Discovery of Mdm2-MdmX E3 ligase inhibitors using a cell-based ubiquitination assay (original) (raw)
Related papers
Functional analysis and consequences of Mdm2 E3 ligase inhibition in human tumor cells
Oncogene, 2012
Mdm2 is the major negative regulator of p53 tumor suppressor activity. This oncoprotein is overexpressed in many human tumors that retain the wild type p53 allele. As such, targeted inhibition of Mdm2 is being considered as a therapeutic anticancer strategy. The N-terminal hydrophobic pocket of Mdm2 binds to p53 and thereby inhibits the transcription of p53 target genes. Additionally, the C-terminus of Mdm2 contains a RING domain with intrinsic ubiquitin E3 ligase activity. By recruiting E2 ubiquitin conjugating enzyme(s), Mdm2 acts as a molecular scaffold to facilitate p53 ubiquitination and proteasome-dependent degradation. Mdmx (Mdm4), an Mdm2 homolog, also has a RING domain and hetero-oligomerizes with Mdm2 to stimulate its E3 ligase activity. Recent studies have shown that C-terminal residues adjacent to the RING domain of both Mdm2 and Mdmx contribute to Mdm2 E3 ligase activity. However, the molecular mechanisms mediating this process remain unclear, and the biological consequences of inhibiting Mdm2/Mdmx cooperation or blocking Mdm2 ligase function are relatively unexplored. This study presents biochemical and cell biological data that further elucidate the mechanisms by which Mdm2 and Mdmx cooperate to regulate p53 level and activity. We use chemical and genetic approaches to demonstrate that functional inhibition of Mdm2 ubiquitin ligase activity is insufficient for p53 activation. This unexpected result suggests that concomitant treatment with Mdm2/Mdmx antagonists may be needed to achieve therapeutic benefit.
Anticancer strategies by upregulating p53 through inhibition of its ubiquitination by MDM2
Medicinal Chemistry Research, 2020
The potentiation of p53 activity through inhibition of its negative regulator MDM2 is an attractive strategy for anticancer therapy. Much progress has been made in the last decade in a diverse range of areas related to p53 and MDM2. This review focuses on the recent progress in developing small-molecule inhibitors of the MDM2 protein by covering the following approaches that inhibit the function of the p53-MDM2 axis: (1) direct binding to MDM2, (2) direct binding to p53, (3) targeted degradation of MDM2 by the PROTAC approach, and (4) inhibition of MDM2/MDM4 interaction. Given the importance of p53 in cancer development, we hope that research in this area will lead to anticancer drugs in the not too distant future.
Small Molecule Antagonists of the MDM2 Oncoprotein as Anticancer Agents
Current Cancer Drug Targets, 2005
In this early phase of the new era of molecularly targeted patient friendly cancer chemotherapy, there is a need for novel viable anticancer molecular targets. The MDM2 oncoprotein has been validated as a potential target for cancer drug development. MDM2 amplification and/or overexpression occur in a wide variety of human cancers, several of which can be treated experimentally with MDM2 antagonists. MDM2 interacts primarily with the p53 tumor suppressor protein in an autoregulatory negative feedback loop to attenuate p53's cell cycle arrest and apoptosis functions. Inhibition of the p53-MDM2 interaction has been shown to cause selective cancer cell death, as well as sensitize cancer cells to chemotherapy or radiation effects. Consequently, this interaction has been the main focus of anticancer drug discovery targeted to MDM2. The promotion of the proteasomal degradation of the p53 protein by MDM2 is central to its repression of the tumor suppressor functions of p53, and many proteins impinge upon this activity, either enhancing or inhibiting it. MDM2 also has oncogenic activity independent of its interaction with p53, but this has so far not been explored for drug discovery. Among the approaches for targeting MDM2 for cancer therapy, small molecule antagonists have recently featured as effective anticancer agents in experimental models, although the repertoire is currently limited and none has yet entered human clinical trials. Small molecules that have been reported to disrupt the p53-MDM2 binding, thereby enhancing p53 activity to elicit anticancer effects include the following: synthetic chalcones, norbornane derivatives, cis-imidazoline derivatives (Nutlins), a pyrazolidinedione sulfonamide and 1,4-benzodiazepine-2,5-diones, as well as tryptophan derivatives. In addition to compounds disrupting p53pMDM2 binding, three compounds have been discovered that are effective in inhibiting the E3 ligase activity of MDM2 towards p53, and should serve as leads for drug discovery targeting this aspect of the p53-MDM2 interaction as well. These compounds were discovered from library screening and/or structure-based rational drug design strategies.
Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif
Journal of Chemical Biology, 2009
The tumor suppressor p53 has evolved a MDM2dependent feedback loop that promotes p53 protein degradation through the ubiquitin-proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or "lid" in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2 S17D ) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg 97 / Lys 98 , and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phosphomimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.
Small molecule inhibitors of HDM2 ubiquitin ligase activity stabilize and activate p53 in cells
Cancer Cell, 2005
The p53 tumor suppressor protein is regulated by its interaction with HDM2, which serves as a ubiquitin ligase (E3) to target p53 for degradation. We have identified a family of small molecules (HLI98) that inhibits HDM2's E3 activity. These compounds show some specificity for HDM2 in vitro, although at higher concentrations effects on unrelated RING and HECT domain E3s are detectable, which could be due, at least in part, to effects on E2-ubiquitin thiol-ester levels. In cells, the compounds allow the stabilization of p53 and HDM2 and activation of p53-dependent transcription and apoptosis, although other p53-independent toxicity was also observed. Ras transformed wild-type and p53 null MEFs, and Dr. Stan Lipkowitz (NCI, anti-rabbit secondary antibodies and enhanced chemiluminescence (Amer-Bethesda) for MDA-MB-468 breast cancer cells expressing Cbl-b. We are sham Biosciences, UK). also grateful to Carlton Briggs for help with generation of the U2OS-pG13 reporter cell line, and Dr. John Beutler and Dr. Kevin Lorick (NCI, Frederick) In vitro autoubiquitylation for helpful discussions. Glutathione sepharose-bound GST fusion proteins (GST-HDM2, GST-Nedd4, and GST-Siah1) were resuspended in 27 l of ubiquitylation reaction buffer (50 mM Tris [pH 7.4], 0.2 mM ATP, 0.5 mM MgCl 2 , 0.1 mM DTT, 16 M creatine phosphokinase, and 1 mM creatine phosphate). Ubiquitylation
Cancer is a class of diseases characterized by out-of-control cell growth. Cancer is a leading cause of death worldwide.The p53 tumor suppressor is one of the principal mediators of cell-cycle arrest and the activation of apoptosis in response to cellular injuries . In normal unstressed cells, p53 is regulated by a feedback loop with the negative regulator protein MDM2 (murine double -minute clone 2, referred to as human double -minute clone 2, HDM2 , in humans) . A well -known mechanism for the loss of wild -type p53 activity in cancer cells is the overexpression of MDM2.The murine double minute 2 (MDM2) protein facilitates G1 to S phase transition by activation of E2F-1 and can enhance cell survival by suppressing wild-type p53 function.Murine DM2 (MDM2) protein is overexpressed in a variety of neoplasms, including acute leukemias, myelodysplastic syndrome, chronic lymphocytic leukemia and lymphomas , multiple myelomas etc. Blocking the MDM2-p53 interaction to reactivate the p53 function is a promising cancer therapeutic strategy. Activation of the p53 protein protects the organism against the propagation of cells that carry damaged DNA with potentially oncogenic mutations. This can be attained by designing a molecule which can bind to P53 transactivation site of Mdm2 and further this Mdm2 protein cannot bind with P53 .The aim of present study is designing a small molecule(antagonist) having capability to bind with the over expressed Mdm2 protein and blocking its path to bind with p53 tumour suppressor protein that is having sufficient absorption and free of hepatotoxicity and carcinogenicity.
Drug Design, Development and Therapy
Background: The tumor suppressor protein p53 plays an important role in preventing tumor formation and progression through its involvement in cell division control and initiation of apoptosis. Mdm2 protein controls the activity of p53 protein through working as ubiquitin E3 ligase promoting p53 degradation through the proteasome degradation pathway. Inhibitors for Mdm2-p53 interaction have restored the activity of p53 protein and induced cancer fighting properties in the cell. Purpose: The objective of this study is to use computer-aided drug discovery techniques to search for new Mdm2-p53 interaction inhibitors. Methods: A set of pharmacophoric features were created based on a standard Mdm2 inhibitor and this was used to screen a commercial drug-like ligand library; then potential inhibitors were docked and ranked in a multi-step protocol using GLIDE. Top ranked ligands from docking were evaluated for their inhibition activity of Mdm2-p53 interaction using ELISA testing. Results: Several compounds showed inhibition activity at the submicromolar level, which is comparable to the standard inhibitor Nutlin-3a. Furthermore, the discovered inhibitors were evaluated for their anticancer activities against different breast cancer cell lines, and they showed an interesting inhibition pattern. Conclusion: The reported inhibitors can represent a starting point for further SAR studies in the future and can help in the discovery of new anticancer agents.