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Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 315 (2003) 256–261 www.elsevier.com/locate/yabio
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 Yasuda3 Research Center Kyoto, Bayer Yakuhin, Ltd., 6-5-1-3, Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-0216, Japan Received 29 September 2002
Abstract Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-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 tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation 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-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell 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 (p72syk ) is a 72-kDa cytoplasmic protein-tyrosine kinase that serves as a critical molecule in the signal transduction pathway from certain receptors of the immunoglobulin Fc regions (FceRI, FccRI, FccRIIA) expressed on many hematopoietic cells [1–6] and from B cell receptor complex on B cells [7]. In response to the engagement of receptors with antigens and immunoglobulins, Src family tyrosine kinases such as Lyn * Corresponding author. Fax: +81-774-75-2507. E-mail address: [email protected] (N. Yamamoto). 1 Current address: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 830 N. University, Ann Arbor, MI 48109-1048, USA, 2 Current address: Antibacterial Research, Bayer AG, PH-Research 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.
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. Activated Syk is involved in the transduction of various signals, like degranulation, lipid mediator release, cytokine production, phagocytosis, superoxide production, apoptosis, and proliferation [1–6,12,13]. Treatment of permeabilized rat basophil leukemia cells (RBL-2H3) with kinase domain-truncated Syk protein inhibited degranulation and leukotriene production in 4 Abbreviations used: AL, activation loop peptide; HTS, highthroughput screening; CV, coefficient of variation; GST, glutathione Stransferase; ITAM, immunoreceptor tyrosine-based activation motif; S/B, signal-to-background ratio; PKC, protein kinase C; PKA, protein kinase A; EGF, epidermal growth factor; BSA, bovine serum albumin; 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
N. Yamamoto et al. / Analytical Biochemistry 315 (2003) 256–261
response to antigen stimulation [1]. A Syk-deficient variant of RBL-2H3 cells and fetal liver-derived mast cells from Syk knockout mice were also reported not to respond to antigen stimulation [2,3]. Macrophages and neutrophils derived from Syk knockout mice were defective in FccR-induced phagocytosis and O 2 production [4,5]. Furthermore, antisense DNA targeting the Syk mRNA inhibited FccRIIA-mediated phagocytosis in monocytes [6]. Since Syk plays such important and indispensable roles in various inflammatory cells, an inhibitor of Syk could be a novel therapy for asthma and other allergic diseases. Much evidence has accumulated that phosphorylation of conserved tyrosine(s) in the activation loop region upregulates the activity of tyrosine kinases, for example, insulin receptor kinase [14,15], nerve growth factor receptor kinase [16], Tec family kinase Itk [17], Src family kinase Lck [18,19], and Syk [12]. The first physical model that accounts for the role of this conserved tyrosine(s) was provided by the crystal structure of the insulin receptor kinase [14,15]. This structure indicates that the activation loop lies in a groove between the catalytic lobe and the ATP binding lobe, with the conserved tyrosine(s) occupying the catalytic site. Furthermore, phosphorylation of tyrosine(s) in the activation loop is considered to induce the movement of the loop to permit substrate access to the active site and ATP access to the ATP binding site, resulting in the efficient kinase activation. Although most of kinase inhibitors known so far are competitive inhibitors against ATP, we aimed to identify both ATP- and substrate-competitive inhibitors in high-throughput screening (HTS). For this purpose, we employed a peptide corresponding to the activation loop domain of Syk itself as a substrate in this assay, since autophosphorylation of tyrosines in the activation loop domain is a key for the activation of Syk kinase [12]. In order to obtain a wide detection range in a nonradioactive format and to apply the assay to robotic operation, a time-resolved fluorescence (europium) technique in the microtiter plate format was employed as previously reported [20]. After examining various parameters, which affect the signal-to-background (S/B) ratio, we have established a nonradioactive quantitative Syk kinase assay, which was applicable to HTS, and have validated this assay by comparing the effects of kinase inhibitors on native and recombinant Syk enzymes.
