Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Development of a Selective Labeling Probe for Bruton’s Tyrosine Kinase Quantification in Live Cells Jiahui Chen, Xiafeng Wang, Fengli He, and Zhengying Pan* State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Shenzhen Graduate School, Peking University, Xili University Town, PKU Campus, Shenzhen 518055, China S Supporting Information *
ABSTRACT: As a key regulator of the B-cell receptor signaling pathway, Bruton’s tyrosine kinase (Btk) has emerged as an important therapeutic target for various malignancies and autoimmune disorders. However, data on the expression profiles of Btk are lacking. Here, we report the discovery of a new, selective Btk probe and of a sandwich-type ELISA quantification method to detect endogenous Btk in live cells. We achieved selective labeling of Btk in vivo and quantified Btk levels in seven types of human lymphoma cell lines. This quantification method provides a powerful tool to study Btk in live cells that may also be useful in clinical settings.
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been used for Btk imaging in live cells13−15 (Figure S1). The CNX-500 probe with the spebrutinib scaffold was applied in cell lysates to measure the amount of Btk not occupied by inhibitor.16 However, little is known about variations in endogenous Btk levels and changes in response to stimulus; therefore, a more selective probe for Btk labeling and accurate quantification is needed. Here, we report a convenient approach combining a highly selective cell permeable fluorescent probe and ELISA technology to quantify Btk in live cells (Figure 1), which should facilitate further studies on Btk and the activity of its inhibitors in native settings.17
INTRODUCTION Bruton’s tyrosine kinase (Btk), a member of the Tec family, is an important regulator of the B cell receptor (BCR) pathway, which plays a key role in B-lymphocyte maintenance and differentiation. Due to its important roles in B cells, Btk has emerged as an important therapeutic target in autoimmune disorders and various malignancies.1,2 Mutations in the Btk gene lead to Btk-deficiency diseases, X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice.3,4 Btk deficiency prevents B cell maturation, and Btk inhibitors can block BCR signaling and induce apoptosis.5−7 Conversely, Btk overexpression may have negative effects on B cell functions. Studies have shown that Btk protein expression is elevated in Chronic Lymphocytic Leukemia (CLL) compared with normal B cells.8,9 Thus, a reliable and convenient assay for measuring Btk levels would help to elucidate the functions of Btk and facilitate both the diagnosis and the prognosis of various cancers in humans. Small molecule probes with excellent cellular activity and selectivity can serve as useful tools for assessing the biological roles of proteins and signal transduction pathways because they can provide greater spatiotemporal control over protein function.10 The presence or absence of the proteins of interest and their subcellular localization are highly valuable pieces of information to understand the physiological and/or pathological functions of proteins. A number of Btk probes have been developed for imaging, drug target engagement, and other biological studies. PCI-33380 (CRA-033380, Figure S1), the first Btk probe, was used to study the connection between the inhibitor binding event and the phenotypic readouts of cellular responses due to the inhibition of Btk functions.11 Other fluorescent probes based on the ibrutinib12 scaffold have also © XXXX American Chemical Society
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RESULTS AND DISCUSSION Synthesis of Probes. An ideal chemical probe for our purpose should be highly selective, cell permeable, and efficient for labeling.18 Even though the ibrutinib scaffold has previously been used to develop probes, its selectivity must be improved to meet our needs. Based on the proposed binding mode of ibrutinib in Btk, a small group of derivatives were designed, focusing on variations in reactive groups to improve selectivity (Figure S2). The synthetic route for fluorescent probes is shown in Scheme 1. Compound 2, the ibrutinib analogue, was synthesized from compound 1 with a base-mediated substitution reaction. Following hydrolysis and coupling reactions with N-(2-aminoethyl) carbamic acid tert-butyl ester, compound 3 was obtained with a good yield. The carboxybenzyl Received: February 26, 2018 Revised: March 19, 2018
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DOI: 10.1021/acs.bioconjchem.8b00137 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
Figure 1. Structure of small-molecule fluorescent probes and diagram of the detection methods.
