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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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Docking of quinolones and diones with ParE/GyrBmutant enzyme.Representative ternary complex interactions were taken from the top20 binding poses obtained from docking moxifloxacin (A), UING5-249(B, C), UING5-157 (D), and UING5-207 (E) into the 2XKK crystal structuremodified from ParE-Glu437 to Cys437. Hydrogen bonds are shown as dashedyellow lines. The top pose for moxifloxacin (A) showed formation ofthe same hydrogen bond to dA-20 as that found with the wild-type structure.However, when UING5-249 was docked into the E437C mutant structure(B, C), the top scoring poses no longer showed hydrogen bonds to the437 position or to R418. Instead, the C-7 amine rotated about 180°to form hydrogen-bond interactions with DNA (dA-20 or dT-19). In thecase of docking UING5-157 (D) into the mutant enzyme, the top twoposes (shown) either form a hydrogen bond to R418 or form no electrostatic/hydrogen-bondinteraction. When UING5-207 (E) was docked, its top pose displayedhydrogen bonds from the C-7 amine to both the R418 side chain andto the deoxyribose oxygen of dG-17.
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fig7: Docking of quinolones and diones with ParE/GyrBmutant enzyme.Representative ternary complex interactions were taken from the top20 binding poses obtained from docking moxifloxacin (A), UING5-249(B, C), UING5-157 (D), and UING5-207 (E) into the 2XKK crystal structuremodified from ParE-Glu437 to Cys437. Hydrogen bonds are shown as dashedyellow lines. The top pose for moxifloxacin (A) showed formation ofthe same hydrogen bond to dA-20 as that found with the wild-type structure.However, when UING5-249 was docked into the E437C mutant structure(B, C), the top scoring poses no longer showed hydrogen bonds to the437 position or to R418. Instead, the C-7 amine rotated about 180°to form hydrogen-bond interactions with DNA (dA-20 or dT-19). In thecase of docking UING5-157 (D) into the mutant enzyme, the top twoposes (shown) either form a hydrogen bond to R418 or form no electrostatic/hydrogen-bondinteraction. When UING5-207 (E) was docked, its top pose displayedhydrogen bonds from the C-7 amine to both the R418 side chain andto the deoxyribose oxygen of dG-17.

Mentions: We next performed dockingwith the GyrB-E466C substitution by introducingthe equivalent change (ParE-E437C) into the 2XKK crystal structureof cleaved complexes formed with A. baumannii topoisomerase IV. With moxifloxacin, the top 20 binding poses weresimilar to the top 20 binding poses generated using wild-type enzyme(Figure 7A; see SupportingInformation Figures S7 and S8 for visualization of all 20 bindingposes), as expected from moxifloxacin lacking a contact with GyrB-Glu466.Thus, the ParE-E437C (GyrB-E466C) substitution appears to have littleeffect on the preferred interaction of the C-7 group of moxifloxacin.In contrast, the top 20 binding poses of the fluoroquinolone UING5-249with the ParE-E437C variant showed greater variation for C-7 grouporientation and binding contacts: the primary amine of the C-7 aminomethylpyrrolidine group formed hydrogen bonds to dA-20, to dT-19, to thebackbone carbonyl of ParE-Arg418, or simply failed to form any polaror electrostatic interaction (two DNA contacts are exemplified inFigures 7B,C). Thus, the Cys substitution severelydisrupts interaction with GyrB-466. Similar differences were seenwith the cognate diones (Figure 7D,E), indicatingthat the C-7 aminomethyl pyrrolidine group might interact with wild-typeGyrB and thus provide a stabilizing effect with GyrB that is not seenwith the C-7 diazabicylco compounds (i.e., moxifloxacin and UING5-157).Finally, a consistent observation throughout comparison of the topbinding poses was that, as compared to the fluoroquinolone, the positioningand orientation of the dione core structures were highly conservedin all binding poses due to the restriction imposed by the hydrogenbond between the dione C-2 carbonyl group and Arg123 of ParC (seeoverlapping structures in Supporting InformationFigures S7 and S8).