Materials and methods Kinase preparation. Human Syk kinase was prepared as a chimeric molecule with glutathione S-transferase (GST). Briefly, syk cDNA was amplified by RT-PCR from the total RNA of a human B cell line, Raji. After
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the identity of the sequence was confirmed as the same as in the previous report [21], it was inserted into the BamHI and SmaI sites of pAcG2T (Pharmingen, San Diego, CA, USA) in frame with GST. This vector was used to generate recombinant baculovirus with BaculoGold (Pharmingen). Virus plaque purification, amplification, and expression of GST-Syk were carried out using Sf21 insect cells essentially according to the manufacturerÕs instructions. GST-Syk was purified using glutathione-affinity chromatography (Amersham Pharmacia Biotech, England, UK) to more than 90% purity by SDS–PAGE according to the manufacturerÕs instructions. Other materials. N-terminally biotinylated activation loop peptide (AL), biotin–KISDFGLSKALRADENY YKAQTHGKWPVKW, was custom synthesized by Peptide Institute (Osaka, Japan). Streptavidin-coated plates were purchased from Labsystems (Helsinki, Finland). Genistein was purchased from Fujicco (Hyogo, Japan), staurosporine was from Wako (Osaka, Japan), piceatannol was from Sigma (St. Louis, MO, USA), and other inhibitors were from Calbiochem (San Diego, CA, USA). Europium labeling of antibody. Anti-phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Lake Placid, NY, USA) was labeled using a europium labeling kit (Perkin–Elmer, Wellesley, MA, USA) according to the manufacturerÕs recommendation. Briefly, after buffer exchange for labeling (50 mM NaHCO3 , pH 8.5, 0.9% NaCl) through a NAP-5 column (Amersham Pharmacia Biotech), antibody sample was mixed with europium labeling reagent and incubated at 22 °C for 1– 3 days. The europium-labeled antibody was purified by gel-filtration column in elution buffer (50 mM Tris–HCl, pH 7.8, 0.9% NaCl, 0.05% NaN3 ) and stored at 4 °C until use. Assay buffers. Assay buffers were prepared as follows: kinase assay buffer, 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 0.1 mM Na3 VO4 , 0.1% BSA, 1 mM DTT; termination buffer, 50 mM Tris–HCl (pH 8.0), 10 mM EDTA, 500 mM NaCl, 0.1% BSA; washing buffer, 50 mM Tris–HCl (pH 8.0), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20; antibody buffer, 50 mM Tris–HCl (pH 8.0), 138 mM NaCl, 2.7 mM KCl, 1% BSA, 60 ng/ml europium-labeled antibody. Enhancement solution was purchased from Perkin–Elmer. Standard assay protocol. GST-Syk (3.2 ng), 0.5 lg AL, 30 lM ATP, and testing compound in the presence of 0.25% Me2 SO were mixed in 50 ll/well of kinase assay buffer in polypropylene U-bottom 96-well microtiter plates. The mixture was incubated for 1 h at room temperature, and the reaction was terminated by the addition of 120 ll of termination buffer. To capture AL, 120 ll of the terminated mixture was transferred to streptavidin-coated plates, followed by incubation at room temperature for more than 30 min. After three
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washes with washing buffer, 100 ll of antibody buffer was added and incubated at room temperature for more than 30 min. After three more washes, 100 ll of enhancement solution was added. Time-resolved fluorescence was measured by multilabel counter ARVO (Wallac Oy, Turku, Finland).
Results and discussion Optimization of basic assay condition. At an early stage of the assay optimization, we first immobilized the substrate to the streptavidin-coated plate prior to the initiation of the reaction by adding kinase and ATP. Despite many manipulations, this assay format yielded only a signal (with ATP) to background (without ATP) ratio of 3 at best (data not shown). Immobilization of the biotinylated AL might fix it in a conformation that is not recognized well by Syk kinase. We found that the simultaneous mixing of all the kinase reaction ingredients in solution phase was effective to improve the S/B to more than 20. This finding would be applicable to other enzyme assays in which fixed substrates are used, to improve reaction efficiency. Using this assay format, we determined the optimal settings for various parameters. First, we tested the pH dependency of the kinase reaction. As shown in Fig. 1, we obtained higher signals at higher pH in the range examined (pH 6.5–9.0). The S/B also increased as the buffer pH increased, and it reached a plateau at around pH 8.0. Therefore, the pH of the buffer was adjusted to 8.0 in later experiments. Since we usually use Me2 SO as a solvent of test compounds, we examined the effect of Me2 SO on the kinase reaction. Increasing concentrations of Me2 SO (ranging from 0 to 1%) were tested at four different ATP concentrations (0, 10, 30, and 100 lM). Me2 SO gradually decreased the signal and maximally 20% decrease at 1% Me2 SO was observed (Fig. 2). Up to 0.25% Me2 SO had no effect on
Fig. 1. Effect of the buffer pH on S/B. To optimize the buffer pH, kinase reaction was carried out under various buffer pH conditions from 6.5 to 9.0 in the presence (open bar) or absence (hatched bar) of 30 lM ATP. The S/B (closed circle) is plotted simultaneously on the right. A representative result is shown.
Fig. 2. Effect of Me2 SO (DMSO) on the Syk kinase reaction. The kinase reaction was performed in the presence of various concentration of Me2 SO, the vehicle of test compounds. Symbols represent means SD of four independent experiments. ATP concentration for each symbol is (closed circle) 100 lM; (dark hatched square) 30 lM; (light hatched triangle) 10 lM; (open triangle) 0 lM.
kinase activity at any ATP concentrations used in this assay. Determination of apparent Km values for ATP and AL. To decide the concentration of AL and ATP for the assay, apparent Km values for them were examined by implementing the kinase assay with various concentrations of AL or ATP. From the Lineweaver–Burk plot analysis shown in Fig. 3, the apparent Km for AL and ATP were calculated as 550 and 76 lM, respectively.
