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Journal of Biomolecular Screening
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A Fluorescence Lifetime–Based Binding Assay to Characterize Kinase Inhibitors

Connie S. Lebakken

Invitrogen Discovery Sciences, Madison, Wisconsin

Hee Chol Kang

Invitrogen Molecular Probes, Eugene, Oregon

Kurt W. Vogel

Invitrogen Discovery Sciences, Madison, Wisconsin, kurt.vogel{at}invitrogen.com

The authors present a fluorescence lifetime—based kinase binding assay that identifies and characterizes compounds that bind to the adenosine triphosphate (ATP)—binding pocket of a range of tyrosine and serine/threonine kinases. The assay is based on displacement of an Alexa Fluor® 647 conjugate of staurosporine from the ATP-binding site of a kinase, which is detected by a change in the fluorescence lifetime of the probe between the free (displaced) and kinase-bound states. The authors screened 257 kinases for specific binding and displacement of the Alexa Fluor® 647-staurosporine probe and found that approximately half of the kinases tested could potentially be assayed with this method. They present inhibitor binding data against 4 selected serine/threonine kinases and 4 selected tyrosine kinases, using 6 commonly used kinase inhibitors. Two of these kinases were chosen for further studies, in which inhibitor binding data were compared to inhibition of kinase activity using 2 separate activity assay formats. Rank-order potencies of compounds were similar, but not identical, between the binding and activity assays. It was postulated that these differences could be caused by the fact that the assays are measuring distinct phenomena, namely, activity versus binding, and in a purified recombinant kinase preparation, there can exist a mixture of active and nonactivated kinases. To explore this possibility, the authors compared binding affinity for the probe using 2 kinases in their respective nonactivated and activated (phosphorylated) forms and found a kinase-dependent difference between the 2 forms. This assay format therefore represents a simple method for the identification and characterization of small-molecule kinase inhibitors that may be useful in screening a wide range of kinases and may be useful in identifying small molecules that bind to kinases in their active or nonactivated states. (Journal of Biomolecular Screening 2007:828-841)

