Identification of Akt Pathway Inhibitors Using Redistribution Screening on the FLIPR and the IN Cell 3000 Analyzer

Lundholt, Betina Kerstin; Linde, Viggo; Loechel, Frosty; Pedersen, Hans Chr.; Möller, Sören; Præstegaard, Morten; Mikkelsen, Ivan; Scudder, Kurt; Bjørn, Sara Petersen; Heide, Morten; Arkhammar, Per; Terry, Robert; Nielsen, Søren

doi:10.1177/1087057104269989

Cited by 52 publications

(23 citation statements)

References 17 publications

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“…The subcellular localization of target proteins that lie downstream of the perturbation provides a visual read-out for measuring the activity of signaling pathways and supports the identification of small molecule compounds or relevant therapeutic targets that might be exploited pharmaceutically to restore or interfere with protein localization and function (Kau et al, 2003;Kau et al, 2004;Zanella et al, 2010c;Zanella et al, 2008). The analysis of the subcellular relocalization of signaling proteins, including NF-B (Taylor, 2010), FOXO (Kau et al, 2003;Link et al, 2009;Zanella et al, 2010c;Zanella et al, 2008;Zanella et al, 2009), NFAT (Wolff et al, 2006), p27Kip1, p53 (Xu et al, 2008), estrogen receptor alpha (Dull et al, 2010), p38 (Trask et al, 2006;Trask et al, 2009), Ets domain transcriptional repressor (ERF1) (Granas et al, 2006), AKT (Lundholt et al, 2005;Rosado et al, 2008;Wolff et al, 2006), glucokinase (Wolff et al, 2008) and HIV1 regulator of virion (REV) (Zanella et al, 2010b), have shed light on molecular mechanisms that underlie the spatiotemporal regulation of protein translocation and led to the identification of small molecule agents that interfere with the corresponding signaling networks.…”

Section: Discussionmentioning

confidence: 99%

Protein localization in disease and therapy

Hung

Link

2011

Journal of Cell Science

375

289

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SummaryThe eukaryotic cell is organized into membrane-covered compartments that are characterized by specific sets of proteins and biochemically distinct cellular processes. The appropriate subcellular localization of proteins is crucial because it provides the physiological context for their function. In this Commentary, we give a brief overview of the different mechanisms that are involved in protein trafficking and describe how aberrant localization of proteins contributes to the pathogenesis of many human diseases, such as metabolic, cardiovascular and neurodegenerative diseases, as well as cancer. Accordingly, modifying the disease-related subcellular mislocalization of proteins might be an attractive means of therapeutic intervention. In particular, cellular processes that link protein folding and cell signaling, as well as nuclear import and export, to the subcellular localization of proteins have been proposed as targets for therapeutic intervention. We discuss the concepts involved in the therapeutic restoration of disrupted physiological protein localization and therapeutic mislocalization as a strategy to inactivate disease-causing proteins. Journal of Cell Sciencecompartment has been associated with human diseases, and in the following sections we will discuss some of the mechanisms that can lead to such changes in protein localization. Mislocalization through alterations of the protein trafficking machineryDysregulation of the protein trafficking machinery can have dramatic effects on general protein transport processes, modifying cell morphology and physiology. Along these lines, changes in the nuclear pore complex (NPC) have been linked to several genetic disorders (Chahine and Pierce, 2009). For example, in patients with familial atrial fibrillation, the homozygous mutation R391H in the nucleoporin NUP155 has been shown to reduce nuclear envelope permeability and affect the export of Hsp70 mRNA and import of HSP70 protein (Zhang et al., 2008). That study was the first to link a nucleoporin defect to cardiovascular disease.Mutations in other components of the NPC, such as the nucleoporin p62 protein and ALADIN (alacrima achalasia adrenal insufficiency neurologic disorder, officially known as AAAS) are thought to cause the neurodegenerative diseases infantile bilateral striatal necrosis and triple A syndrome, respectively (Basel-Vanagaite et al., 2006; Kiriyama et al., 2008). Mutant ALADIN prevents nuclear entry of the DNA repair proteins aprataxin and DNA ligase I and, therefore, results in increased DNA damage and subsequent cell death caused by oxidative stress (Kiriyama et al., 2008). In a similar fashion, protein import into other organelles can be affected by mutations in the trafficking machinery. For instance, mutations in the peroxin gene PEX7, which encodes a peroxisomal import receptor that is responsible for the transport of several essential peroxisomal enzymes, have been found to cause the peroxisome biogenesis disorder rhizomelic chondrodysplasia punctata type 1 (RCDP1) (Braverman e...

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Section: Discussionmentioning

confidence: 99%

Protein localization in disease and therapy

Hung

Link

2011

Journal of Cell Science

375

289

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“…These key features serve to increase the quality of hit compounds and allow meaningful direct progression of compounds through functional characterization without further in vitro ADME/Tox studies. High-content screening assays also have certain disadvantages, including the need for specialized personnel and expensive equipment (see Lundholt et al 17 for discussion), and traditional cell-based assays for cancer drug discovery, such as tumor cell proliferation assays, may also allow the discovery of tumor-selective compounds. However, it is often difficult to find relevant control cell lines as most tumor cell lines have different growth characteristics from corresponding normal cells.…”

Section: Discussionmentioning

confidence: 99%

“…17,21 The MAPK pathway is a key regulator of cellular proliferation. Several proteins in this pathway are known as relevant targets for cancer therapy, and a number of drugs inhibiting this pathway are already in preclinical or clinical development.…”

