Inhibition of ribonucleotide reductase (RR) activity is a well recognized target for rational design of cancer chemotherapeutic and antiviral agents, given the direct role that this enzyme plays in regulating DNA replication as well as its indirect role in regulating other enzymes in the DNA synthesis pathway through its control of the nucleotide pool. In addition, the small subunit of RR, R2, is expressed at a level 4-30 times higher in premalignant breast lesions (noncomedo ductal carcinoma in situ - DCIS) than in normal breast epithelial cells, raising the question of whether R2 expression is correlated with progression to invasive cancer. The work proposed is directed toward developing peptides and peptide mimetics that will be potent and selective inhibitors of mammalian RR (mRR) and, by extension, effective inhibitors of tumor cell proliferation and viral replication. The reaction catalyzed by RR, the reduction of nucleoside diphosphates to deoxynucleoside diphosphosphates, 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 dimers of two different subunits, R12 and R22. The C-terminal peptide of R2 inhibits RR activity. Thus, a lead molecule for an RR inhibitor is the acetylated heptapeptide N-AcFTLDADF, corresponding to the C-terminus of mR2. Starting with this molecule, peptide based inhibitors will be developed that will not only have high affinity for mR1 but also be effective inhibitors in tissue cell culture. Such molecules would be promising candidates as new therapeutic agents. The proposal derives from the PI's recent NMR determination of the structure of N-AcFTLDADF bound to mouse R1, which shares a common reverse turn when compared with the crystallographically determined structure of the C-terminus of E. coli R2 bound to E. coli R1. This commonality leads to the notion that attempts to construct high affinity, peptide-derived inhibitors of type 1 RRs should be based on the common turn structure. The PI will seek to enhance binding affinity and selectivity for mR1 using conformationally constrained peptides and peptide mimetics, through variations in peptide sequence, and by the use of nonnatural hydrophobic amino acids. Homology modeling, based on the x-ray structures of the E. coli R2 C-terminus bound to E. coli R1 will aid in prioritizing among synthetic targets. The PI will also make use of NMR and, as appropriate, X-ray methodologies to rationalize observed binding affinities in terms of peptide structure. Finally, the PI will link high affinity peptides and peptide mimetics with carriers, such as folic acid and enterotoxin B, that allow cellular uptake. These conjugates will be tested as inhibitors of tumor cell proliferation and viral replication in tissue culture.
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