Targeting a Critical Enzyme to Combat Bubonic Plague
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Yersinia pestis is the bacterium that causes plague infection, which continues to be prevalent in rural areas of the world. Moreover, because pneumonic plague has a high mortality rate even with antibiotic therapy, aerosolized Y. pestis is a powerful bioterrorism weapon. As antibiotic-resistant strains of Y. pestis appear, novel therapeutic targets are increasingly needed to combat this disease. YopH is a phosphatase in Y. pestis that is necessary for infection and was therefore considered a novel therapeutic target for plague. The gene for YopH was synthesized via primer overlap PCR, cloned into the pNIC-Bsa4 plasmid, and expressed in E. coli. The enzyme was purified using Ni-NTA affinity chromatography and FPLC gel filtration. Virtual screening in GOLD and ICM was used to dock large chemical libraries into the active site of an X-ray crystallography structure of YopH in order to identify a compound that can serve as an inhibitor of this enzyme, thereby treating plague infection. Top-scoring ligands were identified and purchased for testing via biochemical assays. The inhibition activity of each ligand was measured, and one compound, of 4-[(2-bromo-4-methylphenyl)amino]-4-oxo-2-butenoic acid (Chembridge ID 5303660), showed potential as an inhibitor. Kinetic experiments and differential scanning fluorimetry suggested that this compound may not be a competitive inhibitor of YopH. YopH was crystallized, but crystals formed in clustered star-like patterns which were not ideal for diffraction. Future experiments will focus on optimizing crystals. The ultimate goal is cocrystallizing YopH with the compound of interest in order to further characterize this relationship and determine if the compound may serve as a novel therapeutic for plague.