Key Words: kinase • inhibitors • fluorescence lifetime • drug discovery

References

  • Parker GJ, Law TL, Lenoch FJ, Bolger RE: Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays . J Biomol Screen 2000;5(2): 77-88 .[Abstract/Free Full Text]
  • Morgan AG, McCauley TJ, Stanaitis ML, Mathrubutham M., Millis SZ: Development and validation of a fluorescence technology for both primary and secondary screening of kinases that facilitates compound selectivity and site-specific inhibitor determination . Assay Drug Dev Technol 2004;2(2): 171-181 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Xia W., Rininsland F., Wittenburg SK, Shi X., Achyuthan KE, McBranch DW, et al: Applications of fluorescent polymer superquenching to high throughput screening assays for protein kinases . Assay Drug Dev Technol 2004;2(2): 183-192 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Loomans EE, van Doornmalen AM, Wat JW, Zaman GJ: High-throughput screening with immobilized metal ion affinity-based fluorescence polarization detection, a homogeneous assay for protein kinases . Assay Drug Dev Technol 2003;1(3): 445-453 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Rodems SM, Hamman BD, Lin C., Zhao J., Shah S., Heidary D., et al: A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases . Assay Drug Dev Technol 2002;1(1pt.1): 9-19 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Shults MD, Janes KA, Lauffenburger DA, Imperiali B.: A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates . Nat Methods 2005;2(4): 277-283 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Mathis G.: Probing molecular interactions with homogeneous techniques based on rare earth cryptates and fluorescence energy transfer . Clin Chem 1995;41(9): 1391-1397 .[Abstract/Free Full Text]
  • Peterson MF, Martin WH, Spencer RW, Tate BF: The use of beta, gammamethyleneadenosine 5'-triphosphate to determine ATP competition in a scintillation proximity kinase assay . Anal Biochem 1999;271(2): 131-136 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Slon-Usakiewicz JJ, Dai JR, Ng W., Foster JE, Deretey E., Toledo-Sherman L., et al: Global kinase screening: applications of frontal affinity chromatography coupled to mass spectrometry in drug discovery . Anal Chem 2005;77(5): 1268-1274 .[Medline] [Order article via Infotrieve]
  • Annis DA, Nazef N., Chuang CC, Scott MP, Nash HM: A general technique to rank protein-ligand binding affinities and determine allosteric versus direct binding site competition in compound mixtures . J Am Chem Soc 2004;126(47): 15495-15503 .[Web of Science][Medline] [Order article via Infotrieve]
  • Wang WU, Chen C., Lin KH, Fang Y., Lieber CM: Label-free detection of small-molecule-protein interactions by using nanowire nanosensors . Proc Natl Acad Sci U S A 2005;102(9): 3208-3212 .[Abstract/Free Full Text]
  • Fabian MA, Biggs WH Iii, Treiber DK, Atteridge CE, Azimioara MD, Benedetti MG, et al: A small molecule-kinase interaction map for clinical kinase inhibitors . Nat Biotechnol 2005;23(3): 329-336 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Vainshtein I., Silveria S., Kaul P., Rouhani R., Eglen RM, Wang J.: A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP) . J Biomol Screen 2002;7(6): 507-514 .[Abstract/Free Full Text]
  • Pargellis C., Tong L., Churchill L., Cirillo PF, Gilmore T., Graham AG, et al: Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site . Nat Struct Biol 2002;9(4): 268-272 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Zhang WX, Wang R., Wisniewski D., Marcy AI, LoGrasso P., Lisnock JM, et al: Time-resolved Forster resonance energy transfer assays for the binding of nucleotide and protein substrates to p38alpha protein kinase . Anal Biochem 2005;343(1): 76-83 .[Web of Science][Medline] [Order article via Infotrieve]
  • Kashem MA, Nelson RM, Yingling JD, Pullen SS, Prokopowicz AS III, Jones JW, et al: Three mechanistically distinct kinase assays compared: measurement of intrinsic ATPase activity identified the most comprehensive set of ITK inhibitors . J Biomol Screen 2007;12(1): 70-83 .[Abstract/Free Full Text]
  • Toledo LM, Lydon NB: Structures of staurosporine bound to CDK2 and cAPK—new tools for structure-based design of protein kinase inhibitors . Structure 1997;5(12): 1551-1556 .[Medline] [Order article via Infotrieve]
  • Marks BD, Qadir N., Eliason HC, Shekhani MS, Doering K., Vogel KW: Multiparameter analysis of a screen for progesterone receptor ligands: comparing fluorescence lifetime and fluorescence polarization measurements . Assay Drug Dev Technol 2005;3(6): 613-622 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Popp D., Haupl C., Doering K.: Nanosecond fluorescence lifetime analysis— a new dimension for detection in microplates . Paper presented at: the SBS 8th Annual Conference and Exhibition, The Hague, Netherlands, September 22-26, 2002.
