Core C: Enzymology Core

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ABSTRACT The overarching goal of Core C is to support READDI-AC discovery efforts in collaboration with Discovery Core B and MedChem Core D by validation, characterization, and optimization of compounds in enzyme assays and structural studies, and to provide the Research Projects with detailed mechanisms of action and mechanistic insights to mitigate potential problems with resistance conferring mutations. Enzymology Core C includes seven internationally recognized laboratories that cover complementary expertise at the interfaces of virology, biochemistry, biophysics and structural biology. Three specific aims that describe the central activities of Enzymology Core C: Aim 1: Develop assays to support hit discovery efforts by Discovery Core B. Key activities include the expression and purification of enzymes that the Research Projects and Discovery Core B have selected as targets. Enzymology Core C will support the development of protocols for the expression and purification of new enzyme targets and their corresponding assays to monitor activity and inhibition. The translation of enzyme assays into high density formats to facilitate high throughput screening by Discovery Core B is a second responsibility. Aim 2. Structural Biology to guide hit and lead optimization. Enzymology Core C will focus on solving 3D structures of polymerases and RNA helicases that are currently not available to the scientific community. Priority will also be given to solving the structures of flavivirus and alphavirus polymerases in complex with the nucleotide and non-nucleotide analog inhibitors to provide detailed information on the inhibitor binding sites. Aim 3. Support the Projects in detailed mechanism of action studies for lead compounds. Functional assays will be employed to assess the inhibitory activity against the biologically relevant enzyme target, such as measurements of the half maximal inhibitory concentration to determine compound potency. Promising hits and leads will also be tested against homologous enzymes within the protein family as well as distant enzymes to assess breadth and the potential for off-target activity. Enzymology Core C will provide an array of state-of-the-art biochemical/biophysical approaches to provide MedChem Core D and the Projects with information on the mechanisms associated with inhibition and resistance.