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Access Type
WSU Access
Date of Award
January 2024
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Pharmaceutical Sciences
First Advisor
Steven M. Firestine
Abstract
ABSTRACT
TARGETING UNDEREXPLORED BACTERIAL FOLATE AND DE NOVO PURINE BIOSYNTHETIC PATHWAYS FOR THE DEVELOPMENT OF NOVEL ANTIMICROBIAL AGENTS
by
MARCELLA F. SHARMA
August 2024
Advisor: Dr. Steven M. Firestine Major: Pharmaceutical Sciences Degree: Doctor of Philosophy One mechanism to develop novel antibacterial agents is to target critical pathways that are unique to bacteria. Two such pathways are the focus of this work: de novo purine biosynthesis, and para-aminobenzoic acid (PABA) biosynthesis. Previous studies have shown that both pathways are critical for bacterial growth and survival. De novo purine biosynthesis is responsible for the synthesis of new purine nucleotides. The pathway is present in both eukaryotes and prokaryotes; however, there is a divergence in the pathway between human and bacteria, yeast, and fungi. This difference is centered on the conversion of 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-5- aminoimidazole ribonucleotide (CAIR). In microbes, CAIR synthesis requires two unique enzymes, N5-CAIR synthetase (PurK) and N5-CAIR mutase (PurE); however, humans accomplish the same conversion using a single enzyme AIR carboxylase. While strong evidence has revealed the critical role PurE plays in the survival of bacteria, there are no drug-like and selective inhibitors of the enzyme. To address this problem, a 4,500- fragment library was screened using an optimized thermal binding assay against the E. coli enzyme. Six fragments that showed dose-dependent increases in melting temperature were identified. Five of the six fragments showed inhibition of PurE activity as measured by the CAIR decarboxylation assay. Interestingly, one of the fragments displayed sigmoidal Michaelis-Menten kinetics suggesting that the compound was an allosteric inhibitor of the enzyme. Surprisingly, the fragments identified in our screen are structurally related to fragments recently found to bind to the human AIR carboxylase enzyme. Folate biosynthesis is a well-known target for antibacterial agents. However, only two enzymes in the pathway have been targeted. A key component of folate biosynthesis is PABA, which is prepared from chorismate. This conversion requires two enzymes, aminodeoxychorismate synthase (ADCS), a heterodimeric protein and aminodeoxychorismate lyase (ADCL). Previous studies have shown that ADCS is critical for bacterial viability, and that inhibition of the enzyme is lethal to bacteria. Unfortunately, existing inhibitors of ADCS have limitations. To identify new inhibitors of ADCS, a novel, optimized thermal binding assay was developed. Using this assay, a diverse library of 2,400 molecules consisting of FDA approved drugs, natural products, and other bioactive agents were screened for binding agents. Twelve compounds were identified to have dose-dependent increases in melting temperature with Kd values ranging from 4.5 μM to 58 μM.
Recommended Citation
Sharma, Marcella, "Targeting Underexplored Bacterial Folate And De Novo Purine Biosynthetic Pathways For The Development Of Novel Antimicrobial Agents" (2024). Wayne State University Dissertations. 4063.
https://digitalcommons.wayne.edu/oa_dissertations/4063