MDMX Proteins Research Articles

Correlation between protein conformational flexibility and ligand binding investigated by NMR dynamics

Mdm2 and MdmX can inhibit the activity of the tumor suppressor protein p53 in cancers. MdmX and Mdm2 proteins have highly homologous to each other in terms of their amino acid residues and their three-dimensional structures. However, it is elusive that current small molecule inhibitors exhibit high binding affinity for Mdm2 but very weak for MdmX. In this study, aiming to prepare isotopically-labelled NMR sample for dynamics NMR experiments, we constructed a fusion protein expression system to improve protein expression and facilitate the purification of protein separation.

Studying Homo-oligomerization and Hetero-oligomerization of MDMX and MDM2 Proteins in Single Living Cells by Using In Situ Fluorescence Correlation Spectroscopy.

Protein oligomerization plays a very important role in many physiological processes. p53 acts as a key tumor suppressor by regulating cell cycle arrest, DNA repair, and apoptosis, and its antitumor activity is regulated by the hetero- and homo-oligomerization of MDMX and MDM2 proteins. So far, some traditional methods have been utilized to study the oligomerization of MDMX and MDM2 in vitro, but they have not clarified some controversial issues or whether the extracellular results can represent the intracellular results. Here, we put forward an in situ method for studying protein homo- and hetero-oligomerization in single living cells by using fluorescence correlation spectroscopy. In this study, MDMX and MDM2 were labeled with fluorescent proteins using lentiviral transfection. Autocorrelation spectroscopy and cross-correlation spectroscopy methods were used to study the oligomerization of MDMX and MDM2 in situ and the effect of regulation of MDMX oligomerization on p53-MDMX interactions in single living cells. We observed the homo- and hetero-oligomerization of MDMX and MDM2 in living cells. Meanwhile, the levels of the homo-oligomers of MDMX and MDM2 were increased due to the lack of hetero-oligomerization. Finally, the binding affinity of MDMX for p53 was improved with an increase in the level of MDMX hetero-oligomerization.

The Molecular Docking of Flavonoids Isolated from Daucus carota as a Dual Inhibitor of MDM2 and MDMX.

Cancer is characterized by overexpression of p53 associated proteins, which down-regulate P53 signaling pathway. In cancer therapy, p53 activity can be restored by inhibiting the interaction of MDMX (2N0W) and MDM2 (4JGR) proteins with P53 protein. In the current, study in silico approaches were adapted to use a natural product as a source of cancer therapy. In the current study in silico approaches were adapted to use a natural product as a source of cancer therapy. For in silico studies, Chemdraw and Molecular Operating Environment were used for structure drawing and molecular docking, respectively. Flavonoids isolated from D. carota were docked with cancerous proteins. Based on the docking score analysis, we found that compound 7 was the potent inhibitor of both cancerous proteins and can be used as a potent molecule for inhibition of 2N0W and 4JGR interaction with p53. Thus the compound 7 can be used for the revival of p53 signaling pathway function however, intensive in vitro and in vivo experiments are required to prove the in silico analysis.

A therapeutic patent overview of MDM2/X-targeted therapies (2014–2018)

ABSTRACTIntroduction: MDM2 and MDMX proteins provide the inhibition of p53 tumor suppressor, thus allowing for accelerated mutation-driven cancer microevolution. A pharmacological blockade of MDM2/X-p53 interaction results in p53 reactivation in p53wt cells, leading to cancer growth inhibition. Throughout the past 20 years, multiple chemical entities have been proposed to reactivate p53 by antagonizing MDM2/X proteins.Areas covered: This manuscript reviews 2014–2018 therapeutic patents in the field of MDM2/X antagonists and is a continuation of previous reviews on similar matter. The patents covering the use of MDM2/X antagonists in drug combinations are also presented in this review, as they constitute an important trend in the field of cancer treatment with MDM2/X antagonists.Expert opinion: In the years 2014–2018, several previously-known chemical scaffolds have been further developed and disclosed. Importantly, in the same time period, many lead compounds have entered clinical trials for the treatment of cancer patients. Meanwhile, several important reports have pointed to serious limitations of anticancer properties of MDM2 antagonists. As a result, many efforts have been made to seek for positive, synergistic therapeutic effects of combined anti-cancer treatment strategies. One recent example is a dual targeting of MDM2 and additional protein targets by utilizing the PROTAC technology.Trial registration: ClinicalTrials.gov identifier: NCT02264613.Trial registration: ClinicalTrials.gov identifier: NCT02624986.Trial registration: ClinicalTrials.gov identifier: NCT02670044.Trial registration: ClinicalTrials.gov identifier: NCT02545283.

