CELL-CYCLE ARREST AND INHIBITION OF CDK4 ACTIVITY BY SMALL PEPTIDES BASED ON THE CARBOXY-TERMINAL DOMAIN OF P21(WAF1)

Background: A common event in the development of human neoplasia is th e inactivation of a damage-inducible cell-cycle checkpoint pathway reg ulated by p53. One approach to the restoration of this pathway is to m imic the activity of key downstream effecters, The cyclin-dependent ki nase (Cdk) inhibitor p21(WAF1) is one such molecule, as it is a major mediator of the p53-dependent growth-arrest pathway, and can, by itsel f, mediate growth suppression. The primary function of the p21(WAF1) p rotein appears to be the inhibition of G1 cyclin-Cdk complexes, Thus, if we can identify the region(s) of p21(WAF1) that contain its inhibit or activity they may provide a template from which to develop novel an tiproliferative drugs for use in tumours with a defective p53 pathway. Results: We report on the discovery of small synthetic peptides based on the sequence of p21(WAF1) that bind to and inhibit cyclin D1-Cdk4. The peptides and the full-length protein are inhibitory at similar co ncentrations. A 20 amino-acid peptide based on the carboxy-terminal do main of p21(WAF1) inhibits Cdk4 activity with a concentration for half -maximal inhibition (I-0.5) of 46 nM, and it is only four fold less ac tive than the full-length protein. The length of the peptide has been minimized and key hydrophobic residues forming the inhibitory domain h ave been defined. When introduced into cells, both a 20 amino-acid and truncated eight amino-acid peptide blocked phosphorylation of the ret inoblastoma protein (pRb) and induced a potent G1/S growth arrest. The se data support a physiological role for the carboxyl terminus of p21( WAF1) in the inhibition of Cdk4 activity, Conclusions: We have discove red that a small peptide is Sufficient to mimic p21(WAF1) function and inhibit the activity of a critical G1 cyclin-Cdk complex, preventing pRb phosphorylation and producing a G1 cell-cycle arrest in tissue cul ture cell systems, This makes cyclin D1-Cdk4 a realistic and exciting target for the design of novel synthetic compounds that can act as ant i-proliferative agents in human cells.