Nuclear export inhibitors and kinase inhibitors identified using a MAPK-activated protein kinase 2 redistribution screen

Redistribution (BioImage) A/S, S?borg, Denmark) is a novel high-throughput screening technology that monitors translocation of specific protein components of intracellular signaling pathways within intact mammalian cells, using green fluorescent protein as a tag. A single Redistribution assay can be used to identify multiple classes of compounds that act at, or upstream of, the level of the protein target used in the primary screening assay. Such compounds may include both conventional and allosteric enzyme inhibitors, as well as protein-protein interaction modulators. We have developed a series of Redistribution assays to discover and characterize compounds that inhibit tumor necrosis factor-alpha biosynthesis via modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. A primary assay was designed to identify low-molecular-weight compounds that inhibit the activation-dependent nuclear export of the p38 kinase substrate MAPK-activated protein kinase 2 (MK2). Hits from the primary screen were categorized, using secondary assays, either as direct inhibitors of MK2 nuclear export, or as inhibitors of the upstream p38 MAPK pathway. Activity profiles are presented for a nuclear export inhibitor, and a compound that structurally and functionally resembles a known p38 kinase inhibitor. These results demonstrate the utility of Redistribution technology as a pathway screening method for the identification of diverse and novel compounds that are active within therapeutically important signaling pathways.     

A facile method to screen inhibitors of protein-protein interactions including MDM2-p53 displayed on T7 phage

Protein-protein interactions are essential in many biological processes including cell cycle and apoptosis. It is currently of great medical interest to inhibit specific protein-protein interactions in order to treat a variety of disease states. Here, we describe a facile multiwell plate assay method using T7 phage display to screen for candidate inhibitors of protein-protein interactions. Because T7 phage display is an effective method for detecting protein-protein interactions, we aimed to utilize this technique to screen for small-molecule inhibitors that disrupt these types of interaction. We used the well-characterized interaction between p53 and MDM2 and an inhibitor of this interaction, nutlin 3, as a model system to establish a new screening method. Phage particles displaying p53 interacted with GST-MDM2 immobilized on 96-well plates, and the interaction was inhibited by nutlin 3. Multiwell plate assay was then performed using a natural product library, which identified dehydroaltenusin as a candidate inhibitor of the p53-MDM2 interaction. We discuss the potential applications of this novel T7 phage display methodology, which we propose to call 'reverse phage display'.     

Identification of FDA-approved Drugs that Computationally Bind to MDM2

The integrity of the p53 tumor suppressor pathway is compromised in the majority of cancers. In 7% of cancers p53 is inactivated by abnormally high levels of MDM2 - an E3 ubiquitin ligase that polyubiquitinates p53, marking it for degradation. MDM2 engages p53 through its hydrophobic cleft, and blockage of that cleft by small molecules can re-establish p53 activity. Small molecule MDM2 inhibitors have been developed, but there is likely to be a high cost and long time period before effective drugs reach the market. An alternative is to repurpose FDA-approved drugs. This report describes a new approach, called Computational Conformer Selection, to screen for compounds that potentially inhibit MDM2. This screen was used to computationally generate up to 600 conformers of 3244 FDA-approved drugs. Drug conformer similarities to 41 computationally-generated conformers of MDM2 inhibitor nutlin 3a were ranked by shape and charge distribution. Quantification of similarities by Tanimoto combo scoring resulted in scores that ranged from 0.142 to 0.802. In silico docking of drugs to MDM2 was used to calculate binding energies and to visualize contacts between the top-ranking drugs and the MDM2 hydrophobic cleft. We present 15 FDA-approved drugs predicted to inhibit p53/MDM2 interaction.     

