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Detection of casein kinase II by aggregation-induced emission
Talanta 201 (2019) 450–454
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Detection of casein kinase II by aggregation-induced emission Zhenzhu Luan, Li Zhao, Chao Liu, Weiling Song, Peng He, Xiaoru Zhang
T
∗
Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, Key Laboratory of Biochemical Analysis, Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, 266042, PR China
ARTICLE INFO
ABSTRACT
Keywords: Aggregation-induced emission Protein kinases CKII Click reaction
A novel aggregation-induced emission (AIE) probe comprised of a hydrophilic protein kinase specific peptide and a hydrophobic tetraphenylethene (TPE) unit was synthesized through click reaction. The prepared TPEpeptide probe could be completely degraded by carboxypeptidase Y (CPY) to release hydrophobic TPE part, which aggregated in buffer solution and showed strong TPE emission. In the presence of casein kinase (CKII), the phosphorylation of peptide prevented the complete degradation by CPY producing the nonemissive probe. Thus, the developed probe can be used to detect CKII homogeneously and conveniently. This detection process can be finished within 1.5 h with high sensitivity (0.05 mU/μL) and good selectivity. The developed method can also be used to screen protein kinase inhibitor even in a complex biological system.
1. Introduction Now, more than 510 kinds of protein kinases have been found [1], which can be used to catalyze the transfer of the phosphate groups from adenosine-5′-triphosphate (ATP) to a free hydroxyl group of serine, threonine, or tyrosine residues in a peptide. The phosphorylation of cellular protein by kinases plays a crucial role in many cellular processes, including gene expression, cell differentiation, metabolism and apoptosis [2,3]. Abnormal phosphorylation catalyzed by protein kinases may lead to various diseases, such as HIV [4], cancer [5], Parkinson's disease [6], and Alzheimer's disease [7]. Therefore, developing sensitive and convenient methods for kinase assays and potential inhibitor screening is of great significance in the study of fundamental cellular processes as well as the development of kinase-related drug [8]. As an acidophilic and a ubiquitous serine/threonine kinase [9], CKII was chosen as a proof-of concept targets in this work. Specific artificial peptide substrate GRRRADDSDDDDD [10] was used as a molecule recognition substrate for CKII, where the phosphorylation occurs at the S residues. Traditional protein kinase assay relies on the use of radioactive ATP (32P-ATP) [11], which is a well-established method but suffers from a high risk of harmful radioactive contamination. Recently, several nonradioactive assays have been established, such as mass spectrometry [12], electrogenerated chemiluminescence (ECL) [13], electrochemistry [14], colorimetriy [15], fluorescence [16–20], quartz crystal microbalance [21], and surface-enhanced Raman spectroscopy (SERS) [22,23]. Among them, homogeneous fluorescence assays have
∗
some inherent advantages due to its high sensitivity, simplicity and the avoidance of tedious surface immobilization. However, the light emission of conventional luminophores often suffers from weakened or annihilated emission in their aggregated state, which is known as aggregation-caused quenching (ACQ). To address this concern, a new class of organic fluorogens with aggregationinduced emission (AIE) property was developed by Tang et al., in 2001 [24,25]. Propeller-shaped AIE molecule is nonemissive when dispersed, but distinctly emissive in the aggregated state. So far, the most widely used AIE molecules are tetraphnylethylene (TPE) derivatives, which have the merit of easy synthesis and functionalization [26–28]. Herein, a novel fluorescent probe was designed for the detection of protein kinase by coupling a hydrophilic peptide sequence with a hydrophobic AIE fluorogen. The presence of carboxypeptidase Y (CPY) can hydrolyze a peptide bond from the C-terminal end of the peptide probe and release free TPE molecule, which showed strong TPE emission. While the phosphorylation by CKII can prevent the digestion of CPY. Thus, low fluorescence intensity was observed. This strategy can be used to detect protein kinases CKII as well as screen their inhibitors. 2. Experimental 2.1. Materials and reagents Sodium ascorbate and benzoyl peroxide were obtained from J&K Scientific Ltd (Beijing, China). CKII and adenosine triphosphate (ATP)
Corresponding author. E-mail address: [email protected] (X. Zhang).
https://doi.org/10.1016/j.talanta.2019.04.036 Received 31 December 2018; Received in revised form 12 March 2019; Accepted 14 April 2019 Available online 19 April 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Talanta 201 (2019) 450–454
Z. Luan, et al.
Fig. 1. Schematic diagram of the synthesis process for probe TPE-Peptide 5.
were obtained from New England Biolabs (Beverly, MA, USA). CPY was purchased from Worthington Biochemical Co., Ltd (Lakewood, USA). Fmoc-propargyl-Gly-OH was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). 4,5,6,7-tetrabromobenzotriazole (TBB) were obtained from Aladdin (Shanghai, China). Deionized water (Millipore Milli-Q grade) with a resistivity of 18.2 MΩ cm−1 was used in all the experiments. All reagents used in the experiment were analytically pure.