Calculation of hot spots for protein-protein interaction in p53/PMI-MDM2/MDMX complexes.

The recently developed MM/GBSA_IE method is applied to computing hot and warm spots in p53/PMI-MDM2/MDMX protein-protein interaction systems. Comparison of the calculated hot (>2 kcal/mol) and warm spots (>1 kcal/mol) in P53 and PMI proteins interacting with MDM2 and MDMX shows a good quantitative agreement with the available experimental data. Further, our calculation predicted hot spots in MDM2 and MDMX proteins in their interactions with P53 and PMI and they help elucidate the interaction mechanism underlying this important PPI system. In agreement with the experimental result, the present calculation shows that PMI has more hot and warm spots and binds stronger to MDM2/MDMX. The analysis of these hot and warm spots helps elucidate the fundamental difference in binding between P53 and PMI to the MDM2/MDMX systems. Specifically, for p53/PMI-MDM2 systems, p53 and PMI use essentially the same residues (L54, I61, Y67, Q72, V93, H96, and I99) of MDM2 for binding. However, PMI enhanced interactions with residues L54, Y67, and Q72 of MDM2. For the p53/PMI-MDMX system, p53 and PMI use similar residues (M53, I60, Y66, Q71, V92, and Y99) of MDMX for binding. However, PMI exploited three extra residues (M61, K93, and L98) of MDMX for enhanced binding. In addition, PMI enhanced interaction with four residues (M53, Y66, Q71, and Y99) of MDMX. These results gave quantitative explanation on why the binding affinities of PMI-MDM2/MDMX interactions are stronger than that of p53-MDM2/MDMX although their binding modes are similar. © 2018 Wiley Periodicals, Inc.

Synthetic 10FN3-based mono- and bivalent inhibitors of MDM2/X function.

Engineered non-antibody scaffold proteins constitute a rapidly growing technology for diagnostics and modulation/perturbation of protein function. Here, we describe the rapid and systematic development of high-affinity 10FN3 domain inhibitors of the MDM2 and MDMX proteins. These are often overexpressed in cancer and represent attractive drug targets. Using facile in vitro expression and pull-down assay methodology, numerous design iterations addressing insertion site(s) and spacer length were screened for optimal presentation of an MDM2/X dual peptide inhibitor in the 10FN3 scaffold. Lead inhibitors demonstrated robust, on-target cellular inhibition of MDM2/X leading to activation of the p53 tumor suppressor. Significant improvement to target engagement was observed by increasing valency within a single 10FN3 domain, which has not been demonstrated previously. We further established stable reporter cell lines with tunable expression of EGFP-fused 10FN3 domain inhibitors, and showed their intracellular location to be contingent on target engagement. Importantly, competitive inhibition of MDM2/X by small molecules and cell-penetrating peptides led to a readily observable phenotype, indicating significant potential of the developed platform as a robust tool for cell-based drug screening.

Molecular Simulations Identify Binding Poses and Approximate Affinities of Stapled α-Helical Peptides to MDM2 and MDMX.

