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

DprE2 is an essential enzyme in the synthesis of decaprenylphosphoryl-ß-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.

New natural amino acid-bearing prodrugs boost pterostilbene's oral pharmacokinetic and distribution profile.

The biomedical effects of the natural phenol pterostilbene are of great interest but its bioavailability is negatively affected by the phenolic group in position 4' which is an ideal target for the conjugative enzymes of phase II metabolism. We report the synthesis and characterization of prodrugs in which the hydroxyl moiety is reversibly protected as a carbamate ester linked to the N-terminus of a natural amino acid. Prodrugs comprising amino acids with hydrophobic side chains were readily absorbed after intragastric administration to rats. The Area Under the Curve for pterostilbene in blood was optimal when prodrugs with isoleucine or ß-alanine were used. The prodrug incorporating isoleucine was used for further studies to map distribution into major organs. When compared to pterostilbene itself, administration of the isoleucine prodrug afforded increased absorption, reduced metabolism and higher concentrations of pterostilbene, sustained for several hours, in most of the organs examined. Experiments using Caco-2 cells as an in vitro model for human intestinal absorption suggest that the prodrug could have promising absorption profiles also in humans; its uptake is partly due to passive diffusion, and partly mediated by H+-dependent transporters expressed on the apical membrane of enterocytes, such as PepT1 and OATP.