色谱  2013, Vol. 31 Issue (7): 646-655   PDF (854KB)    
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张倩倩
张雪佩
张含智
康经武
细管电泳-激光诱导荧光检测用于多个胞内蛋白激酶的抑制剂筛选和选择性评价
张倩倩, 张雪佩, 张含智, 康经武     
中国科学院上海有机化学研究所, 上海 200032
收稿日期：2013-04-18
基金项目：National Natural Science Foundations of China (90713021, 20975109, 20675086); National Key Laboratory of the Organic Biochemistry Opening Foundation.
通讯作者：康经武.E-mail:jingwu.kang@sioc.ac.cn.
摘要：发展了一种基于毛细管电泳(CE)-激光诱导荧光(LIF)检测的多个细胞内源激酶的抑制剂平行筛选及选择性评价方法。CE高效的分离能力和LIF检测器的高选择性,使得同时测试多个胞内激酶的活性成为可能。共4种细胞系、3种特异性蛋白激酶底物肽、2种选择性蛋白激酶抑制剂和1种非选择性蛋白激酶抑制剂用于方法的建立。特异性底物肽与细胞裂解液混合后孵育,被其相应的激酶选择性地磷酸化,利用CE-LIF分离检测磷酸化产物和底物肽。同时测定一个抑制剂对几种蛋白激酶的抑制活性,用于评价抑制剂的选择性。与传统的单靶标筛选模式相比,这种基于细胞裂解液的多靶标筛选方法能提供更多的信息,更加高效,且细胞裂解液作为一种廉价的激酶来源大大降低了筛选成本。
关键词： 毛细管电泳     激光诱导荧光检测     蛋白激酶抑制剂筛选     选择性评价    
Inhibitor screening and selectivity assessment against multiple cellular protein kinases by capillary electrophoresis with laser-induced fluorescence detection
ZHANG Qianqian, ZHANG Xuepei, ZHANG Hanzhi, KANG Jingwu     
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
Received: 2013-04-18
Abstract: A method that can be used for screening protein kinase inhibitors (PKIs) and simultaneously assessing their selectivity is described. The method is based on simultaneously assaying multiple cellular protein kinases by performing capillary electrophoresis (CE) separation and measuring the peak areas of the phosphorylated substrate peptides. The powerful separation capability of CE combined with the highly sensitive and selective laser-induced fluorescence (LIF) detector enables the direct screening of PKIs against cell lysates, which are used as an inexpensive source of enzymes. Four cell lines, three specific substrate peptides labeled with 5-carboxyfluorescein (5-FAM), two relative specific PKIs (TBB and H-89) and one non-specific PKI (staurosporine) were utilized to prove the methodology. With this method, the inhibitory activity of the tested compounds against multiple protein kinases was identified in parallel by comparing the peak areas of the phosphorylated substrates with those obtained in the absence of any inhibitors. The reduced peak area of the phosphorylated substrate definitively represents a positive screening result. Simultaneously, assaying the inhibition of one inhibitor against mutiple cellular protein kinases enables the assessment of its selectivity. Compared to the conventional, single-target screening format, the cell lysate-based multi-target method is more informative, more straightforward and more cost effective.
Key words: capillary electrophoresis (CE)     laser-induced fluorescence detection     protein kinase inhibitor screening     selectivity assessment    
Dysregulated cell signaling through protein kinases frequently contributes to the pathogenesis of diseases such as cancer, diabetes and rheumatoid arthritis[ 1, 2, 3, 4, 5 ]. Therefore, protein kinases have become the second largest group of therapeutic targets, after G-protein coupled receptors[ 1, 6 ]. To date, nine small molecular protein kinase inhibitors (PKIs) have been approved by the US Food and Drug Administration (FDA) for cancer treatment, and approximately 80 inhibitors have been in various stages of clinic trials [ 7 ]. Despite such a success, compared to the number of the clinically validated protein kinase-based drug targets, PKIs having high potency and reasonable selectivity toward their target kinases are quite rare [ 8 ]. Moreover, none of the kinase-targeted drugs responsible for treating immunological, neurological, metabolic and infectious diseases have yet been proved by US FDA, although these diseases also involve dysregulated protein kinases[ 7, 9 ]. Therefore, it is imperative to develop more effective techniques for inhibitor screening to gain more high quality PKIs [ 7 ].

