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Access Type

WSU Access

Date of Award

January 2020

Degree Type

Thesis

Degree Name

M.S.

Department

Pharmaceutical Sciences

First Advisor

Steven M. Firestine

Abstract

ABSTRACT

THE DEVELOPMENT AND APPLICATION OF A FLUORESCENCE BASED ASSAY FOR BACTERIAL N5-CAIR MUTASE

by

MARCELLA F. SHARMA

May 2020

Advisor: Dr. Steven M. Firestine

Major: Pharmaceutical Sciences

Degree: Master of Science

The lack of development in the antimicrobial drug pipeline has led us to focus on novel drug targets. The bacterial enzymes of the de novo purine biosynthesis pathway serve as a novel target for antimicrobial drug discovery. Within this pathway, there is a divergence between humans and bacteria in the step that converts 5-aminoimidazole ribonucleotide (AIR) to carboxy-5-aminoimidazole ribonucleotide (CAIR). Humans require only one enzyme, AIR carboxylase to accomplish this conversion while bacteria utilize two distinctive enzymes, N5-CAIR synthetase and N5-CAIR mutase. Unfortunately, a robust activity-based assay does not exist for N5-CAIR mutase. To address this problem, a novel, fluorescence based activity screen was developed in the Firestine lab that is based on the reaction of enzymatically produced AIR with fluorescein-labeled isatin (I-FITC). Reaction of AIR with I-FITC results in a large increase in the fluorescence signal. The assay is robust (Z’ 0.79) and we have used it to conduct a small screen of 80 fragments. No reproducible hits were identified. A high-throughput screen was conducted on N5-CAIR mutase using the 2,400 compound Microsource Spectrum library. Again, no verified hits were identified. Future studies will explore larger, focused fragment library to identify inhibitors of N5-CAIR mutase.

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