Fig. 3. Determination of apparent Km values for AL (a) and ATP (b). The kinase reaction was performed with various concentrations of AL and ATP around their apparent Km values. Increase in fluorescence was divided by the reaction time length to calculate initial velocity (v). The apparent Km values were calculated as the reciprocals of the intercepts. Representative results are shown.
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The apparent Km value for AL was similar to that for a short AL peptide substrate (DENYYKA) in a previous publication in which purified Syk from rat spleen was used (Km ¼ 750 lM) [22]. We decided to use concentrations of AL (3 lM) and ATP (30 lM) that were lower than their apparent Km values in order to effectively find AL- or ATP-competitive inhibitors. Although several other peptide substrates for Syk with low micromolar Km values have been reported [22,23], we decided to use AL, which has relatively high apparent Km value. The reason was that autophosphorylation of tyrosines in the activation loop domain is a critical step for Syk activation [12]. Optimization of other assay parameters. To optimize the reaction time and the amount of enzyme, we carried out the Syk kinase assay under various conditions. As shown in Fig. 4a, 1-h reaction with 3.2 ng/well of GSTSyk, 3 lM of AL, and 30 lM of ATP was almost in the linear range of reaction for all four parameters. Fluorescence in the absence of AL (6.51% of control) was slightly high compared to that without ATP (0% of control), but it was not statistically significant. The fluorescence intensity obtained under optimized condition was less than 10% of that obtained with 1000 lM ATP (data not shown). Assuming that the kinase reaction in the presence of 1000 lM ATP results in complete AL phosphorylation, this suggested that consumption of AL was less than 10% of total, which might not affect the initial velocity of reaction. Intraplate data scattering was examined in a HTS robotic system and calculated as
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less than 10% CV, suggesting the stability of this kinase assay. Effects of kinase inhibitors. Under the protocol optimized as discussed above, this assay exerted a S/B of 20 to 30. Under this condition, 21 various tyrosine kinase inhibitors were tested (Fig. 5). Staurosporine, piceatannol, Go 6976, calphostin C, and ST638 inhibited the kinase reaction with IC50 values of 92 pM, 38 lM, 73 pM, 1.1 lM, and 27 lM, respectively. Sixteen other compounds had little or no inhibitory effect. Immunoprecipitated Syk from human monocytic U937 cells showed similar sensitivity to staurosporine (IC50 ¼ 0:3 nM), piceatannol (IC50 ¼ 25 lM), and genistein (IC50 ¼ 230 lM) (Fig. 5a), indicating that recombinant GST-Syk might have pharmacological characters similar to those of native immunoprecipitated Syk from human cells. Staurosporine and its analogue Go 6976 were very potent inhibitors for Syk kinase, but another analogue, Ro 318220, was not (Fig. 5). Because Ro 31-8220 was reported to have a selectivity for protein kinase C (PKC) over protein kinase A [24], there is a possibility that Ro 318220 also has a selectivity over Syk kinase. On the other hand, Ro 31-8220 could be a useful selective inhibitor for PKC over Syk and PKA in cellular and in vivo assays. Further, these results suggest that the structure–activity relationship (SAR) of staurosporine analogs might be different for each tyrosine kinase. Analysis of various staurosporine analogs in this Syk kinase assay would reveal the SAR for Syk kinase and it might be helpful for further drug development. Calphostin C, a PKC-selective
Fig. 4. Optimization of the assay condition. The Syk kinase reaction was performed under various conditions slightly different from ‘‘standard assay condition’’ (3.2 ng/well GST-Syk, 3 lM AL, and 30 lM ATP were incubated for 60 min). In (a) reaction time, in (b) concentration of GST-Syk, in (c) concentration of AL, and in (d) concentration of ATP was varied. Data were plotted as the percentage of control (0%, without ATP in the standard assay condition; 100%, standard assay condition). Each symbol means average SE of three independent experiments.
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Fig. 5. Effects of various inhibitors on the Syk kinase reaction. The Syk kinase reaction was performed in the presence of various kinase inhibitors. Data were expressed as percentages of controls as described in the legend for Fig. 4. Each point or bar represents the mean SE of two to four independent experiments. Recombinant GST-Syk was used in most of the experiments. In (a) native Syk was immunoprecipitated (open symbols) from human monocytic cells, U937, and compared with recombinant GST-Syk (closed symbols). (a) Circles, staurosporine; squares, piceatannol; triangles, genistein. (b) Closed circle, Go 6976; hatched square, calphostin C; open triangle, ST 638. The concentrations of inhibitors used in (c) were all 10 lM.