Traditionally, computing the binding affinities of proteins to even relatively small and rigid ligands by free-energy methods has been challenging due to large computational costs and significant errors. Here, we apply a new molecular simulation acceleration method called MELD (Modeling by Employing Limited Data) to study the binding of stapled α-helical peptides to the MDM2 and MDMX proteins. We employ free-energy-based molecular dynamics simulations (MELD-MD) to identify binding poses and calculate binding affinities. Even though stapled peptides are larger and more complex than most protein ligands, the MELD-MD simulations can identify relevant binding poses and compute relative binding affinities. MELD-MD appears to be a promising method for computing the binding properties of peptide ligands with proteins.

Dual function of MDM2 and MDMX toward the tumor suppressors p53 and RB

The orchestrated crosstalk between the retinoblastoma (RB) and p53 pathways contributes to preserving proper homeostasis within the cell. The deregulation of one or both pathways is a common factor in the development of most types of human cancer. The proto-oncoproteins MDMX and MDM2 are the main regulators of the well- known tumor suppressor p53 protein. Under normal conditions, MDM2 and MDMX inhibit p53, either via repression of its transcriptional activity by protein-protein interaction, or via polyubiquitination as a result of MDM2-E3 ubiquitin ligase activity, for which MDM2 needs to dimerize with MDMX. Under genotoxic stress conditions, both become positive regulators of p53. The ATM-dependent phosphorylation of MDM2 and MDMX allow them to bind p53 mRNA, these interactions promote p53 translation. MDM2 and MDMX are also being revealed as effective regulators of the RB protein. MDM2 is able to degrade RB by two different mechanisms, that is, by ubiquitin dependent and independent pathways. MDMX enhances the ability of MDM2 to bind and degrade RB protein. However, MDMX also seems to stabilize RB through interaction and competition with MDM2. Here, we will contextualize the findings that suggest that the MDM2 and MDMX proteins have a dual function on both p53 and RB.

Discovery of novel dual inhibitors against Mdm2 and Mdmx proteins by in silico approaches and binding assay

AimsThe p53 protein, also called guardian of the genome, has a key role in cell cycle regulation. It is activated under stressful circumstances, such as DNA damage which results in permanent arrest or cell death. The protein is disabled in several types of human cancer due to over-expression of the two regulators, Mdm2 and Mdmx. As a result, inhibiting Mdm subtypes could reactivate p53 and bring about a promising therapeutic strategy in cancers. Main methodsHere a structure-based pharmacophore search and docking simulation are presented in order to filter our in-house library which contains 1035 compounds to find novel scaffolds that inhibit Mdm2 and Mdmx concomitantly. Afterwards, fluorescence polarization binding assay was used to obtain inhibition constant of final compounds. Key findingsThirty two ligands were introduced to bioassay as a result of in-silico methods. Twelve of them inhibit both proteins with almost balanced Ki value ranging from 18 to 162μM for Mdm2 and 18 to 233μM for Mdmx. It was observed that all compounds fill Phe19 and Trp23 pockets of Mdm2/x binding sites and form a hydrogen bond with Trp23 pocket's neighbor amino acids in a manner similar to p53 protein. Additionally, it was concluded that Trp23 pocket of Mdmx has a bigger hydrophobic volume comparing with the one of Mdm2. SignificanceThree structure–activity relationship patterns are supposed which one of them presents usefulness features and can be used in future studies. This study presents first qualitative SAR for dual inhibitors against Mdm2/x.

Structural basis of how stress-induced MDMX phosphorylation activates p53.

