Doctor of Philosophy (PhD)
Donald R. Ronning
Tuberculosis, Staphylococcus aureus
Intrinsic drug resistance in bacteria predates the clinical use of antibiotics. Additionally, heterogeneous metabolic states and the ability of many bacteria to form biofilms further enhances bacterial survival during infection and exacerbates treatment challenges. This necessitates the identification of novel drugs against new drug targets. These studies focus on the development of compounds targeting enzymes in Mycobacterium tuberculosis (Mtb) and Staphylococcus aureus that avoid intrinsic antibiotic defenses. The Mtb-encoded homoserine transacetylase enzyme (HTA) catalyzes the first step of the methionine biosynthesis pathway that produces methionine and S-adenosyl methionine. These compounds are shown to be essential for both dormant and actively dividing Mtb. The presented results focus on understanding the structure-function relationship of this enzyme as well as identifying chemotypes that afford the development of novel HTA inhibitors. Mycothiol is the major cytosolic, thiol-containing redox modulator in mycobacteria. The Mycothiol S-transferase enzyme (MST) proposed to catalyze transfer of Mycothiol (MSH) to electrophilic acceptors. The MST-catalyzed reaction initiates the multistep processing of xenobiotics leading to drug efflux. The presented results identified MST as a metalloenzyme and determined X-ray crystal structures highlight the metal-dependent binding of MSH and offer tantalizing clues about the catalytic mechanism and possible identification of MSH acceptors. The S. aureus D-alanine D-alanine ligase enzyme (Ddl) synthesizes the D-alanine D-alanine dipeptide and is therefore essential for cell wall biosynthesis. Steady-state enzyme kinetics and co-crystallization experiments were used to understand the known but unpublished inhibitory effect caused by high concentrations of carboxylic acids in S. aureus, confirming Ddl as one of the targets of these compounds. Inhibition of this enzyme by D-cycloserine causes cell wall disruption leading to bacterial death. This presents identified interactions that support D-cycloserine inhibition by determining the S. aureus Ddl/D-cycloserine co-crystal structure. This structure will afford structure-guided drug discovery of novel Ddl inhibitors that possess higher potency and minimal toxicity. Taken together, the initial functional and structural insights open new perspectives for novel drug development and modification of current drugs to reduce toxicity.
Jayasinghe Arachchige, Yahani Pankaja, "Structural and Biochemical Studies of Three Enzymes Involved in Amino Acid Metabolism to Develop Novel Drugs" (2022). Theses & Dissertations. 719.
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