Cell-cycle arrest and inhibition of Cdk4 activity by small peptides based on the carboxy-terminal domain of p21WAF1

Background: A common event in the development of human neoplasia is the inactivation of a damage-inducible cell-cycle checkpoint pathway regulated by p53. One approach to the restoration of this pathway is to mimic the activity of key downstream effectors. The cyclin-dependent kinase (Cdk) inhibitor p21WAF1 is one such molecule, as it is a major mediator of the p53-dependent growth-arrest pathway, and can, by itself, mediate growth suppression. The primary function of the p21WAF1 protein appears to be the inhibition of G1 cyclin–Cdk complexes. Thus, if we can identify the region(s) of p21WAF1 that contain its inhibitor activity they may provide a template from which to develop novel anti-proliferative 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 p21WAF1 that bind to and inhibit cyclin D1–Cdk4. The peptides and the full-length protein are inhibitory at similar concentrations. A 20 amino-acid peptide based on the carboxy-terminal domain of p21WAF1 inhibits Cdk4 activity with a concentration for half-maximal inhibition (l0.5) of 46 nM, and it is only four-fold less active than the full-length protein. The length of the peptide has been minimized and key hydrophobic residues forming the inhibitory domain have been defined. When introduced into cells, both a 20 amino-acid and truncated eight amino-acid peptide blocked phosphorylation of the retinoblastoma protein (pRb) and induced a potent G1/S growth arrest. These data support a physiological role for the carboxyl terminus of p21WAF1 in the inhibition of Cdk4 activity. Conclusions: We have discovered that a small peptide is sufficient to mimic p21WAF1 function and inhibit the activity of a critical G1 cyclin–Cdk complex, preventing pRb phosphorylation and producing a G1 cell-cycle arrest in tissue culture cell systems. This makes cyclin D1–Cdk4 a realistic and exciting target for the design of novel synthetic compounds that can act as anti-proliferative agents in human cells.