Dihydroorotase from Bacillus anthracis and Staphylococcus aureus

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

The de novo pyrimidine biosynthesis pathway is a potential target for antibacterial drug development and has been implicated in bacterial survival and proliferation during host infections. The third enzyme in the pathway, dihydroorotase (DHOase), is an attractive target for drug design, as it has been previously identified as critical for parasites and certain bacteria. DHOase is divided into two classes, with Class II DHOase from gram-negative strains, such as Escherichia coli, being the most extensively studied. The research presented here focuses on Class I DHOase from two gram-positive bacteria, Bacillus anthracis and Staphylococcus aureus. The first portion of the research focuses on the structure of DHOase from B. anthracis. A major difference between the two classes of DHOase is a long catalytic loop found in Class II counterparts that stabilizes the substrate by hydrogen bond interactions. Class I DHOases lack this catalytic loop as well as residues near the loop site that could contribute to hydrogen bonding. In our studies, the structure of B. anthracis DHOase (BaDHOase) with its substrate in the active site was solved at 2.45 Å and compared with that of E. coli and human. There is little difference between the apo and substrate-bound structure of BaDHOase, and the substrate, carbamyl-aspartate, binds in a similar manner in BaDHOase as it does in E. coli. However, the newer structure provides some insight into substrate-loop interactions, and suggests that the peptide backbone of a glycine residue could be contributing the hydrogen bonding interaction and stabilizing the substrate. The second portion of this research focuses on high-throughput screening of several compound libraries to identify potential lead inhibitors. A robust, end-point assay based on enzymatic activity was optimized for 384-well format, and compared with previously used assay methods for DHOase. Various enzyme assay buffer components were tested and compared between a historically used assay method and our optimized method. Using the optimized assay, 28,000 compounds and fragments were screened against BaDHOase, followed by an orthogonal binding assay using fluorescence thermal shift. The resulting hits were further analyzed by surface plasmon resonance to compare the binding equilibration constant between hits and analogs to develop a preliminary structure-activity relationship map. Last, a smaller high-throughput screen of against a 3,000 fragment library was performed against Staphylococcus aureus DHOase (SaDHOase), using the optimized enzymatic assay and surface plasmon resonance only. The KD was determined for analogs of the resulting hits and a preliminary structure-activity-relationship map was created based on three different scaffolds. Surface plasmon resonance was also used for competition analysis to confirm if the hits were binding in the enzyme active site. Overall, high-throughput screening resulted in one compound each for BaDHOase and SaDHOase, which can be further optimized as a potential inhibitor against gram-positive bacteria.