Small molecule protein-protein inhibitors for the p53-MDM2 interaction

This article describes recent progress in the development of small molecule protein-protein inhibitors of the p53-MDM2 (purine double minute 2, or HDM2 for the human congener) protein-protein interaction, with special attention to the diversity of chemotypes reported to disrupt this protein-protein interaction. In >50% of all human cancers, the tumor suppressor 53 KDa phospho-protein p53 is either mutated or deleted. The discovery that MDM2 (HDM2) negatively regulates p53 and therefore inhibits the tumor-suppressor activity of p53 has instigated numerous drug discovery campaigns aimed at disrupting this protein-protein interaction as a potential cancer therapy. Once regarded as intractable targets disrupted by only large macromolecules, protein-protein interactions (PPI) are now mainstream targets due in large part to the intensive effort applied to the study of p53 and the surprising diversity of small molecules (peptides, natural products, terphenyl and other alpha-helix mimetics, chalcones, piperidines, piperazines, fused indoles, isoindolinones, spiro-oxindoles, cis-imidazolines (nutlins), quinolinol and benzodiazepines) capable of disrupting the p53-HDM2 PPI. In addition, drug discovery researchers have employed a number of screening approaches and technologies to identify SMPPIs of the p53-HDM2 interaction, and these discovery paradigms will be discussed. This review will detail the biology of the p53-MDM2 interaction, the major classes of SMPPIs and key medicinal chemistry and in vitro/in vivo biological data reported through October 2006.     

Small molecule inhibitors of p53/MDM2 interaction

The discovery of the key negative regulator MDM2 (mouse double minute 2, also termed HDM2 for its human equivalent) provided a great opportunity to manipulate the levels of the tumor suppressor p53 in cancer cells. Activation of p53 in tumor cells by inhibiting the interaction of MDM2 with p53 has therefore been the focus of a large effort in drug discovery. The modulation of protein-protein interactions, however, has historically been very difficult to achieve owing to the large surface area of interaction. In this article, we review the recent accomplishments in this area and our quest for a clinically viable MDM2 inhibitor.     

Small molecule inhibitors of the p53-MDM2

Recent researches have discovered that MDM2 (murine double minute 2, or HDM2 for the human congener) protein is the main negative regulator of p53, which is an attractive therapeutic target in oncology because its tumor-suppressor activity which can be stimulated to eradicate tumor cells. Inhibiting the p53-MDM2 interaction is a promising approach for activating p53, because this association is well characterized at the structural and biological levels. A number of drug screening approaches and technologies have been used to identity novel inhibitors of the p53-MDM2 interaction. This review will detail the development history of MDM2 protein and the p53-MDM2 interaction, the major classes of novel small-molecular p53-MDM2 binding inhibitors, key medicinal action with the protein-protein interaction and in vitro or in vivo biological activities.     

Small molecule antagonists of the MDM2 oncoprotein as anticancer agents

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.     

Network perspectives on HDM2 inhibitor chemotherapy combinations

The discovery of small molecule inhibitors of HDM2-p53 interaction is considered one of the most significant therapeutic developments in the area p53 research. Intensive work on different classes of HDM2 inhibitors has proven their therapeutic utility as activators of p53 in multiple tumor models. Many laboratories have shown that HDM2 inhibitors can synergize with chemotherapeutic agents resulting in enhanced efficacy through both p53-dependent and independent mechanisms. In our hands HDM2 inhibitor and platinum drug combination showed remarkable antitumor activity that led tumor free survival in one of the most resistant and complex pancreatic xenograft models. Although antitumor efficacy of such combinations has been studied in detail, not much is known on the molecular mechanisms governing this synergy. This is partly due to complexity of multiple pathways modulated by p53 and HDM2. We are of the view that in order to decode this complexity, an integrated approach is needed that considers both HDM2 and p53 as components of a network and not in isolation. This review highlights recent advancements in our understanding of HDM2 inhibitor combination therapy based on network modeling and systems biology driven science. Our recent findings support such a network view as integrated gene expression profiling and pathway network modeling on MI-219-oxaliplatin treated cells revealed activation of multiple and closely knit biological networks. We anticipate that in the near future such network-centric approaches will benefit clinical development of HDM2 inhibitors for genetically predefined subsets of cancer patients and this will be a step towards personalized medicine.     

