Abstract
Due to their important roles in cellular signaling and their dysfunctions being linked to diseases, kinases have become a class of proteins being actively pursued as potential drug targets. Biochemical assays for kinases have been developed in various formats to facilitate inhibitor screening and selectivity profiling. Here, we focus on one such technology: homogeneous time-resolved fluorescence (HTRF). In this chapter, we describe the methods of developing an HTRF kinase assay using mutant EGFR enzyme as an example. We show how to determine the kinetic parameter of the enzyme (ATP K m), as well as how to study the inhibitor mechanism of action (MoA) exemplified by inhibitors of different MoAs. All methods described here can be readily applied to other kinases with minor modifications.
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References
Manning G, Whyte DB, Martinez R et al (2002) The protein kinase complement of the human genome. Science 298:1912–1934
Cohen P (1999) The development and therapeutic potential of protein kinase inhibitors. Curr Opin Chem Biol 3:459–465
Hunter T (2000) Signaling—2000 and beyond. Cell 100:113–127
Paez JG, Janne PA, Lee JC et al (2004) EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304:1497–1500
Pao W, Girard N (2011) New driver mutations in non-small-cell lung cancer. Lancet Oncol 12:175–180
Soda M, Choi YL, Enomoto M et al (2007) Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer. Nature 448:561–566
Davies H, Bignell GR, Cox C et al (2002) Mutations of the BRAF gene in human cancer. Nature 417:949–954
Fabbro D, Garcia-Echeverria C (2002) Targeting protein kinases in cancer therapy. Curr Opin Drug Discov Dev 5:701–712
Myers MR, He W, Hulme C (1997) Inhibitors of tyrosine kinases involved in inflammation and autoimmune disease. Curr Pharm Des 3:473–502
Minor L (2005) Assays for membrane tyrosine kinase receptors: methods for high-throughput screening and utility for diagnostics. Expert Rev Mol Daign 5:561–571
Sittampalam GS, Kahl SD, Janzen WP (1997) High-throughput screening: advances in assay technologies. Curr Opin Chem Biol 1:384–391
Zaman GJR, Garritsen A, de Boer TH et al (2003) Fluorescence assays for high-throughput screening of protein kinases. Comb Chem High Throughput Screen 6:313–320
von Ahsen O, Bömer U (2005) High-throughput screening for kinase inhibitors. Chem Bio Chem 6:481–490
Jia Y, Gu X, Brinker A et al (2008) Measuring the tyrosine kinase activity: a review of biochemical and cellular assay technologies. Expert Opin Drug Discov 3:959–978
Jia Y, Quinn CM, Kwak S et al (2008) Current in vitro kinase assay technologies: the quest for a universal format. Curr Drug Discov Technol 5:59–69
Zegzouti H, Zdanovskaia M, Hsiao K, Goueli SA (2009) ADP-Glo: A bioluminescent and homogeneous ADP monitoring assay for kinases. Assay Drug Dev Technol 7:560–572
Mathis G (1999) HTRF technology. J Biomol Screen 4:309–313
Mathis G (1995) Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer. Clin Chem 41:1391–1397
Mathis G (1993) Rare earth cryptate and homogeneous fluoroimmunoassays with human sera. Clin Chem 39:1953–1959
Kobayashi S, Boggon TJ, Dayaram T et al (2005) EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 352:786–792
Pao W, Miller VA, Politi KA et al (2005) Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2, e73
Yun C-H, Boggon TJ, Li Y et al (2007) Structure of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell 11:217–227
Jia Y, Quinn CM, Gagnon AI (2006) Homogeneous time-resolved fluorescence and its applications for kinase assays in drug discovery. Anal Biochem 356:273–281
Hong L, Quinn CM, Jia Y (2009) Evaluating the utility of the HTRF Transcreener™ ADP assay technology: A comparison with the standard HTRF assay technology. Anal Biochem 391:31–38
Li D, Ambrogio L, Shimamura T et al (2008) BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene 27:4702–4711
Moyer JD, Barbacci EG, Iwata KK et al (1997) Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res 57:4838–4848
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Jia, Y., Manuia, M., Juarez, J. (2016). HTRF Kinase Assay Development and Methods in Inhibitor Characterization. In: Zegzouti, H., Goueli, S. (eds) Kinase Screening and Profiling. Methods in Molecular Biology, vol 1360. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3073-9_1
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DOI: https://doi.org/10.1007/978-1-4939-3073-9_1
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3072-2
Online ISBN: 978-1-4939-3073-9
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