The tumor-suppressor protein p53 is tightly controlled in normal cells by its two negative regulators--the E3 ubiquitin ligase MDM2 and its homolog MDMX. Under stressed conditions such as DNA damage, p53 escapes MDM2- and MDMX-mediated functional inhibition and degradation, acting to prevent damaged cells from proliferating through induction of cell cycle arrest, DNA repair, senescence or apoptosis. Ample evidence suggests that stress signals induce phosphorylation of MDM2 and MDMX, leading to p53 activation. However, the structural basis of stress-induced p53 activation remains poorly understood because of the paucity of technical means to produce site-specifically phosphorylated MDM2 and MDMX proteins for biochemical and biophysical studies. Herein, we report total chemical synthesis, via native chemical ligation, and functional characterization of (24-108)MDMX and its Tyr99-phosphorylated analog with respect to their ability to interact with a panel of p53-derived peptide ligands and PMI, a p53-mimicking but more potent peptide antagonist of MDMX, using FP and surface plasmon resonance techniques. Phosphorylation of MDMX at Tyr99 weakens peptide binding by approximately two orders of magnitude. Comparative X-ray crystallographic analyses of MDMX and of pTyr99 MDMX in complex with PMI as well as modeling studies reveal that the phosphate group of pTyr99 imposes extensive steric clashes with the C-terminus of PMI or p53 peptide and induces a significant lateral shift of the peptide ligand, contributing to the dramatic decrease in the binding affinity of MDMX for p53. Because DNA damage activates c-Abl tyrosine kinase that phosphorylates MDMX at Tyr99, our findings afford a rare glimpse at the structural level of how stress-induced MDMX phosphorylation dislodges p53 from the inhibitory complex and activates it in response to DNA damage.

An observational study on the expression levels of MDM2 and MDMX proteins, and associated effects on P53 in a series of human liposarcomas

BackgroundInactivation of wild type P53 by its main cellular inhibitors (MDM2 and MDMX) is a well recognised feature of tumour formation in liposarcomas. MDM2 over-expression has been detected in approximately 80% of liposarcomas but only limited information is available about MDMX over-expression. To date, we are not aware of any study that has described the patterns of MDM2 and MDMX co-expression in liposarcomas. Such information has become more pertinent as various novel MDM2 and/or MDMX single and dual affinity antagonist compounds are emerging as an alternative approach for potential targeted therapeutic strategies.MethodsWe analysed a case series of 61 fully characterized liposarcomas of various sub-types by immunohistochemistry, to assess the expression levels of P53, MDM2 and MDMX, simultaneously. P53 sequencing was performed in all cases that expressed P53 protein in 10% or more of cells to rule out mutation-related over-expression.Results50 cases over-expressed MDM2 and 42 of these co-expressed MDMX at varying relative levels. The relative expression levels of the two proteins with respect to each other were subtype-dependent. This apparently affected the detected levels of P53 directly in two distinct patterns. Diminished levels of P53 were observed when MDM2 was significantly higher in relation to MDMX, suggesting a dominant role for MDM2 in the degradation of P53. Higher levels of P53 were noted with increasing MDMX levels suggesting an interaction between MDM2 and MDMX that resulted in a reduced efficiency of MDM2 in degrading P53. Of the 26 cases of liposarcoma with elevated P53 expression, 5 were found to have a somatic mutation in the P53 gene.ConclusionsThe results suggest that complex dynamic interactions between MDM2 and MDMX proteins may directly affect the cellular levels of P53. This therefore suggests that careful characterization of both these markers will be necessary in tumours when considering in vivo evaluation of novel blocker compounds for MDM proteins, as a therapeutic strategy to restore wild type P53 function.

In vitro and in silico studies of MDM2/MDMX isoforms predict Nutlin-3A sensitivity in well/de-differentiated liposarcomas

