The p53 tumor suppressor plays a key role in maintaining genomic stability and protection against malignant transformation. MDM2 and MDMX are both p53-binding proteins that regulate p53 stability and activity. Recent development of the MDM2 inhibitor Nutlin 3 has greatly facilitated functional analysis of MDM2-p53 binding. We found that although MDMX is homologous to MDM2 and binds to the same region on p53 N terminus, Nutlin does not disrupt p53-MDMX interaction. The ability of Nutlin to activate p53 is compromised in tumor cells overexpressing MDMX. Combination of Nutlin with MDMX siRNA resulted in synergistic activation of p53 and growth arrest. These results suggest that MDMX is also a valid target for p53 activation in tumor cells. Development of novel compounds that are MDMX-specific or optimized for dual-inhibition of MDM2 and MDMX are necessary to achieve full activation of p53 in tumor cells.p53 is a transcription factor mutated in ϳ50% of human tumors. In unstressed normal cells, p53 is present at very low levels due to rapid degradation through the ubiquitin-dependent proteasome pathway. MDM2 is an important regulator of p53 turnover by binding p53 and acting as a ubiquitin E3 ligase. Overexpression of MDM2 abrogates the ability of p53 to induce cell cycle arrest and apoptosis. In about 30% of human osteogenic sarcomas and soft tissue sarcomas, MDM2 is overexpressed due to gene amplification. In tumors without MDM2 amplification, hyperactivation of MDM2 due to silencing of ARF expression also leads to p53 inactivation. Therefore, MDM2 is a key factor in tolerance of wild type p53 in nearly 50% of tumors, making it an attractive target for the development of novel anti-tumor agents (1).MDMX is a p53-binding protein with significant sequence homology to MDM2 (2, 3). Unlike MDM2, MDMX does not have intrinsic E3 ligase activity and does not promote p53 degradation. However, MDMX binds to MDM2 through C-terminal RING domain interaction (4, 5) and stimulates the ability of MDM2 to ubiquitinate and degrade p53 (6, 7). MDMX-MDM2 interaction can also lead to ubiquitination and degradation of MDMX (8 -10); this may be an important mechanism for elimination of MDMX during DNA damage response. MDMX knock-out mice die in utero despite having endogenous MDM2 (11). This suggests that MDMX has a unique role in regulating p53 during embryonic development. MDMX overexpression has been found in a number of primary tumors or tumor cell lines with wild-type p53 (12, 13), suggesting that MDMX may contribute to p53 inactivation during tumorigenesis.MDM2 and MDMX are both targeted by stress signaling pathways that activate p53. DNA damage by ionizing irradiation induces phosphorylation of MDM2 by ATM and c-Abl kinases and inhibits its ability to ubiquitinate p53. Recent studies showed that DNA damage also induces MDMX phosphorylation at the C-terminal region by ATM (14), Chk1 (15), and Chk2 (16). MDMX phosphorylation stimulates 14-3-3 binding (17, 18) and promotes MDMX nuclear translocation and degradation by MDM2 (19,20). M...

-The design and solution-phase synthesis of an α-helix mimetic library as an integral component of a small molecule library targeting protein-protein interactions are described. The iterative design, synthesis, and evaluation of the candidate α-helix mimetic was initiated from a precedented triaryl template and refined by screening the designs for inhibition of MDM2/p53 binding. Upon identifying a chemically and biologically satisfactory design and consistent with the screening capabilities of academic collaborators, the corresponding complete library was assembled as 400 mixtures of 20 compounds (20 × 20 × 20-mix), where the added subunits are designed to mimic all possible permutations of the naturally occurring i, i+4, i+7 amino acid side chains of an α-helix. The library (8000 compounds) was prepared using a solution-phase synthetic protocol enlisting acid/base liquid-liquid extractions for purification on a scale that insures its long term availability for screening campaigns. Screening of the library for inhibition of MDM2/p53 binding not only identified the lead α-helix mimetic upon which the library was based, but also suggests that a digestion of the initial screening results that accompany the use of such a comprehensive library can provide insights into the nature of the interaction (e.g., an α-helix mediated protein-protein interaction) and define the key residues and their characteristics responsible for recognition.