inhibitor, exerts its inhibitory action on PKC by diacylglycerol competitiveness with an IC50 value of 50 nM [25]. Therefore, the inhibition of Syk kinase activity by calphostin C (IC50 ¼ 1:1 lM, Fig. 5b) might be a nonspecific activity of this compound. In fact, when we implemented the kinase assay with a plate-coated T cell receptor f-chain as a substrate, calphostin C did not show any inhibition of Syk activity up to 10 lM (data not shown). This was contrasted with the fact that staurosporine, piceatannol, ST638, and genistein showed IC50 values similar to those when AL was used (0.16 nM, 10 lM, 100 lM, and 230 lM, respectively) (data not shown). From another point of view, AL was shown to be as useful as large protein substrates such as T cell receptor
f-chain to screen inhibitors. Tyrphostins, which have IC50 values in the micromolar range for EGF receptor kinase [26], were without effect on Syk (Fig. 5c). ST638, which has an IC50 value of 0.37–1.1 lM for EGF receptor kinase [27,28], was less potent for Syk (IC50 ¼ 27 lM, Fig. 5b). These results suggest that Syk, a nonreceptortype tyrosine kinase, might have structural features different from those of a receptor-type tyrosine kinase. In conclusion, we have established a nonradioactive quantitative Syk kinase assay utilizing the activation loop peptide as a substrate with a fluoroimmunoassay format for high-throughput screening. Baculovirus-expressed recombinant Syk enzyme tagged with GST showed a pharmacological profile similar to that of
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native immunoprecipitated Syk from a human cell line, and AL was as useful as T cell receptor f-chain, supporting the relevance of this assay system for the drug discovery purposes.
Acknowledgments We thank Dr. Michitaka Shichijo, Ms. Satomi Kobayashi, and Ms. Masako Sato for helping and supporting the experiments.
References [1] J.A. Taylor, J.L. Karas, M.K. Ram, O.M. Green, C.S. Dugan, Activation of high-affinity immunoglobulin E receptor Fc(epsilon)RI in RBL-2H3 cells is inhibited by Syk SH2 domains, Mol. Cell. Biol. 15 (1995) 4149–4157. [2] J. Zhang, E.H. Berenstein, R.L. Evans, R.P. Siraganian, Transfection of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 cells, J. Exp. Med. 184 (1996) 71–79. [3] P.S. Costello, M. Turner, A.E. Walters, C.N. Cunningham, P.H. Bauer, J. Downward, V.L.J. Tybulewicz, Critical role for the tyrosine kinase Syk in signaling through the high affinity IgE receptor of mast cells, Oncogene 13 (1996) 2595–2605. [4] M.T. Crowley, P.S. Costello, C.J.F. Attas, M. Turner, F. Meng, C. Lowell, V.L.J. Tybulewicz, A.L. DeFranco, A critical role for Syk in signal transduction and phagocytosis mediated by Fc(gamma) receptors on macrophages, J. Exp. Med. 186 (1997) 1027–1039. [5] F. Kiefer, J. Brumell, N.A. Alawi, S. Latour, A. Cheng, A. Veillette, S. Grinstein, T. Pawson, The Syk protein tyrosine kinase is essential for Fc(gamma) receptor signaling in macrophages and neutrophils, Mol. Cell. Biol. 18 (1998) 4209–4220. [6] M. Matsuda, J.G. Park, D.C. Wang, S. Hunter, P. Chien, A.D. Schreiber, Abrogation of the Fc(gamma) receptor IIA-mediated phagocytic signal by stem–loop syk antisense oligonucleotides, Mol. Biol. Cell 7 (1996) 1095–1106. [7] M. Takata, H. Sabe, A. Hata, T. Inazu, Y. Homma, T. Nukada, H. Yamamura, T. Kurosaki, Tyrosine kinase Lyn and Syk regulate B cell receptor-coupled Ca2þ mobilization through distinct pathways, EMBO J. 13 (1994) 1341–1349. [8] A.M. Scharenberg, S. Lin, B. Cuenod, H. Yamamura, J.P. Kinet, Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering, EMBO J. 14 (1995) 3385–3394. [9] H. Kihara, R.P. Siraganian, Src homology 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor, J. Biol. Chem. 269 (1994) 22427–22432. [10] L. Shiue, J. Green, O.M. Green, J.L. Karas, J.P. Morgenstern, M.K. Ram, M.K. Taylor, M.J. Zoller, L.D. Zydowsky, J.B. Bolen, J.S. Brugge, Interaction of p72syk with beta and gamma subunits of the high-affinity receptor for immunoglobulin E, Fc(epsilon)RI, Mol. Cell. Biol. 15 (1995) 272–281. [11] T. Kurosaki, S.A. Johnson, L. Pao, K. Sada, H. Yamamura, J.C. Cambier, Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling, J. Exp. Med. 182 (1995) 1815–1823.