wavelength of 312 nm and emission wavelengths ranged from 350 to 600 nm [29]. Both of the excitation and emission slit widths were set at 5.0 nm. As for CKII inhibition assay, the phosphorylation reaction solution containing 100 mU/μL CKII and different concentrations of TBB (0–25 μM) were used in the test. The rest steps were the same as mentioned above. 2.4. Detection of protein kinase activity in cancer cell lysate
2.2. Synthesis of probe Fmoc-Gly-TPE (4) and TPE-peptide (5)
Human cervical carcinoma cells (HeLa cells) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and grown in a humidified atmosphere of 5% CO2 in air at 37 °C. HeLa cells were cultured with serum-free medium for 4 h. Then dimethyl sulfoxide (DMSO) and different concentrations of TBB were added to the medium. After cultured in the incubator for 30 min, cell lysates were obtained by scraping and sonication in Dulbecco's Phosphate Buffered Saline (2 s × 60 times at an interval of 3 s). The obtained solution was centrifuged at 15000 rpm, 4 °C for 60 min, and the resulting supernatant was stored at −20 °C for subsequent experiments. The Bradford protein assay kit (protein standard was bovine serum albumin) was used to measure the protein concentration of the cell lysate. Finally, the protein concentration of the cell lysate was diluted to 8 μg/mL.
Compound 3 in Fig. 1 (0.7741 g, 2.0 mmol, synthesis process seeing Supplementary Material 1.4) and Fmoc-propargyl-Gly-OH (0.6631 g, 1.977 mmol) were dissolved in a mixture of DMSO/DMF/H2O (10/7/6, v/v/v) under N2. Then sodium ascorbate (8.8 mmol) and CuSO4·5H2O (4.4 mmol) were added and the mixture was stirred overnight at room temperature. After the reaction was completed, a large amount of water was added, and the solution was extracted three times with ethyl acetate. The organic layers were combined, dried and concentrated. The resulting crude product was further purified by column chromatography using dichloromethane (DCM)/methanol (v/v = 20:1) + 1% acetic acid as eluent to give compound Fmoc-Gly-TPE 4 as a brown solid (0.5302 g, 37.14% yield). 1H NMR (DMSO, 500 MHz), δ (TMS, ppm):7.77-7.68 (m, 10H), 7.10 (s, 4H), 5.44 (s, 1H), 4.24 (s, 4H), 3.34 (s, 5H), 2.53-2.52 (m, 5H), 1.68-1.26 (m, 8H), 0.93 (s, 4H). ESI MS [M +H]+ m/z: calcd. 723.29, found 723.30. TPE-peptide 5 (TPE-GRRRADDSDDDD) was obtained by polypeptide synthesis using compound Fmoc-Gly-TPE 4 as a raw material. This process was conducted by Chinapeptides (Shanghai, China). ESI MS [M +3H]3+ m/z: found 645.27; [M+2H]2+ found 967.40; calcd. 1932.92.
3. Results and discussion 3.1. The design of AIE probe To obtain the probe TPE-peptide 5, firstly diphenylmethane reacted with 4-methylbenzophenone to give TPE skeleton structure 1 through McMurry cross coupling reaction. Then compound 2 was synthesized by bromination with N-bromosuccinimide (NBS). After azidation with NaN3, intermediate TPE-N3 3 was obtained. Click reaction between 3 and Fmoc-propargyl-Gly-OH produced Fmoc-Gly-TPE 4 under the catalysis of CuSO4/sodium ascorbate. The overall yield of these four steps was about 10%. The synthesis processes were illustrated in Fig. 1 and described in detail in Supplementary Material. Finally, TPE-peptide 5 was synthesized by the extension of the peptide chain using compound 4 as a starting material. The last step was completed by a commercial company. The structures of compounds were characterized by 1H-
2.3. Detection the activity and inhibition of CKII For phosphorylation process, 100 μL reaction mixture containing different concentration of CKII (0–100 mU/μL), 10 μM TPE-peptide 5, 1 mM ATP and CKII reaction solution (20 mM Tris-HCl, 10 mM MgCl2 and 50 mM KCl, pH 7.5) was incubated at 30 °C for 1 h. Then CPY was added bringing its final concentration to 3.5 mU/μL. The hydrolysis process was carried out at 25 °C for 30 min. The fluorescence intensities were measured at an excitation 451
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Fig. 2. Schematic diagram of detecting CKII by aggregation-induced emission.
