This Article Abstract Free Full Text (Free PDF) Free Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Ishii, T. Articles by Noumi, T. Search for Related Content PubMed PubMed Citation Articles by Ishii, T. Articles by Noumi, T. Social Bookmarking                         What's this?

A Robust, Target-Driven, Cell-Based Assay for Checkpoint Kinase 1 Inhibitors

GlaxoSmithKline K.K., Tsukuba-shi Ibaraki, Japan

Hiroshi Sootome

Bioscience Department GlaxoSmithKline K.K. 43 Wadai, Tsukuba-shi Ibaraki 300-4247, Japan, hiroshi_sootome{at}merck.com

Alastair J. King

GlaxoSmithKline, Upper Providence, Pennsylvania

Mikiya Suda

GlaxoSmithKline K.K., Tsukuba-shi Ibaraki, Japan, Molecular Medicine Research Laboratories, Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki, Japan

Nobuhiro Noro

GlaxoSmithKline K.K., Tsukuba-shi Ibaraki, Japan

Keizo Yamashita

GlaxoSmithKline K.K., Tsukuba-shi Ibaraki, Japan

Takato Noumi

GlaxoSmithKline K.K., Tsukuba-shi Ibaraki, Japan

Checkpoint kinase 1 (Chk1), a serine/threonine kinase, plays an important role in DNA damage checkpoint control and is an attractive target for cancer treatment. To develop a Chk1-specific cell-based assay, stable clones were established in which Chk1 kinase domain fused at its N-terminus with p53 through 4 tandem repeats of Gly-Gly-Gly-Gly-Ser was expressed in an inducible manner. Chk1 kinase specificity of the phosphorylation of fused p53 was confirmed by the experiments with a kinase-inactive Chk1. Only in the presence of an inducer molecule was phosphorylation of p53 at Ser-15 in the stable clones induced. Furthermore, its assay performance proved acceptable for high-throughput screening applications, judging from the Z' factor values (> 0.77). Finally, the cell-based assay thus established yielded structure-activity relationship data for a small set of test inhibitors of Chk1 within cells. Collectively, these results demonstrate that the established cell-based assay provides a novel and highly sensitive cellular platform for Chk1 inhibitor discovery. (Journal of Biomolecular Screening 2007:809-817)

Key Words: checkpoint kinase 1 • GeneSwitch system • target specific • cell-based assay