Currently, several technologies based on the cell and biochemical assays are available for PKI screening. An advantage of the cell-based assays is that the targets are both in the natural cellular environment and in their native configuration. However, the cell-based assays often suffer from off-target hits, especially when the particular targets are involved in a complex signal-transduction pathway. Moreover, the toxicity of the compounds can cause false positive results [ 10 ]. Biochemical assays have been accepted as the gold standard in the pharmaceutical industry during the early stages of drug discovery due to their high throughput nature [ 10 ]. The performance of the biochemical assays requires purified enzymes and antiphospho-antibodies[ 11, 12, 13, 14 ]. Nevertheless, the preparation of purified protein kinases and phosphospecific antibodies with high affinities and specificities is a tough task. Additionally, the major biochemical assays is based on one recombinant purified kinase and one single substrate [ 15 ]. This kind of assay format may neglect an important fact that one small molecule may interact simultaneously with multiple cellular proteins. A good example is the newly developed anticancer drug dasatinib [ 16 ]. To this point, the use of the crude cell lysates may be a desirable choice to discover the multi-targeted inhibitors [ 17, 18 ]. Furthermore, quantifying protein kinase activities directly in complex cell lysates may be helpful to understand the biological regulatory pathways of the PKIs.

Synthetic peptides with specific sequences are commonly used as the substrates for PKI screening in biochemical assay format[ 19, 20, 21, 22 ]. Compared to protein substrates, using peptide substrates can simplify protein kinase assay methods due to the advantages such as ease of storage, handling and protocol modification [ 5 ]. Several substrate peptide-based techniques such as fluorescence polarization and time-resolved fluorescence are widely used for PKI screening due to their advantages, e.g., homogeneous mix-and-measure assay and high throughput capacity[ 11, 12, 13, 23 ]. However, these techniques are not suitable for assaying the complex mixture such as the cell lysate[ 9, 10 ]. Most recently, mass spectrometry (MS) emerged as a promising technique for both rapid and quantitative evaluation of the protein kinase activity in cell lysate and inhibitor screening using synthesized substrate peptides[ 5, 24 ]. However, MS procedures require complex sample pretreatments, such as desalination, protein elimination and targeted phosphorylated peptide enrichment. Moreover, stable isotope-labeled peptides are necessary for accurate quantitation of the proteins. Therefore, to develop simpler and more straightforward method for multiplex PKI screening is still in demand. Capillary electrophoresis (CE) has proven to be a versatile separation tool for both enzyme analysis and enzyme inhibitor screening due to its high separation performance, high speed separation, extremely low sample and reagent consumption, and capacity for automation[ 25, 26, 27, 28, 29, 30, 31 ]. CE coupled with laser-induced fluorescence (LIF) detection permits detection sensitivity as high as 10-12 mol/L. Compared with the above-mentioned techniques, CE, as a separation-based technique, is suitable for multiple kinase assays due to the following reasons: (1) sample pretreatment is very simple; (2) LIF detection permits very high detection
sensitivity and selectivity when a complex biological sample is analyzed. Thus far, several CE methods have been developed for PKI screening. High throughput PKI screenings with a 48 multiplexed CE system with UV detection have been developed by He and Yeung [ 32 ]. Sano et al. [ 33 ] have developed micellar electrokinetic chromatography (MEKC) with LIF detection for a protein kinase assay. However, both methods employ purified Protein kinase A (PKA) as a model enzyme in a one enzyme-one substrate assay format.

Here, we report a CE-LIF based method for screening PKIs against multiple cellular protein kinases. The specific substrate peptides can be phosphorylated after mixed with cell lysates. The kinase activities were determined by measuring the concentration of the phosphorylated peptides. The powerful separation capacity of CE and the high sensitivity of LIF detection permit us to directly use cell lysates as the enzyme source. This will greatly facilitate PKI screening and reduce the reagent costs, which are currently the bottleneck in high throughput PKI screening processes. Moreover, the inhibitor screening against multiple kinases simultaneously provides a selectivity assessment for the identified inhibitors. This work has the potential to be a useful platform for the study of cellular kinases in signal regulation and novel drug discovery.