Scheme 1. Synthetic Route of Probesa
a Reagents and conditions: (a) H-Pyrazolo[3,4-d pyrimidin-4-amine, 3-(4-phenoxyphenyl), K2CO3, DMF, 80 °C; (b) 1 N LiOH, THF:MeOH (3:1) then 1 N HCl to pH 2; (c) 1,2-Ethanediamine, N1-(1,1-dimethylethoxy), EDCI, HOBt, DIPEA, DCM; (d) Pd(OH)2/C, H2, EtOAc, some drop MeOH; (e) R-COOH, HATU, HOBt, DIPEA, DMF; (f) TFA, DCM; (g) BODIPY FL acid, HATU, HOBt, DIPEA, DMF; (h) Cy5-dye acid, HATU, HOBt, DIPEA, DMF.
facilitate the attack of the Michael acceptor by the thiol group on Cys481. Selectivity of Probes in Live Cells. All four probes were examined for their ability to selectively detect Btk in live cells. Gratifyingly, we found that probe 10 not only showed high labeling efficiency of Btk protein in vitro but also presented excellent selectivity in a cellular environment (Figure 2). The labeling of recombinant Btk protein was saturated by probe 10 at 1 μM for 1 h, and the same result was achieved in OCI-Ly7 (3 million cells). This probe could also label Btk highly selectively in live cells, even at high concentrations. In contrast, probe 8 labeled two lines in cells even at lower concentrations; probes 9 and 11 showed good selectivity at low concentrations but not at high concentrations (Figure S4). Furthermore, probe
group was removed, and the liberated amino group was coupled with four types of acids to yield the corresponding probe precursors 4, 5, 6, and 7. After deprotecting the tertbutoxycarbony group, the condensation reactions with BODIPYFL acid or Cy5 dye acid yielded the target probes 8, 9, 10, 11, and 12. The four probe precursors were used to predict probe reactivity (Figure 2a and Figure S3). Compound 4 (IC50 = 6 nM) showed similar activity to ibrutinib, indicating that the five-membered ring and the linker moiety had little structural impact. Compound 6 had a higher IC50 value than compound 4, which may be due to the steric hindrance of the terminal methyl group, whereas compound 5 (IC50 = 31 nM) with a tertiary amino group may provide a basic environment and B
DOI: 10.1021/acs.bioconjchem.8b00137 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry
Figure 2. (a) IC50 values of four probe precursors. (b) Probe 10 labeled recombinant Btk protein. (c) Btk in OCI-Ly7 cells were selectively labeled by probe 10. (d) Probe 10 labeled Btk in different human lymphocytes.
10 efficiently labeled Btk in multiple human lymphocyte cells with similar selectivity (Figure 2d). Quantitative Analysis of Btk in Cells with EnzymeLinked Immunosorbent Assay. Next, we attempted, based on the selective labeling of Btk by probe 10, to establish a quantification method combining a specific cellular probe and a sensitive ELISA method. As shown in Figure 1, after Btk is labeled by probe 10 in live cells, a sandwich-type ELISA format is adopted. Btk is pulled down from cell lysates incubated in specific anti-Btk antibody-coated wells of multiwell plates; then, the anti-BODIPY antibody is used to detect the fluorescent probe, followed by incubation with an HRP-conjugated second antibody, which recognize the BODIPY antibody and allow weak signals to be amplified and accurately measured. A standard curve was first constructed using calibrated Btk proteins (Figure 3a and Figure S5). The Btk murine monoclonal antibody (1:500 dilution) was coated onto a 96well plate at 4 °C overnight. Different concentrations of Btk fully labeled by probe 10 were added to the wells and incubated for 1 h. Then, BODIPY-FL antibody (1:5000 dilution) was added and incubated for 2 h. The HRP-conjugated antibody (1:12000 dilution) was incubated for another 30 min. Subsequently, the color developing agent 3,3′,5,5′-tetramethylbenzidine and the stop solution were added to stop the reaction. A 3-order linear detection range of 5.9−3000 nanogram per milliliter was achieved. To confidently use this ELISA detection method in cells, we must also answer two questions. The first question is whether all cellular Btk can be labeled by probe 10. The second question is whether all labeled Btk can be captured by the antibody coated on the wells. To answer the first question, we synthesized a new probe 12 with a different fluorescent group Cy5 (Figure 1). OCI-Ly7 cells were first incubated with probe 10 (1 μM). Then, the cells were lysed, and the cell lysate was incubated by probe 12 at a 16-fold concentration. The results indicated that all Btk proteins were labeled with probe 10 in cells and that neither remnants nor newly synthesized Btk protein could be identified
Figure 3. (a) Standard curve of the ELISA method constructed by Btk protein labeling with probe 10. All the data are mean ± s.e.m. (n = 2). (b) Detection of Btk by probe 10 in cellular samples. A, Labeling by probe 10 (1 μM) in OCI-Ly7 cells; B, Labeling by Cy5 probe 12 (1 μM) in OCI-Ly7 cells; C, Cells were first labeled with probe 10 (1 μM), and then its lysate was incubated with probe 12 (16 μM); D, Cell lysate without prior treatment with probe 10 was labeled by probe 12 (1 μM). (c) Detection of the residual Btk protein in cellular samples after the ELISA experiment, 400* represents the total content of Btk in 400-thousand cells.
