A Scintillation Proximity Assay for the Raf/MEK/ERK Kinase Cascade: High-Throughput Screening and Identification of Selective Enzyme Inhibitors

McDonald, Octerloney B.; Chen, Wen Ji; Ellis, Byron; Hoffman, Christine R.; Overton, Laurie K.; Rink, Martin J.; Smith, Andrew N.; Marshall, Christopher J.; Wood, Edgar R.

doi:10.1006/abio.1998.3030

Cited by 63 publications

(41 citation statements)

References 45 publications

(23 reference statements)

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“…Expression vectors for ERK2 and ERK5 expression in yeast were constructed by PCR amplifying the coding sequences of ERK2 (from plasmid pGEX4T-ERK2) (33) and ERK5a (from image clone 4300124; GenBank accession no. BC009963) (primer sequences available on request).…”

Section: Methodsmentioning

confidence: 99%

Expressed in the Yeast Saccharomyces cerevisiae , Human ERK5 Is a Client of the Hsp90 Chaperone That Complements Loss of the Slt2p (Mpk1p) Cell Integrity Stress-Activated Protein Kinase

et al. 2006

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ERK5 is a mitogen-activated protein (MAP) kinase regulated in human cells by diverse mitogens and stresses but also suspected of mediating the effects of a number of oncogenes. Its expression in the slt2⌬ Saccharomyces cerevisiae mutant rescued several of the phenotypes caused by the lack of Slt2p (Mpk1p) cell integrity MAP kinase. ERK5 is able to provide this cell integrity MAP kinase function in yeast, as it is activated by the cell integrity signaling cascade that normally activates Slt2p and, in its active form, able to stimulate at least one key Slt2p target (Rlm1p, the major transcriptional regulator of cell wall genes). In vitro ERK5 kinase activity was abolished by Hsp90 inhibition. ERK5 activity in vivo was also lost in a strain that expresses a mutant Hsp90 chaperone. Therefore, human ERK5 expressed in yeast is an Hsp90 client, despite the widely held belief that the protein kinases of the MAP kinase class are non-Hsp90-dependent activities. Two-hybrid and protein binding studies revealed that strong association of Hsp90 with ERK5 requires the dual phosphorylation of the TEY motif in the MAP kinase activation loop. These phosphorylations, at positions adjacent to the Hsp90-binding surface recently identified for a number of protein kinases, may cause a localized rearrangement of this MAP kinase region that leads to creation of the Hsp90-binding surface. Complementation of the slt2⌬ yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5.Mitogen-activated protein kinase (MAPK) modules consist of 3 protein kinases that stimulate each other in series (MAP3K 3 MAP2K 3 MAPK), resulting in the activation of the terminal and often multifunctional MAPK. These signaling cascades are key components of the highly interactive protein kinase networks in eukaryotic cells. As their name implies, some MAPKs initiate a proliferative response. Others control pathways of embryogenesis, differentiation, stress responses, and cell death (42). Association of MAPK modules with scaffold proteins appears to be one way of ensuring that each MAPK only becomes activated in response to the correct extracellular stimuli or stress signals (52). Once activated, this MAPK can then proceed to phosphorylate its substrates, the latter being often involved in both short-term and longer-term (e.g., transcription-mediated) cellular changes. Many MAPKs have substrates in both the cytoplasm and the nucleus, such that nuclear import/export mechanisms frequently govern their accessibility to their substrates. The final outcome of any MAPK activation event is presumably dictated by this substrate availability in any given cell type, by an intrinsic substrate specificity directed by the docking interaction of the MAPK with its substrates, and by signal attenuation. The latter involves the intervention of the protein phosphatases that, by dephosphorylating and thereby deactivating the MAPK, modulate both the intensity and the dura...

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Section: Methodsmentioning

confidence: 99%

Expressed in the Yeast Saccharomyces cerevisiae , Human ERK5 Is a Client of the Hsp90 Chaperone That Complements Loss of the Slt2p (Mpk1p) Cell Integrity Stress-Activated Protein Kinase

et al. 2006

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“…The majority of these early biochemical assays relied on the detection of radiolabeled phosphate incorporation into peptide and protein substrates using filter binding and the more recent scintillation proximity assay (SPA) technology. 11,12 Although filtration technology has evolved into a much higher throughput format and remains the gold standard by which other formats are judged, a number of newer technologies that offer the promise of higher throughput, nonradioactive detection are available. Fluorescence polarization (FP), enhanced chemiluminescence (ECL), and microfluidics (MF) are a few of the technologies that the drug discovery industry is evaluating in an effort to increase kinase lead generation.…”

