosine. Fluoroquinolone derivatives having positively charged or aromatic moieties extended from the N-1 position were designed to probe for binding contacts with the phosphotyrosine residue in ternary complex. While alkylamine, alkylphthalimide, and alkylphenyl groups introduced at the N-1 position afforded derivatives that maintained modest inhibition of the supercoiling activity of DNA gyrase, none retained ability to poison DNA gyrase. Thus, the addition of a large and/or long moiety at the N-1 position disrupts ternary complex formation, and retained ability to inhibit supercoiling is likely through interference with the strand breakage reaction. Two derivatives were found to possess inhibitory effects on the decatenation activity of human topoisomerase II.

Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg(2+) to the second metal binding site.

BACKGROUND: Fluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase-DNA covalent complex as a topoisomerase-fluoroquinolone-DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction. METHODS: We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg(2+)-, Mn(2+)-, or Ca(2+)-supported DNA cleavage activity of Escherichia coli Topo IV. RESULTS: In the absence of any drug, 20-30 mM Mg(2+) was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1mM of either Mn(2+) or Ca(2+) was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg(2+) concentrations where Topo IV alone could not efficiently cleave DNA. CONCLUSIONS AND GENERAL SIGNIFICANCE: At low Mg(2+) concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg(2+) binding to metal binding site B through the structural distortion in DNA. As Mg(2+) concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg(2+) at site B or inhibition the binding of Mg(2+) to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg(2+) binding.

Bypassing fluoroquinolone resistance with quinazolinediones: studies of drug-gyrase-DNA complexes having implications for drug design.

Widespread fluoroquinolone resistance has drawn attention to quinazolinediones (diones), fluoroquinolone-like topoisomerase poisons that are unaffected by common quinolone-resistance mutations. To better understand differences between quinolones and diones, we examined their impact on the formation of cleaved complexes (drug-topoisomerase-DNA complexes in which the DNA moiety is broken) with gyrase, one of two bacterial targets of the drugs. Formation of cleaved complexes, measured by linearization of a circular DNA substrate, required lower concentrations of quinolone than dione. The reverse reaction, detected as resealing of DNA breaks in cleaved complexes, required higher temperatures and EDTA concentrations for quinolones than diones. The greater stability of quinolone-containing complexes was attributed to the unique ability of the quinolone C3/C4 keto acid to complex with magnesium and form a previously described drug-magnesium-water bridge with GyrA-Ser83 and GyrA-Asp87. A nearby substitution in GyrA (G81C) reduced activity differences between quinolone and dione, indicating that resistance due to this variation derives from perturbation of the magnesium-water bridge. To increase dione activity, we examined a relatively small, flexible C-7-3-(aminomethyl)pyrrolidinyl substituent, which is distal to the bridging C3/C4 keto acid substituent of quinolones. The 3-(aminomethyl)pyrrolidinyl group at position C-7 was capable of forming binding interactions with GyrB-Glu466, as indicated by inspection of crystal structures, computer-aided docking, and measurement of cleaved-complex formation with mutant and wild-type GyrB proteins. Thus, modification of dione C-7 substituents constitutes a strategy for obtaining compounds active against common quinolone-resistant mutants.