The molecular marker of well-differentiated/de-differentiated liposarcomas is MDM2 gene amplification coupled with protein overexpression and wild-type TP53. MDMX is a recently identified MDM2 homolog and its presence in this tumor is unexplored. Our aim was to investigate the role of full-length MDM2 and MDMX proteins and their isoforms in surgical specimens of well-differentiated/de-differentiated liposarcomas in view of Nutlin-3A (a MDM2 inhibitor) treatment. Frozen and matched formalin-fixed, paraffin-embedded material from surgical specimens was examined by means of: (1) fluorescence in situ hybridization to determine MDM2 and MDMX gene copy numbers; (2) RT-PCR and densitometry to analyze alternative splicing forms of mdm2 and mdmx; (3) immunoblotting and immunohistochemistry to assess the corresponding translated proteins; and (4) in vitro and in silico assays to determine their affinity for Nutlin-3A. All these cases showed MDM2 gene amplification with an MDMX disomic pattern. In all cases, the full-length mdm2 transcript was associated with the mdm2-b transcript, with ratios ranging from 0.07 to 5.6, and both were translated into protein; mdmx and mdmx-s were co-transcripted, with ratios ranging from 0.1 to 5.6. MDMX-S was frequently more upregulated than MDMX at both transcriptional and protein level. Each case showed different amounts of mdm2, mdm2-b, mdmx, and mdmx-s transcripts and the corresponding proteins. In vitro assays showed that Nutlin-3A was ineffective against MDM2-B and was unable to disrupt the MDMX/TP53 and MSMX-S/TP53 complexes. Molecular simulations confirmed these in vitro findings by showing that MDM2 has high Nutlin-3A affinity, followed by MDMX-S, MDMX, and MDM2-B. Nutlin-3A is predicted to be a good therapeutic option for well-differentiated/de-differentiated liposarcomas. However, our findings predict heterogeneous responses depending on the relative expression of mdm2, mdm2-b, mdmx, and mdmx-s transcripts and proteins.

Affinity-based screening of MDM2/MDMX–p53 interaction inhibitors by chemical array: Identification of novel peptidic inhibitors

MDM2 and MDMX are oncoproteins that negatively regulate the activity and stability of the tumor suppressor protein p53. The inhibitors of protein–protein interactions (PPIs) of MDM2–p53 and MDMX–p53 represent potential anticancer agents. In this study, a novel approach for identifying MDM2–p53 and MDMX–p53 PPI inhibitor candidates by affinity-based screening using a chemical array has been established. A number of compounds from an in-house compound library, which were immobilized onto a chemical array, were screened for interaction with fluorescence-labeled MDM2 and MDMX proteins. The subsequent fluorescent polarization assay identified several compounds that inhibited MDM2–p53 and MDMX–p53 interactions.

MDM2, MDMX and p53 in oncogenesis and cancer therapy

The MDM2 and MDMX (also known as HDMX and MDM4) proteins are deregulated in many human cancers and exert their oncogenic activity predominantly by inhibiting the p53 tumour suppressor. However, the MDM proteins modulate and respond to many other signalling networks in which they are embedded. Recent mechanistic studies and animal models have demonstrated how functional interactions in these networks are crucial for maintaining normal tissue homeostasis, and for determining responses to oncogenic and therapeutic challenges. This Review highlights the progress made and pitfalls encountered as the field continues to search for MDM-targeted antitumour agents.

Stochastic and Deterministic Models of Cellular p53 Regulation

The protein p53 is a key regulator of cellular response to a wide variety of stressors. In cancer cells inhibitory regulators of p53 such as MDM2 and MDMX proteins are often overexpressed. We apply in silico techniques to better understand the role and interactions of these proteins in a cell cycle process. Furthermore we investigate the role of stochasticity in determining system behavior. We have found that stochasticity is able to affect system behavior profoundly. We also derive a general result for the way in which initially synchronized oscillating stochastic systems will fall out of synchronization with each other.