1 Experimental section
1.1 Reagents and chemicals
Peptide substrates of kinases PKA, AKT, CK2 (listed in Table 1) were purchased from AnaSpec (San Jose, CA). N- -5-isoquinoline sulfonamide dihydrochloride (H-89), staurosporine from Streptomyces sp, adenosine 5′-triphosphate disodium salt (ATP), polyoxyethylene lauryl ether (Brij 35), tris(hydroxymethyl)aminomethane (Tris), sodium deoxycholate, adenosine 3′,5′-cyclic monophosphate (cAMP), fluorescein sodium (FS), Triton X-100, dimethyl sulfoxide (DMSO), sodium orthovanadate (Na3VO4), phenylmethanesulfonyl fluoride (PMSF), β -glycerophosphate disodium, phorbol-12-myristate-13-acetate (PMA), glycerol, insulin, trypsin, bovine serum albumin (BSA), and 2-hydroxypropyl- β -cyclodextrin ( β -HP-CD) were purchased from Sigma-Aldrich (St. Louis, MO). Nonidet P-40 (NP-40), ethylenediaminetetraacetic acid (EDTA) and ethylene glycol tetraacetic acid (EGTA) were purchased from Amresco (Solon, OH). N-2-hydroxyethyl-piperazine-N′-2-ethane sulfonic acid (HEPES) was from Farco Chemical Supplies (Hong Kong, China). Dithiothreitol (DTT), protease inhibitor cocktail and 4,5,6,7-tetrabromobenzotriazole (TBB) were purchased from Merck (Darmstadt, Germany). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s liquid medium (DMEM), RPMI-1640 liquid medium, 0.25% (w/v) trypsin and PBS were purchased from Invitrogen (Carlsbad, CA).

The CE running buffer consisted of 100 mmol/L Hepes adjusted to pH 7.5 with 1 mol/L NaOH. Tris buffer (25 mmol/L) was prepared and adjusted to pH 7.5 with 1 mol/L HCl. The assay buffer contained 25 mmol/L Tris, pH 7.5, 20 mmol/L MgCl2, 2 mmol/L DTT, 20% (w/v) glycerol, 2.0×10-7 mol/L fluorescein as an internal standard, 2 mmol/L ATP and 4 μ mol/L of each substrate peptide, the type of which varied in each of the different assays. The assay buffer (25 μ L) and cell lysate (15 μ g) were mixed and supplemented with Tris buffer to constitute a total reaction volume of 50 μ L. The cell lysis solution was composed of 25 mmol/L Tris, 0.5% (w/v) NP-40, 0.1% (w/v) Brij 35 P, 0.1% (w/v) sodium deoxycholate, 10% (w/v) glycerol, 1 mmol/L EDTA, 5 mmol/L EGTA, 10 mmol/L DTT, 150 mmol/L NaCl, 1 mmol/L Na3VO4, 50 mmol/L β -glycerophosphate disodium, 1 mmol/L PMSF and 1% (v/v) protease inhibitor cocktail, with pH of 7.5. All solutions were prepared in deionized water purified by a Millipore water purification system (Milford, WA). The solutions were filtered through 0.45 μ m membrane filters prior to use.

1.2 Instrumentation and operation
All CE analyses were performed on a P/ACE MDQ capillary electrophoresis system (Beckman Coulter, CA) equipped with a laser-induced fluorescence detector. An argon ion laser was used as the excitation source (488 nm), and the electropherograms were recorded by monitoring the emission of fluorescence at 520 nm. Fused-silica capillaries with the dimension of 40 cm (effective length 29.5 cm from the inlet to the detection window)×50 μ m i.d. (370 μ m o.d.) were purchased from Polymicro Technologies (Phoenix, AZ).

The new capillaries were pretreated by flushing with 1 mol/L NaOH for 20 min, followed by deionized water and running buffer for 5 min under 207 kPa of pressure. Prior to each run, the capillary was flushed with 1 mol/L NaOH for 3 min and deionized water and the running buffer for 1 min at a pressure of 207 kPa. The samples were injected for 5 s with 1.38 kPa. A voltage of 25 kV was applied to the capillary to perform the separation. The column was incubated at 25 ℃. Fluorescein sodium (FS) was used as an internal standard (IS) spiked into the substrate solution to calibrate for the variations in the injection volume. After several runs, the separation suffered from peak tailing and poor reproducibility of the migration time. This phenomenon, most likely due to the adsorption of proteins on the capillary wall, can be improved by flushing the capillary with 150 mmol/L NaCl.

