Homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assays represent a highly sensitive and robust high-throughput screening (HTS) method for the quantification of kinase activity. Traditional TR-FRET kinase assays detect the phosphorylation of an exogenous substrate. The authors describe the development and optimization of a TR-FRET technique that measures the autophosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) kinase and extend its applicability to a variety of other kinases. The VEGFR-2 assay demonstrated dose-dependent inhibition by compounds known to modulate the catalytic activity of this receptor. In addition, kinetic analysis of a previously characterized VEGFR-2 inhibitor was performed using the method, and results were consistent with those obtained using a different assay format. Because of the known involvement of VEGFR-2 in angiogenesis, this assay should facilitate HTS for antiangiogenic agents. In addition, this general technique should have utility for the screening for inhibitors of kinases as potential therapeutic agents for many other disease indications.
References
1.
1. Cohen P: Protein kinases—the major drug targets of the twenty-first century? Nat Rev Drug Disc2002;1(4): 309-315 .
2.
2. Farley K, Mett H, McGlynn E, Murray B, Lydon NB: Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c-src tyrosine protein kinase activity . Anal Biochem1992;203(1): 151-157 .
3.
3. Cook ND: Scintillation proximity assay: a versatile high-throughput screening technology . Drug Discov Today1996;1(7): 287-294 .
4.
4. Seethala R, Menzel R: A homogeneous, fluorescence polarization assay for Src-family tyrosine kinases . Anal Biochem1997;253: 210-218 .
5.
5. Kolb AJ, Kaplita PV, Hayes DJ, Park Y-W, Pernell C, Major JS, et al: Tyrosine kinase assays adapted to homogeneous time-resolved fluorescence . Drug Discov Today1998;3(7): 333-342 .
6.
6. Pope AJ: Introduction: LANCETM vs. HTRF® technologies (orvice versa) . J Biomol Screen1999;4(6): 301-302 .
7.
7. Folkman J: Angiogenesis in cancer, vascular, rheumatoid and other disease . Nat Med1995;1(1): 27-31 .
8.
8. Ferrara N: VEGF and the quest for tumor angiogenesis factors . Nat Rev Canc2002;2: 795-803 .
10. Scappaticci FA: Mechanisms and future directions for angiogenesis-based cancer therapies . J Clin Onc2002;20(18): 3906-3927 .
11.
11. Quinn TP, Peters KG, De Vries C, Ferrara N, Williams LT: Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium . Proc Natl Acad Sci USA1993;90(16): 7533-7537 .
12.
12. Boyer SJ: Small molecule inhibitors of KDR (VEGFR-2) kinase: an overview of structure activity relationships . Curr Top Med Chem2002;2: 973-1000 .
13.
13. Sun L, Liang C, Shirazian S, Zhou Y, Miller T, Cui J, et al: Discovery of 5-[5-fluoro-2-oxo-1, 2-dihydroindol-(3Z)-ylidenemethyl]-2, 4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethyl)amide, a novel tyrosine kinase inhibitor targeting vascular endothelial and platelet-derived growth factor receptor tyrosine kinase . J Med Chem2003;46(7): 1116-1119 .
14.
14. Shawver LK, Schwartz DP, Mann E, Chen H, Tsai J, Chu L, et al: Inhibition of platelet-derived growth factor-mediated signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl]5-methylisoxazole-4-carboxamide . Clin Canc Res1997;3(7): 1167-1177 .
15.
15. Blake RA, Broome MA, Liu X, Wu J, Gishizky M, Sun L, et al: SU6656, a selective src family kinase inhibitor, used to probe growth factor signaling . Mol Cell Biol2000;20(23): 9018-9027 .
16.
16. Mendel DB, Laird AD, Xin X, Louie SG, Christensen JG, Li G, et al: In vivo anti-tumor activity of SU11248, a novel tyrosine kinase inhibitor targeting VEGF and PDGF receptors: determination of a pharmacokinetic/pharmacodynamic relationship . Clin Canc Res2003;9: 327-337 .
17.
17. Bader B, Butt E, Palmetshofer A, Walter U, Jarchau T, Drueckes P: A cGMP-dependent protein kinase assay for high throughput screening based on time-resolved fluorescence resonance energy transfer . J Biomol Screen2001;6(4): 255-264 .
18.
18. Biazzo-Ashnault DE, Park Y-W, Cummings RT, Ding V, Moller DE, Zhang BB, et al: Detection of insulin receptor tyrosine kinase activity using time-resolved fluorescence energy transfer technology . Anal Biochem2001;291: 155-158 .
19.
19. Ohmi N, Wingfield JM, Yazawa H, Inagaki O: Development of a homogeneous time-resolved fluorescence assay for high-throughput screening to identify Lck inhibitors: comparison with scintillation proximity assay and streptavidin-coated plate assay . J Biomol Screen2000;5(6): 463-470 .
20.
20. Park Y-W, Cummings RT, Wu L, Zheng S, Cameron PM, Woods A, et al: Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence . Anal Biochem1999;269: 94-104 .
21.
21. Dougher-Vermazen M, Hulmes JD, Bohlen P, Terman BI: Biological activity and phosphorylation sites of the bacterially expressed cytosolic domain of the KDR VEGF-receptor . Biochem Biophys Res Comm1994;205(1): 728-738 .
22.
22. Dougher M, Terman BI: Autophosphorylation of KDR in the kinase domain is required for maximal VEGF-stimulated kinase activity and receptor internalization . Oncogene1999;18(8): 1619-1627 .
23.
23. Parast CV, Mroczkowski B, Pinko C, Misialek S, Khambatta G, Appelt K: Characterization and kinetic mechanism of catalytic domain of human vascular endothelial growth factor receptor-2 tyrosine kinase (VEGFR2 TK), a key enzyme in angiogenesis . Biochemistry1998;37(47): 16788-16801 .
