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.

Indoline-6-Sulfonamide Inhibitors of the Bacterial Enzyme DapE

“…This structure uncovered the role of His194B of the opposite peptide chain in the dimeric enzyme, which moves 10.1 Å to provide a key H-bond in the active site, activating the substrate for enzymatic cleavage [ 14 ]. This new insight into the enzymatic mechanism also explains the observed inactivity of monomeric DapE constructs [ 10 ]. This products-bound DapE structure has enabled further refinement of a mechanistic hypothesis for amide bond cleavage by DapE enzymes, supported by our products-bound transition state modeling (PBTSM) approach [ 14 , 15 ], which in turn will facilitate inhibitor identification.…”

“…To conveniently measure the inhibitory potency of test compounds against DapE, we recently developed a ninhydrin-based assay employing the unnatural but well-tolerated substrate N 6 -methyl- l,l -SDAP ( 1b ) [ 10 ]. Enzymatic cleavage of 1b affords primary amine product 3b ( Figure 1 ), which can be quantified spectrophotometrically following treatment with ninhydrin.…”

Aspectos estructurales y funcionales de la N-Succinil-L, L-diaminopimelato desuccinilasa, una enzima clave para el crecimiento bacteriano y un blanco para el control antimicrobiano

“…NSDAP con marca radioactiva (Lin, Myhrman, Schrag & Gelb, 1988), sin embargo, este método es aún más costoso y requiere una variedad de pasos para lograr medir la reacción, por lo que en estudios cinéticos es impráctico. Recientemente se desarrolló un derivado metilado del sustrato NSADP, en donde se bloquea al grupo amino libre logrando medir sin incertidumbre la reacción con ninhidrina (Heath et al, 2018); este derivado se propuso para la identificación de nuevos inhibidores en ensayos de cribado de compuestos, pero su obtención tiene el inconveniente de que su costo es elevado. También se emplea el monitoreo espectroscópico en el UV del enlace amida (Gillner, Armoush, Holz & Becker, 2009b), pero numerosos ligandos e incluso la propia enzima introducen un alto ruido y falsos positivos en la hidrólisis.…”

“…Se han realizado estudios de búsqueda de compuestos que contengan grupos reactivos sobre el Zn; derivado de esto, se han encontrado tioles, ácidos carboxílicos, ácidos bóricos, fosfonatos e hidroximatos (Gillner et al, 2013), (Figura 10). Los derivados de grupos fenilos (Gillner et al, 2009a) y derivados de índoles (Heath et al, 2018) también son agentes potenciales. Dentro de los más sobresalientes resulta el L-captopril, un fármaco que se utiliza para tratar la hipertensión arterial y que inhibe a DapE a concentraciones submicromolares (Starus et al, 2015;Uda & Creus, 2011) así como a otros compuestos relativamente pequeños como el ácido 3-mercaptobenzoico, el ácido fenil bórico y el ácido 2-tiofenobórico (Gillner et al, 2009a).…”

Transporters and Efflux Pumps Are the Main Mechanisms Involved in Staphylococcus epidermidis Adaptation and Tolerance to Didecyldimethylammonium Chloride

“…The dapE gene (SE2220, 3-4.0-fold) encodes succinyl-diaminopimelate desuccinylase (Table 3). This enzyme is involved in the biosynthesis of lysine, an important amino acid in bacterial cell-wall synthesis [57]. SE1079 was highly down-regulated (-16.9-fold) and encodes alanine racemase, which is involved in cell-wall synthesis and growth in S. aureus [58].…”