Assay development and inhibition of the Mt-DprE2 essential reductase from Mycobacterium tuberculosis
Sarah M. Batt, Szilvi Toth, Beatriz Rodríguez, Katherine A. Abrahams, Natacha Veerapen, Giacomo Chiodarelli, Liam R. Cox, Patrick J. Moynihan, Joël Lelièvre, Klaus Fütterer, Gurdyal S. Besra
Abstract
DprE2 is an essential enzyme in the synthesis of decaprenylphosphoryl-β- d -arabinofuranose (DPA) and subsequently arabinogalactan, and is a significant new drug target for M. tuberculosis . Two compounds from the GSK-177 box set, GSK301A and GSK032A, were identified through Mt -DprE2-target overexpression studies. The Mt -DprE1-DprE2 complex was co-purified and a new in vitro DprE2 assay developed, based on the oxidation of the reduced nicotinamide adenine dinucleotide cofactor of DprE2 (NADH/NADPH). The Mt -DprE1-DprE2 complex showed interesting kinetics in both the DprE1 resazurin-based assay, where Mt -DprE2 was found to enhance Mt -DprE1 activity and reduce substrate inhibition; and also in the DprE2 assay, which similarly exhibited substrate inhibition and a difference in kinetics of the two potential cofactors, NADH and NADPH. Although, no inhibition was observed in the DprE2 assay by the two GSK set compounds, spontaneous mutant generation indicated a possible explanation in the form of a pro-drug activation pathway, involving fgd1 and fbiC .