Identification of Novel Lead Inhibitors for Bacillus anthracis Adenylosuccinate Synthetase
thesisposted on 2016-11-05, 00:00 authored by Siddharth Jindal
Anthrax is an infectious disease caused by the bacterium Bacillus anthracis. It came into public notability in 2001 during the bioterrorism attack in the United States. Because of the potential threat and the emergence of antibiotic resistance B. anthracis, novel antibiotics are needed to overcome this problem. One way is to develop antibiotics that inhibit novel targets. One such target is the de novo purine biosynthesis pathway. The enzyme, Adenylosuccinate synthetase (PurA), catalyses the first committed step in the synthesis of adenosine, an important component for bacterial purine biosynthesis pathway. This metallo-enzyme is a homo-dimer which catalyses formation of adenylosuccinate from IMP in presence of GTP and aspartate and releases an inorganic phosphate (Pi). PurA is found in all life forms with few exceptions and because of its essential role in the biosynthesis of adenine nucleotides and ultimately nucleic acids, it is potentially an excellent target for anti-bacterial drug development. The goal of this project is to identify inhibitors that exhibit antibacterial activity against the Category A pathogen, B. anthracis. We used combination of different approaches to identify novel lead inhibitors against B. anthracis adenylosuccinate syntheatse. The purA gene was cloned and the enzyme was expressed and purified. MESG assay was employed to determine the substrate kinetics for PurA. Subsequently, malachite green assay was optimized for high throughput screening of the Chembridge and the Life Chemicals library totaling 75,000 compounds to identify inhibitors against PurA. The hits were validated and assay interference from the hits from high throughput screening was corrected using the artifact assay. To confirm the hits from artifact assay, an independent binding assay was performed to determine the binding and equilibrium dissociation constant of these compounds. Here we present data describing the optimization of the assays and various strategies for identification and characterization of inhibitors of PurA. We have identified two compounds as promising hits for further development in a hit-to-lead optimization process. Both these compounds have good inhibition in HTS, a good MIC and bind to the enzyme.