Small-Molecule Antagonists of p53-MDM2 Binding: Research Tools and Potential Therapeutics

p53 regulates a key pathway which protects normal tissues from tumor development that may result from diverse forms of stress. In the absence of stress, growth suppressive and proapoptotic activity of p53 is inhibited by MDM2 which binds p53 and negatively regulates its activity and stability. MDM2 antagonists could activate p53 and may offer a novel therapeutic approach to cancer. Recently, we identified the first potent and selective low molecular weight inhibitors of MDM2-p53 binding, the Nutlins. These molecules activate the p53 pathway and suppress tumor growth in vitro and in vivo. Nutlins represent valuable new tools for studying the p53 pathway and its defects in cancer. Their potent activity against osteosarcoma xenogrfts suggests that MDM2 antagonists may have a clinical utility in the treatment of tumors with wild-type p53.     

Synthesis and evaluation of an imidazole derivative–fluorescein conjugate

The murine double minute (MDM2) oncogene a negative regulator of protein 53 (p53) tumor suppressor, is found overexpressed in many different types of cancer and the interaction between MDM2 and p53 has become the target of intensive research. MDM2 inhibitors represent a promising class of p53 activating compounds that may be effective in cancer treatment and diagnostic imaging. Nutlins, a family of cis-imidazoline analogues and small-molecule MDM2 antagonists, have the potential use in cancer therapies. We have synthesized an imidazole derivative (Nutlin–Glycine) conjugated to the commonly used fluorophore, 6-carboxyfluorescein (FAM) and evaluated its possible use as an imaging agent. Cellular uptake studies demonstrated that the fluorescence intensity in human osteosarcoma (SJSA-1) and colon carcinoma (HCT116) cells were significantly increased with the treatment of Nutlin–Glycine–FAM when compared with FAM (control). Blocking studies also confirmed that our imidazole–fluorescein conjugate may be a good candidate for imaging tumors, suggesting the need for further in vivo evaluation by positron emission tomography.     

p53蛋白泛素化修饰的研究进展

p53具有抑制肿瘤细胞增殖的作用,但是细胞内p53蛋白的堆积反而加速细胞衰老或凋亡,因此对p53进行严格的调控显得格外重要.泛素化、磷酸化和乙酰化是p53蛋白最主要的几种修饰形式,但近来研究表明泛素化对p53调控发挥着中心作用.MDM2是主要的负调节因子,其具有泛素连接酶的活性,早先的研究认为MDM2的作用主要是特异性结合p53并介导其在蛋白酶作用下降解,但近来的研究发现MDM2还可以介导p53的核-浆交换,这种现象在DNA损伤时尤为明显.推测MDM2介导p53的泛素化在体内可能发挥着多种调控功能.     

Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.     

Identification of novel p53 pathway activating small-molecule compounds reveals unexpected similarities with known therapeutic agents

Manipulation of the activity of the p53 tumor suppressor pathway has demonstrated potential benefit in preclinical mouse tumor models and has entered human clinical trials. We describe here an improved, extensive small-molecule chemical compound library screen for p53 pathway activation in a human cancer cell line devised to identify hits with potent antitumor activity. We uncover six novel small-molecule lead compounds, which activate p53 and repress the growth of human cancer cells. Two tested compounds suppress in vivo tumor growth in an orthotopic mouse model of human B-cell lymphoma. All compounds interact with DNA, and two activate p53 pathway in a DNA damage signaling-dependent manner. A further screen of a drug library of approved drugs for medicinal uses and analysis of gene-expression signatures of the novel compounds revealed similarities to known DNA intercalating and topoisomerase interfering agents and unexpected connectivities to known drugs without previously demonstrated anticancer activities. These included several neuroleptics, glycosides, antihistamines and adrenoreceptor antagonists. This unbiased screen pinpoints interference with the DNA topology as the predominant mean of pharmacological activation of the p53 pathway and identifies potential novel antitumor agents.     

Activation of p53 by nutlin-3a induces apoptosis and cellular senescence in human glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients.     