The separation was performed on an Agilent 1260 liquid chromatography system equipped with a variable wavelength detector (VWD) and a fluorescence detector (FLD). An Agilent ZORBAX C18 column (150 mm×4.6 mm) with a guard column was applied. The VWD was set at 232 nm; the FLD was set at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The HPLC was connected to an LCQ-Fleet ion-trap mass spectrometer (Thermo Scientific, CA). All ESI mass spectra were acquired in the positive ion mode. The spray voltage was set to 4.5 kV. The capillary voltage was 33 V and the capillary temperature was 320 ℃. The injection volume was 5 μ L. The column was eluted at a flow rate of 1 mL/min and maintained at 25 ℃. Eluents A and B were water and methyl cyanide (MeCN), respectively. Both of the eluents contained 0.1% (v/v) HCOOH. Gradient condition for analyzing the reaction resolution of FPKA (5-carboxyfluorescein (5-FAM) labeled PKA): 0-15 min, 5%B-80%B; 15-15.1 min, 80%B-100%B; 15.1-20 min, 100%B. Gradient condition for analyzing the reaction resolution of FAKT (5-FAM labeled Protein kinase B (AKT)): 0-3 min, 5%B; 3-10 min, 5%B-15%B; 10-10.1 min, 15%B-80%B; 10.1-15 min, 80%B; 15-15.1 min, 80%B-5%B; 15.1-20 min, 5%B. Gradient condition for analyzing the reaction resolution of FCK2 (5-FAM labeled Casein kinase 2 (CK2)): 0-10 min, 30%B; 10-10.1 min, 30%B-90%B; 10-15 min, 90%B; 15-15.1 min, 90%B-5%B; 15.1-20 min, 5%B.

1.3 Cell culture, stimulation and lysis
Four cell lines Hela, H-1299, HepG2 and Jurkat were obtained from Cell Resource Center, Shanghai Institute of Biological Science, CAS (Shanghai, China). The Hela and HepG2 cells were grown in DMEM containing 0.584 g/L glutamine, 100 U/mL penicillin, 0.1 g/L streptomycin and 10% FBS. The H-1299 and Jurkat cells were maintained in RPMI 1640 containing 0.3 g/L glutamine, 100 U/mL penicillin, 0.1 g/L streptomycin and 10% FBS. All cells (approximately 106 cells/100 mm diameter dish) were cultured in a 37 ℃, 5% CO2 humidified atmosphere. Before lysis, all cells were serum starved for 16 h and then treated with 100 nmol/L insulin for 5 min [ 34 ].

The treated cells were washed thrice with cold PBS buffer, followed by the addition of the ice-cold lysis buffer. The cells were lysed for 20 min. All the lysis manipulations were performed on ice. The homogenates were centrifuged at 10000 r/min for 10 min at 4 ℃. The aliquots of the supernatant were quickly frozen in liquid nitrogen and stored in a liquid nitrogen canister until use. The protein mass concentrations were determined as 2.8-3.4 g/L by bicinchoninic acid assay (Sigma, St. Louis, MO).

1.4 Kinase activity assays and inhibitor screening
The schematic diagram of the assay process is shown in Fig. 1. After removed from the liquid nitrogen, the cell lysates were quickly thawed at 37 ℃ and then kept on ice. The cell lysates (15 μ g) and 25 μ L of the assay buffer were diluted with Tris buffer to yield a final reaction volume of 50 μ L. After the incubation at 37 ℃, the reaction was quenched by the addition of 10 μ L of 0.1 mol/L HCl. The reaction was then neutralized with 10 μ L of 0.1 mol/L NaOH. The kinase activities can be quantitatively measured by performing CE separation and measuring the corrected peak areas of the phosphorylated peptides (i.e., peak area ratios between the phosphorylated peptides and the internal standard). For the inhibition studies or screenings, the compounds of interest were spiked into the reaction solution. The inhibitory activities of the compounds can be easily observed by a reduction in the peak area of the phosphorylated substrate compared to a control assay without inhibitors. The percent inhibition (I) was then calculated according to Equation (1):

where Ax and A0 are the peak areas of the phosphorylated peptide produced in the presence and absence of an inhibitor, respectively.