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[12] O.E. El-Hillal, T. Kurosaki, H. Yamamura, J.P. Kinet, A.M. Scherenberg, syk kinase activation by src kinase-initiated activation loop phosphorylation chain reaction, Proc. Natl. Acad. Sci. USA 94 (1997) 1919–1924. [13] T. Kurosaki, Molecular dissection of B cell antigen receptor signaling, Int. J. Mol. Med. 1 (1998) 515–527. [14] S.R. Hubbard, L. Wei, L. Ellis, W.A. Hendrickson, Crystal structure of the tyrosine kinase domain of the human insulin receptor, Nature 372 (1994) 746–754. [15] S.R. Hubbard, Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog, EMBO J. 16 (1997) 5572–5581. [16] M.E. Cunningham, R.M. Stephens, D.R. Kaplan, L.A. Greene, Autophosphorylation of activation loop tyrosine regulates signaling by the TRK nerve growth factor receptor, J. Biol. Chem. 272 (1997) 10957–10967. [17] S.D. Heyeck, H.M. Wilcox, S.C. Bunnell, L.J. Berg, Lck phosphorylates the activation loop tyrosine of the Itk kinase domain and activates Itk kinase activity, J. Biol. Chem. 272 (1997) 25401–25408. [18] H. Yamaguchi, W.A. Hendrickson, Structural basis for activation of human lymphocyte kinase Lck upon tyrosine phosphorylation, Nature 384 (1996) 484–489. [19] J.S. Hardwick, B.M. Sefton, The activated form of the Lck tyrosine protein kinase in cells exposed to hydrogen peroxide is phosphorylated at both Tyr-394 and Tyr-505, J. Biol. Chem. 272 (1997) 25429–25432. [20] A.F. Braunwalder, D.R. Yarwood, M.A. Sills, K.E. Lipson, Measurement of the protein tyrosine kinase activity of c-src using time-resolved fluorometry of europium chelates, Anal. Biochem. 238 (1996) 159–164. [21] C.L. Law, S.P. Sidorenko, K.A. Chandran, K.E. Draves, A.C. Chan, A. Weiss, S. Edelhoff, C.M. Disteche, E.A. Clark, Molecular cloning of human Syk: A B cell protein-tyrosine kinase associated with the surface immunoglobulin M–B cell receptor complex, J. Biol. Chem. 269 (1994) 12310–12319. [22] A.M. Brunati, A. Donella-Deana, M. Ruzzene, O. Marin, L.A. Pinna, Site specificity of p72syk protein tyrosine kinase: efficient phosphorylation of motifs recognized by Src homology 2 domains of the Src family, FEBS Lett. 367 (1995) 149–152. [23] A. Donella-Deana, P. Ruzza, L. Cesaro, A.M. Brunati, A. Calderan, G. Borin, L.A. Pinna, Specific monitoring of Syk protein kinase activity by peptide substrates including constrained analogs of tyrosine, FEBS Lett. 523 (2002) 48–52. [24] P.D. Davis, L.H. Elliott, W. Harris, C.H. Hill, S.A. Hurst, E. Keech, M.K.H. Kumar, G. Lawton, J.S. Nixon, S.E. Wilkinson, Inhibitors of protein kinase C. 2. Substituted bisindolylmaleimides with improved potency and selectivity, J. Med. Chem. 35 (1992) 994–1001. [25] E. Kobayashi, H. Nakano, M. Morimoto, T. Tamaoki, Calphostin C (UCN-1028C), a novel microbial compound, is a highly potent and specific inhibitor of protein kinase C, Biochem. Biophys. Res. Commun. 159 (1989) 548–553. [26] P. Yaish, A. Gazit, C. Gilon, A. Levitzki, Blocking of EGFdependent cell proliferation by EGF receptor kinase inhibitors, Science 242 (1988) 933–935. [27] T. Shiraishi, T. Domoto, N. Imai, Y. Shimada, K. Watanabe, Specific inhibitors of tyrosine-specific protein kinase, synthetic 4hydroxycinnamamide derivatives, Biochem. Biophys. Res. Commun. 147 (1987) 322–328. [28] T. Shiraishi, M.K. Owada, M. Tatsuka, T. Yamashita, K. Watanabe, T. Kakunaga, Specific inhibitors of tyrosine-specific protein kinase: properties of 4-hydroxycinnamamide derivatives in vitro, Cancer Res. 49 (1989) 2374–2378.
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 Yasuda3 Research Center Kyoto, Bayer Yakuhin, Ltd., 6-5-1-3, Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-0216, Japan Received 29 September 2002
Abstract Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-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 tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation 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-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell 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 (p72syk ) is a 72-kDa cytoplasmic protein-tyrosine kinase that serves as a critical molecule in the signal transduction pathway from certain receptors of the immunoglobulin Fc regions (FceRI, FccRI, FccRIIA) expressed on many hematopoietic cells [1–6] and from B cell receptor complex on B cells [7]. In response to the engagement of receptors with antigens and immunoglobulins, Src family tyrosine kinases such as Lyn * Corresponding author. Fax: +81-774-75-2507. E-mail address: [email protected] (N. Yamamoto). 1 Current address: Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 830 N. University, Ann Arbor, MI 48109-1048, USA, 2 Current address: Antibacterial Research, Bayer AG, PH-Research 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.
