RESUMEN
The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53--a cellular process initiated by MDM2 and/or MDMX binding to the N-terminal transactivation domain of p53. MDM2 and MDMX in many tumors confer p53 inactivation and tumor survival, and are important molecular targets for anticancer therapy. We screened a duodecimal peptide phage library against site-specifically biotinylated p53-binding domains of human MDM2 and MDMX chemically synthesized via native chemical ligation, and identified several peptide inhibitors of the p53-MDM2/MDMX interactions. The most potent inhibitor (TSFAEYWNLLSP), termed PMI, bound to MDM2 and MDMX at low nanomolar affinities--approximately 2 orders of magnitude stronger than the wild-type p53 peptide of the same length (ETFSDLWKLLPE). We solved the crystal structures of synthetic MDM2 and MDMX, both in complex with PMI, at 1.6 A resolution. Comparative structural analysis identified an extensive, tightened intramolecular H-bonding network in bound PMI that contributed to its conformational stability, thus enhanced binding to the 2 oncogenic proteins. Importantly, the C-terminal residue Pro of PMI induced formation of a hydrophobic cleft in MDMX previously unseen in the structures of p53-bound MDM2 or MDMX. Our findings deciphered the structural basis for high-affinity peptide inhibition of p53 interactions with MDM2 and MDMX, shedding new light on structure-based rational design of different classes of p53 activators for potential therapeutic use.
Asunto(s)
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Oligopéptidos/farmacología , Péptidos/farmacología , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Biblioteca de Péptidos , Péptidos/química , Péptidos/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53 and are important molecular targets for anticancer therapy. Grafting four residues of p53 critical for MDM2/MDMX binding to the N-terminal alpha-helix of BmBKTx1, a scorpion toxin isolated from the venom of the Asian scorpion Buthus martensi Karsch, converts the miniature protein into an effective inhibitor of p53 interactions with MDM2 and MDMX. Additional mutations enable the 27-residue miniprotein inhibitor to traverse the cell membrane and selectively kill tumor cells in a p53 dependent manner.