Ribonucleotide Reductase, (RNR) is a multi-subunit enzyme that catalyzes the rate-limiting step of de novo precursor DMA synthesis by converting nucleotide diphosphates to deoxynucleotide diphosphates. Crucial for rapidly proliferating cells, RNR is a target for anti-cancer and anti-viral therapy. Recently, we solved the first X -ray structures of eukaryotic ribonucleotide reductase 1 from Saccharomyces cerevisiae. The twelve structures solved reveal two new domains, the structural basis for substrate selection in eukaryotes and the mode of binding of the anti-cancer drug Gemcitabine and ribonucleotide reductase based peptides;the latter complex provides a framework for designing anti-cancer drugs that disrupt the enzyme's multi-subunit assembly. We have expressed and purified the human Rnr1 for crystallization. Until the mammalian Rnr1 structure is solved, the yeast structures provide an invaluable starting point for designing inhibitors that target Rnr1. We will solve the X-ray structures of Rnr1 complexed with inhibitors that target the effector sites (Eg. Clofarabine) and catalytic site (3NUDP) as well as bifunctional molecules that target both effector and catalytic sites (dGTP-ADP linked covalently), and peptidomimetics that disrupt RNR assembly. These structures will provide a starting point for knowledge based drug design. As a proof of principle we have shown that a mouse Rnr2 based inhibitor binds yeast Rnr1. The compounds have been provided by our collaborators Dr. Barry Cooperman (UPENN), Dr. Vasha Ghandi (MD Anderson), and Dr. Willam Parker at the Southern Research Institute. We will also study how SmM binds Rnr1 using cross-linking, limited proteolysis, surface mapping in tandem with mass spectrometry. The structures of intact Sml1-Rnr1 and Rnr1-Sml1peptide complexes will be determined. We will use site-directed mutagenesis to confirm the MS results on the Sml1 binding site, identify the function of the newly identified insert domains and the role of crucial residues identified by our structures that confer substrate specificity. Finally, we will investigate the structural basis of the synthetically lethal mismatch repair mutants identified by Dr. Julian Simon at the Fred Hutchninson Cancer Center. The work proposed will further our understanding on how the vital enzyme ribonucleotide reductase is regulated and the structure based design of inhibitors against it will be important for the therapeutic intervention of proliferative diseases such as cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA100827-09
Application #
8113439
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Knowlton, John R
Project Start
2003-06-12
Project End
2013-07-31
Budget Start
2011-08-01
Budget End
2013-07-31
Support Year
9
Fiscal Year
2011
Total Cost
$258,247
Indirect Cost
Name
Case Western Reserve University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Misko, Tessianna A; Wijerathna, Sanath R; Radivoyevitch, Tomas et al. (2016) Inhibition of yeast ribonucleotide reductase by Sml1 depends on the allosteric state of the enzyme. FEBS Lett 590:1704-12
Ahmad, Md Faiz; Huff, Sarah E; Pink, John et al. (2015) Identification of Non-nucleoside Human Ribonucleotide Reductase Modulators. J Med Chem 58:9498-509
Ahmad, Md Faiz; Dealwis, Chris G (2013) The structural basis for the allosteric regulation of ribonucleotide reductase. Prog Mol Biol Transl Sci 117:389-410
Ahmad, Md Faiz; Wan, Qun; Jha, Shalini et al. (2012) Evaluating the therapeutic potential of a non-natural nucleotide that inhibits human ribonucleotide reductase. Mol Cancer Ther 11:2077-86
Ahmad, Md Faiz; Kaushal, Prem Singh; Wan, Qun et al. (2012) Role of arginine 293 and glutamine 288 in communication between catalytic and allosteric sites in yeast ribonucleotide reductase. J Mol Biol 419:315-29
Wan, Qun; Ahmad, Md Faiz; Fairman, James et al. (2011) X-ray crystallography and isothermal titration calorimetry studies of the Salmonella zinc transporter ZntB. Structure 19:700-10
Fairman, James Wesley; Wijerathna, Sanath Ranjan; Ahmad, Md Faiz et al. (2011) Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization. Nat Struct Mol Biol 18:316-22
Wijerathna, Sanath R; Ahmad, Md Faiz; Xu, Hai et al. (2011) Targeting the Large Subunit of Human Ribonucleotide Reductase for Cancer Chemotherapy. Pharmaceuticals (Basel) 4:1328-1354
Sun, Dianqing; Xu, Hai; Wijerathna, Sanath R et al. (2009) Structure-Based Design, Synthesis, and Evaluation of 2'-(2-Hydroxyethyl)-2'-deoxyadenosine and the 5'-Diphosphate Derivative as Ribonucleotide Reductase Inhibitors. ChemMedChem 4:1649-56
Bennett, Brad C; Wan, Qun; Ahmad, Md Faiz et al. (2009) X-ray structure of the ternary MTX.NADPH complex of the anthrax dihydrofolate reductase: a pharmacophore for dual-site inhibitor design. J Struct Biol 166:162-71

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