An antibody‐free strategy for screening putative HDM2 inhibitors using crude bacterial lysates expressing GST‐HDM2 recombinant protein

Targeting the interaction of p53 with its natural inhibitor MDM2 by the use of small synthetic molecules has emerged as a promising pharmacological approach to restore p53 oncosuppressor function in cancers retaining wild‐type p53. The first critical step in the experimental validation of newly synthesized small molecules developed to inhibit MDM2‐p53 interaction is represented by the evaluation of their efficacy in preventing the formation of the MDM2‐p53 complex. This can be achieved using the in vitro reconstructed recombinant MDM2‐p53 complex in cell‐free assays. A number of possible approaches have been proposed, which are however not suitable for screening large chemical libraries, due to the high costs of reagents and instrumentations, or the need of large amounts of highly pure recombinant proteins. Here we describe a rapid and cheap method for high‐throughput screening of putative inhibitors of MDM2‐p53 complex formation – based on the use of GST‐recombinant proteins – that does not require antibodies and recombinant protein purification steps from bacterial cell lysates. Copyright © 2013 John Wiley & Sons, Ltd.     

Patented inhibitors of p53–Mdm2 interaction (2006 – 2008)

Importance of the field: Induction of apoptosis by reactivation of p53 in cancer cells is an emerging therapeutic concept for the treatment of cancer. The discovery and design of novel small molecules that block the p53–Mdm2 protein interaction, thereby activating p53, has provided interesting drug candidates that have currently entered clinical trials or are at the preclinical development stage.

Areas covered in this review: A selection of the interesting patents focusing on small molecule inhibitors of the p53–Mdm2 interaction, recorded from 2006 until 2009, is presented together with a review of the related structural chemistry space.

What the reader will gain: Readers will rapidly gain an overview of the majority of patented scaffolds of small molecule inhibitors of the p53–Mdm2 protein–protein interaction and learn about current limitations and properties of these compounds.

Take home message: The discovery p53–Mdm2 protein–protein interaction inhibitors have delivered new potential options for a targeted cancer therapy with drug-like, non-toxic small molecules. If successful, this approach could gain considerably more attention in the pharmaceutical industry by targeting a variety of validated intracellular protein–protein interactions.     

Pharmacophore model for novel inhibitors of ubiquitin isopeptidases that induce p53-independent cell death

The tumor suppressor p53 is mutated in more than 50% of all cancers. Importantly, most clinically useful antineoplastic agents are less potent and efficacious in the context of mutant p53. This situation has prompted a search for agents that cause tumor cell death via molecular mechanisms independent of p53. Our recent investigations with electrophilic prostaglandins enabled us to devise a pharmacophore and mechanism of action hypothesis relevant to this problem: a cross-conjugated alpha,beta-unsaturated dienone with two sterically accessible electrophilic beta-carbons is a molecular determinant that confers activity among this class of ubiquitin isopeptidases inhibitors, and that inhibitors of ubiquitin isopeptidases cause cell death in vitro independently of p53. Here, we report the use of the National Cancer Institute's Developmental Therapeutics Database to identify compounds to test this hypothesis. Shikoccin (a diterpene), dibenzylideneacetone, and curcumin fit the pharmacophore hypothesis, inhibit cellular isopeptidases, and cause cell death independently of p53 in isogenic pairs of RKO and HCT 116 cells with differential p53 status. The sesquiterpene achillin and 2,6-diphenyl-4H-thiopyran-4-one, which have cross-conjugated dienones with sterically hindered electrophilic beta-carbons, do not inhibit isopeptidases or cause significant cell death. Furthermore, we show that a catalytic-site proteasome inhibitor causes cell death independently of p53. Combined, these data verify the p53-independence of cell death caused by inhibitors of the proteasome pathway and support the proposition that the ubiquitin-dependent proteasome pathway may contain molecular targets suitable for antineoplastic drug discovery.     

Determination of the potency and subunit-selectivity of ribonucleotide reductase inhibitors with a recombinant-holoenzyme-based in vitro assay

