IMP Dehydrogenase and the Hydra of Cancer Chemotherapy

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DESCRIPTION (provided by applicant): lnosine-5'-monophosphate dehydrogenase (IMPDH, IMP:NAD oxidoreductase, E.G. 1.2.1.14) catalyzes the rate-limiting step in guanine nucleotide biosynthesis, the oxidation of IMP to xanthosine monophosphate (XMP). IMPDH has a central role in DNA and RNA syntheses, in G-protein mediated signal transduction, and in intermediary metabolism. IMPDH is a target for anticancer, anti-viral, immunosuppressive and anti-microbial chemotherapeutic inhibitors, but is an activator of other drugs. An integrated combination of kinetic, spectroscopic and genetic methods will be used to understand the catalytic mechanism, including the contribution of protein flexibility, and the metabolic role of the evolutionary conserved subdomain of unknown function. The catalytic mechanism of human IMPDH will be characterized by kinetic and UV spectroscopic methods. The reaction of the anticancer agent 6-mercaptopurine ribotide (6MPRT), for which IMPDH catalyzes an essential metabolic activation, will be emphasized. How 6MPRT affects inhibition by clinically used inhibitors that bind at the NAD site will be determined. The reverse of the IMPDH reaction will be investigated to provide insight into the interconversion of XMP and the covalent enzyme-XMP* intermediate. Whether the monovalent cation activator K+, and NAD binding site inhibitors, activate oxygen isotope exchange between XMP and water or restrict water accessibility will enhance the understanding of this reaction. The contribution of protein dynamics to the catalytic efficiency of this flexible enzyme will be determined from the viscosity dependence of the reaction rates. How ligands modulate the flexibility of the protein will be assessed by site-directed spin labeling and EPR spectroscopy. The metabolic role of the subdomain will be determined from the in vivo consequences of deletion of the subdomain within the chromosome of the E. coli model organism. A complementary study will employ a computational docking screen between the crystal structure of the domain and metabolite structures. Interactions with potential ligands will be tested by NMR and for modulation of IMPDH activity. These results will reveal the inner working of an essential enzyme and whether there is promise for enhancing clinical efficacy through combinations of known drugs or the discovery of novel subdomain ligands.