Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Abstract

The bacterial enzyme DapE is a hydrolase in the late stage of the mDAP/lysine biosynthetic pathway that catalyzes the hydrolysis of N-succinyl-L,L-diaminopimelic acid (L,L-SDAP) to succinic acid and L,L-diaminopimelate (DAP). The product DAP is ultimately converted to m-DAP and then to lysine which is used in the construction of proteins, whereas for humans, lysine is an essential amino acid obtained through diet. DapE has been shown to be conserved across many strains of bacteria. The m-DAP produced through the succinylase pathway is used by both gram negative and gram-positive bacteria in the construction of the essential peptidoglycan cell wall. Because of conservation across bacterial strains and its absence in humans, DapE has been identified as an ideal drug target for antibiotics with a new mechanism of action1. Inhibitors of this enzyme may serve as broad-spectrum antibiotics with selective toxicity towards bacterial strains without mechanism-based toxicity in humans. Previously, the enzymatic activity was determined by monitoring the cleavage of the substrate's amide bond at 225 nm.2 The development of a new assay which can be observed at a wavelength of 570 nm through detection of Ruhemann's purple complex produced by reaction of ninhydrin and the hydrolyzed primary amine product of a newly synthesized L,L- SDAP substrate analog enables identification of new inhibitors and establishing SAR. Inhibitory data of ligands identified through a HTS screen and their analogs have been obtained using the new ninhydrin-based enzymatic assay. A High-Throughput Screening (HTS) of over 33,000 compounds licensed from by Chembridge Corporation utilizing the enzyme coupled assay identified five lead ligands as inhibitors with inhibition of >20% at 12 μM of DapE. The results consist of four series of inhibitors: a simple amide with a β-sulfonyl moiety, a difluoromethyl sulfonamide ligand, two N-acyl-6-sulfonamide indolines and a chiral phenyltetrazole thioether. Inhibitory data of ligands identified through a HTS screen and their analogs have been obtained using the new ninhydrin-based enzymatic assay. Herein, we report the synthesis, inhibitory data and SAR of analogs from the last three series of inhibitors as well as a novel synthesis for the formation of 7-sulfonamide indolines as a new series of inhibitors of DapE.

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