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

Drlica K, Mustaev A, Towle TR, Luan G, Kerns RJ, Berger JM - ACS Chem. Biol. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Public Health Research Institute and Department of Microbiology & Molecular Genetics, New Jersey Medical School, Rutgers Biomedical and Health Sciences , 225 Warren Street, Newark, New Jersey 07103, United States.

ABSTRACT
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.

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Effect of GyrA-G81C gyrase on dione and quinoloneactivity. (A)Compound concentration effects on complex formation: (i) wild-typegyrase and (ii) GyrA-G81C gyrase. Symbols: empty circles, fluoroquinoloneUING5-249; filled circles, dione UING5-207. Similar results were obtainedwith four replicate experiments. (B) DNA resealing stimulated by EDTA.Reaction mixtures containing cleaved complex were treated with theindicated concentrations of EDTA and analyzed as in Figure 2. Linear DNA was quantified and expressed as a percentof the zero-EDTA control. Symbols: filled circles, dione UING5-207at 80 μM; empty circles, fluoroquinolone UING5-249 at 40 μM.Similar results were obtained with three independent replicate experiments.(C) DNA resealing stimulated by high temperature. Reaction mixturescontaining cleaved complexes were treated and analyzed as in Figure 3. (i) Complexes formed with fluoroquinolone UING5-249;(ii) complexes formed with dione UING5-207. Symbols: empty symbols,wild-type gyrase; filled symbols, GyrA81C gyrase. Similar resultswere obtained with four replicate experiments.
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fig4: Effect of GyrA-G81C gyrase on dione and quinoloneactivity. (A)Compound concentration effects on complex formation: (i) wild-typegyrase and (ii) GyrA-G81C gyrase. Symbols: empty circles, fluoroquinoloneUING5-249; filled circles, dione UING5-207. Similar results were obtainedwith four replicate experiments. (B) DNA resealing stimulated by EDTA.Reaction mixtures containing cleaved complex were treated with theindicated concentrations of EDTA and analyzed as in Figure 2. Linear DNA was quantified and expressed as a percentof the zero-EDTA control. Symbols: filled circles, dione UING5-207at 80 μM; empty circles, fluoroquinolone UING5-249 at 40 μM.Similar results were obtained with three independent replicate experiments.(C) DNA resealing stimulated by high temperature. Reaction mixturescontaining cleaved complexes were treated and analyzed as in Figure 3. (i) Complexes formed with fluoroquinolone UING5-249;(ii) complexes formed with dione UING5-207. Symbols: empty symbols,wild-type gyrase; filled symbols, GyrA81C gyrase. Similar resultswere obtained with four replicate experiments.

Mentions: X-ray analysis of crystalstructures for cleaved complexes suggests that drug binding occursclose enough to GyrA-Gly81 for a Cys substitution to sterically perturbquinolone-mediated magnesium bridging.11,20 Thus, cleavedcomplexes formed with GyrA-G81C gyrase and quinolones were expectedto have properties similar to those of complexes formed with diones.Indeed, when dione UING5-207 and its cognate quinolone (UING5-249)were compared for complex formation with wild-type gyrase, 10- to20-times more dione was required to generate the same amount of linearDNA (Figure 4Ai). By contrast, the differencewas only 2-fold when GyrA-G81C was used (Figure 4Aii). Similarly, examination of DNA resealing by GyrA-G81C gyraseat various EDTA concentrations revealed no difference between dione-and fluoroquinolone-containing complexes at low EDTA concentration(Figure 4B) unlike the result obtained withwild-type gyrase, Figure 2A). The GyrA-G81Csubstitution also lowered DNA resealing temperature more for the fluoroquinolonethan for the dione, bringing them to similar levels (Figure 4C). Overall, these results are consistent with theGyrA-G81C substitution exerting a destabilizing effect on the fluoroquinolone-mediatedmagnesium linkage that is absent in dione-containing complexes. Thus,the GyrA-G81C substitution joins substitutions at GyrA-83 and GyrA-87in acting by impeding the formation of the quinolone–magnesium–water–enzymebridge.9,16