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. Activated Syk is involved in the transduction of various signals, like degranulation, lipid mediator release, cytokine production, phagocytosis, superoxide production, apoptosis, and proliferation [1–6,12,13]. Treatment of permeabilized rat basophil leukemia cells (RBL-2H3) with kinase domain-truncated Syk protein inhibited degranulation and leukotriene production in 4 Abbreviations used: AL, activation loop peptide; HTS, highthroughput screening; CV, coefficient of variation; GST, glutathione Stransferase; ITAM, immunoreceptor tyrosine-based activation motif; S/B, signal-to-background ratio; PKC, protein kinase C; PKA, protein kinase A; EGF, epidermal growth factor; BSA, bovine serum albumin; 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
N. Yamamoto et al. / Analytical Biochemistry 315 (2003) 256–261
response to antigen stimulation [1]. A Syk-deficient variant of RBL-2H3 cells and fetal liver-derived mast cells from Syk knockout mice were also reported not to respond to antigen stimulation [2,3]. Macrophages and neutrophils derived from Syk knockout mice were defective in FccR-induced phagocytosis and O 2 production [4,5]. Furthermore, antisense DNA targeting the Syk mRNA inhibited FccRIIA-mediated phagocytosis in monocytes [6]. Since Syk plays such important and indispensable roles in various inflammatory cells, an inhibitor of Syk could be a novel therapy for asthma and other allergic diseases. Much evidence has accumulated that phosphorylation of conserved tyrosine(s) in the activation loop region upregulates the activity of tyrosine kinases, for example, insulin receptor kinase [14,15], nerve growth factor receptor kinase [16], Tec family kinase Itk [17], Src family kinase Lck [18,19], and Syk [12]. The first physical model that accounts for the role of this conserved tyrosine(s) was provided by the crystal structure of the insulin receptor kinase [14,15]. This structure indicates that the activation loop lies in a groove between the catalytic lobe and the ATP binding lobe, with the conserved tyrosine(s) occupying the catalytic site. Furthermore, phosphorylation of tyrosine(s) in the activation loop is considered to induce the movement of the loop to permit substrate access to the active site and ATP access to the ATP binding site, resulting in the efficient kinase activation. Although most of kinase inhibitors known so far are competitive inhibitors against ATP, we aimed to identify both ATP- and substrate-competitive inhibitors in high-throughput screening (HTS). For this purpose, we employed a peptide corresponding to the activation loop domain of Syk itself as a substrate in this assay, since autophosphorylation of tyrosines in the activation loop domain is a key for the activation of Syk kinase [12]. In order to obtain a wide detection range in a nonradioactive format and to apply the assay to robotic operation, a time-resolved fluorescence (europium) technique in the microtiter plate format was employed as previously reported [20]. After examining various parameters, which affect the signal-to-background (S/B) ratio, we have established a nonradioactive quantitative Syk kinase assay, which was applicable to HTS, and have validated this assay by comparing the effects of kinase inhibitors on native and recombinant Syk enzymes.
Materials and methods Kinase preparation. Human Syk kinase was prepared as a chimeric molecule with glutathione S-transferase (GST). Briefly, syk cDNA was amplified by RT-PCR from the total RNA of a human B cell line, Raji. After
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the identity of the sequence was confirmed as the same as in the previous report [21], it was inserted into the BamHI and SmaI sites of pAcG2T (Pharmingen, San Diego, CA, USA) in frame with GST. This vector was used to generate recombinant baculovirus with BaculoGold (Pharmingen). Virus plaque purification, amplification, and expression of GST-Syk were carried out using Sf21 insect cells essentially according to the manufacturerÕs instructions. GST-Syk was purified using glutathione-affinity chromatography (Amersham Pharmacia Biotech, England, UK) to more than 90% purity by SDS–PAGE according to the manufacturerÕs instructions. Other materials. N-terminally biotinylated activation loop peptide (AL), biotin–KISDFGLSKALRADENY YKAQTHGKWPVKW, was custom synthesized by Peptide Institute (Osaka, Japan). Streptavidin-coated plates were purchased from Labsystems (Helsinki, Finland). Genistein was purchased from Fujicco (Hyogo, Japan), staurosporine was from Wako (Osaka, Japan), piceatannol was from Sigma (St. Louis, MO, USA), and other inhibitors were from Calbiochem (San Diego, CA, USA). Europium labeling of antibody. Anti-phosphotyrosine monoclonal antibody 4G10 (Upstate Biotechnology, Lake Placid, NY, USA) was labeled using a europium labeling kit (Perkin–Elmer, Wellesley, MA, USA) according to the manufacturerÕs recommendation. Briefly, after buffer exchange for labeling (50 mM NaHCO3 , pH 8.5, 0.9% NaCl) through a NAP-5 column (Amersham Pharmacia Biotech), antibody sample was mixed with europium labeling reagent and incubated at 22 °C for 1– 3 days. The europium-labeled antibody was purified by gel-filtration column in elution buffer (50 mM Tris–HCl, pH 7.8, 0.9% NaCl, 0.05% NaN3 ) and stored at 4 °C until use. Assay buffers. Assay buffers were prepared as follows: kinase assay buffer, 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 0.1 mM Na3 VO4 , 0.1% BSA, 1 mM DTT; termination buffer, 50 mM Tris–HCl (pH 8.0), 10 mM EDTA, 500 mM NaCl, 0.1% BSA; washing buffer, 50 mM Tris–HCl (pH 8.0), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20; antibody buffer, 50 mM Tris–HCl (pH 8.0), 138 mM NaCl, 2.7 mM KCl, 1% BSA, 60 ng/ml europium-labeled antibody. Enhancement solution was purchased from Perkin–Elmer. Standard assay protocol. GST-Syk (3.2 ng), 0.5 lg AL, 30 lM ATP, and testing compound in the presence of 0.25% Me2 SO were mixed in 50 ll/well of kinase assay buffer in polypropylene U-bottom 96-well microtiter plates. The mixture was incubated for 1 h at room temperature, and the reaction was terminated by the addition of 120 ll of termination buffer. To capture AL, 120 ll of the terminated mixture was transferred to streptavidin-coated plates, followed by incubation at room temperature for more than 30 min. After three
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washes with washing buffer, 100 ll of antibody buffer was added and incubated at room temperature for more than 30 min. After three more washes, 100 ll of enhancement solution was added. Time-resolved fluorescence was measured by multilabel counter ARVO (Wallac Oy, Turku, Finland).