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)

Docking of quinolones and diones with ParE/GyrBmutant enzyme.Representative ternary complex interactions were taken from the top20 binding poses obtained from docking moxifloxacin (A), UING5-249(B, C), UING5-157 (D), and UING5-207 (E) into the 2XKK crystal structuremodified from ParE-Glu437 to Cys437. Hydrogen bonds are shown as dashedyellow lines. The top pose for moxifloxacin (A) showed formation ofthe same hydrogen bond to dA-20 as that found with the wild-type structure.However, when UING5-249 was docked into the E437C mutant structure(B, C), the top scoring poses no longer showed hydrogen bonds to the437 position or to R418. Instead, the C-7 amine rotated about 180°to form hydrogen-bond interactions with DNA (dA-20 or dT-19). In thecase of docking UING5-157 (D) into the mutant enzyme, the top twoposes (shown) either form a hydrogen bond to R418 or form no electrostatic/hydrogen-bondinteraction. When UING5-207 (E) was docked, its top pose displayedhydrogen bonds from the C-7 amine to both the R418 side chain andto the deoxyribose oxygen of dG-17.
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fig7: Docking of quinolones and diones with ParE/GyrBmutant enzyme.Representative ternary complex interactions were taken from the top20 binding poses obtained from docking moxifloxacin (A), UING5-249(B, C), UING5-157 (D), and UING5-207 (E) into the 2XKK crystal structuremodified from ParE-Glu437 to Cys437. Hydrogen bonds are shown as dashedyellow lines. The top pose for moxifloxacin (A) showed formation ofthe same hydrogen bond to dA-20 as that found with the wild-type structure.However, when UING5-249 was docked into the E437C mutant structure(B, C), the top scoring poses no longer showed hydrogen bonds to the437 position or to R418. Instead, the C-7 amine rotated about 180°to form hydrogen-bond interactions with DNA (dA-20 or dT-19). In thecase of docking UING5-157 (D) into the mutant enzyme, the top twoposes (shown) either form a hydrogen bond to R418 or form no electrostatic/hydrogen-bondinteraction. When UING5-207 (E) was docked, its top pose displayedhydrogen bonds from the C-7 amine to both the R418 side chain andto the deoxyribose oxygen of dG-17.
Mentions: We next performed dockingwith the GyrB-E466C substitution by introducingthe equivalent change (ParE-E437C) into the 2XKK crystal structureof cleaved complexes formed with A. baumannii topoisomerase IV. With moxifloxacin, the top 20 binding poses weresimilar to the top 20 binding poses generated using wild-type enzyme(Figure 7A; see SupportingInformation Figures S7 and S8 for visualization of all 20 bindingposes), as expected from moxifloxacin lacking a contact with GyrB-Glu466.Thus, the ParE-E437C (GyrB-E466C) substitution appears to have littleeffect on the preferred interaction of the C-7 group of moxifloxacin.In contrast, the top 20 binding poses of the fluoroquinolone UING5-249with the ParE-E437C variant showed greater variation for C-7 grouporientation and binding contacts: the primary amine of the C-7 aminomethylpyrrolidine group formed hydrogen bonds to dA-20, to dT-19, to thebackbone carbonyl of ParE-Arg418, or simply failed to form any polaror electrostatic interaction (two DNA contacts are exemplified inFigures 7B,C). Thus, the Cys substitution severelydisrupts interaction with GyrB-466. Similar differences were seenwith the cognate diones (Figure 7D,E), indicatingthat the C-7 aminomethyl pyrrolidine group might interact with wild-typeGyrB and thus provide a stabilizing effect with GyrB that is not seenwith the C-7 diazabicylco compounds (i.e., moxifloxacin and UING5-157).Finally, a consistent observation throughout comparison of the topbinding poses was that, as compared to the fluoroquinolone, the positioningand orientation of the dione core structures were highly conservedin all binding poses due to the restriction imposed by the hydrogenbond between the dione C-2 carbonyl group and Arg123 of ParC (seeoverlapping structures in Supporting InformationFigures S7 and S8).

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