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Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG.

Mori G, Chiarelli LR, Esposito M, Makarov V, Bellinzoni M, Hartkoorn RC, Degiacomi G, Boldrin F, Ekins S, de Jesus Lopes Ribeiro AL, Marino LB, Centárová I, Svetlíková Z, Blaško J, Kazakova E, Lepioshkin A, Barilone N, Zanoni G, Porta A, Fondi M, Fani R, Baulard AR, Mikušová K, Alzari PM, Manganelli R, de Carvalho LP, Riccardi G, Cole ST, Pasca MR - Chem. Biol. (2015)

Bottom Line: Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG.Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides.Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy.

No MeSH data available.


Related in: MedlinePlus

EthA Converts the 7947882 and 7904688 Compounds into Active PyrG Inhibitors(A) Inhibition of PyrG activity during the co-incubation with EthA and 7947882. Gray bars correspond to the activities of the blank controls in the absence of NADPH, and black bars represent the residual activities after incubation with working EthA.(B) UV-Vis spectra of the re-purified PyrG after co-incubation with EthA reaction with 7947882 compound. Solid line is the spectrum of PyrG incubated with full EthA reaction; dashed line is the spectrum of PyrG from blank reaction; dotted line is the spectrum of the compound at 20 μM.(C and D) Co-incubation of PyrG with EthA and 7904688 compound. Conditions are the same as for (A) and (B), respectively.(E and F) Identification of in vitro EthA metabolites of 7947882 compound. Mass spectrometry analysis (from top to bottom) of the 7947882 compound, the partially purified products of EthA reaction M1 and M2, and the synthetic metabolite 11426026. (E) Full electrospray ionization mass spectrometry of the compounds recorded in negative mode. (F) Fragmentation pattern of the compounds.See also Figures S1 and S2; Table S2.
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fig1: EthA Converts the 7947882 and 7904688 Compounds into Active PyrG Inhibitors(A) Inhibition of PyrG activity during the co-incubation with EthA and 7947882. Gray bars correspond to the activities of the blank controls in the absence of NADPH, and black bars represent the residual activities after incubation with working EthA.(B) UV-Vis spectra of the re-purified PyrG after co-incubation with EthA reaction with 7947882 compound. Solid line is the spectrum of PyrG incubated with full EthA reaction; dashed line is the spectrum of PyrG from blank reaction; dotted line is the spectrum of the compound at 20 μM.(C and D) Co-incubation of PyrG with EthA and 7904688 compound. Conditions are the same as for (A) and (B), respectively.(E and F) Identification of in vitro EthA metabolites of 7947882 compound. Mass spectrometry analysis (from top to bottom) of the 7947882 compound, the partially purified products of EthA reaction M1 and M2, and the synthetic metabolite 11426026. (E) Full electrospray ionization mass spectrometry of the compounds recorded in negative mode. (F) Fragmentation pattern of the compounds.See also Figures S1 and S2; Table S2.

Mentions: Thus, to confirm that EthA produces metabolites that might act on PyrG, the EthA enzymatic reaction was performed with either 7947882 or 7904688 in the presence of PyrG, and the activity of the latter enzyme was monitored during the course of the reaction. The blank control was performed omitting reduced nicotinamide adenine dinucleotide phosphate (NADPH) to hinder the EthA-catalyzed reaction, and under these conditions PyrG maintained full activity for up to 6 hr of incubation. By contrast, in the presence of an actively working EthA, PyrG lost full activity within 4 hr when incubated with 7947882, and about 80% of its activity in 6 hr when incubated with 7904688 (Figures 1A and 1C).


Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG.

Mori G, Chiarelli LR, Esposito M, Makarov V, Bellinzoni M, Hartkoorn RC, Degiacomi G, Boldrin F, Ekins S, de Jesus Lopes Ribeiro AL, Marino LB, Centárová I, Svetlíková Z, Blaško J, Kazakova E, Lepioshkin A, Barilone N, Zanoni G, Porta A, Fondi M, Fani R, Baulard AR, Mikušová K, Alzari PM, Manganelli R, de Carvalho LP, Riccardi G, Cole ST, Pasca MR - Chem. Biol. (2015)

EthA Converts the 7947882 and 7904688 Compounds into Active PyrG Inhibitors(A) Inhibition of PyrG activity during the co-incubation with EthA and 7947882. Gray bars correspond to the activities of the blank controls in the absence of NADPH, and black bars represent the residual activities after incubation with working EthA.(B) UV-Vis spectra of the re-purified PyrG after co-incubation with EthA reaction with 7947882 compound. Solid line is the spectrum of PyrG incubated with full EthA reaction; dashed line is the spectrum of PyrG from blank reaction; dotted line is the spectrum of the compound at 20 μM.(C and D) Co-incubation of PyrG with EthA and 7904688 compound. Conditions are the same as for (A) and (B), respectively.(E and F) Identification of in vitro EthA metabolites of 7947882 compound. Mass spectrometry analysis (from top to bottom) of the 7947882 compound, the partially purified products of EthA reaction M1 and M2, and the synthetic metabolite 11426026. (E) Full electrospray ionization mass spectrometry of the compounds recorded in negative mode. (F) Fragmentation pattern of the compounds.See also Figures S1 and S2; Table S2.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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fig1: EthA Converts the 7947882 and 7904688 Compounds into Active PyrG Inhibitors(A) Inhibition of PyrG activity during the co-incubation with EthA and 7947882. Gray bars correspond to the activities of the blank controls in the absence of NADPH, and black bars represent the residual activities after incubation with working EthA.(B) UV-Vis spectra of the re-purified PyrG after co-incubation with EthA reaction with 7947882 compound. Solid line is the spectrum of PyrG incubated with full EthA reaction; dashed line is the spectrum of PyrG from blank reaction; dotted line is the spectrum of the compound at 20 μM.(C and D) Co-incubation of PyrG with EthA and 7904688 compound. Conditions are the same as for (A) and (B), respectively.(E and F) Identification of in vitro EthA metabolites of 7947882 compound. Mass spectrometry analysis (from top to bottom) of the 7947882 compound, the partially purified products of EthA reaction M1 and M2, and the synthetic metabolite 11426026. (E) Full electrospray ionization mass spectrometry of the compounds recorded in negative mode. (F) Fragmentation pattern of the compounds.See also Figures S1 and S2; Table S2.
Mentions: Thus, to confirm that EthA produces metabolites that might act on PyrG, the EthA enzymatic reaction was performed with either 7947882 or 7904688 in the presence of PyrG, and the activity of the latter enzyme was monitored during the course of the reaction. The blank control was performed omitting reduced nicotinamide adenine dinucleotide phosphate (NADPH) to hinder the EthA-catalyzed reaction, and under these conditions PyrG maintained full activity for up to 6 hr of incubation. By contrast, in the presence of an actively working EthA, PyrG lost full activity within 4 hr when incubated with 7947882, and about 80% of its activity in 6 hr when incubated with 7904688 (Figures 1A and 1C).

Bottom Line: Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG.Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides.Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy.

No MeSH data available.


Related in: MedlinePlus