NMRor MS (see Figs. S1 and S3 in Supplementary Material). Further characterization of compounds 1–3 can be found in the references [25–27]. Since there was an azide group in compound 3, IR spectrum was used to demonstrate this characteristic group at about 2100 cm−1 (Fig. S2 in Supplementary Material). The fluorescent intensity for TPEpeptide 5 was stable for at least two months without spontaneous aggregation. 3.2. Detection principle The detection mechanism was shown in Fig. 2. TPE, as a well-known AIE fluorogen, is a propeller-shaped compound. It is almost insoluble in water. When conjugated with peptide (GRRRADDSDDDDD), due to the high hydrophilic property of the peptide part, the water solubility of the whole TPE-peptide 5 complex increased dramatically. The free rotation of the four phenyl rings in this complex led to the nonradiative relaxation of excited electrons. As a result, TPE-peptide 5 complex showed very weak fluorescence in an aqueous environment. Here, we designed a protocol for detecting kinase activity according to the features of CPY and protein kinase CKII using TPE-peptide 5 complex as a probe. CPY as an exopeptidase could be used to degrade the peptide bonds at the C-terminal end of a peptide. When treated the synthesized TPE-peptide 5 probe with CPY, fluorescent TPE part was released into the solution due to the completed hydrolysis of non-phosphorylated peptide. Then small molecules TPE released in solution were aggregated together due to their hydrophobicity. So, high fluorescent signal could be observed according to the AIE effect. However, under the treatment of protein kinase CKII in the presence of ATP, the phosphorylation of serine on peptide produced phosphorylated substrate TPE-GRRRADDpSDDDDD, which could inhibit the hydrolysis ability of CPY. Thus, the TPE-peptide 5 could not be digested completely and the fluorescence signal was very weak due to the still strong hydrophilicity of TPE-GRRRADDpS. Therefore, the fluorescence intensity could be used to measure the activity of kinase CKII.
Fig. 3. Fluorescence signal for a series of experimental conditions: only TPEpeptide 5 (black curve); TPE-peptide 5+CKII+CPY (red curve); TPE-peptide 5+CKII+ATP (blue curve); TPE-peptide 5+CKII+ ATP+CPY (pink curve); TPE-P+CPY (green curve). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
dramatically (green curve), indicating the hydrolysis of TPE-peptide 5 substrate by CPY and releasing the hydrophobic small molecule TPE. ATP was used to provide γ-phosphoryl to a free hydroxyl group of serine under the catalysis of by protein kinase CKII. So, the digestion of CPY would not be hindered in the absence of ATP and high fluorescence intensity was still observed (red curve). However, in the absence of CPY, the phosphorylated substrate obtained by treated the TPE-peptide 5 with CKII and ATP could not be digested, and weak fluorescence signal was showed (blue curve). When CKII, ATP for phosphorylation, CPY for cleavage coexisted in the system simultaneously, phosphorylation reaction was catalyzed, thus, the digestion reaction could only proceed to the middle position of the polypeptide chain. Therefore, the fluorescence intensity of the system was still very low (pink curve). Through the above series of experiments, it could be verified that this experimental protocol was feasible for the detecting the activity of protein kinase CKII.
3.3. Feasibility of the assay In order to verify the feasibility of the protocol, the following experiments were designed: As shown in the Fig. 3, TPE-peptide 5 had lower fluorescence intensity due to the hydrophilicity of the TPE-peptide 5 probe as described above (black curve); After treated the TPEpeptide 5 substrate with CPY, the fluorescence intensity increased 452
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Fig. 4. (A) Fluorescence spectra for different concentrations of CKII (curve a to m: 0, 0.01, 0.1, 0.5, 1, 5, 8, 10, 20, 50, 80, 100, 200 mU/μL). (B) Plot of the relative fluorescence change vs concentration of CKII. Inset: The linear relationship between the relative fluorescence change and CKII concentrations. F0 is the fluorescence intensity of TPE-peptide 5 alone and F is the fluorescence intensity after addition proteins or enzymes.
3.4. CKII activity detection To obtain the best detection effect, the assay conditions were optimized. As shown in Figs. S4–S6 (Supplementary Material), the optimal ATP, CPY and probe TPE-peptide 5 dosages were selected as 1.0 mM, 3.5 U/mL and 10 μM, respectively. Under optimized detection condition, the activity of CKII was quantitatively investigated with increasing concentrations of CKII (0–200 mU/μL). Since the presence of CKII led to the phosphorylation in the middle position of TPE-peptide 5, the hydrolysis of CPY was stopped and the remaining peptide prevented the aggregation of TPE molecule. Thus, the fluorescence intensity decreased along with the increase of CKII concentration (see Fig. 4A). The relative fluorescence intensity ratio showed a linear correlation to the logarithmic CKII concentration within 0.1–80 mU/μL (see Fig. 4B and its insert), with the linear regression equation of (F-F0)/ F0 = 4.435–1.9596 lgC (F0 referred to the fluorescence intensity of TPEpeptide 5 alone and F referred to the fluorescence intensity after addition different concentration of CKII. C was the concentration of CKII (U mL−1), R2 = 0.9971). The EC50 value (enzyme concentration at which 50% substrate is converted) was determined to be 9.38 mU/μL. The detection limit was estimated to be 0.05 mU/μL (3σ), which is in accordance with the results of other reported methods [14,30–32]. This homogeneous detection system is quite simple and can complete within 1.5 h indicating it is a promising method for the analysis of protein kinase.