Fig. 1 Schematic diagram of the assay process
2 Results and discussion
2.1 Method development for multiple cellular protein kinase activity assays
In our initial experiment, the phosphorylation of three substrate peptides with the protein kinase in the cell lysates was individually monitored by CE. The specificities of these substrate peptides against their corresponding cellular protein kinases were reported in literature[ 17, 32, 35, 36 ]. Their sequences and enzymatic kinetic parameters are given in Table 1. The electropherograms for each separation are shown in Fig. 2. Unfortunately, as can be seen in Fig. 2, in the cases of FAKT and FCK2, the obtained electropherograms were complex due to the digestion of the peptides and the presence of the impurities. The peak of the phosphorylated product could be identified as depicted in literature by comparing the samples with and without ATP [ 20 ]. The peaks of the phosphorylated product were further confirmed by using HPLC/FLD and electrospray ion-trap mass spectrometry (ESI/MS). The substrates and the products could be identified by the FLD chromatograms and the extracted ion chromatograms (EICs) (shown in Fig. 3). Compared with HPLC/FLD chromatograms, CE provided more powerful resolution. The stabilities of peptides FPKA, FAKT and FCK2 were investigated with blank experiments. In the blank experiments, the sample solutions without cellular proteins were analyzed with HPLC/FLD after incubation at 37 ℃ for 24 h. No significant degradation of the peptides was observed.
Table 1 Properties of the selected substrate peptides used for kinase assays
Fig. 2 Electropherograms of the separation and identification of the phosphorylated products P-FPKA, P-FAKT and P-FCK2 refer to phosphorylated FPKA, FAKT and FCK2 respectively.
Fig. 3 Separation and identification of the phosphorylated products by HPLC/FLD/ESI-MS a. FLD chromatogram of PKA reaction solution, extracted ion chromatography (EIC) of FPKA and EIC of P-FPKA; b. FLD chromatogram of AKT reaction solution, EIC of FAKT and EIC of P-FAKT; c. FLD chromatogram of CK2 reaction solution, EIC of FCK2 and EIC of P-FCK2.
The concentration of the substrate peptides was linear ranging from 5×10-9 to 5×10-6 mol/L. Under the condition lacking phosphorylated product standards, we assumed the phosphorylated products with the same linear range owing to their very similar structures. For cellular protein kinase assay, the peak area of the phosphorylated product in the electropherogram was used as the criterion to express the production yield. To ensure the accurate measurement of the peak area, the corrected peak area, which is the ratio between the peak area of the phosphorylated substrate and that of the internal standard, was used. The electropherogram of the separation of the substrate peptides FPKA, FAKT, FCK2, as well as the phosphorylated products and the internal standard is shown in Fig. 4. Baseline separation with the satisfactory peak shape in a short period of time can be achieved by using a running buffer consisting of 100 mmol/L Hepes (pH 7.5) with a separation voltage of 25 kV. The method was validated with PKA as a mode in terms of repeatability of migration time and peak area. The RSDs for the intra-day (n=6) and the inter-day (n=3) repeatability of the migration time were determined as 1.4% and 3.6% , respectively; while the RSDs for the intra-day (n=6) and the inter-day (n=3) repeatability of the corrected peak areas of the phosphorylated peptide substrates were determined as 8.3% and 13.3% , respectively. The relatively high inter-day-repeatability of the corrected peak area is most likely caused by the gradually decreased activity of the cellular enzyme.

Fig. 4 Electropherogram of the separation of the multiple substrates and their phosphorylated products Peaks: 1. FAKT; 2. FPKA; 3. P-FAKT; 4. P-FPKA; 5. FCK2; 6. P-FCK2; 7. IS.
2.2 Effect of cell treatment and detergents in the cell lysis buffer on enzyme activity assays
It has been reported that the serum starving and then re-activating cells with protein kinase activators will increase the activities of protein kinases[ 17 ]. In our study, we found that the activity of AKT can be significantly enhanced by this way. The enzyme reaction (expressed as the corrected peak area of the product) was enhanced almost three-fold compared to the untreated cells after the serum starvation for 16 h and re-activation with insulin. For other protein kinases, such enhancement was not observed.

