Peptide inhibition of the interactions of the tumor suppressor protein P53 with its negative regulators MDM2 and MDMX activates P53 in vitro and in vivo,representing a viable therapeutic strat-egy for cancer treatment.Using phage display techniques,we previously identified a potent peptide acti-vator of P53,termed PMI(TSFAEYWNLLSP),with binding affinities for both MDM2 and MDMX in the low nanomolar concentration range.Here we report an ultrahigh affinity,dual-specificity peptide antagonist of MDM2 and MDMX obtained through systematic mutational analysis and additivity-based molecular design.Functional assays of over 100 peptide analogs of PMI using surface plasmon resonance and fluorescence polarization techniques yielded a dodecameric peptide termed PMI-M3(LTFLEYWAQLMQ)that bound to MDM2 and MDMX with Kd values in the low picomolar concen-tration range as verified by isothermal titration calorimetry.Co-crystal structures of MDM2 and of MDMX in complex with PMI-M3 were solved at 1.65 and 3.0 ? resolution,respectively.Similar to PMI,PMI-M3 occupied the P53-binding pocket of MDM2/MDMX,which was dominated energetically by intermolecular interactions involving Phe3,Tyr6,Trp7,and Leu 10.Notable differences in binding between PMI-M3 and PMI were observed at other positions such as Leu4 and Met11 with MDM2,and Leu1 and Met11 with MDMX,collectively contributing to a significantly enhanced binding affinity of PMI-M3 for both proteins.By adding lysine residues to both ends of PMI and PMI-M3 to improve their cellular uptake,we obtained modified peptides termed PMI-2K(KTSFAEYWNLLSPK)and M3-2K(KLTFLEYWAQLMQK).Compared with PMI-2K,M3-2K exhibited significantly improved anti-tumor activities in vitro and in vivo in a P53-dependent manner.This super-strong peptide inhibitor of the P53-MDM2/MDMX interactions may become,in its own right,a powerful lead compound for anticancer drug development,and can aid molecular design of other classes of P53 activators as well for anticancer therapy.