Fig. 5. Selectivity of the method for the detection of 0.1 U/μL of CKII, ALP and HRP, 3% BSA and thrombin. F0 is the fluorescence intensity of TPE-peptide 5 alone and F is the fluorescence intensity after addition proteins or enzymes.
concentration producing 50% inhibition) of TBB was determined to be 1.8 μM, which was comparable to the reported values (1.5 μM) [33]. This demonstrated that the developed method could be used for the screening of CKII inhibitors conveniently. Protein kinases play an important role in cell signalling. So, detection the inhibition effect of TBB in cell lysates is very necessary for monitoring the regulation of kinases in cell systems. After treated cell with different concentrations of TBB (0, 5, 10, 15, 25 μM), the fluorescence measurement was carried out using extracted cell lysates, which have many varieties of proteins. As shown in Fig. 6, the fluorescence intensity was positively correlated with the concentration of TBB, indicating the potential application of this protocol in the screening of protein kinase inhibitor in biological fluids.
3.5. Selectivity Control experiments were carried out to test the selectivity of the developed biosensor. From the results shown in Fig. 3A, the fluorescence intensity change of different proteins or enzymes, including bovine serum albumin (BSA), alkaline phosphatase (ALP), horseradish peroxidase (HRP) and thrombin were much lower than that of CKII. These results suggest that the proposed method possesses high selectivity to CKII.
4. Conclusions
3.6. Inhibition evaluation
In conclusion, a novel, simple and sensitive fluorescence assay was developed for homogeneous detection of protein kinases CKII using AIE fluorescent probe. The probe was synthesized by click reaction between TPE-N3 and Fmoc-propargyl-Gly-OH firstly. Then the peptide chain was extended to give TPE-Peptide 5. This probe was non-fluorescent in aqueous buffers but became emissive when cleaved by CPY. The presence of CKII led to phosphorylation on the peptide, which prevented further digestion of CPY. Hence the fluorescent intensity could be used to monitor the concentration of protein kinases CKII. The whole detection process could be finished within 1.5 h with high sensitivity (0.05 mU/μL). This method was used in protein kinase inhibitor screening even in a complex biological system, which was very useful in the discovery of anticancer drugs. What's more, after switching the
Since protein kinase inhibitors are of great significance in drug discovery. To investigate the potential applicability of the proposed protocol in the inhibition assay, a small molecule 4,5,6,7-tetrabromobenzotriazole (TBB) was chosen to confirm the inhibition effect. A series of concentrations of TBB (0, 1, 2, 5, 8, 10, 15, 20, 25 μM) were added to 80 mU/μL CKII reaction solution before phosphorylation. After the CPY digestion, fluorescence signals were measured. Fig. 5 showed that the fluorescent intensity increased gradually with the increase of TBB concentration, which demonstrated the inhibition effect for CKII. When the concentration of TBB reached 15 μM, the activity of protein kinase CKII could be inhibited completely and the fluorescence signal was identical to that without CKII. The IC50 (inhibitor 453
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Fig. 6. (A) The relationship between TBB concentration and fluorescence intensity; (B) The fluorescence intensity for different concentrations of TBB in cell lysates. Error bars were derived from the standard deviation of three measurements.
peptide substrates sequences, this method can also be used to detection other protein kinases and screen their inhibitor. This strategy extends the application of the AIE effect and provides an alternative method for the detection of protein kinases.