Results and discussion Optimization of basic assay condition. At an early stage of the assay optimization, we first immobilized the substrate to the streptavidin-coated plate prior to the initiation of the reaction by adding kinase and ATP. Despite many manipulations, this assay format yielded only a signal (with ATP) to background (without ATP) ratio of 3 at best (data not shown). Immobilization of the biotinylated AL might fix it in a conformation that is not recognized well by Syk kinase. We found that the simultaneous mixing of all the kinase reaction ingredients in solution phase was effective to improve the S/B to more than 20. This finding would be applicable to other enzyme assays in which fixed substrates are used, to improve reaction efficiency. Using this assay format, we determined the optimal settings for various parameters. First, we tested the pH dependency of the kinase reaction. As shown in Fig. 1, we obtained higher signals at higher pH in the range examined (pH 6.5–9.0). The S/B also increased as the buffer pH increased, and it reached a plateau at around pH 8.0. Therefore, the pH of the buffer was adjusted to 8.0 in later experiments. Since we usually use Me2 SO as a solvent of test compounds, we examined the effect of Me2 SO on the kinase reaction. Increasing concentrations of Me2 SO (ranging from 0 to 1%) were tested at four different ATP concentrations (0, 10, 30, and 100 lM). Me2 SO gradually decreased the signal and maximally 20% decrease at 1% Me2 SO was observed (Fig. 2). Up to 0.25% Me2 SO had no effect on
Fig. 1. Effect of the buffer pH on S/B. To optimize the buffer pH, kinase reaction was carried out under various buffer pH conditions from 6.5 to 9.0 in the presence (open bar) or absence (hatched bar) of 30 lM ATP. The S/B (closed circle) is plotted simultaneously on the right. A representative result is shown.
Fig. 2. Effect of Me2 SO (DMSO) on the Syk kinase reaction. The kinase reaction was performed in the presence of various concentration of Me2 SO, the vehicle of test compounds. Symbols represent means SD of four independent experiments. ATP concentration for each symbol is (closed circle) 100 lM; (dark hatched square) 30 lM; (light hatched triangle) 10 lM; (open triangle) 0 lM.
kinase activity at any ATP concentrations used in this assay. Determination of apparent Km values for ATP and AL. To decide the concentration of AL and ATP for the assay, apparent Km values for them were examined by implementing the kinase assay with various concentrations of AL or ATP. From the Lineweaver–Burk plot analysis shown in Fig. 3, the apparent Km for AL and ATP were calculated as 550 and 76 lM, respectively.
Fig. 3. Determination of apparent Km values for AL (a) and ATP (b). The kinase reaction was performed with various concentrations of AL and ATP around their apparent Km values. Increase in fluorescence was divided by the reaction time length to calculate initial velocity (v). The apparent Km values were calculated as the reciprocals of the intercepts. Representative results are shown.
