Inhibition of mammalian cell proliferation by genetically selected peptideaptamers that functionally antagonize E2F activity

The p16-cyclin D-pRB-E2F pathway is frequently deregulated in human tumors. This critical regulatory pathway controls the G1/S transition of the mamma lian cell cycle by positive and negative regulation of E2F-responsive genes required for DNA replication. To assess the value of the transcription fac tors E2Fs as targets for antiproliferative strategies, we have initiated a program aiming to develop inhibitors targeting specifically these proteins in vitro and in vivo. The cellular activity of E2F is the result of the het erodimeric association of two families of proteins, E2Fs and DPs, which the n bind DNA. Here, we use a two hybrid approach to isolate from combinatoria l libraries peptide aptamers that specifically interact with E2Fs DNA bindi ng and dimerization domains. One of these is a potent inhibitor of E2F bind ing activity ill vitro and in mammalian fibroblasts, blocks cells in G1, an d the free variable region from this aptamer has the same effect. Our exper iments argue that the variable region of this aptamer is structured, and th at it functions by binding E2F with a motif that resembles a DP heterodimer ization region, and blocking E2F's association with DP, Those results show that cell proliferation can be inhibited using genetically-selected synthet ic peptides that specifically target protein-protein interaction motifs wit hin cell cycle regulators. These results also emphasize the critical role o f the E2F pathway for cell proliferation and might allow the design of nove l antiproliferative agents targeting the cyclin/CDK-pRB-E2F pathway.