beyond our detection limit during the process (Figure 3b). To answer the second question, we first assessed the saturation time required for the antibody to capture all of target Btk protein, which was 2.5 h (Figure S6). Although a fluorescent band started to appear in the sample of 800 000 cells indicating that not all of Btk had been retained by the coated wells, Btk in 400 000 cells could be fully captured in the anti-Btk antibodycoated wells (Figure 3c). Finally, Btk was transfected into Jurkat cells, a type of T cell that does not express Btk, and could also be quantified using C
DOI: 10.1021/acs.bioconjchem.8b00137 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 4. (a) Labeling of Btk as shown in the Western blots of different types of cells incubated with probe 10 (1 μM). (b) Quantities of Btk in different cell lines (400 thousand cells each). (c) The Btk content of Ramos cell after BCR activation. (d) The relative mRNA expression levels of Ramos cells after BCR activation. Differences were evaluated using Student’s t test comparing the value of T = 0 h with the later time points; ns, nonsignificant (c,d). All the data are mean ± s.e.m. of two independent experiments measured in duplicate (n = 2).
activation in Ramos and OCI-Ly7 cells, thus suggesting that mechanisms other than changes in protein quantity are more important in signal transduction by Btk. Moreover, our approach is not restricted to Btk and can serve as a general method for other biological targets by varying different recognizing groups and covalent anchoring points.
our ELISA method (Figure S7). After establishing the ELISA method, the Btk contents of seven human lymphocytes were determined. Using the standard curve, the Btk content in 400 000 cells can be calculated from the fluorescence values (Figure 4b). The Btk content ranged from approximately 30 to 38 ng/400 000 cells in the different cell lines examined, which shows that Btk is a relatively low abundance protein, and its concentration varies as much as approximately 25% among various human cancer cell lines. To further understand the response mode of Btk upon stimulation in live cells, the BCR pathway of Ramos and OCILy7 cells was activated by incubating with anti-IgM antibody. Cell samples at different time points, as long as 48 h after stimulation, were subjected to the ELISA method to quantify the Btk content. The results clearly indicated no significant variations in Btk content, in both Ramos and OCI-Ly7 cells (Figure 4c and Figure S8). PCR experiments were also performed to detect the mRNA expression levels of Btk, whose results also showed (Figure 4d) no significant changes in Btk content upon activation of the BCR pathway. These results confirm that cells do not respond to BCR pathway activation by changing the quantity of the key regulator Btk.