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

Drlica K, Mustaev A, Towle TR, Luan G, Kerns RJ, Berger JM - ACS Chem. Biol. (2014)

Effect of GyrA-G81C gyrase on dione and quinoloneactivity. (A)Compound concentration effects on complex formation: (i) wild-typegyrase and (ii) GyrA-G81C gyrase. Symbols: empty circles, fluoroquinoloneUING5-249; filled circles, dione UING5-207. Similar results were obtainedwith four replicate experiments. (B) DNA resealing stimulated by EDTA.Reaction mixtures containing cleaved complex were treated with theindicated concentrations of EDTA and analyzed as in Figure 2. Linear DNA was quantified and expressed as a percentof the zero-EDTA control. Symbols: filled circles, dione UING5-207at 80 μM; empty circles, fluoroquinolone UING5-249 at 40 μM.Similar results were obtained with three independent replicate experiments.(C) DNA resealing stimulated by high temperature. Reaction mixturescontaining cleaved complexes were treated and analyzed as in Figure 3. (i) Complexes formed with fluoroquinolone UING5-249;(ii) complexes formed with dione UING5-207. Symbols: empty symbols,wild-type gyrase; filled symbols, GyrA81C gyrase. Similar resultswere obtained with four replicate experiments.
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Related In: Results  -  Collection

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fig4: Effect of GyrA-G81C gyrase on dione and quinoloneactivity. (A)Compound concentration effects on complex formation: (i) wild-typegyrase and (ii) GyrA-G81C gyrase. Symbols: empty circles, fluoroquinoloneUING5-249; filled circles, dione UING5-207. Similar results were obtainedwith four replicate experiments. (B) DNA resealing stimulated by EDTA.Reaction mixtures containing cleaved complex were treated with theindicated concentrations of EDTA and analyzed as in Figure 2. Linear DNA was quantified and expressed as a percentof the zero-EDTA control. Symbols: filled circles, dione UING5-207at 80 μM; empty circles, fluoroquinolone UING5-249 at 40 μM.Similar results were obtained with three independent replicate experiments.(C) DNA resealing stimulated by high temperature. Reaction mixturescontaining cleaved complexes were treated and analyzed as in Figure 3. (i) Complexes formed with fluoroquinolone UING5-249;(ii) complexes formed with dione UING5-207. Symbols: empty symbols,wild-type gyrase; filled symbols, GyrA81C gyrase. Similar resultswere obtained with four replicate experiments.
Mentions: X-ray analysis of crystalstructures for cleaved complexes suggests that drug binding occursclose enough to GyrA-Gly81 for a Cys substitution to sterically perturbquinolone-mediated magnesium bridging.11,20 Thus, cleavedcomplexes formed with GyrA-G81C gyrase and quinolones were expectedto have properties similar to those of complexes formed with diones.Indeed, when dione UING5-207 and its cognate quinolone (UING5-249)were compared for complex formation with wild-type gyrase, 10- to20-times more dione was required to generate the same amount of linearDNA (Figure 4Ai). By contrast, the differencewas only 2-fold when GyrA-G81C was used (Figure 4Aii). Similarly, examination of DNA resealing by GyrA-G81C gyraseat various EDTA concentrations revealed no difference between dione-and fluoroquinolone-containing complexes at low EDTA concentration(Figure 4B) unlike the result obtained withwild-type gyrase, Figure 2A). The GyrA-G81Csubstitution also lowered DNA resealing temperature more for the fluoroquinolonethan for the dione, bringing them to similar levels (Figure 4C). Overall, these results are consistent with theGyrA-G81C substitution exerting a destabilizing effect on the fluoroquinolone-mediatedmagnesium linkage that is absent in dione-containing complexes. Thus,the GyrA-G81C substitution joins substitutions at GyrA-83 and GyrA-87in acting by impeding the formation of the quinolone–magnesium–water–enzymebridge.9,16

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Public Health Research Institute and Department of Microbiology & Molecular Genetics, New Jersey Medical School, Rutgers Biomedical and Health Sciences , 225 Warren Street, Newark, New Jersey 07103, United States.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus