Practical Spectrophotometric Assay for the \u3cem\u3edapE\u3c/em\u3e-Encoded \u3cem\u3eN\u3c/em\u3e-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

A Racial/Ethnic Performance Disparity on the Facial Recognition Test

The Facial Recognition Test is a widely used psychometric instrument for assessing visuoperceptual functioning. Only two prior studies have examined the effects of race/ethnicity on this test. Given that the United States has become more culturally diverse since the creation of the test, it is important to re-visit the effects of this demographic variable on performance. Participants were 75 males and 75 females between the ages of 18 and 43 years (M = 21.91, SD = 5.33). Racial/ethnic categories utilized by the U.S. Census Bureau were equally represented. No gender differences were observed. The race/ethnicity main effect was significant. The gender x race/ethnicity interaction was not significant. The data revealed a clear racial/ethnic performance disparity on the Facial Recognition Test

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

Hydrolysis of L,L-SDAP and analogs by <i>Hi</i>DapE.

<p>L,L-SDAP (<b>1a</b>) and analogs <i>N</i>⁶-methyl SDAP (<b>1b</b>) and <i>N</i>⁶-acetyl-SDAP (<b>1c</b>) with formation of hydrolysis products succinate (<b>2</b>) and L,L-diaminopimelic acid derivatives (<b>3a-c</b>). Enzyme-mediated hydrolysis was not observed for <i>N-</i>acetyl analog <b>1c</b> which would afford <b>3c</b>.</p

Circular dichroism thermal denaturation study of <i>Hi</i>DapE.

<p>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 <i>°</i>C, and (B) percent secondary structure remaining with continued heating at 80 <i>°</i>C.</p

Asymmetric synthesis of <i>N</i>⁶-methyl-L,L-SDAP 1b.

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

Enzyme saturation curve of <i>Hi</i>DapE using 2 mM of <i>N</i>⁶-methyl-L,L-SDAP as the substrate (50 mM HEPES buffer at pH 7.5).

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

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

<p>Glutamic acid control standard curve for the development of ninhydrin and primary amine in 50 mM HEPES buffer at pH 7.5.</p