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EXPERIMENTAL PROCEDURES Materials. All chemicals were purchased from commercial vendors and used without further purification. Anhydrous DMF was distilled from calcium hydride. Anhydrous DCM was distilled from calcium hydride. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.2 mm Jiangyou silica gel plates (HSGF254) using UV light as visualizing agent or ninhydrin as developing agent. Flash column chromatography was carried out using Puke silica (ZCX-II, 200−300 mesh). Albumin Bovine V was purchased from Scientific Research Special. SDS-polyacrylamide gel preparing reagents and Protein marker (CST 10022139) were purchased from Bio-Rad. The ELISA antibody pair buffer kit (CNB0011) was from Invitrogen. Btk plasmid (SO16110718) was provided by Sino Biological Inc. Recombinant kinases Btk were purchased from Carna Biosciences (08− 080). phosphatase inhibitors (5872S), Anti-Btk (8547S) antibodies, and the secondary antibody against rabbit (7074s) were purchased from Cell Signaling Technology. Anti-Btk (sc28387) antibodies were purchased from Santa Cruz Biotechnology. BODIPY-FL antibody (A5770) was purchased from Invitrogen. Cell Culture. OCI-Ly7 (CoBioer CBP60559), Namalwa (SGST TcHu-75), Jurkat (SGST TcHu123), WSU-NHL (DSMZ ACC-58), Toledo (ATCC CRL-2631), Ramos (ATCC CRL-1596), Daudi (ATCC CCL-213), and Raji
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CONCLUSION In summary, the fluorescent probe-based method for the detection and quantification of target proteins is a promising tool to study quantity variations of endogenous proteins in native samples. We successfully developed a cell permeable and highly selective fluorescent probe for Btk. A rapid, convenient, and accurate quantification method was then established. We demonstrated the possibility of quantifying endogenous Btk in live cells and have successfully detected variations in Btk in different cell lines and their unresponsiveness to BCR pathway D
DOI: 10.1021/acs.bioconjchem.8b00137 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Transfer system (Bio-Rad). Total Btk was detected using antiBtk antibody and standard Western blotting techniques. Establishment of the Standard Curve. The 96-well plate was coated with monoclonal Btk antibody (sc28387 1:100) in coating buffer (pH 9.6 Invitrogen CB01100). The plate was covered with adhesive plastic and incubated overnight at 4 °C. The coating solution was removed and washed three times by filling the wells with 200 μL washing buffer (Invitrogen WB 01). The remaining drops were removed by patting the plate on a paper towel. The protein-binding sites in wells were blocked with nonfat milk in PBS (10%) by adding 300 μL per well. The plate was covered and incubated for 1 h in 37 °C. 300 μL washing buffer was added into the plate and washed three times. Different concentrations of Btk protein or different numbers of cell samples were labeled with probes, and diluted in each well with 100 μL assay buffer. After incubation for 3 h at 250 rpm 25 °C, samples were removed and washed three times with 300 μL washing buffer. 100 μL diluted BODIPY FL antibody (1:1000) was added to each well and incubated for 2 h at 37 °C. The plate was washed three times with washing buffer again, and 100 μL conjugated secondary antibody (Abcam ab6721, 1:12000) was added. The plate was covered and incubated for 30 min at 37 °C, then washed three times. 100 μL TMB solution (Invitrogen SB01) was added to each well, incubated for 5 min before 100 μL stop solution (Invitrogen SS03100) was added. The optical density was measured at 450 nm. The standard curve was established and the Btk concentration in samples can be obtained by interpolating the fluorescence values from the standard curve. Quantification of Btk by ELISA. Quantification of Btk in Different Cells. The different cells at a density of 400 × 103 cells/mL were labeled with probe 10 (2 μM) for 1 h 37 °C. Their lysates were used to detect the Btk content by ELISA. Quantification of Btk with BCR Activation. 20 × 103 Ramos and OCI-Ly7 cells were incubated in 1 mL of RPMI 1640 medium containing 9% fetal bovine serum and activated with 200 μg/mL IgM (R&D G-105-C) for 0, 1, 2, 4, 9, 12, and 48 h. All the cells were labeled with probe10 (2 μM) for 1 h at 37 °C. Their lysates were used to detected the Btk content by ELISA.