The P53 Pathway Is Inactive in Acute Myeloid Leukemia

Abstract 5122The pathway controlled by the p53 tumor-suppressor protein is altered in most, if not all, human cancers and the TP53 gene is mutated in half of all human tumors. Such mutations are rare in human hematological malignancies, leading to the assumption that the p53 pathway is inactivated by alternative mechanisms. However, to date, the state of activity of the p53 pathway in hematological malignancies is not well understood. We investigated the functional status of the p53 pathway in Acute Myeloid Leukemia (AML) patients, particularly in patients with cytogenetically normal AML (CN-AML) and patients with Acute Promyelocytic Leukemia (APL).We performed bioinformatic analysis of p53 pathway-related gene expression. For this purpose, we first assembled a list that, to the best of our knowledge, is the most comprehensive list to date of genes related to the p53 pathway. The list consists of 1153 p53 pathway-related genes: 916 p53-related genes and 582 partially overlapping genes related to important components of the p53 pathway (Mdm2, Mdmx, Puma, Slug and Chk2). The list of p53 pathway-related genes was constructed based on gene and protein web databases and literature search. Only genes with proven biochemical relationships to the p53 pathway were included.Publically available Affymetrix gene expression array data was analyzed which included 290 CN-AML and 34 APL patients at diagnosis in comparison to 63 normal bone marrow (nBM) samples. Differentially expressed genes (DEGs) were identified out of 1153 p53 pathway-related genes using a linear statistical model that produced gene expression contrasts between leukemic samples and nBM. Study effect differences were also corrected by this model. One hundred forty seven DEGs were identified in CN-AML and 172 in APL (fold change>2. 8, p value<0. 01). We found a significant over-representation of p53 pathway related DEGs above the genomic background in both leukemias. Our analysis demonstrated homogeneity of gene expression in APL patients and discovered that CN-AML patients were further divided into 3 sub-groups by hierarchical clustering analysis. Most of the DEGs were down regulated both in CN-AML (108/147) and in APL (135/172) patients. We analyzed the DEGs and concluded that in both leukemias there was no p53-dependent induction of canonical cell cycle arrest genes, canonical pro-apoptotic genes, p53-related antioxidant defense genes, DNA damage repair genes and anti-glycolysis genes.We compared our bioinformatic results to gene expression signatures related to p53 activation by various stimuli from the literature. This analysis demonstrated that p53 protein did not exert transcriptional activation of the majority of its target genes in CN-AML and APL, implying that p53 pathway is not activated in these leukemias.We found downregulation of p300, PCAF and CARM1 genes in patient samples compared to nBM. Deregulation of these genes points to decreased acetylation and methylation of the p53 protein that can result in the inhibition of p53 transcriptional activity.We examined protein levels of p53 and its main inhibitors Mdmx and Mdm2 by immunohistochemistry in 25 CN-AML and 23 APL patients in comparison to 36 nBM biopsies. We found that the fraction of cells expressing p53, Mdmx and Mdm2 proteins was significantly higher in leukemias (70–90%) compared to nBM (10–30%). However, the intensity of Mdm2 staining was not elevated in leukemic blasts compared to nBM and p53 levels were similarly low in both nBM and leukemias. Importantly, Mdmx protein level was significantly upregulated in leukemia cells, offering an explanation for inhibition of p53 transcriptional activity in leukemia. The increased level of Mdmx protein together with low levels of p53 protein is in agreement with inhibition of p53 transcriptional activity in CN-AML and APL demonstrated by our bioinformatic analysis.Inactivation of p53 pathway shown here may be one of the important leukomogenic events in AML development. Importantly, gene expression and thus the functional status of p53 pathway is very similar in CN-AML and APL patients compared to nBM, despite the different underlying molecular etiology of these diseases. This finding may have important therapeutic implications in that similar reactivation of the p53 pathway may be a therapeutic modality applicable to these two biologically different types of leukemia. Disclosures:No relevant conflicts of interest to declare.

The new function of &lt;I&gt;p53&lt;/I&gt; family and its pathway related pro-teins in female reproduction