Effect of the detergents in the cell lysis solution on the assay of the enzymatic activity was also investigated. Several non-ionic detergents, including Triton X-100, NP-40, Brij 35 and sodium deoxycholate in lysis buffer solution, were often evaluated for this purpose. An enhanced fluorescence signal of the substrate peptides was observed when Triton X-100 was used in the cell lysis solution. However, this enhancement effect was fluctuated with the RSD as high as 29.2% (n=6) for the corrected peak area of the substrate peptide of PKA. On the contrary, we found that a three-detergent system [ 24 ], consisting of NP-40, Brij 35 and sodium deoxycholate, permitted a stable fluorescence signal (RSD 2.9% , n=6).

Effect of the amount of the cell lysates ranging from 7.5 to 45 μ g on the enzyme activity assays was investigated using PKA as a model. The yield (represented as the peak area of the phosphorylated substrate peptide) increased with increasing the amounts of the cell lysate, up to a maximum amount of 30 μ g. The turnover decreased when further increased the amount of the cell lysate because the digestion of the substrate peptide by cellular proteases became worse. Severe peak tailing of P-FPKA occurred when the amount of the cell lysate exceeded 15 μ g due to the adsorption of proteins on the capillary wall. Therefore, 15 μ g cell lysate was considered to be optimal for kinase assays.

2.3 Kinase assays and kinetics measurements
As the substrate peptide of PKA was not digested in the assay process, H-89 (an inhibitor of PKA) was utilized to measure the kinetics parameters and the inhibition kinetics. Because PKA is a bi-substrate enzyme (e.g., ATP and the peptide substrate FPKA), the Michaelis-Menten constant Km can be measured by maintaining one substrate to be saturated and varying the concentration of other substrate. The enzymatic reaction time was fixed at 5 min to ensure that the phosphorylated product was less than 10% (the reaction was in the range of the initial reaction velocity).

The Lineweaver-Burk plots are shown in Fig. 5a and b. By keeping the FPKA concentration at 20 μ mol/L and varying the ATP concentration in the range of 0.01 to 0.5 mmol/L, the Km value of ATP was measured as 50 μ mol/L. Similarly, the measured Km value of FPKA was 20.2 μ mol/L when the concentration of ATP was kept at 0.5 mmol/L and the FPKA concentration was varied in the range of 2 to 20 μ mol/L. Our results are consistent with the values reported in the literature (3-60 μ mol/L for FPKA and 10-30 μ mol/L for ATP)[ 38, 39, 40 ]. This investigation indicated that it is feasible to use cellular protein kinases as the enzyme source for enzyme assay if the specificity of the substrate is good enough and if there is no interference from the digestion of the substrate. For in vitro inhibition studies, a substrate concentration close to the Km is preferred to obtain the maximum assay sensitivity[ 41 ]. Therefore, 50 μ mol/L ATP and 2 μ mol/L FPKA was used for the following inhibition study. The inhibition curve of H-89 against PKA is shown in Fig. 5c, and IC50 is determined as 3.5 μ mol/L, which is in agreement with the value reported in the literature (0.135-7.01 μ mol/L)[ 42, 43, 44 ]. This implies that a reasonable kinetics parameter can be measured using the crude cellular enzyme if the specificity of the substrate peptide is good enough [ 32 ].

Fig. 5 Enzymatic kinetic plots of PKA a. Lineweaver-Burk plot of ATP with saturated FPKA; b. Lineweaver-Burk plot for FPKA with a saturated concentration of ATP; c. IC50 plot of PKA against H-89.Conditions: the concentration of ATP was fixed at 0.5 mmol/L; the concentration of FPKA varied from 2 to 20 μ mol/L.
2.4 Inhibitor screening and selectivity assessment against multiple protein kinases
The reactions of the three peptide substrates FPKA, FCK2 and FAKT with their corresponding cellular protein kinases (PKA, CK2 and AKT) from the four human cell lines (Hela, H1299, HepG2 and Jurkat) were monitored from 0 to 60 min. In this experiment, the ATP concentration was kept at 1 mmol/L, which is near the cellular concentration of ATP so as to produce a similar activity profiles as those like in vivo. Because the final concentration of the endogenous ATP in the reaction solution was approximately 7 μ mol/L after dilution, the effect of the native ATP on the enzyme activity assay was negligible. The initial reaction rates increased linearly with increase of the reaction time within 25 min. Therefore, 25 min was selected as the maximum reaction time for measuring the initial reaction rate. For the three tested substrate peptides, their corresponding cellular kinase in the HepG2 cell lysate displayed the highest activities. Therefore, HepG2 cell lysate was used for the following experiments.