Ribonucleotide reductase (RR) is an important therapeutic target for anticancer drugs. The structure of human RR features a 1:1 complex of two homodimeric subunits, hRRM1 and hRRM2. p53R2 is a newly identified homologue of hRRM2. We have devised a holoenzyme-based in vitro assay for the determination of the potency and subunit-selectivity of small-molecule inhibitors of RR. The assay was implemented using two forms of recombinant RR (hRRM2/hRRM1 and p53R2/hRRM1) and based on their [(3)H]CDP reduction activity. Hydroxyurea was used to standardize the assay. We found that the activities of hRRM2/hRRM1 and p53R2/hRRM1 were decreased by hydroxyurea in a dose-dependent manner. The -NH-OH segment of hydroxyurea was shown to be essential for inhibition. In the presence of Fe(III) and reductants, less inhibition of enzymatic activity by hydroxyurea was observed, especially for p53R2/hRRM1. The potency of four hydroxyurea analogues (Schiff bases of hydroxysemicarbazide, SB-HSC) decreased in the order SB-HSC 21 > SB-HSC 24 > SB-HSC 2 > hydroxyurea (HU) > SB-HSC 29. SB-HSC 2 and SB-HSC 24 inhibited p53R2/hRRM1 significantly more than hRRM2/hRRM1, whereas SB-HSC 21 and SB-HSC 29 showed low subunit-selectivity. Electron paramagnetic resonance (EPR) measurements showed that inhibition of RR was accompanied by reduction of its tyrosyl radical. The method was validated by comparison with data obtained using cell-based assays. We suggest that this novel recombinant-holoenzyme-based in vitro assay is a useful tool for the discovery of more potent and subunit-selective inhibitors of RR.     

Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells

Traditional drug discovery efforts have resulted in the approval of a handful of receptor tyrosine kinase (RTK) inhibitors; however, their discovery relied solely on screening recombinant kinases, often with poor cellular activity outcome. The ability to screen RTKs in their natural environment is sought as an alternative approach. We have adapted a novel strategy utilizing a green fluorescent protein-labeled SRC homology 2 domain-based biosensor as a surrogate reporter of endogenous epidermal growth factor receptor (EGFR) activity in A549 cells. Upon activation of the receptor, EGFR function in live cells is measured by the number of green granules that form. Here we describe assay miniaturization and demonstrate specificity for EGFR through its chemical inhibition and RNAi-dependent knockdown resulting in complete abrogation of granule formation. Gefitinib and PD 153035 were identified as hits in a pilot screen. This approach allows for the identification of novel EGFR modulators in high-throughput formats for screening chemical and RNAi libraries.     

靶向MDM2小分子抑制药:肿瘤治疗的新方法

癌蛋白MDM2可与p53蛋白结合形成复合物,抑制p53基因的反式激活,而p53的缺失可诱导肿瘤发生。设计针对MDM2与p53蛋白间相互作用的小分子抑制药(如Nutlins),可再激活p53基因,利于肿瘤治疗。本文综述了MDM2小分子抑制药的特点、作用机制与治疗潜力等最新进展。     

Understanding small-molecule binding to MDM2: insights into structural effects of isoindolinone inhibitors from NMR spectroscopy

The interaction between murine double minute (MDM2) and p53 is a major target in anticancer drug design. Several potent compound series, including the nutlins and spirooxindoles, have previously been established as high-affinity antagonists of MDM2. In this paper, we describe the interaction of isoindolinone inhibitors with MDM2, as characterized by nuclear magnetic resonance spectroscopy. Isoindolinone inhibitors bind specifically to the MDM2 p53 binding site and exploit all sub-pockets used by p53, nutlins and spirooxindoles. Furthermore, isoindolinones bind with low micromolar to high nanomolar affinities, with the best compound approaching the potency of nutlin-3.     

Reactivation of p53 by inhibiting Mdm2 E3 ligase: a novel antitumor approach

The p53 tumor suppressor has been pursued as a cancer therapeutic target based on its ability to induce cell cycle arrest and apoptosis. Reactivation of p53 in the approximately 50% of tumors that retain a functional p53 has served as potential approach in the development of cancer drug therapy. Mdm2 is a major negative regulator of p53 and has long been thought to inhibit p53 in two ways: through ubiquitination of p53, signaling for its degradation by the proteasome, and through directly binding to p53, masking its transactivation domain. Research on Mdm2 E3 function and regulation has important implications for the feasibility of targeting Mdm2 in cancer treatment. By targeting Mdm2 in cancers, especially those harboring wild-type p53, it may be possible to restore p53 function to control tumor growth. Several inhibitors for Mdm2 have been developed and have shown promise in restoring p53 function. This review will summarize the current progress of targeting Mdm2 in cancer treatment with a focus on regulating Mdm2 E3 ubiquitin ligase activity via a number of small Mdm2 binding proteins and the post-translational modification of Mdm2 itself. The potential of inhibitors of Mdm2 E3 ligase as a new novel class of anticancer drugs will also be discussed.