mentioning

confidence: 99%

Data Concordance from a Comparison between Filter Binding and Fluorescence Polarization Assay Formats for Identification of ROCK-II Inhibitors

et al. 2003

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The Rho-associated coiled-coil-containing protein serine/threonine kinases ROCK-I and ROCK-II are thought to play a major role in cytoskeletal dynamics by serving as downstream effectors of the Rho/Rac family of cytokine-and growth factoractivated small GTPases. As such, the ROCK family members are attractive intervention targets for a variety of pathologies, including cancer and cardiovascular disease. The authors developed a high-throughput screen to identify ROCK-II inhibitors and report results from a direct comparison of 2 screening campaigns for ROCK-II inhibitors using fluorescence polarization (FP) and filter binding (FB). Screening protocols to identify inhibitors of ROCK-II were developed in FB and FP formats under similar assay and kinetic conditions. A 30,000-member compound library was screened using FB ( 33 P) and FP detection systems, and compounds that were active in either assay were retested in 5-point curve confirmation assays. Analysis of these data showed an approximate 95% agreement of compounds identified as active in both assay formats. Also, compound potency determinations from FB and FP had a high degree of correlation and were considered equivalent. These data suggest that the assay methodology has little impact on the quality and productivity of the screen, provided that the assays are developed to standardize kinetic conditions. (Journal of Biomolecular Screening 2003:399-409)

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“…Excellent combinatorial chemistry approaches and high-throughput screening assays have led to successful identification of many selective enzyme inhibitors (Lam et al, 1991;McDonald et al, 1999;Su et al, 2000;Wilhelm et al, 2006). For drug development, the universal goal is to find specific kinase inhibitors with selectivity toward a particular malignant cell type.…”

Section: Discussionmentioning

confidence: 99%

Molecular Characteristics of CTA056, a Novel Interleukin-2-Inducible T-Cell Kinase Inhibitor that Selectively Targets Malignant T Cells and Modulates Oncomirs

et al. 2012

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Interleukin-2-inducible T-cell kinase (Itk) is a member of the Btk (Bruton's tyrosine kinase) family of tyrosine kinases. Itk plays an important role in normal T-cell functions and in the pathophysiology of both autoimmune diseases and T-cell malignancies. Here, we describe the initial characterization of a selective inhibitor, 7-benzyl-1-(3-(piperidin-1-yl)propyl)-2-(4-(pyridin-4-yl)phenyl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one (CTA056), that was developed through screening a 9600-compound combinatorial solution phase library, followed by molecular modeling, and extensive structure-activity relationship studies. CTA056 exhibits the highest inhibitory effects toward Itk, followed by Btk and endothelial and epithelial tyrosine kinase. Among the 41 cancer cell lines analyzed, CTA056 selectively targets acute lymphoblastic T-cell leukemia and cutaneous T-cell lymphoma. Normal T cells are minimally affected. Incubation of Jurkat and MOLT-4 cells with CTA056 resulted in the inhibition of the phosphorylation of Itk and its effectors including PLC-␥, Akt, and extracellular signal-regulated kinase, as well as the decreased secretion of targeted genes such as interleukin-2 and interferon-␥. Jurkat cells also underwent apoptosis in a dose-dependent manner when incubated with CTA056. The potent apoptosis-inducing potential of CTA056 is reflected by the significant modulation of microRNAs involved in survival pathways and oncogenesis. The in vitro cytotoxic effect on malignant T cells is further validated in a xenograft model. The selective expression and activation of Itk in malignant T cells, as well as the specificity of CTA056 for Itk, make this molecule a potential therapeutic agent for the treatment of T-cell leukemia and lymphoma.

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A Scintillation Proximity Assay for the Raf/MEK/ERK Kinase Cascade: High-Throughput Screening and Identification of Selective Enzyme Inhibitors

Cited by 63 publications

References 45 publications

Expressed in the Yeast Saccharomyces cerevisiae , Human ERK5 Is a Client of the Hsp90 Chaperone That Complements Loss of the Slt2p (Mpk1p) Cell Integrity Stress-Activated Protein Kinase

Expressed in the Yeast Saccharomyces cerevisiae , Human ERK5 Is a Client of the Hsp90 Chaperone That Complements Loss of the Slt2p (Mpk1p) Cell Integrity Stress-Activated Protein Kinase

Data Concordance from a Comparison between Filter Binding and Fluorescence Polarization Assay Formats for Identification of ROCK-II Inhibitors

Molecular Characteristics of CTA056, a Novel Interleukin-2-Inducible T-Cell Kinase Inhibitor that Selectively Targets Malignant T Cells and Modulates Oncomirs

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