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The apparent Km value for AL was similar to that for a short AL peptide substrate (DENYYKA) in a previous publication in which purified Syk from rat spleen was used (Km ¼ 750 lM) [22]. We decided to use concentrations of AL (3 lM) and ATP (30 lM) that were lower than their apparent Km values in order to effectively find AL- or ATP-competitive inhibitors. Although several other peptide substrates for Syk with low micromolar Km values have been reported [22,23], we decided to use AL, which has relatively high apparent Km value. The reason was that autophosphorylation of tyrosines in the activation loop domain is a critical step for Syk activation [12]. Optimization of other assay parameters. To optimize the reaction time and the amount of enzyme, we carried out the Syk kinase assay under various conditions. As shown in Fig. 4a, 1-h reaction with 3.2 ng/well of GSTSyk, 3 lM of AL, and 30 lM of ATP was almost in the linear range of reaction for all four parameters. Fluorescence in the absence of AL (6.51% of control) was slightly high compared to that without ATP (0% of control), but it was not statistically significant. The fluorescence intensity obtained under optimized condition was less than 10% of that obtained with 1000 lM ATP (data not shown). Assuming that the kinase reaction in the presence of 1000 lM ATP results in complete AL phosphorylation, this suggested that consumption of AL was less than 10% of total, which might not affect the initial velocity of reaction. Intraplate data scattering was examined in a HTS robotic system and calculated as
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less than 10% CV, suggesting the stability of this kinase assay. Effects of kinase inhibitors. Under the protocol optimized as discussed above, this assay exerted a S/B of 20 to 30. Under this condition, 21 various tyrosine kinase inhibitors were tested (Fig. 5). Staurosporine, piceatannol, Go 6976, calphostin C, and ST638 inhibited the kinase reaction with IC50 values of 92 pM, 38 lM, 73 pM, 1.1 lM, and 27 lM, respectively. Sixteen other compounds had little or no inhibitory effect. Immunoprecipitated Syk from human monocytic U937 cells showed similar sensitivity to staurosporine (IC50 ¼ 0:3 nM), piceatannol (IC50 ¼ 25 lM), and genistein (IC50 ¼ 230 lM) (Fig. 5a), indicating that recombinant GST-Syk might have pharmacological characters similar to those of native immunoprecipitated Syk from human cells. Staurosporine and its analogue Go 6976 were very potent inhibitors for Syk kinase, but another analogue, Ro 318220, was not (Fig. 5). Because Ro 31-8220 was reported to have a selectivity for protein kinase C (PKC) over protein kinase A [24], there is a possibility that Ro 318220 also has a selectivity over Syk kinase. On the other hand, Ro 31-8220 could be a useful selective inhibitor for PKC over Syk and PKA in cellular and in vivo assays. Further, these results suggest that the structure–activity relationship (SAR) of staurosporine analogs might be different for each tyrosine kinase. Analysis of various staurosporine analogs in this Syk kinase assay would reveal the SAR for Syk kinase and it might be helpful for further drug development. Calphostin C, a PKC-selective
Fig. 4. Optimization of the assay condition. The Syk kinase reaction was performed under various conditions slightly different from ‘‘standard assay condition’’ (3.2 ng/well GST-Syk, 3 lM AL, and 30 lM ATP were incubated for 60 min). In (a) reaction time, in (b) concentration of GST-Syk, in (c) concentration of AL, and in (d) concentration of ATP was varied. Data were plotted as the percentage of control (0%, without ATP in the standard assay condition; 100%, standard assay condition). Each symbol means average SE of three independent experiments.
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Fig. 5. Effects of various inhibitors on the Syk kinase reaction. The Syk kinase reaction was performed in the presence of various kinase inhibitors. Data were expressed as percentages of controls as described in the legend for Fig. 4. Each point or bar represents the mean SE of two to four independent experiments. Recombinant GST-Syk was used in most of the experiments. In (a) native Syk was immunoprecipitated (open symbols) from human monocytic cells, U937, and compared with recombinant GST-Syk (closed symbols). (a) Circles, staurosporine; squares, piceatannol; triangles, genistein. (b) Closed circle, Go 6976; hatched square, calphostin C; open triangle, ST 638. The concentrations of inhibitors used in (c) were all 10 lM.
inhibitor, exerts its inhibitory action on PKC by diacylglycerol competitiveness with an IC50 value of 50 nM [25]. Therefore, the inhibition of Syk kinase activity by calphostin C (IC50 ¼ 1:1 lM, Fig. 5b) might be a nonspecific activity of this compound. In fact, when we implemented the kinase assay with a plate-coated T cell receptor f-chain as a substrate, calphostin C did not show any inhibition of Syk activity up to 10 lM (data not shown). This was contrasted with the fact that staurosporine, piceatannol, ST638, and genistein showed IC50 values similar to those when AL was used (0.16 nM, 10 lM, 100 lM, and 230 lM, respectively) (data not shown). From another point of view, AL was shown to be as useful as large protein substrates such as T cell receptor
f-chain to screen inhibitors. Tyrphostins, which have IC50 values in the micromolar range for EGF receptor kinase [26], were without effect on Syk (Fig. 5c). ST638, which has an IC50 value of 0.37–1.1 lM for EGF receptor kinase [27,28], was less potent for Syk (IC50 ¼ 27 lM, Fig. 5b). These results suggest that Syk, a nonreceptortype tyrosine kinase, might have structural features different from those of a receptor-type tyrosine kinase. In conclusion, we have established a nonradioactive quantitative Syk kinase assay utilizing the activation loop peptide as a substrate with a fluoroimmunoassay format for high-throughput screening. Baculovirus-expressed recombinant Syk enzyme tagged with GST showed a pharmacological profile similar to that of
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native immunoprecipitated Syk from a human cell line, and AL was as useful as T cell receptor f-chain, supporting the relevance of this assay system for the drug discovery purposes.
Acknowledgments We thank Dr. Michitaka Shichijo, Ms. Satomi Kobayashi, and Ms. Masako Sato for helping and supporting the experiments.
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