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Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgement This work has been financially supported by the National Natural Science Foundation of China (Nos.: 21775080, 21705086, 21505082); Key Research and Development Project of Shandong Province, China (No. 2017GSF221004), A project of Shandong Province Higher Educational Science and Technology Program (Grant J16LC10). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.04.036. References [1] K. Mitch, E. Jessie, M. Vincent, G. Ferdous, W. Luquan, Q. Ping, G. Jonathan, L. Thomas, Human members of the eukaryotic protein kinase family, Genome Biol. 3 (0043) (2002) 1–12. [2] T. Hunter, Protein kinases and phosphatases: the Yin and Yang of protein phosphorylation and signalling, Cell 80 (1995) 225–236. [3] A.L. Bauman, J.D. Scott, Kinase and phosphatase-anchoring proteins: harnessing the dynamic duo, Nat. Cell Biol. 4 (2002) E203–E206. [4] K. Shirakawa, A. Takaori-Kondo, M. Yokoyama, T. Izumi, M. Matsui, K. Io, T. Sato, H. Sato, T. Uchiyama, Phosphorylation of APOBEC3G by protein kinase A regulates its interaction with HIV-1 Vif, Nat. Struct. Mol. Biol. 15 (2008) 1184–1191. [5] H. Wang, A. Davis, S. Yu, K. Ahmed, Response of cancer cells to molecular interruption of the CK2 signal, Mol. Cell. Biochem. 227 (2001) 167–174. [6] A.B. West, D.J. Moore, S. Biskup, A. Bugayenko, W.W. Smith, C.A. Ross, V.L. Dawson, T.M. Dawson, Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 16842–16847. [7] S. Leclerc, M. Garnier, R. Hoessel, D. Marko, J.A. Bibb, G.L. Snyder, P. Greengard, J. Biernat, Y.Z. Wu, E.M. Mandelkow, G. Eisenbrand, L. Meijer, Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors, J. Biol. Chem. 276 (2001) 251–260. [8] J.S. Sebolt-Leopold, J.M. English, Mechanisms of drug inhibition of signalling molecules, Nature 441 (2006) 457–462. [9] J.S. Kim, J.I. Eom, J.W. Cheong, A.J. Choi, J.K. Lee, W.I. Yang, Y.H. Min, Protein kinase CK2α as an unfavorable prognostic marker and novel therapeutic target in acute myeloid leukemia, Clin. Cancer Res. 13 (2007) 1019–1028. [10] O. Marin, F. Meggio, L.A. Pinna, Design and synthesis of two new peptide substrates for the specific and sensitive monitoring of casein kinases-1 and -2, Biochem. Biophys. Res. Commun. 198 (1994) 898–905. [11] M. Flajolet, G. He, M. Heiman, A. Lin, A.C. Nairn, P. Greengard, Regulation of Alzheimer's disease amyloid-β formation by casein kinase I, Proc. Natl. Acad. Sci. Unit. States Am. 104 (2006) 4159–4164.
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Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Detection of casein kinase II by aggregation-induced emission Zhenzhu Luan, Li Zhao, Chao Liu, Weiling Song, Peng He, Xiaoru Zhang
T
∗
Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, Key Laboratory of Biochemical Analysis, Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, 266042, PR China
ARTICLE INFO
ABSTRACT
Keywords: Aggregation-induced emission Protein kinases CKII Click reaction
A novel aggregation-induced emission (AIE) probe comprised of a hydrophilic protein kinase specific peptide and a hydrophobic tetraphenylethene (TPE) unit was synthesized through click reaction. The prepared TPEpeptide probe could be completely degraded by carboxypeptidase Y (CPY) to release hydrophobic TPE part, which aggregated in buffer solution and showed strong TPE emission. In the presence of casein kinase (CKII), the phosphorylation of peptide prevented the complete degradation by CPY producing the nonemissive probe. Thus, the developed probe can be used to detect CKII homogeneously and conveniently. This detection process can be finished within 1.5 h with high sensitivity (0.05 mU/μL) and good selectivity. The developed method can also be used to screen protein kinase inhibitor even in a complex biological system.
1. Introduction Now, more than 510 kinds of protein kinases have been found [1], which can be used to catalyze the transfer of the phosphate groups from adenosine-5′-triphosphate (ATP) to a free hydroxyl group of serine, threonine, or tyrosine residues in a peptide. The phosphorylation of cellular protein by kinases plays a crucial role in many cellular processes, including gene expression, cell differentiation, metabolism and apoptosis [2,3]. Abnormal phosphorylation catalyzed by protein kinases may lead to various diseases, such as HIV [4], cancer [5], Parkinson's disease [6], and Alzheimer's disease [7]. Therefore, developing sensitive and convenient methods for kinase assays and potential inhibitor screening is of great significance in the study of fundamental cellular processes as well as the development of kinase-related drug [8]. As an acidophilic and a ubiquitous serine/threonine kinase [9], CKII was chosen as a proof-of concept targets in this work. Specific artificial peptide substrate GRRRADDSDDDDD [10] was used as a molecule recognition substrate for CKII, where the phosphorylation occurs at the S residues. Traditional protein kinase assay relies on the use of radioactive ATP (32P-ATP) [11], which is a well-established method but suffers from a high risk of harmful radioactive contamination. Recently, several nonradioactive assays have been established, such as mass spectrometry [12], electrogenerated chemiluminescence (ECL) [13], electrochemistry [14], colorimetriy [15], fluorescence [16–20], quartz crystal microbalance [21], and surface-enhanced Raman spectroscopy (SERS) [22,23]. Among them, homogeneous fluorescence assays have
∗
some inherent advantages due to its high sensitivity, simplicity and the avoidance of tedious surface immobilization. However, the light emission of conventional luminophores often suffers from weakened or annihilated emission in their aggregated state, which is known as aggregation-caused quenching (ACQ). To address this concern, a new class of organic fluorogens with aggregationinduced emission (AIE) property was developed by Tang et al., in 2001 [24,25]. Propeller-shaped AIE molecule is nonemissive when dispersed, but distinctly emissive in the aggregated state. So far, the most widely used AIE molecules are tetraphnylethylene (TPE) derivatives, which have the merit of easy synthesis and functionalization [26–28]. Herein, a novel fluorescent probe was designed for the detection of protein kinase by coupling a hydrophilic peptide sequence with a hydrophobic AIE fluorogen. The presence of carboxypeptidase Y (CPY) can hydrolyze a peptide bond from the C-terminal end of the peptide probe and release free TPE molecule, which showed strong TPE emission. While the phosphorylation by CKII can prevent the digestion of CPY. Thus, low fluorescence intensity was observed. This strategy can be used to detect protein kinases CKII as well as screen their inhibitors. 2. Experimental 2.1. Materials and reagents Sodium ascorbate and benzoyl peroxide were obtained from J&K Scientific Ltd (Beijing, China). CKII and adenosine triphosphate (ATP)
Corresponding author. E-mail address: [email protected] (X. Zhang).
