Miniature protein inhibitors of the p53-hDM2 interaction.

We have developed a strategy for the design of miniature proteins that bind DNA[1-3] or protein surfaces[4-7] with high affinity and selectivity. This strategy, which is often called protein grafting,[8-11] involves dissecting a functional recognition epitope from its native α-helical or polyproline type II (PPII) helical context and presenting it on a small but structured protein scaffold (Figure 1A). Here we describe the development and characterization of miniature proteins that bind the human double minute 2 oncoprotein (hDM2) in the nanomolar concentration range and inhibit its interaction with a peptide derived from the activation domain of p53 (p53AD).[12-14] hDM2 is the principal cellular antagonist of the tumor suppressor protein p53.[15] Elevated hDM2 levels are found in many solid tumors that express wild-type p53 and there is considerable interest in hDM2 ligands capable of up-regulating p53 activity in vitro or in vivo.[16] The high-resolution structure of the p53AD•hDM2 complex reveals a recognition epitope composed primarily of three p53AD residues (F19, W23 and L26) located on one face of a short α-helix.[14] Although the p53AD peptide possesses little α-helical structure in the absence of hDM2,[14,17] augmenting the level of intrinsic α-helix structure in p53AD using constrained, non-natural amino acids dramatically increases affinity for hDM2 in vitro and activity in vivo.[18,19] In addition, several other scaffolds have been used to display the p53AD epitope, including large proteins,[20] cyclic β-hairpin peptides,[21] retroinverso peptides,[22] and β-peptides.[13,23] The first highly active small molecule inhibitors were reported in 2004, with IC50’s for inhibiting the p53h D M 2 interaction of 100 300 nM;[24] these molecules also