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

Giorgia Mori (1) , Laurent Chiarelli (1) , Marta Esposito (1) , Vadim Makarov (2) , Marco Bellinzoni (3) , Ruben Hartkoorn (4) , Giulia Degiacomi (5) , Francesca Boldrin (5) , Sean Ekins (6) , Ana Luisa de Jesus Lopes Ribeiro (1) , Leonardo Marino (7, 8) , Ivana Centárová (9) , Zuzana Svetlíková (9) , Jaroslav Blaško (9) , Elena Kazakova (2) , Alexander Lepioshkin (2) , Nathalie Barilone (3) , Giuseppe Zanoni (1) , Alessio Porta (1) , Marco Fondi (10) , Renato Fani (10) , Alain Baulard (11) , Katarína Mikušová (9) , Pedro Alzari (3) , Riccardo Manganelli (5) , Luiz Pedro S de Carvalho (7) , Giovanna Riccardi (1) , Stewart Cole (4) , Maria Rosalia Pasca (1)

1 UNIPV - Università degli Studi di Pavia = University of Pavia
2 RAS - Russian Academy of Sciences [Moscow]
3 Microb. Struc. (UMR_3528 / U-Pasteur_5) - Microbiologie structurale - Structural Microbiology
4 EPFL - Ecole Polytechnique Fédérale de Lausanne
5 Unipd - Università degli Studi di Padova = University of Padua
6 Collaborative Drug Discovery
7 The Francis Crick Institute [London]
8 UNESP - Universidade Estadual Paulista Júlio de Mesquita Filho = São Paulo State University
9 Comenius University in Bratislava
10 UniFI - Università degli Studi di Firenze = University of Florence
11 Institut Pasteur de Lille

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.

Domains: Cristallography; Structural Biology [q-bio.BM]; Biochemistry, Molecular Biology; Bioinformatics [q-bio.QM]; Biological Physics [physics.bio-ph]; Cellular Biology

Giorgia Mori, Laurent Chiarelli, Marta Esposito, Vadim Makarov, Marco Bellinzoni, et al.. Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG. Chemistry and Biology, 2015, 22 (7), pp.917-927. ⟨10.1016/j.chembiol.2015.05.016⟩. ⟨pasteur-03096090⟩