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 N6-methyl-N2-succinyl-L,L-diaminopimelic acid (N6-methyl-L,L-SDAP) as the substrate with ninhydrin used to detect cleavage of the amide bond of the modified substrate, wherein N6-methylation enables selective detection of the primary amine enzymatic product. Molecular modeling supported preparation of the mono-N6-methylated-L,L-SDAP as 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, N6-methyl-L,L-SDAP, was synthesized from the tert-butyl 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] μM, 3-mercaptobenzoic acid (IC50 = 21.8 [±2.2] μM, phenylboronic acid (IC50 = 316 [± 23.6] μM, and 2-thiopheneboronic acid (IC50 = 111 [± 16] μM. 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.本研究报道了一种针对细菌来源琥珀酰二氨基庚二酸脱琥珀酰酶（succinyl-diaminopimelate desuccinylase，DapE，E.C. 3.5.1.18）的新型酶学检测方法。该检测体系以N6-甲基-N2-琥珀酰-L,L-二氨基庚二酸（N6-methyl-L,L-SDAP）作为底物，利用茚三酮检测修饰底物的酰胺键裂解产物；其中N6-甲基化修饰可实现对酶解产生的伯胺的选择性检测。鉴于该底物在DapE活性位点的结合模式与内源底物相当，通过分子模拟辅助设计合成了单-N6-甲基化-L,L-SDAP作为本检测体系的替代底物。本检测体系的替代底物N6-甲基-L,L-SDAP以叔丁氧羰基（tert-butyloxycarbonyl，Boc）-L-谷氨酸的叔丁酯为原料，经霍纳尔-沃兹沃思-埃蒙斯（Horner-Wadsworth-Emmons）烯化反应，再以铑(I)(1,5-环辛二烯)(S,S)-乙基-DuPHOS（Rh(I)(COD)(S,S)-Et-DuPHOS）作为手性催化剂进行对映选择性还原制得。研究利用来自流感嗜血杆菌（Haemophilus influenza，HiDapE）的已知DapE抑制剂对该新型茚三酮检测体系进行了验证，包括卡托普利（IC₅₀ = 3.4 [± 0.2] μM）、3-巯基苯甲酸（IC₅₀ = 21.8 [±2.2] μM）、苯基硼酸（IC₅₀ = 316 [± 23.6] μM）以及2-噻吩硼酸（IC₅₀ = 111 [± 16] μM）。基于上述实验数据，该检测方法简便易行且稳定性优异，可适配高通量筛选；这一重要进展为开发可作为新型抗生素的DapE抑制剂的药物化学研究铺平了道路。