Section: Redistributionmentioning

confidence: 99%

“…On the day of the experiment, cells were treated for 1 h at 37 °C, 5% CO 2 , and 95% humidity, in the presence or absence of the appropriate compounds and controls. Samples from treated cells were prepared and immunoblotted as described previously by Lundholt et al 17 The primary antibodies used were B-RAF (SC-5284; diluted 1:3000) and C-RAF (RAF-1), and C-12 (SC-133; diluted 1:500) from Santa Cruz Biotechnology or MEK1/2 (#9122; diluted 1:2000), p44/42 MAP kinase antibody (#9102; diluted 1:1000), and phospho-p44/42 MAP kinase (Thr202/Tyr204) (#9101; diluted 1:1000) or the Pathscan ® Multiplex Western Cocktail I (diluted 1:1000) in blocking buffer from Cell Signaling Technology (Beverly, MA). This cocktail simultaneously detects levels of phospho-p90RSK, phospho-Akt, phospho-p44/42 MAPK (ERK1/2), and phospho-S6 ribosomal protein and also contains an eIF4E antibody to monitor protein loading.…”

Section: Western Blot Analysismentioning

confidence: 99%

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Identification of RAS-Mitogen-Activated Protein Kinase Signaling Pathway Modulators in an ERF1 Redistribution® Screen

Grånäs¹,

Lundholt²,

Loechel³

et al. 2006

SLAS Discovery

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The RAS-mitogen-activated protein kinase (MAPK) signaling pathway has a central role in regulating the proliferation and survival of both normal and tumor cells. This pathway has been 1 focus area for the development of anticancer drugs, resulting in several compounds, primarily kinase inhibitors, in clinical testing. The authors have undertaken a cell-based, highthroughput screen using a novel ERF1 Redistribution ® assay to identify compounds that modulate the signaling pathway. The hit compounds were subsequently tested for activity in a functional cell proliferation assay designed to selectively detect compounds inhibiting the proliferation of MAPK pathway-dependent cancer cells. The authors report the identification of 2 cell membrane-permeable compounds that exhibit activity in the ERF1 Redistribution ® assay and selectively inhibit proliferation of MAPK pathway-dependent malignant melanoma cells at similar potencies (IC 50 = < 5 µM). These compounds have drug-like structures and are negative in RAF, MEK, and ERK in vitro kinase assays. Drugs belonging to these compound classes may prove useful for treating cancers caused by excessive MAPK pathway signaling. The results also show that cell-based, high-content Redistribution ® screens can detect compounds with different modes of action and reveal novel targets in a pathway known to be disease relevant. 1 The pathway is deregulated in various cancer types, and pathway-associated proteins are important targets in cancer drug discovery. Overexpression or mutations in receptor tyrosine kinases (RTKs) that result in excessive pathway activation are common in various types of carcinomas, and approximately 50% of several cancers, especially melanoma and colorectal and pancreatic cancers, exhibit aberrant MAPK pathway signaling.2-4 The MAPK pathway is activated by various growth factors that bind to RTKs, resulting in the activation of the RAS proteins. These proteins are GTPases that, when active, form a complex with RAF protein kinases. 5 This complex translocates to the plasma membrane where the RAF proteins subsequently activate MEK proteins. 5The downstream MEK1 and 2 proteins are dual-specificity protein kinases and constitute a focal point in the pathway because the kinases ERK1 and 2 (also named p44 and p42 MAPKs) are the only identified MEK substrates. Inactive ERK proteins are sequestered in the cytoplasm by the MEK kinases. Activated MEK1/2 release ERK1/2 from their anchorage in the cytoplasm by phosphorylating ERK1/2 on tyrosine and threonine residues.1,6 Activated ERK proteins phosphorylate multiple downstream effectors and also move into the nucleus to regulate gene expression by phosphorylating certain transcription factors.7,8 As a result of stimulating these transcriptional regulators, key cell cycle regulatory proteins are expressed, which enable the cell to progress through the G1 phase of the cell cycle. 9 In the nucleus, ERK1/2 is inactivated by the MAPK phosphatases (MKPs). 10-12The Ets domain transcriptional repressor ERF1 is a ubiquitously ex...

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“…Examples of published screens at large and medium scale include de-orphanizing of GPCR receptors in a 750,000-compound screen 24 and complex mechanistic investigation of 45,000 compounds using protein translocation assays to identify and profile Akt1 inhibitors. 25 So what are the evolutionary forces and drivers in action today? What is the future of HCS when pharma philosophy has shifted toward "fail early" 26 and the economic climate is very different from more profligate times when new technologies were seized upon in the race to screen more and more, faster and The evolution of bench-top HCS instruments from the first widely available commercial system from Cellomics continuing through the latest releases from each of the major HCS vendors.…”

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confidence: 99%

High-Content Screening: A Decade of Evolution

Thomas

2010

SLAS Discovery

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Identification of Akt Pathway Inhibitors Using Redistribution Screening on the FLIPR and the IN Cell 3000 Analyzer

Cited by 52 publications

References 17 publications

Protein localization in disease and therapy

Protein localization in disease and therapy

Identification of RAS-Mitogen-Activated Protein Kinase Signaling Pathway Modulators in an ERF1 Redistribution® Screen

High-Content Screening: A Decade of Evolution

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