Biophysical Studies of Enzymes in the Purine Biosynthetic Pathway of Pathogens for Antibiotic Development

The de novo purine biosynthetic pathway is essential for most organisms. Structure based drug design of critical enzymes of this pathway, such as PurE and PurC, could lead to an effective antibiotic by gaining molecular information. Biophysical and structural studies of these two enzymes have been utilized to gain insight on the active site details, protein stability and conformation, and enzymatic inhibition. PurC from Streptococcus pneumoniae was studied for molecular information of its active site. Our X-ray structure of PurC is the first to contain aspartic acid, a co-substrate. ADP and AIR, similar to PurC substrates ATP and CAIR, were found in the active site. This active site structural information allowed us to propose a reaction mechanism involving critical active site residues. Biochemical and biochemical studies of PurE from Bacillus anthracis in solution were assessed for secondary structure, hydrodynamic radius, thermal denaturation and enzymatic activity under different conditions. The results of these experiments led to the development of a thermal shift assay for the identification of compounds that bind to PurE. The optimized assay was evaluated and applied to high throughput screening of a 25,000-compound library, resulting in detection of more than 500 compounds that bind to PurE. Subsequent studies, by others in the lab, identified several compounds with excellent activity in inhibiting the growth of B. anthracis as well as E. coli, S. aureus and Y. pestis. These small molecules are comparable to ciprofloxacin, an existing antibiotic treatment of anthrax infection. In summary, molecular information of PurC from Streptococcus pneumoniae has given us an active site structure with a proposed mechanism, while biochemical and biophysical studies of PurE from Bacillus anthracis has provided us with a robust high throughput screening method to eventually identify effective antimicrobial compounds.