Our work is directed toward developing peptides, peptide mimetics, and bivalent nucleotides that will be potent and selective inhibitors of mammalian ribonucleotide reductase (mRR), a type 1 RR, and by extension, effective inhibitor of tumor cell proliferation and viral replication. The reaction catalyzed by RR, the reduction of nucleoside diphosphates (NDPs) to deoxynucleosides diphosphates (dNDPs), is the rate-limiting step in the de novo synthesis of dNTPs, and hence of DNA, and as such is a clear target for therapeutic agents directed against these diseases. Active RR depends on the association of two different subunits, mR1 and mR2. The oligomeric structures of the active enzyme (mR1mR2)n is dependent on allosteric effectors, with n ranging from two to six. The C-terminal peptide of mR2 inhibits RR activity. Thus, a lead molecule for an mRR inhibitor is the acetylated heptapeptide AcFTLDADF, denoted P7, corresponding to the C-terminus of mR2. Recently, we have found that a cyclic peptide, mimicking P7 and denoted cycP7, and the simple protected amino acids FmocTrp and FmocPhe, are also effective inhibitors. In addition, within the R1 dimer, the active site of RR lies close to an allosteric specificity site, raising the possibility that these sites can be effectively bridged by bivalent nucleotide inhibitors. We propose to continue efforts begun in the current grant period to develop peptide-based and bivalent nucleotide inhibitors that will not only have high affinity for mR1 but also be effective inhibitors in tissue cell culture. Such molecules would be promising candidates for new therapeutic agents. In carrying out this work we will make extensive use of trNOE NMR and modeling, employing a feedback logic to the design of new peptides and peptidomimetics and the evolving model for the mR1 peptide binding site. We will also attempt to obtain crystals of mR1 and of the mR1.mR2 complex suitable for high resolution structure determination as an aid to inhibitor design. Finally, high affinity peptides and peptidomimetics will be converted into inhibitors of tumor cell proliferation and viral replication in cell culture by conjugation with carriers mediating delivery into intact cells. Such carriers, along with bivalent nucleosides, and phosphorylated bivalent nucleoside prodrugs, will permit in vivo testing of our compounds.
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