Development of a high throughput assay for the screening of acyl-CoA carboxylase inhibitors.

BAZET LYONNET, B; DIACOVICH, L.; GAGO, G.; GRAMAJO, H

Mar del Plata

Congreso; LI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2015

SAIB

Acyl-CoA carboxylases (ACCases) commit acyl-CoAs to the biosynthesis of lipids in Mycobacterium tuberculosis.This pathogen has several genes coding for ACCase subunits in its genome: three α subunits (accA1?3), six βsubunits (accD1?6) and one ε subunit (accE5). ACCase 5 complex is formed by the biotinylated α subunit AccA3,the carboxyltransferase β subunit AccD5 and the small ε subunit AccE5. To gain insight about the metabolicrelevance of this enzyme in mycobacteria, we obtained M. smegmatis mutants in accD5-accE5, the two subunitsspecifically associated with ACCase 5. The analysis of this conditional mutant demonstrated that AccD5 and AccE5are part of an essential ACCase involved in lipid biosynthesis, and proposed ACCase 5 as an attractive target fortuberculosis drug discovery. In this sense, we developed an enzyme-based assay to identify inhibitors of ACCase 5,and we optimized it for high throughput screening. We used this assay towards a library containing 11,000compounds and found 34 candidates. We further analyzed these candidates by conventional methods and we found 6compounds that inhibit ACCase 5 with different potency and now need to be characterized in more detail. Theseresults validated the high throughput screening assay as a powerful tool for identifying novel enzyme inhibitors thatcould be developed as anti-tuberculosis drugs.