References

• Liu Q., Guntuku S., Cui XS, Cortez G., Tamai K., Luo G., et al: Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint . Genes Dev 2000;14: 1448-1459 .[Abstract/Free Full Text]
• Dixon H., Norbury CJ: Therapeutic exploitation of checkpoint defects in cancer cells lacking p53 function . Cell Cycle 2002;1: 362-368 .[Medline] [Order article via Infotrieve]
• Tao ZF, Lin NH: Chk1 inhibitors for novel cancer treatment . Anticancer Agents Med Chem 2006;6: 377-388 .[Medline] [Order article via Infotrieve]
• Sausville EA, Arbuck SG, Messmann R., Headlee D., Bauer KS, Lush RM, et al: Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms . J Clin Oncol 2001;19: 2319-2333 .[Abstract/Free Full Text]
• Ou YH, Chung PH, Sun TP, Shieh SY: p53 C-terminal phosphorylation by CHK1 and CHK2 participates in the regulation of DNA-damage-induced C-terminal acetylation . Mol Biol Cell 2005;16: 1684-1695 .[Abstract/Free Full Text]
• Maki CG, Huibregtse JM, Howley PM: In vivo ubiquitination and proteasome-mediated degradation of p53(1) . Cancer Res 1996;56: 2649-2654 .[Abstract/Free Full Text]
• Honda R., Yasuda H.: Association of p19(ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53 . EMBO J 1999; 18: 22-27 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Oren M.: Regulation of the p53 tumor suppressor protein . J Biol Chem 1999;274: 36031-36034 .[Free Full Text]
• Ashcroft M., Kubbutat MH, Vousden KH: Regulation of p53 function and stability by phosphorylation . Mol Cell Biol 1999;19: 1751-1758 .[Abstract/Free Full Text]
• Liu AM, Ho MK, Wong CS, Chan JH, Pau AH, Wong YH: Galpha(16/z) chimeras efficiently link a wide range of G protein-coupled receptors to calcium mobilization . J Biomol Screen 2003;8: 39-49 .[Abstract/Free Full Text]
• Storz P., Doppler H., Horn-Muller J., Groner B., Pfizenmaier K., Muller G.: A cellular reporter assay to monitor insulin receptor kinase activity based on STAT 5-dependent luciferase gene expression . Anal Biochem 1999; 276: 97-104 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Wood JM, Bold G., Buchdunger E., Cozens R., Ferrari S., Frei J., et al: PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration . Cancer Res 2000;60: 2178-2189 .[Abstract/Free Full Text]
• Quinones A., Rainov NG: Identification of genotoxic stress in human cells by fluorescent monitoring of p53 expression . Mutat Res 2001;494: 73-85 .[Web of Science][Medline] [Order article via Infotrieve]
• Dikov MM, Reich MB, Dworkin L., Thomas JW, Miller GG: A functional fibroblast growth factor-1 immunoglobulin fusion protein . J Biol Chem 1998;273: 15811-15817 .[Abstract/Free Full Text]
• Sakurai H., Nishi A., Sato N., Mizukami J., Miyoshi H., Sugita T.: TAK1-TAB1 fusion protein: a novel constitutively active mitogen-activated protein kinase kinase kinase that stimulates AP-1 and NF-kappaB signaling pathways . Biochem Biophys Res Commun 2002;297: 1277-1281 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Zheng C., Xiang J., Hunter T., Lin A.: The JNKK2-JNK1 fusion protein acts as a constitutively active c-Jun kinase that stimulates c-Jun transcription activity . J Biol Chem 1999;274: 28966-28971 .[Abstract/Free Full Text]
• Zentrich E., Han SY, Pessoa-Brandao L., Butterfield L., Heasley LE: Collaboration of JNKs and ERKs in nerve growth factor regulation of the neurofilament light chain promoter in PC12 cells . J Biol Chem 2002; 277: 4110-4118 .[Abstract/Free Full Text]
• Huston JS, Levinson D., Mudgett-Hunter M., Tai MS, Novotny J., Margolies MN, et al: Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli . Proc Natl Acad Sci U S A 1988;85: 5879-5883 .[Abstract/Free Full Text]
• Hazen RJ, Harvey RJ, St. Clair MH, Ferris RG, Freeman GA, Tidwell JH, et al: Anti-human immunodeficiency virus type 1 activity of the nonnucleoside reverse transcriptase inhibitor GW678248 in combination with other antiretrovirals against clinical isolate viruses and in vitro selection for resistance . Antimicrob Agents Chemother 2005;49: 4465-4473 .[Abstract/Free Full Text]
• Gollin SM: Chromosomal instability . Curr Opin Oncol 2004;16: 25-31 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Suda M., Ishii T., Sootome H., Shibahara M., Yamashita K., Nobuhiro N., et al: Cell-based screening for specifically detecting kinase activity . Biotechniques. Forthcoming.
• Zhao B., Bower MJ, McDevitt PJ, Zhao H., Davis ST, Johanson KO, et al: Structural basis for Chk1 inhibition by UCN-01 . J Biol Chem 2002; 277: 46609-46615 .[Abstract/Free Full Text]
• Warren GL, Andrews CW, Capelli AM, Clarke B., LaLonde J., Lambert MH, et al: A critical assessment of docking programs and scoring functions . J Med Chem 2006;49: 5912-5931 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Yue TL, Gu JL, Wang C., Reith AD, Lee JC, Mirabile RC, et al: Extracellular signal-regulated kinase plays an essential role in hypertrophic agonists, endothelin-1 and phenylephrine-induced cardiomyocyte hypertrophy . J Biol Chem 2000;275: 37895-37901 .[Abstract/Free Full Text]
• Wang XJ, Liefer KM, Tsai S., O'Malley BW, Roop DR: Development of gene-switch transgenic mice that inducibly express transforming growth factor beta1 in the epidermis . Proc Natl Acad Sci U S A 1999;96: 8483-8488 .[Abstract/Free Full Text]
• Zhang JH, Chung TD, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high-throughput screening assays . J Biomol Screen 1999;4: 67-73 .[Abstract/Free Full Text]
• Cheng Y., Prusoff WH: Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction . Biochem Pharmacol 1973;22: 3099-3108 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Knight ZA, Shokat KM: Features of selective kinase inhibitors . Chem Biol 2005;12: 621-637 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
• Kubbutat MH, Vousden KH: Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability . Mol Cell Biol 1997;17: 460-468 .[Abstract]
• Oliner JD, Pietenpol JA, Thiagalingam S., Gyuris J., Kinzler KW, Vogelstein B.: Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53 . Nature 1993;362: 857-860 .[CrossRef][Medline] [Order article via Infotrieve]

This version was published on September 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 6, 809-817 (2007)
DOI: 10.1177/1087057107303323


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This Article Abstract Free Full Text (Free PDF) Free Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Ishii, T. Articles by Noumi, T. Search for Related Content PubMed PubMed Citation Articles by Ishii, T. Articles by Noumi, T. Social Bookmarking                         What's this?