Identification of novel inhibitors of Mycobacterium tuberculosis ACCase5 via high-throughput screening.

BAZET LYONNET, B; GRAMAJO, H; COLACCINI, F; GAGO, G.; FIORITO, M.; BANCHIO, C

Salta

Congreso; XIV PABMB Congress and LV Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB).; 2019

Tuberculosis (TB) is one of the top ten causes of death and the leading cause from a single infectious agent. The emergence of multidrug-resistant TB (MDR-TB) presents an increasingly difficult therapeutic challenge and is now the main cause of death due to antimicrobial resistance. In this context, the effective control of this major health problem requires the identification of novel drug targets and new chemical entities suitable for the development of new anti-mycobacterial drugs. Mycobacterium tuberculosis (Mtb) produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases (ACCase). Bioinformatic, biochemical and structural analysis of Mtb ACCase 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. Previous studies indicated that this enzyme complex is essential for the viability of mycobacteria, inferring that the ACCase 5 complex has an essential activity for this bacterium. Moreover, 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, high-throughput screening assays were implemented to test millions of compounds belonging to Glaxo SmithKline and Novartis, and found several molecules that inhibited more that 85% of the ACCase 5 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, MIC on Mtb H37Ra and cytotoxicity assays were obtained. To start elucidating the mechanism of action of these compounds in vivo, we studied their effect on lipid biosynthesis by analyzing 14C-acetate incorporation on fatty acids and mycolic acids and found that all of them inhibited lipid biosynthesis. These results validated the high-throughput screening assay as a powerful tool for identifying novel enzyme inhibitors that could be developed as anti-tuberculosis drugs