Practical spectrophotometric assay for the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase, a potential antibiotic target

Abstract

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.

Fig 1. Hydrolysis of L,L-SDAP and analogs by HiDapE.

L,L-SDAP (1a) and analogs N⁶-methyl SDAP (1b) and N⁶-acetyl-SDAP (1c) with formation of hydrolysis products succinate (2) and L,L-diaminopimelic acid derivatives (3a-c). Enzyme-mediated hydrolysis was not observed for N-acetyl analog 1c which would afford 3c.

Fig 2. Minimized substrate analogs docked and modeled in the HiDapE active site.

The diaminopimelate moiety is depicted in yellow and the succinate in turquoise. A) Native substrate L,L-SDAP, B) N⁶-methyl-L,L-SDAP, and C) N⁶-acetyl-SDAP. The catalytic domain of Chain A is depicted in green, whereas the dimerization domain of Chain B is shown in orange.

Fig 3. Asymmetric synthesis of N⁶-methyl-L,L-SDAP 1b.

Synthetic route for preparation of monomethyl substrate analog as the hydrochloride salt (1b.HCl) via the methyl ester or the trifluoroacetate salt (1b.TFA) via the benzyl ester.

Fig 4. Circular dichroism thermal denaturation study of HiDapE.

The α-helical structures are represented in red and β-sheets are represented in blue with (A) percent secondary structure observed over the course of heating from 20–80 °C, and (B) percent secondary structure remaining with continued heating at 80 °C.

Fig 5. Glutamic acid control standard curve for the development of ninhydrin and primary amine in 50 mM HEPES buffer at pH 7.5.

Fig 6. Enzyme saturation curve of HiDapE using 2 mM of N⁶-methyl-L,L-SDAP as the substrate (50 mM HEPES buffer at pH 7.5).

Optimal enzyme concentration selected for absorbance of primary amine product at or around 1 AU (absorbance unit).