p53 is an important tumor suppressor gene and one of the key genes in sensing and regulating responses to the environmental stress. Recent study showed that cold winter temperature naturally selected p53 Arg72 in eastern Asian population, suggesting that p53 plays a role in reproduction. It has also been reported that some SNPs of p53, Mdm2(Murine double minute 2), MdmX and Hausp (Herpes virus-associated ubiquitin-specific protease) in p53 pathway are associated with the risk of the women's reproduction disorder. p53 regulates the LIF (leukaemia inhibitory factor) expression level by its DBD domain, and thus contributes to female reproduction by affecting the embryo implantation process. The MDM2, MDMX, and HAUSP proteins regulates the level and activity of p53 protein, which are critical for the appropriate p53 response in the embryo implantation process. The members of p53 family, p63 and p73, also play roles in female reproduction through other pathways. p63 has been implicated as a major regulator of oocyte death following treatment with irradiation and chemotherapeutic drugs, which prevents fetal malformation. p73 regulates the formation of spindle assembly complex(SAC). The dysfunction of SAC results in poor blastocyst quality and defects in kinetochore-microtubule associations, which leads to aneuploidy. This review summarized the function of p53 family and its pathway related proteins in female reproduction, pointed out a new method in improving the success rate in IVF-ET, and provided a new diagnosis idea for unexplained infertile women. It will facilitate personalized strategies in the infertility therapy.

Activation of the p53 pathway by small-molecule-induced MDM2 and MDMX dimerization

Activation of p53 tumor suppressor by antagonizing its negative regulator murine double minute (MDM)2 has been considered an attractive strategy for cancer therapy and several classes of p53-MDM2 binding inhibitors have been developed. However, these compounds do not inhibit the p53-MDMX interaction, and their effectiveness can be compromised in tumors overexpressing MDMX. Here, we identify small molecules that potently block p53 binding with both MDM2 and MDMX by inhibitor-driven homo- and/or heterodimerization of MDM2 and MDMX proteins. Structural studies revealed that the inhibitors bind into and occlude the p53 pockets of MDM2 and MDMX by inducing the formation of dimeric protein complexes kept together by a dimeric small-molecule core. This mode of action effectively stabilized p53 and activated p53 signaling in cancer cells, leading to cell cycle arrest and apoptosis. Dual MDM2/MDMX antagonists restored p53 apoptotic activity in the presence of high levels of MDMX and may offer a more effective therapeutic modality for MDMX-overexpressing cancers.

Abstract C128: Small-molecule antagonists of MDM2 and MDMX.

Abstract The p53 tumor suppressor is controlled by MDM2 and MDMX that negatively modulate its activity. Both proteins possess p53-binding sites within their N-terminal domains and can inhibit the transcriptional activity of p53. However, only MDM2 can serve as E3 ubiquitin ligase for p53 and is thus responsible for its stability. MDMX does not have intrinsic ligase activity but can modulate the ligase activity of MDM2. Many human tumors overproduce MDM2 or MDMX to impair p53 function. Small-molecule MDM2 antagonists, the nutlins, interact specifically with the p53-binding pocket of MDM2 and can release p53 from negative control. Treatment of cancer cells expressing wild-type p53 with nutlins stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest and apoptosis in vitro and in vivo. However, nutlins and other published MDM2 antagonists do not inhibit the p53-MDMX interaction and their effectiveness can be compromised in tumors overexpressing MDMX. We identify the first small molecules that potently block p53 interaction with both MDM2 and MDMX (in vitro IC50 of 17 nM and 24 nM, respectively) by inhibitor-driven homo- and/or hetero-dimerization of MDM2 and MDMX proteins. Structural studies revealed that these idole-hydantoin compounds bind into and occlude p53 pockets of MDM2 and/or MDMX by inducing the formation of dimeric protein complexes kept together by a dimeric small-molecule core. This novel mode of inhibiting protein-protein interactions effectively stabilized p53 and activated p53 signaling in cancer cells, leading to cell cycle arrest and apoptosis. MDM2/MDMX antagonist, RO-5963, restored p53 apoptotic activity in cultured osteosarcoma cells in the presence of high levels of MDMX. RO-5963 showed a significantly better apoptotic activity against MCF7 and other solid tumor cell lines with higher MDMX levels than the MDM2-specific inhibitor, nutlin-3a, suggesting that dual antagonists may offer a more effective therapeutic modality for MDMX-overexpressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C128.

MdmX Protein Is Essential for Mdm2 Protein-mediated p53 Polyubiquitination

Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.