Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors Noriyuki Yamamoto, * Haruki Hasegawa, 1 Hitomi Seki, Karl Ziegelbauer, 2 and Takahiro Yasuda 3 Research Center Kyoto, Bayer Yakuhin, Ltd., 6-5-1-3, Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-0216, Japan Received 29 September 2002 Abstract SykisatyrosinekinasewhichisindispensableinimmunoglobulinFcreceptor-andBcellreceptor-mediatedsignaltransduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactivekinaseassaytoidentifyinhibitorsofSyk.WeusedrecombinantGST-taggedSykpurifiedfrombaculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated ty- rosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphory- lation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-de- pendentinhibitionofrecombinantSykkinaseactivitywithalmostthesameefficacyforimmunoprecipitatedSykfromahumancell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Syk; Europium-based fluoroimmunoassay; High-throughput screening; Asthma Syk(p72 syk )isa72-kDacytoplasmicprotein-tyrosine kinase that serves as a critical molecule in the signal transduction pathway from certain receptors of the im- munoglobulin Fc regions (FceRI, FccRI, FccRIIA) expressedonmanyhematopoieticcells[1–6]andfromB cell receptor complex on B cells [7]. In response to the engagement of receptors with antigens and immuno- globulins, Src family tyrosine kinases such as Lyn phosphorylate a conserved cytoplasmic portion of the receptors, the so-called immunoreceptor tyrosine-based activation motif (ITAM) 4 [8]. Syk is then recruited to the doubly phosphorylated ITAM region of receptors through its Src-homology domain [9–11] and becomes activated by autophosphorylation on tyrosine residues. ActivatedSykisinvolvedinthetransductionofvarious signals, like degranulation, lipid mediator release, cyto- kine production, phagocytosis, superoxide production, apoptosis, and proliferation [1–6,12,13]. Treatmentofpermeabilizedratbasophilleukemiacells (RBL-2H3) with kinase domain-truncated Syk protein inhibited degranulation and leukotriene production in Analytical Biochemistry 315 (2003) 256–261 www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: +81-774-75-2507. E-mail address: noriyuki.yamamoto.ny@bayer.co.jp (N. Yamam- oto). 1 Current address: Department of Molecular, Cellular, and Devel- opmental Biology, University of Michigan, 830 N. University, Ann Arbor, MI 48109-1048, USA, 2 Current address: Antibacterial Research, Bayer AG, PH-Re- search Anti-infectives, P.O. Box 10 17 09, D-42096 Wuppertal, Germany. 3 Current address: Department of Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, 4072, Queensland, Australia. 4 Abbreviations used: AL, activation loop peptide; HTS, high- throughputscreening;CV,coefficientofvariation;GST,glutathione S- transferase; ITAM, immunoreceptor tyrosine-based activation motif; S/B,signal-to-backgroundratio;PKC,proteinkinaseC;PKA,protein kinaseA;EGF,epidermalgrowthfactor;BSA,bovineserumalbumin; DTT, dithiothreitol; SAR, structure–activity relationship. 0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00026-5