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Abstract of [Han_phdthesis]

The Application of X-ray Crystallography Towards the Design of Novel Inhibitors of MurA and CDK2

Huijong Han

Ph.D. Thesis, University of Kansas (2008)

Infectious diseases and cancers are the second and third largest causes of death worldwide. UDP-N-acetyl-glucosamine (UNAG) enolpyruvyl transferase (MurA) and Cyclin-dependent kinases (CDKs) are proven as antibiotic and anticancer targets, respectively. MurA belongs to the enolpyruvyl transferase family of enzymes. MurA catalyzes the first committed step in the biosynthesis of cell wall peptidoglycan, and is the target of fosfomycin, a naturally occurring broad-spectrum antibiotic. Ever increasing resistance of bacteria to fosfomycin has placed an emphasis on the identification and characterization of novel MurA inhibitors. Knowledge of the detailed enzymatic mechanism is essential for the discovery of potent MurA inhibitors. The studies on the mutant MurA enzymes Arg91Lys, Asp123Ala, Arg120Ala, and Cys115Asp, revealed key catalytic residues and residues important for the conformational changes in the enzymatic reaction. Several new inhibitors of MurA were identified by High-Throughput Screening (HTS), and kinetically characterized. It appears that most of these compounds bind to the open conformation of MurA, and thus the inhibitor binding site is largely solvent exposed. These results suggest that MurA inhibitors need to be designed to induce the open-closed transformation of the enzyme, like the natural substrate UNAG. Such inhibitors should be much more potent than the inhibitors discovered in this work. CDK2 plays a critical role in the G1- to S-phase checkpoint of the cell cycle. Only a few drugs targeting CDK2 are in clinical trials, thus there is a need for the discovery of novel CDK2 inhibitors. Six CDK2 inhibitor scaffolds were identified by HTS, and the molecular modes of action of four of them were thoroughly characterized by steady-state kinetics and crystallography. Structure-Activity Relationship (SAR) analysis of the four scaffolds gave rise to the design of analogs with excellent potency. In addition, computational studies were performed, and novel CDK2 inhibitor scaffolds were designed. The selectivities and cytotoxic properties of these inhibitors are not known yet.

Tags: MurA, CDK2, KU
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