Presenter Information

Elliot GildFollow

Major

Chemistry

Anticipated Graduation Year

2020

Access Type

Open Access

Abstract

The previously most widely used DapE assay monitors amide bond cleavage of the L,L-SDAP substrate spectrophotometrically at 225 nm.1 This assay is straightforward and reliable for structurally simple inhibitors, but it cannot be used to test potential inhibitors that absorb strongly in the UV region, precluding its use in testing many preferred medicinal chemistry leads and analogs. Ninhydrin-based assays provide ease of application and reliability to detect primary amino groups such as that formed upon the hydrolysis of L,L-SDAP. We synthesized N6-methylated and N6-acetylated L,L-SDAP derivatives to prevent ninhydrin side reactions in the assay and also tested them as viable substrates for the Haemophilus influenza DapE (HiDapE) used in the assay. The data showed that the N-methylated derivative was both less reactive with ninhydrin and also was a better suited substrate for the HiDapE. In addition to the development of the assay itself we have also used a high throughput coupled screening to identify a number of lead HiTS (Figure 2) that show binding potential to DapE. These compounds will be tested as inhibitors themselves, while also being the basis for potential derivatives. The synthesis of analogs of these derivatives will be guided by structure activity relationships (SAR) as well as Lipinski’s rule of five, a general guide for predicting orally activity of potential drugs.

Faculty Mentors & Instructors

Dr. Daniel Becker

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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Spectrophotometric assay for dapE-encoded N-succinyl-L,L-di aminopimelic acid desuccinylase, a potential antibiotic target

The previously most widely used DapE assay monitors amide bond cleavage of the L,L-SDAP substrate spectrophotometrically at 225 nm.1 This assay is straightforward and reliable for structurally simple inhibitors, but it cannot be used to test potential inhibitors that absorb strongly in the UV region, precluding its use in testing many preferred medicinal chemistry leads and analogs. Ninhydrin-based assays provide ease of application and reliability to detect primary amino groups such as that formed upon the hydrolysis of L,L-SDAP. We synthesized N6-methylated and N6-acetylated L,L-SDAP derivatives to prevent ninhydrin side reactions in the assay and also tested them as viable substrates for the Haemophilus influenza DapE (HiDapE) used in the assay. The data showed that the N-methylated derivative was both less reactive with ninhydrin and also was a better suited substrate for the HiDapE. In addition to the development of the assay itself we have also used a high throughput coupled screening to identify a number of lead HiTS (Figure 2) that show binding potential to DapE. These compounds will be tested as inhibitors themselves, while also being the basis for potential derivatives. The synthesis of analogs of these derivatives will be guided by structure activity relationships (SAR) as well as Lipinski’s rule of five, a general guide for predicting orally activity of potential drugs.