https://doi.org/10.1016/j.talanta.2019.04.036 Received 31 December 2018; Received in revised form 12 March 2019; Accepted 14 April 2019 Available online 19 April 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.
Talanta 201 (2019) 450–454
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Fig. 1. Schematic diagram of the synthesis process for probe TPE-Peptide 5.
were obtained from New England Biolabs (Beverly, MA, USA). CPY was purchased from Worthington Biochemical Co., Ltd (Lakewood, USA). Fmoc-propargyl-Gly-OH was purchased from Tokyo Chemical Industry Co., Ltd (Tokyo, Japan). 4,5,6,7-tetrabromobenzotriazole (TBB) were obtained from Aladdin (Shanghai, China). Deionized water (Millipore Milli-Q grade) with a resistivity of 18.2 MΩ cm−1 was used in all the experiments. All reagents used in the experiment were analytically pure.
wavelength of 312 nm and emission wavelengths ranged from 350 to 600 nm [29]. Both of the excitation and emission slit widths were set at 5.0 nm. As for CKII inhibition assay, the phosphorylation reaction solution containing 100 mU/μL CKII and different concentrations of TBB (0–25 μM) were used in the test. The rest steps were the same as mentioned above. 2.4. Detection of protein kinase activity in cancer cell lysate
2.2. Synthesis of probe Fmoc-Gly-TPE (4) and TPE-peptide (5)
Human cervical carcinoma cells (HeLa cells) were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and grown in a humidified atmosphere of 5% CO2 in air at 37 °C. HeLa cells were cultured with serum-free medium for 4 h. Then dimethyl sulfoxide (DMSO) and different concentrations of TBB were added to the medium. After cultured in the incubator for 30 min, cell lysates were obtained by scraping and sonication in Dulbecco's Phosphate Buffered Saline (2 s × 60 times at an interval of 3 s). The obtained solution was centrifuged at 15000 rpm, 4 °C for 60 min, and the resulting supernatant was stored at −20 °C for subsequent experiments. The Bradford protein assay kit (protein standard was bovine serum albumin) was used to measure the protein concentration of the cell lysate. Finally, the protein concentration of the cell lysate was diluted to 8 μg/mL.
Compound 3 in Fig. 1 (0.7741 g, 2.0 mmol, synthesis process seeing Supplementary Material 1.4) and Fmoc-propargyl-Gly-OH (0.6631 g, 1.977 mmol) were dissolved in a mixture of DMSO/DMF/H2O (10/7/6, v/v/v) under N2. Then sodium ascorbate (8.8 mmol) and CuSO4·5H2O (4.4 mmol) were added and the mixture was stirred overnight at room temperature. After the reaction was completed, a large amount of water was added, and the solution was extracted three times with ethyl acetate. The organic layers were combined, dried and concentrated. The resulting crude product was further purified by column chromatography using dichloromethane (DCM)/methanol (v/v = 20:1) + 1% acetic acid as eluent to give compound Fmoc-Gly-TPE 4 as a brown solid (0.5302 g, 37.14% yield). 1H NMR (DMSO, 500 MHz), δ (TMS, ppm):7.77-7.68 (m, 10H), 7.10 (s, 4H), 5.44 (s, 1H), 4.24 (s, 4H), 3.34 (s, 5H), 2.53-2.52 (m, 5H), 1.68-1.26 (m, 8H), 0.93 (s, 4H). ESI MS [M +H]+ m/z: calcd. 723.29, found 723.30. TPE-peptide 5 (TPE-GRRRADDSDDDD) was obtained by polypeptide synthesis using compound Fmoc-Gly-TPE 4 as a raw material. This process was conducted by Chinapeptides (Shanghai, China). ESI MS [M +3H]3+ m/z: found 645.27; [M+2H]2+ found 967.40; calcd. 1932.92.