Finally, a relative specific inhibitor (TBB), a non-specific inhibitor (staurosporine) and three substrate peptides corresponding to kinases PKA, CK2 and AKT were used to demonstrate inhibitor screening and selectivity assessment against the multiple cellular protein kinases simultaneously. The electropherograms for the inhibition investigation on staurosporine are shown in Fig. 6a. The peak areas of the phosphorylated products P-FAKT and P-FPKA decreased with increasing inhibitor concentrations, in the range of 1 to 10 μ mol/L; while no significant inhibition on the activity of CK2 was observed. Staurosporine at 10 μ mol/L only exhibited 13% inhibition against CK2. These results agreed well with those on the literature [ 45 ]. The IC50 values of staurosporine for PKA and AKT were determined as 7 μ mol/L and 0.88 μ mol/L, respectively (Table 2). These results provide the information that staurosporine can be an inhibitor against PKA and AKT. As shown in Fig. 6b, the peak areas of P-FCK2 decreased with increasing TBB concentration in the range of 1 to 100 μ mol/L, while the peak areas of P-FAKT and P-FPKA were not affected. The IC50 of TBB against CK2 was determined as 33 μ mol/L, but IC50 of TBB against PKA and AKT were both more than 100 μ mol/L (Table 2). These results confirmed that TBB has good selectivity against its target CK2 (Table 2).

Fig. 6 Electropherograms of the inhibitor screen and selectivity assessments against multiple cellular protein kinases a. inhibition study of staurosporine against PKA, AKT and CK2; b. inhibition study of TBB against PKA, AKT and CK2.
Table 2 IC50 values of the inhibitors determined with protein kinases in crude cell lysates
In our experiment, IC50 of staurosporine against AKT was 0.88 μ mol/L close to the reported value of 0.83 μ mol/L [ 46 ]; whereas IC50 of TBB against CK2 (33 μ mol/L) and IC50 of staurosporine against PKA (7 μ mol/L) were both higher than the values reported in the literature (1.6 μ mol/L [ 47 ] and 15 nmol/L [ 45 ], respectively). These discrepancies can be explained by the Cheng-Prusoff equation[ 37, 48 ]:

where Ki is the dissociation constant of the complex formed between the inhibitor and its target; Km, ATP represents the Michaelis-Menten constant of the enzyme in terms of ATP; and represents the ATP concentration. The IC50 increases with increased ATP concentration. When the ATP concentration is close to the cellular ATP concentration (1-5 mmol/L)[ 49, 50 ], the potency of an inhibitor depends critically on the Km, ATP of its target. Because AKT has a higher Km, ATP (Table 1), the inhibition of AKT by staurosporine does not change much as the concentration of ATP is increased. In contrast, the IC50 of TBB against CK2 and the IC50 of staurosporine against PKA are strongly influenced by the ATP concentration, due to the lower Km, ATP values. On the other hand, compared to those observed in the in vitro assays, these higher IC50 values may be due to the cross-phosphorylation by other kinases [ 32 ].
3 Conclusions
A CE-LIF-based method for screening PKIs against multiple cellular protein kinases has been developed. Owing to the high separation efficiency and detection sensitivity of CE, the CE-based inhibitor screening method can be a promising method for simultaneously screening inhibitors against the multiple cellular protein kinases and assessing the selectivity in a single assay, although getting substrate peptides with high specifity and suppressing digestion of the substrate peptides by the proteases in the cell lysate remain challenges. One benefit of this method is that the cell lysate can be directly used as the enzyme source, which greatly reduces the overall cost of the assay. In addition, using cell lysates for PKI screening may produce screening results that is similar to those obtained in vivo because the cellular protein kinases are still in their native structure. The CE method for PKI screening is simple, fast, straightforward, and cost-effective.
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