Graduation Date

Fall 12-15-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Professor Donald R. Ronning

Abstract

Tuberculosis and Cancer treatments share common challenges due to prolonged therapy, the necessity of combination therapy and severe side effects. Characterization of novel drug targets and identification of new drugs modulating such targets within the context of multidrug treatments will contribute to overcoming current therapeutic challenges.

Tuberculosis (TB) is one of the challenging diseases to treat due to the persistence of Mycobacterium tuberculosis (Mtb) in the dormant phase. Current TB drugs are more effective toward actively dividing bacteria and less effective toward dormant bacteria. The metabolic transition between actively dividing bacteria and dormant bacteria is driven by pivotal metabolic enzymes that respond immediately to changes in the environment. Such enzymes are termed metabolic switches. Modulation of these metabolic switches forcing bacterial metabolism to maintain active bacterial division is a promising approach to enhance Mtb susceptibility to current TB drugs and decrease treatment time.

The overexpression of Citrate Synthase (CitA) in Mtb promotes bacterial growth and division under hypoxic conditions. The first study described here focuses on understanding the structure and function of CitA to afford small molecule discovery efforts ensuring high CitA activity under all growth conditions. The results suggest that pyruvate allosterically regulates Mtb CitA instead of the typical allosteric regulator of citrate synthases, NADH. This study also highlights the functional impact of residue C143 modification, which is known to undergo mycothiolation upon oxidative stress. We show that modification of C143 fully inhibits CitA enzymatic activity. As the protein cap covering the pyruvate binding pocket harbors C143, these data illustrate a direct connection between this region of CitA and enzymatic activity suggesting it is a CitA regulatory domain that can be targeted with small molecule compounds capable of ensuring that CitA retains enzymatic activity.

The Trehalose synthase (TreS) maintains high enzyme activity during an Mtb infection, which facilitates a catalytic shift in Mtb to promote metabolic changes that encourage actively dividing bacteria to transition to a dormant phase. The second study focuses on structural and biophysical approaches to understand the inhibition of the TreS enzyme by trehalose analogs. The solved Cryo-EM structure of TreS in complex with 6- TreAz reveals a conformational change that supports binding of the trehalose analog and intermediate retention as expected for this type of isomerase reaction.

All cells consist of DNA damage checkpoints to minimize or eliminate DNA damage by activating repair mechanisms to maintain genomic integrity. Unrecoverable DNA damage results in apoptosis of the cells. Inhibition of DNA damage repair to accumulate damaged DNA and induce cell death is an intriguing approach for the treatment of Cancer. The third project focuses on the inhibition of RHNO1 binding to the RAD9-HUS1-RAD1 complex. This complex recruits various proteins to sites of double-strand DNA breaks to promote repair. In this study, we developed a fluorescence polarization assay that facilitates high-throughput screening to identify protein-protein interface inhibitors of the RAD1/RHNO1 complex. A modest inhibitor was identified by screening a small library of compounds derived from a molecular docking study. The developed assay will afford the identification of more efficacious inhibitors and determine the Ki for further validation.

Comments

2023 Copyright, the authors

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