3. Results and discussion 3.1. The design of AIE probe To obtain the probe TPE-peptide 5, firstly diphenylmethane reacted with 4-methylbenzophenone to give TPE skeleton structure 1 through McMurry cross coupling reaction. Then compound 2 was synthesized by bromination with N-bromosuccinimide (NBS). After azidation with NaN3, intermediate TPE-N3 3 was obtained. Click reaction between 3 and Fmoc-propargyl-Gly-OH produced Fmoc-Gly-TPE 4 under the catalysis of CuSO4/sodium ascorbate. The overall yield of these four steps was about 10%. The synthesis processes were illustrated in Fig. 1 and described in detail in Supplementary Material. Finally, TPE-peptide 5 was synthesized by the extension of the peptide chain using compound 4 as a starting material. The last step was completed by a commercial company. The structures of compounds were characterized by 1H-
2.3. Detection the activity and inhibition of CKII For phosphorylation process, 100 μL reaction mixture containing different concentration of CKII (0–100 mU/μL), 10 μM TPE-peptide 5, 1 mM ATP and CKII reaction solution (20 mM Tris-HCl, 10 mM MgCl2 and 50 mM KCl, pH 7.5) was incubated at 30 °C for 1 h. Then CPY was added bringing its final concentration to 3.5 mU/μL. The hydrolysis process was carried out at 25 °C for 30 min. The fluorescence intensities were measured at an excitation 451
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Fig. 2. Schematic diagram of detecting CKII by aggregation-induced emission.
NMRor MS (see Figs. S1 and S3 in Supplementary Material). Further characterization of compounds 1–3 can be found in the references [25–27]. Since there was an azide group in compound 3, IR spectrum was used to demonstrate this characteristic group at about 2100 cm−1 (Fig. S2 in Supplementary Material). The fluorescent intensity for TPEpeptide 5 was stable for at least two months without spontaneous aggregation. 3.2. Detection principle The detection mechanism was shown in Fig. 2. TPE, as a well-known AIE fluorogen, is a propeller-shaped compound. It is almost insoluble in water. When conjugated with peptide (GRRRADDSDDDDD), due to the high hydrophilic property of the peptide part, the water solubility of the whole TPE-peptide 5 complex increased dramatically. The free rotation of the four phenyl rings in this complex led to the nonradiative relaxation of excited electrons. As a result, TPE-peptide 5 complex showed very weak fluorescence in an aqueous environment. Here, we designed a protocol for detecting kinase activity according to the features of CPY and protein kinase CKII using TPE-peptide 5 complex as a probe. CPY as an exopeptidase could be used to degrade the peptide bonds at the C-terminal end of a peptide. When treated the synthesized TPE-peptide 5 probe with CPY, fluorescent TPE part was released into the solution due to the completed hydrolysis of non-phosphorylated peptide. Then small molecules TPE released in solution were aggregated together due to their hydrophobicity. So, high fluorescent signal could be observed according to the AIE effect. However, under the treatment of protein kinase CKII in the presence of ATP, the phosphorylation of serine on peptide produced phosphorylated substrate TPE-GRRRADDpSDDDDD, which could inhibit the hydrolysis ability of CPY. Thus, the TPE-peptide 5 could not be digested completely and the fluorescence signal was very weak due to the still strong hydrophilicity of TPE-GRRRADDpS. Therefore, the fluorescence intensity could be used to measure the activity of kinase CKII.
Fig. 3. Fluorescence signal for a series of experimental conditions: only TPEpeptide 5 (black curve); TPE-peptide 5+CKII+CPY (red curve); TPE-peptide 5+CKII+ATP (blue curve); TPE-peptide 5+CKII+ ATP+CPY (pink curve); TPE-P+CPY (green curve). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
dramatically (green curve), indicating the hydrolysis of TPE-peptide 5 substrate by CPY and releasing the hydrophobic small molecule TPE. ATP was used to provide γ-phosphoryl to a free hydroxyl group of serine under the catalysis of by protein kinase CKII. So, the digestion of CPY would not be hindered in the absence of ATP and high fluorescence intensity was still observed (red curve). However, in the absence of CPY, the phosphorylated substrate obtained by treated the TPE-peptide 5 with CKII and ATP could not be digested, and weak fluorescence signal was showed (blue curve). When CKII, ATP for phosphorylation, CPY for cleavage coexisted in the system simultaneously, phosphorylation reaction was catalyzed, thus, the digestion reaction could only proceed to the middle position of the polypeptide chain. Therefore, the fluorescence intensity of the system was still very low (pink curve). Through the above series of experiments, it could be verified that this experimental protocol was feasible for the detecting the activity of protein kinase CKII.
3.3. Feasibility of the assay In order to verify the feasibility of the protocol, the following experiments were designed: As shown in the Fig. 3, TPE-peptide 5 had lower fluorescence intensity due to the hydrophilicity of the TPE-peptide 5 probe as described above (black curve); After treated the TPEpeptide 5 substrate with CPY, the fluorescence intensity increased 452
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Fig. 4. (A) Fluorescence spectra for different concentrations of CKII (curve a to m: 0, 0.01, 0.1, 0.5, 1, 5, 8, 10, 20, 50, 80, 100, 200 mU/μL). (B) Plot of the relative fluorescence change vs concentration of CKII. Inset: The linear relationship between the relative fluorescence change and CKII concentrations. F0 is the fluorescence intensity of TPE-peptide 5 alone and F is the fluorescence intensity after addition proteins or enzymes.
