Practical Spectrophotometric Assay For The Dape-Encoded N-Succinyl-L, L-Diaminopimelic Acid Desuccinylase, A Potential Antibiotic Target

A new enzymatic assay for the bacterial enzyme succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) is described. This assay employs N-6-methyl-N-2-succinyl-L, L-diamino-pimelic acid (N-6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N-6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N-6-methylated-L,L-SDAPas an alternate substrate for the assay, given binding in the active site of DapE predicted to be comparable to the endogenous substrate. The alternate substrate for the assay, N-6-methyl-L, L-SDAP, was synthesized from the tertbutyl ester of Boc-L-glutamic acid employing a Horner-Wadsworth-Emmons olefination followed by an enantioselective reduction employing Rh(I)(COD)(S,S)-Et-DuPHOS as the chiral catalyst. Validation of the new ninhydrin assay was demonstrated with known inhibitors of DapE from Haemophilus influenza (HiDapE) including captopril (IC50 = 3.4 [+/- 0.2] mu M, 3-mercaptobenzoic acid (IC50 = 21.8 [+/- 2.2] mu M, phenylboronic acid (IC50 = 316 [+/- 23.6] mu M, and 2-thiopheneboronic acid (IC50 = 111 [+/- 16] mu M. Based on these data, this assay is simple and robust, and should be amenable to high-throughput screening, which is an important step forward as it opens the door to medicinal chemistry efforts toward the discovery of DapE inhibitors that can function as a new class of antibiotics.