Assay development and inhibition of the Mt -DprE2 essential reductase from Mycobacterium tuberculosis

• Published in: Microbiology (Society for General Microbiology) Vol. 169; no. 1
• Main Authors: Batt, Sarah M, Toth, Szilvi, Rodriguez, Beatriz, Abrahams, Katherine A, Veerapen, Natacha, Chiodarelli, Giacomo, Cox, Liam R, Moynihan, Patrick J, Lelievre, Joel, Fütterer, Klaus, Besra, Gurdyal S
• Format: Journal Article
• Language: English
• Published: England Microbiology Society 01.01.2023
• Subjects: Antimicrobials and AMR; Antitubercular Agents - metabolism; Antitubercular Agents - pharmacology; Bacterial Proteins - chemistry; Mycobacterium tuberculosis; NAD - metabolism; NADP - metabolism; Oxidoreductases - genetics; Oxidoreductases - metabolism; decaprenylphosphoryl-D-arabinose; Mycobacterium tuberculosis; substrate inhibition; Mt-DprE1; Mt-DprE2; mycobacterial cell wall; enzyme kinetics
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.001288

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 . Two compounds from the GSK-177 box set, GSK301A and GSK032A, were identified through -DprE2-target overexpression studies. The -DprE1-DprE2 complex was co-purified and a new DprE2 assay developed, based on the oxidation of the reduced nicotinamide adenine dinucleotide cofactor of DprE2 (NADH/NADPH). The -DprE1-DprE2 complex showed interesting kinetics in both the DprE1 resazurin-based assay, where -DprE2 was found to enhance -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 and .