DNAJB1 stabilizes MDM2 and contributes to cancer cell proliferation in a p53-dependent manner

Both MDM2 and MDMX regulate p53, but these proteins play different roles in this process. To clarify the difference, we performed a yeast 2 hybrid (Y2H) screen using the MDM2 acidic domain as bait. DNAJB1 was found to specifically bind to MDM2, but not MDMX, in vitro and in vivo. Further investigation revealed that DNAJB1 stabilizes MDM2 at the post-translational level. The C-terminus of DNAJB1 is essential for its interaction with MDM2 and for MDM2 accumulation. MDM2 was degraded faster by a ubiquitin-mediated pathway when DNAJB1 was depleted. DNAJB1 inhibited the MDM2-mediated ubiquitination and degradation of p53 and contributed to p53 activation in cancer cells. Depletion of DNAJB1 in cancer cells inhibited activity of the p53 pathway, enhanced the activity of the Rb/E2F pathway, and promoted cancer cell growth in vitro and in vivo. This function was p53 dependent, and either human papillomavirus (HPV) E6 protein or siRNA against p53 was able to block the contribution caused by DNAJB1 depletion. In this study, we discovered a new MDM2 interacting protein, DNAJB1, and provided evidence to support its p53-dependent tumor suppressor function.     

Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction

MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.     

mRNA display selection of an optimized MDM2-binding peptide that potently inhibits MDM2-p53 interaction

p53 is a tumor suppressor protein that prevents tumorigenesis through cell cycle arrest or apoptosis of cells in response to cellular stress such as DNA damage. Because the oncoprotein MDM2 interacts with p53 and inhibits its activity, MDM2-p53 interaction has been a major target for the development of anticancer drugs. While previous studies have used phage display to identify peptides (such as DI) that inhibit the MDM2-p53 interaction, these peptides were not sufficiently optimized because the size of the phage-displayed random peptide libraries did not cover all of the possible sequences. In this study, we performed selection of MDM2-binding peptides from large random peptide libraries in two stages using mRNA display. We identified an optimal peptide named MIP that inhibited the MDM2-p53 and MDMX-p53 interactions 29- and 13-fold more effectively than DI, respectively. Expression of MIP fused to the thioredoxin scaffold protein in living cells by adenovirus caused stabilization of p53 through its interaction with MDM2, resulting in activation of the p53 pathway. Furthermore, expression of MIP also inhibited tumor cell proliferation in a p53-dependent manner more potently than DI. These results show that two-stage, mRNA-displayed peptide selection is useful for the rapid identification of potent peptides that target oncoproteins.     

Nucleolar protein GLTSCR2 stabilizes p53 in response to ribosomal stresses

p53 is a key regulator of cell growth and death by controlling cell cycle progression and apoptosis under conditions of stress such as DNA damage or oncogenic stimulation. As these processes are critical for cell function and inhibition of tumor development, p53 regulatory pathways are strictly monitored in cells. Recently, it was recognized that nucleolar proteins, including nucleophosmin/B23, ribosomal protein L11, and alternate reading frame (ARF), form the nucleolus-ARF-murine double minute 2 (MDM2) axis in p53 regulatory pathways, which increases p53 stability by suppressing the activity of MDM2. In this work, we show that nucleolar protein glioma tumor-suppressor candidate region gene 2 (GLTSCR2) translocates to the nucleoplasm under ribosomal stress, where it interacts with and stabilizes p53 and inhibits cell cycle progression without the involvement of the major upstream p53 regulator, ARF. Furthermore, ectopic expression of GLTSCR2 significantly suppressed growth of cancer cells in a xenograft animal model via p53-dependent pathway. Our data identify GLTSCR2 as a new member of the nucleolus-nucleoplasmic axis for p53 regulation. ARF-independent direct regulation of p53 by GLTSCR2 may be a key mechanism and therapeutic target for cell death or growth inhibition when nucleolus-ARF-p53 pathways are inactivated by genetic or epigenetic modifications of ARF, which are the second most common types of genetic change observed in human cancers.     

The p53-MDM2 network: from oscillations to apoptosis

The p53 protein is well-known for its tumour suppressor function. The p53-MDM2 negative feedback loop constitutes the core module of a network of regulatory interactions activated under cellular stress. In normal cells, the level of p53 proteins is kept low by MDM2, i.e. MDM2 negatively regulates the activity of p53. In the case of DNA damage, the p53-mediated pathways are activated leading to cell cycle arrest and repair of the DNA. If repair is not possible due to excessive damage, the p53-mediated apoptotic pathway is activated bringing about cell death. In this paper, we give an overview of our studies on the p53-MDM2 module and the associated pathways from a systems biology perspective. We discuss a number of key predictions, related to some specific aspects of cell cycle arrest and cell death, which could be tested in experiments.