The proposed project joins the synthetic and pharmacological expertise of the Kerwin group (Division of Medicinal Chemistry, College of Pharmacy) with the analytical expertise of the Brodbelt group (Department of Chemistry and Biochemistry) and the molecular biology expertise of the Ellington group (Department of Chemistry and Biochemistry, Institute of Cellular and Molecular Biology) to provide a strategy for the design and characterization of DNA-interactive agents. The overall goal of the project is to develop the capabilities of electrospray ionization mass spectrometry (ESI-MS) for characterization of metal-mediated drug/DNA and drug/quadruplex interactions along with rapid in vitro selection methods to provide insights into the processes by which drug-metal and drug-drug association affect drug/DNA binding, selectivity and enzyme inhibition. Specific objectives include: 1) the design and synthesis of metal-mediated DNA interactive compounds and G-quadruplex DNA binding compounds with a focus on addressing specific structural aspects of metal-mediated drug/DNA binding and drug/G-quadruplex DNA binding and for use as directed libraries for specific ESI-MS based screening applications. Individual compounds and libraries of analogs of the Mg 2+ ion-mediated DNA binding antitumor agents UK-1 and A62176, perylene diimides, and benzannulated UK-1and A-62176 analogs will be prepared by solution and solid-phase methods. 2) the development and application of ESI-MS and in vitro selection methods for investigation of metal-mediated drug/DNA complexes and drug/quadruplex complexes. ESI-MS and spectrophotometric/isothermal calorimetric measurements of binding affinities and selectivities will be undertaken. Energy-variable collision activated dissociation methods will be used to investigate the fragmentation of drug/DNA complexes, and an array of novel gas-phase footprinting methods will be developed to map the binding sites. Rapid selection methods based on affinity capture and release involving immobilized DNA or drugs will be developed. 3) the characterization of the consequences of metal-mediated DNA binding of drug/DNA and drug/G-quadruplex complexes to establish functional correlates of DNA binding in biologically relevant systems. One aspect involves determining the ability of the metal-mediated DNA binding agents and G-quadruplex DNA ligands to inhibit specific DNA processing enzymes. Separate SV40 large T antigen double-stranded DNA and Gquadruplex DNA helicase inhibition studies will be carried out on select DNA ligands. Human topoiomerase II inhibition studies will be carried out on the UK-1 and A-62176 analogs in order to establish the relationship between metal ion-mediated DNA binding and enzyme inhibition. Human telomerase inhibition assays will be performed with the perylene diimide analogs using DNA primers. The anticancer and antibacterial effects of UK-1 and A-62176 analogs will be examined in a range of human cancer cell lines and Gram negative and Gram positive bacteria.
Showing the most recent 10 out of 33 publications
