SCREENING AND CHARACTERIZATION OF MYCOBACTERIUM TUBERCULOSIS ACYL-COA CARBOXYLASE INHIBITORS

COLLACCINI F, MICAELA FIORITO, BAZET LYONNET B, ENSINCK D, DIACOVICH L, GAGO G, GRAMAJO H

Congreso; SAIB 2018; 2018

SCREENING AND CHARACTERIZATION OF Mycobacterium tuberculosisACYL-COA CARBOXYLASE INHIBITORS Collaccini F; Fiorito M; BazetLyonnet B; Lara J; CrottaAsis A; Ensinck E, Diacovich L; Gago G; Gramajo HInstituto de Biología Molecular y Celular de Rosario (IBR - CONICET). FBIOyF- UNR. E-mail: fcolaccini@gmail.comTuberculosis (TB) represents a major public health problem worldwide. This disease remains today one of the leading causes of death from an infectious agent. The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains has challenged TB control. Therefore, the discovery of novel pharmacological targets for the development of a new generation of drugs is urgently needed. Acyl-CoA carboxylases (ACCases) are key enzymes providing acyl-CoAs, substrates for biosynthesis of all the unique lipids present in Mtb cell envelope. Bioinformatic, biochemical and structural analysis of MtbACCase 5 complex indicated that the main catalytic activity of this enzyme corresponds to that of a propionyl-CoA/acetyl-CoA carboxylase. This complex is formed by the biotinylated α subunit AccA3, the carboxyltransferase β subunit AccD5 and the small ε subunit AccE5. Studies carried out by high-density mutagenesis in Mtb and directed mutagenesis in M. smegmatis indicate that this enzyme complex is essential for the viability of mycobacteria, inferring that the ACCase 5complex has an essential activity for this bacterium. Also the analysis of a conditional mutant demonstrated that AccD5 and AccE5 are part of an essential ACCase involved in lipid biosynthesis, and proposed ACCase 5 as an attractive target for tuberculosis drug discovery. In this work, we used two different enzyme-based assays to identify inhibitors of ACCase 5. High throughput screening assays were implemented to test millons of compounds belonging to Glaxo SmithKline and Novartis, and found several molecules that inhibited more that 85% of the ACCase 5 PCC activity. We further analyzed these candidates by conventional enzymatic methods and found five compounds that inhibited ACCase 5 at low μM concentrations. For these compounds IC50 and MIC on Mtb H37Ra were obtained, and their influence in the biosynthesis of fatty and mycolic acids was evaluated. These results validated the high