  • Turconi S., Bingham RP, Haupts U., Pope AJ: Developments in fluorescence lifetime-based analysis for ultra-HTS. Drug Discovery Today 2001; 6(12 suppl.): S27-S39 .
  • Hoefelschweiger BK, Pfeifer L., Wolfbeis OS: Screening scheme based on measurement of fluorescence lifetime in the nanosecond domain . J Biomol Screen 2005;10(7): 687-694 .[Abstract]
  • Wallrabe H., Periasamy A.: Imaging protein molecules using FRET and FLIM microscopy . Curr Opin Biotechnol 2005;16(1): 19-27 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Riddle SM, Vedvik KL, Hanson GT, Vogel KW: Time-resolved fluorescence resonance energy transfer kinase assays using physiological protein substrates: applications of terbium-fluorescein and terbium-green fluorescent protein fluorescence resonance energy transfer pairs . Anal Biochem 2006;356(1): 108-116 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Grinvald A., Steinberg IZ: On the analysis of fluorescence decay kinetics by the method of least-squares . Anal Biochem 1974;59(2): 583-598 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Moger J., Gribbon P., Sewing A., Winlove CP: The application of fluorescence lifetime readouts in high-throughput screening . J Biomol Screen 2006;11(7): 765-772 .[Abstract/Free Full Text]
  • Zhang JH, Chung TD, Oldenberg KR: A simple statistical parameter for use in evaluation and validation of high-throughput screening assays . J Biomol Screen 1999;4(2): 67-73 .[Abstract/Free Full Text]
  • Levitzki A., Gazit A.: Tyrosine kinase inhibition: an approach to drug development . Science 1995;267(5205): 1782-1788 .[Abstract/Free Full Text]
  • Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S., Green D., et al: A protein kinase involved in the regulation of inflammatory cytokine biosynthesis . Nature 1994;372(6508): 739-746 .[CrossRef][Medline] [Order article via Infotrieve]
  • Davies SP, Reddy H., Caivano M., Cohen P.: Specificity and mechanism of action of some commonly used protein kinase inhibitors . Biochem J 2000;351(pt. 1): 95-105 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Gschwendt M., Dieterich S., Rennecke J., Kittstein W., Mueller HJ, Johannes FJ: Inhibition of protein kinase C mu by various inhibitors: differentiation from protein kinase C isoenzymes . FEBS Lett 1996;392(2): 77-80 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Goekjian PG, Jirousek MR: Protein kinase C in the treatment of disease: signal transduction pathways, inhibitors, and agents in development . Curr Med Chem 1999;6(9): 877-903 .[Web of Science][Medline] [Order article via Infotrieve]
  • Buolamwini JK, Kamath S.: Molecule-kinase interaction map . Nat Biotechnol 2005;23(11): 1346 .[Web of Science][Medline] [Order article via Infotrieve]
  • Neuweiler H., Schulz A., Vaiana AC, Smith JC, Kaul S., Wolfrum J., et al: Detection of individual p53-autoantibodies by using quenched peptide-based molecular probes . Angew Chem Int Ed Engl 2002;41(24): 4769-4773 .[CrossRef]
  • Visser AJ: Kinetics of stacking interactions in flavin adenine dinucleotide from time-resolved flavin fluorescence . Photochem Photobiol 1984;40(6): 703-706 .[Web of Science][Medline] [Order article via Infotrieve]
  • Vedvik KL, Eliason HC, Hoffman RL, Gibson JR, Kupcho KR, Somberg RL, et al: Overcoming compound interference in fluorescence polarization-based kinase assays using far-red tracers . Assay Drug Dev Technol 2004; 2(2): 193-203 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Jager S., Brand L., Eggeling C.: New fluorescence techniques for high-throughput drug discovery . Curr Pharm Biotechnol 2003;4(6): 463-476 .[CrossRef][Medline] [Order article via Infotrieve]
  • Harpur AG, Wouters FS, Bastiaens PI: Imaging FRET between spectrally similar GFP molecules in single cells . Nat Biotechnol 2001;19(2): 167-169 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  • Huang X.: Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand . J Biomol Screen 2003;8(1): 34-38 .[Abstract/Free Full Text]
  • Comley J.: Assay interference: a limiting factor in HTS ? Drug Discov World 2003(summer): 91-98 .
  • Turek-Etienne TC, Lei M., Terracciano JS, Langsdorf EF, Bryant RW, Hart RF, et al: Use of red-shifted dyes in a fluorescence polarization AKT kinase assay for detection of biological activity in natural product extracts . J Biomol Screen 2004;9(1): 52-61 .[Abstract/Free Full Text]
  • Hennig A., Florea M., Roth D., Enderle T., Nau WM: Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore . Anal Biochem 2007;360(2): 255-265 .[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

This version was published on September 1, 2007

Journal of Biomolecular Screening, Vol. 12, No. 6, 828-841 (2007)
DOI: 10.1177/1087057107304480
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[Abstract] [PDF]

This Article
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