Development of Novel Topoisomerase and Replication Initiator Assays

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DESCRIPTION (provided by applicant): The reliable measurement of enzyme activity is a cornerstone of biochemistry. Biochemical assays are used in nearly all studies of proteins to answer fundamental questions about protein/ligand interactions, catalytic mechanism, and the control of biological processes. Assays also comprise a critical component of high-throughput screens for small molecule agents that disrupt or modulate function. Compounds isolated or re-engineered from such efforts allow researchers to analyze enzyme action in vitro and in vivo, and provide essential therapeutics for the treatment of disease. To serve as a reporter for high-throughput screens, an assay must satisfy stringent technical demands. An inability to meet these requirements - e.g., low cost, high sensitivity and reliability, ease of use, and portability to 96-well or higher formats - can significantly impede the identification of small molecule agonists and antagonists. This problem is particularly acute if the type of reaction catalyzed by a target enzyme is difficult to characterize without the use of classic methods such as gel electrophoresis or radiolabeling. Many proteins responsible for copying and packaging genomic information fall into this class of challenging targets. DNA replication is a defining event in cell proliferation that has long attracted the attention of the basic research sector. Replication factors also have served as valuable targets for inhibitors with beneficial antibiotic, antiviral, or anti-tumorigenic properties. Unfortunately, resistance to and/or toxic side effects from these agents require that new drugs be found. There is likewise a pressing need to develop new small molecule effectors of replication enzymes for probing molecular mechanism. The identification of such agents requires screening chemical libraries against appropriate reporter assays via high-throughput approaches. The goal of this application, developed in synergy with program announcement PA-07-320, is to take advantage of recent structural and conceptual breakthroughs from my lab and the field to design and benchmark a suite of new high-throughput assays for two essential bacterial enzymes: type II topoisomerases and the replication initiator, DnaA. We expect that this effort will not only provide much-needed new tools for studying these proteins, but also will pave the way for small molecule screens to identify new probes for biochemical function and novel leads for antibiotic-discovery effort. PUBLIC HEALTH RELEVANCE: The pathway to identifying drugs for the treatment of disease requires specialized biochemical assays that report on specific enzymatic activities and their inhibition by clinical agents. This effort will develop several new assays that monitor key reactions carried out by two essential bacterial proteins in support of DNA replication and cell growth. These assays will serve as valuable tools for high-throughput screening programs aimed at identifying new antibiotics for the treatment of acute and chronic infections.