Identification of Bacillus anthracis PurE inhibitors with antimicrobial activity

Abstract

N(5)-carboxy-amino-imidazole ribonucleotide (N(5)-CAIR) mutase (PurE), a bacterial enzyme in the de novo purine biosynthetic pathway, has been suggested to be a target for antimicrobial agent development. We have optimized a thermal shift method for high-throughput screening of compounds binding to Bacillus anthracis PurE. We used a low ionic strength buffer condition to accentuate the thermal shift stabilization induced by compound binding to Bacillus anthracis PurE. The compounds identified were then subjected to computational docking to the active site to further select compounds likely to be inhibitors. A UV-based enzymatic activity assay was then used to select inhibitory compounds. Minimum inhibitory concentration (MIC) values were subsequently obtained for the inhibitory compounds against Bacillus anthracis (ΔANR strain), Escherichia coli (BW25113 strain, wild-type and ΔTolC), Francisella tularensis, Staphylococcus aureus (both methicillin susceptible and methicillin-resistant strains) and Yersinia pestis. Several compounds exhibited excellent (0.05-0.15μg/mL) MIC values against Bacillus anthracis. A common core structure was identified for the compounds exhibiting low MIC values. The difference in concentrations for inhibition and MIC suggest that another enzyme(s) is also targeted by the compounds that we identified.

Keywords: 2-Carboxamido-1,3,4-oxadiazole; De novo purine biosynthesis; High-throughput screening; PurE; Sypro Orange; Thermal shift assay.