3.4. CKII activity detection To obtain the best detection effect, the assay conditions were optimized. As shown in Figs. S4–S6 (Supplementary Material), the optimal ATP, CPY and probe TPE-peptide 5 dosages were selected as 1.0 mM, 3.5 U/mL and 10 μM, respectively. Under optimized detection condition, the activity of CKII was quantitatively investigated with increasing concentrations of CKII (0–200 mU/μL). Since the presence of CKII led to the phosphorylation in the middle position of TPE-peptide 5, the hydrolysis of CPY was stopped and the remaining peptide prevented the aggregation of TPE molecule. Thus, the fluorescence intensity decreased along with the increase of CKII concentration (see Fig. 4A). The relative fluorescence intensity ratio showed a linear correlation to the logarithmic CKII concentration within 0.1–80 mU/μL (see Fig. 4B and its insert), with the linear regression equation of (F-F0)/ F0 = 4.435–1.9596 lgC (F0 referred to the fluorescence intensity of TPEpeptide 5 alone and F referred to the fluorescence intensity after addition different concentration of CKII. C was the concentration of CKII (U mL−1), R2 = 0.9971). The EC50 value (enzyme concentration at which 50% substrate is converted) was determined to be 9.38 mU/μL. The detection limit was estimated to be 0.05 mU/μL (3σ), which is in accordance with the results of other reported methods [14,30–32]. This homogeneous detection system is quite simple and can complete within 1.5 h indicating it is a promising method for the analysis of protein kinase.
Fig. 5. Selectivity of the method for the detection of 0.1 U/μL of CKII, ALP and HRP, 3% BSA and thrombin. F0 is the fluorescence intensity of TPE-peptide 5 alone and F is the fluorescence intensity after addition proteins or enzymes.
concentration producing 50% inhibition) of TBB was determined to be 1.8 μM, which was comparable to the reported values (1.5 μM) [33]. This demonstrated that the developed method could be used for the screening of CKII inhibitors conveniently. Protein kinases play an important role in cell signalling. So, detection the inhibition effect of TBB in cell lysates is very necessary for monitoring the regulation of kinases in cell systems. After treated cell with different concentrations of TBB (0, 5, 10, 15, 25 μM), the fluorescence measurement was carried out using extracted cell lysates, which have many varieties of proteins. As shown in Fig. 6, the fluorescence intensity was positively correlated with the concentration of TBB, indicating the potential application of this protocol in the screening of protein kinase inhibitor in biological fluids.
3.5. Selectivity Control experiments were carried out to test the selectivity of the developed biosensor. From the results shown in Fig. 3A, the fluorescence intensity change of different proteins or enzymes, including bovine serum albumin (BSA), alkaline phosphatase (ALP), horseradish peroxidase (HRP) and thrombin were much lower than that of CKII. These results suggest that the proposed method possesses high selectivity to CKII.
4. Conclusions
3.6. Inhibition evaluation
In conclusion, a novel, simple and sensitive fluorescence assay was developed for homogeneous detection of protein kinases CKII using AIE fluorescent probe. The probe was synthesized by click reaction between TPE-N3 and Fmoc-propargyl-Gly-OH firstly. Then the peptide chain was extended to give TPE-Peptide 5. This probe was non-fluorescent in aqueous buffers but became emissive when cleaved by CPY. The presence of CKII led to phosphorylation on the peptide, which prevented further digestion of CPY. Hence the fluorescent intensity could be used to monitor the concentration of protein kinases CKII. The whole detection process could be finished within 1.5 h with high sensitivity (0.05 mU/μL). This method was used in protein kinase inhibitor screening even in a complex biological system, which was very useful in the discovery of anticancer drugs. What's more, after switching the
Since protein kinase inhibitors are of great significance in drug discovery. To investigate the potential applicability of the proposed protocol in the inhibition assay, a small molecule 4,5,6,7-tetrabromobenzotriazole (TBB) was chosen to confirm the inhibition effect. A series of concentrations of TBB (0, 1, 2, 5, 8, 10, 15, 20, 25 μM) were added to 80 mU/μL CKII reaction solution before phosphorylation. After the CPY digestion, fluorescence signals were measured. Fig. 5 showed that the fluorescent intensity increased gradually with the increase of TBB concentration, which demonstrated the inhibition effect for CKII. When the concentration of TBB reached 15 μM, the activity of protein kinase CKII could be inhibited completely and the fluorescence signal was identical to that without CKII. The IC50 (inhibitor 453
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Fig. 6. (A) The relationship between TBB concentration and fluorescence intensity; (B) The fluorescence intensity for different concentrations of TBB in cell lysates. Error bars were derived from the standard deviation of three measurements.
peptide substrates sequences, this method can also be used to detection other protein kinases and screen their inhibitor. This strategy extends the application of the AIE effect and provides an alternative method for the detection of protein kinases.
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