title: Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG.
abstract: To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). 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. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. 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.
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy.
authors.forename: Giorgia
authors.initials: G
authors.lastname: Mori
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy.
authors.forename: Laurent R
authors.initials: LR
authors.lastname: Chiarelli
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Esposito
authors.affiliation: A. N. Bakh Institute of Biochemistry, Russian Academy of Science, 119071 Moscow, Russia.
authors.forename: Vadim
authors.initials: V
authors.lastname: Makarov
authors.affiliation: Institut Pasteur, Unité de Microbiologie Structurale, CNRS-UMR3528, Université Paris Diderot, Sorbonne Paris Cité, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Marco
authors.initials: M
authors.lastname: Bellinzoni
authors.affiliation: Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Station 19, 1015 Lausanne, Switzerland.
authors.forename: Ruben C
authors.initials: RC
authors.lastname: Hartkoorn
authors.affiliation: Department of Molecular Medicine, University of Padova, 35128 Padua, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Degiacomi
authors.affiliation: Department of Molecular Medicine, University of Padova, 35128 Padua, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Boldrin
authors.affiliation: Collaborative Drug Discovery, 1633 Bayshore Highway, Suite 342, Burlingame, CA 94010, USA.
authors.forename: Sean
authors.initials: S
authors.lastname: Ekins
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy.
authors.forename: Ana Luisa
authors.initials: AL
authors.lastname: de Jesus Lopes Ribeiro
authors.affiliation: Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, UK; Faculty of Pharmaceutical Sciences, UNESP - Univ Estadual Paulista, Araraquara, São Paulo 14801-902, Brazil.
authors.forename: Leonardo B
authors.initials: LB
authors.lastname: Marino
authors.affiliation: Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovičova 6, Mlynská dolina, 84215 Bratislava, Slovakia.
authors.forename: Ivana
authors.initials: I
authors.lastname: Centárová
authors.affiliation: Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovičova 6, Mlynská dolina, 84215 Bratislava, Slovakia.
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Svetlíková
authors.affiliation: Institute of Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovičova 6, Mlynská dolina, 84215 Bratislava, Slovak Republic.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Blaško
authors.affiliation: A. N. Bakh Institute of Biochemistry, Russian Academy of Science, 119071 Moscow, Russia.
authors.forename: Elena
authors.initials: E
authors.lastname: Kazakova
authors.affiliation: A. N. Bakh Institute of Biochemistry, Russian Academy of Science, 119071 Moscow, Russia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Lepioshkin
authors.affiliation: Institut Pasteur, Unité de Microbiologie Structurale, CNRS-UMR3528, Université Paris Diderot, Sorbonne Paris Cité, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Barilone
authors.affiliation: Department of Chemistry, University of Pavia, 27100 Pavia, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Zanoni
authors.affiliation: Department of Chemistry, University of Pavia, 27100 Pavia, Italy.
authors.forename: Alessio
authors.initials: A
authors.lastname: Porta
authors.affiliation: Department of Biology, University of Florence, Sesto Fiorentino, Florence 50019, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Fondi
authors.affiliation: Department of Biology, University of Florence, Sesto Fiorentino, Florence 50019, Italy.
authors.forename: Renato
authors.initials: R
authors.lastname: Fani
authors.affiliation: Institut Pasteur de Lille, Center for Infection and Immunity, 59019 Lille, France.
authors.forename: Alain R
authors.initials: AR
authors.lastname: Baulard
authors.affiliation: Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovičova 6, Mlynská dolina, 84215 Bratislava, Slovakia.
authors.forename: Katarína
authors.initials: K
authors.lastname: Mikušová
authors.affiliation: Institut Pasteur, Unité de Microbiologie Structurale, CNRS-UMR3528, Université Paris Diderot, Sorbonne Paris Cité, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Alzari
authors.affiliation: Department of Molecular Medicine, University of Padova, 35128 Padua, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Manganelli
authors.affiliation: Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Luiz Pedro S
authors.initials: LP
authors.lastname: de Carvalho
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Riccardi
authors.affiliation: Global Health Institute, Ecole Polytechnique Fédérale de Lausanne, Station 19, 1015 Lausanne, Switzerland. Electronic address: stewart.cole@epfl.ch.
authors.forename: Stewart T
authors.initials: ST
authors.lastname: Cole
authors.affiliation: Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, 27100 Pavia, Italy. Electronic address: mariarosalia.pasca@unipv.it.
authors.forename: Maria Rosalia
authors.initials: MR
authors.lastname: Pasca
mesh_terms: D065767:Activation, Metabolic; D000818:Animals; D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods; D056945:Hep G2 Cells; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D051379:Mice; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013876:Thiophenes / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D013876:Thiophenes; D010088:Oxidoreductases; C480352:ethA protein, Mycobacterium tuberculosis; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase