Label-Free and Real-Time Cell-Based Kinase Assay for Screening Selective and Potent Receptor Tyrosine Kinase Inhibitors Using Microelectronic Sensor Array

Abstract

Kinases are the 2nd largest group of therapeutic targets in the human genome. In this article, a label-free and real-time cell-based receptor tyrosine kinase (RTK) assay that addresses limitation of existing kinase assays and can be used for high-throughput screening and lead optimization studies was validated and characterized. Using impedance, growth factor-induced morphological changes were quantitatively assessed in real time and used as a measure of RTK activity. COS7 cells treated with epidermal growth factor (EGF) and insulin results in a rapid increase in cell impedance. Assessment of these growth factor-induced morphological changes and levels of receptor autophosphorylation using fluorescent microscopy and enzyme-linked immunosorbent assay, respectively, demonstrates that these changes correlate with changes in impedance. This assay was used to screen, identify, and characterize a potent EGF receptor inhibitor from a compound library. This report describes an assay that is simple in that it does not require intensive optimization or special reagents such as peptides, antibodies, or probes. More important, because the assay is cell based, the studies are done in a physiologically relevant environment, allowing for concurrent assessment of a compound’s solubility, stability, membrane permeability, cytotoxicity, and off-target interaction effects. (Journal of Biomolecular Screening 2006:634-643)

Keywords

impedance cell-based assay receptor tyrosine kinase kinase growth factor

References

Manning G , Whyte DB , Martinez R , Hunter T , Sudarsanam S : The protein kinase complement of the human genome. Science 2002;298;1912-1934.

Schlessinger J : Cell signaling by receptor tyrosine kinases. Cell 2000; 103: 211-225.

Ullrich A , Coussens L , Hayflick JS , Dull TJ , Gray A , Tam AW , et al : Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 1984;309: 418-425.

Nicholson RI , Gee JM , Harper ME : EGFR and cancer prognosis. Eur J Cancer 2001;37(Suppl 4): S9-S15.

Salomon DS , Brandt R , Ciardiello F , Normanno N : Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995;19: 183-232.

Slamon DJ , Clark GM , Wong SG , Levin WJ , Ullrich A , McGuire WL : Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235: 177-182.

Lohrisch C , Piccart M : HER2/neu as a predictive factor in breast cancer. Clin Breast Cancer 2001;2: 129-135.

Gschwind A , Fischer OM , Ullrich A : The discovery of receptor tyrosine kinases: targets for cancer therapy. Nat Rev Cancer 2004;4: 361-370.

Pawson T , Schlessinger J : SH2 and SH3 domains. Curr Biol 1993;3: 434-442.

10.

Ridley AJ , Hall A : The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992;70: 389-399.

11.

Ridley AJ , Paterson HF , Johnston CL , Diekmann D , Hall A : The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992;70: 401-410.

12.

Hall A : Rho GTPases and the actin cytoskeleton. Science 1998;279: 509-514.

13.

Chinkers M , McKanna JA , Cohen S : Rapid induction of morphological changes in human carcinoma cells A-431 by epidermal growth factors. J Cell Biol 1979;83: 260-265.

14.

Miyata Y , Nishida E , Koyasu S , Yahara I , Sakai H : Protein kinase C-dependent and -independent pathways in the growth factor-induced cytoskeletal reorganization. J Biol Chem 1989;264: 15565-15568.

15.

Welsh JB , Gill GN , Rosenfeld MG , Wells A : A negative feedback loop attenuates EGF-induced morphological changes. J Cell Biol 1991;114: 533-543.

16.

Yip SC , El-Sibai M , Hill KM , Wu H , Fu Z , Condeelis JS , et al : Over-expression of the p110beta but not p110alpha isoform of PI 3-kinase inhibits motility in breast cancer cells. Cell Motil Cytoskeleton 2004;59: 180-188.

17.

Kurokawa K , Itoh RE , Yoshizaki H , Nakamura YO , Matsuda M : Coactivation of Rac1 and Cdc42 at lamellipodia and membrane ruffles induced by epidermal growth factor. Mol Biol Cell 2004;15: 1003-1010.

18.

Giaever I , Keese CR : Monitoring fibroblast behavior in tissue culture with an applied electric field. Proc Natl Acad Sci U S A 1984;81: 3761-3764.

19.

Solly K , Wang X , Xu X , Strulovici B , Zheng W. Application of real-time cell electronic sensing (RT-CES) technology to cell-based assays. Assay Drug Dev Technol 2004;2: 363-372.

20.

Xing JZ , et al. Dynamic monitoring of cytotoxicity on microelectronic sensors. Chem Res Toxicol 2005;18: 154-161.

21.

Wegener J , Keese CR , Giaever I : Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces. Exp Cell Res 2000;259: 158-166.

22.

Atienza JM , Zhu J , Wang X , Xu X , Abassi Y : Dynamic monitoring of cell adhesion and spreading on microelectronic sensor arrays. J Biomol Screen 2005;10: 795-805.

23.

Keese CR , Bhawe K , Wegener J , Giaever I. Real-time impedance assay to follow the invasive activities of metastatic cells in culture. Biotechniques 2002;33: 842-844, 846-846, 848-850.

24.

Ciambrone GJ , Liu VF , Lin DC , McGuinness RP , Leung GK , Pitchford S : Cellular dielectric spectroscopy: a powerful new approach to label-free cellular analysis. J Biomol Screen 2004;9: 467-480.

25.

Cockerill S , Stubberfield C , Stables J , Carter M , Guntrip S , Smith K , et al : Indazolylamino quinazolines and pyridopyrimidines as inhibitors of the EGFr and C-erbB-2. Bioorg Med Chem Lett 2001;11: 1401-1405.

26.

Chen P , Murphy-Ullrich JE , Wells A. A role for gelsolin in actuating epidermal growth factor receptor-mediated cell motility. J Cell Biol 1996; 134: 689-698.

27.

Wells A , Ware MF , Allen FD , Lauffenburger DA : Shaping up for shipping out: PLCgamma signaling of morphology changes in EGF-stimulated fibroblast migration. Cell Motil Cytoskeleton 1999;44: 227-233.

28.

Rodriguez-Viciana P , Warne PH , Khwaja A , Marte BM , Pappin D , Das P , et al : Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras. Cell 1997;89: 457-467.

29.

Wennstrom S , Hawkins P , Cooke F , Hara K , Yonezawa K , Kasuga M , et al : Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Curr Biol 1994;4: 385-393.

30.

Kotani K , Yonezawa K , Hara K , Ueda H , Kitamura Y , Sakaue H , et al : Involvement of phosphoinositide 3-kinase in insulin-or IGF-1-induced membrane ruffling. Embo J 1994;13: 2313-2321.

31.

Sun XG , Rotenberg SA : Overexpression of protein kinase Calpha in MCF-10A human breast cells engenders dramatic alterations in morphology, proliferation, and motility. Cell Growth Differ 1999;10: 343-352.

32.

Davies SP , Reddy H , Caivano M , Cohen P : Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 2000;351: 95-105.

33.

Burridge K , Wennerberg K : Rho and Rac take center stage. Cell 2004; 116: 167-179.

34.

Ayscough KR , et al. High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A. J Cell Biol. 1997;137: 399-416.

35.

Morton WM , Ayscough KR , McLaughlin PJ : Latrunculin alters the actinmonomer subunit interface to prevent polymerization. Nat Cell Biol 2000;2: 376-378.

36.

Bridges AJ , Zhou H , Cody DR , Rewcastle GW , McMichael A , Showalter HD , et al : Tyrosine kinase inhibitors. 8. An unusually steep structure-activity relationship for analogues of 4-(3-bromoanilino)-6,7-dimethoxyquinazoline (PD 153035), a potent inhibitor of the epidermal growth factor receptor. J Med Chem 1996;39: 267-276.

37.

Rewcastle GW , Palmer BD , Bridges AJ , Showalter HD , Sun L , Nelson J , et al : Tyrosine kinase inhibitors. 9. Synthesis and evaluation of fused tricyclic quinazoline analogues as ATP site inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor. J Med Chem 1996;39: 918-928.

38.

Soler C , Beguinot L , Carpenter G : Individual epidermal growth factor receptor autophosphorylation sites do not stringently define association motifs for several SH2-containing proteins. J Biol Chem 1994;269: 12320-12324.

39.

Wyckoff JB , Insel L , Khazaie K , Lichtner RB , Condeelis JS , Segall JE : Suppression of ruffling by the EGF receptor in chemotactic cells. Exp Cell Res 1998;242: 100-109.