(SGST TcHu-44) cell lines were cultured in IMDM (12440053, Invitrogen, Carlsbad, CA) or RPMI 1640 medium (A10491, Invitrogen, Carlsbad, CA) containing 10% heatinactivated fetal bovine serum (10099−141, Invitrogen, Carlsbad, CA) at 37 °C in 5% CO2. Synthesis of Probe 10. The important intermediate compound 3 (220 mg, 0.32 mmol) was dissolved in 3 mL ethyl acetate and a few drops of methanol. A catalytic palladium hydroxide on carbon was added. The mixture was stirred under hydrogen at room temperature for 4 h. The mixture was filtered with celatom and concentrated to give intermediate which was directly used in the next reaction without purification. Crotonic acid (31 mg, 0.36 mmol), HOBt (25 mg, 0.18 mmol), EDCI (86 mg, 0.45 mmol), and Et3N (45 mg, 0.45 mmol) were added to the intermediate in 3 mL DCM and stirred for 6 h. The mixture was concentrated and extracted with ethyl acetate and brine three times. The organic layers were combined and dried over anhydrous Na2SO4, filtered, and concentrated by rotary evaporator in vacuo. The concentrate was purified by flash column chromatography (EtOAc:Hexanes = 3:1) to give the compound 6 as white solid (yield 76%). HRMS (m/z): [M + H]+ calcd. for C33H39N8O5+, 627.3038; found: 627.3071. Trifluoroacetic acid (35 mg, 0.31 mmol) was added to compound 6 (15 mg, 0.017 mmol) dissolved in 1 mL anhydrous DCM. The reaction was stirred at room temperature for 3 h. The mixture was concentrated (theoretical: 8.8 mg, 0.017 mmol) and dissolved in 2 mL DMF. HATU (19.4 mg, 0.051 mmol), BODIPYFL (5 mg, 0.017 mmol), and DIPEA (11 mg, 15 μL, 0.085 mmol) were added to the intermediate at 0 °C under argon atmosphere. The reaction was stirred for 6 h at room temperature. The mixture was concentrated in vacuo and extracted with ethyl acetate and brine three times. The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The mixture was purified by HPLC (gradient elution of acetonitrile from 50% to 95% in 15 min, purity 98%). The product was lyophilized to give probe 10 as red solids (4 mg, yield 25%). HRMS (m/z): [M + H]+ calcd. for C42H44BF2N10O4+, 801.3603; found: 801.3657. Synthetic procedures of compounds are provided in Supporting Information. Labeling of Recombinant Btk and Btk in Live Cells. Recombinant Btk kinases were diluted to the final concentrations in reaction buffer (50 mM HEPES, 250 mM NaCl, 5% glycerol, 10 mM MgCl2, 2 mM DTT, pH 8) and incubated with various concentrations of probes 8, 9, 10, and 11 for 1 h at 25 °C; reaction was stopped by the addition of LDS Sample Buffer (NP0007, Invitrogen) and Sample Reducing Agent (NP0004, Invitrogen). Cells (at a density of 3 000 000 cells/mL) in standard growth media were incubated with different concentrations of probes at 37 °C in 5% CO2 for 1 h. Cells were washed with PBS three times to remove excess probes, and lysed in lysis buffer (Beyotime P0013, 1 mM PMSF, 10 mM NaF, 100× phosphatase inhibitors, and 40× Protease inhibitors). The samples for recombinant protein and live cell labeling experiment were heated at 100 °C for 10 min. Then the denatured samples were loaded onto the SDS-page gel (10%), and the gel was scanned by fluorescence gel scanning with PharasFX Plus Molecular Imager (Bio-Rad, Ex = 480 nm and Em = 530 nm for BODIPY porbe or Ex = 635 nm and Em = 695 nm for Cy5 probe). The gel after scanning was transferred to polyvinylidene difluoride membrane using Trans-Blot Turbo
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00137. Synthesis of compounds and detailed experimental procedures of biological assays (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Zhengying Pan: 0000-0002-4312-7103 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank funding support from The National Natural Science Foundation of China (81373270), Shenzhen Science and Technology Innovation Commission E
DOI: 10.1021/acs.bioconjchem.8b00137 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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based detection of human carbonic anhydrase II and IX. MedChemComm 7, 2045−2062.
(JCYJ20160226105227446), 973 Program (2013CB910704), and Peking University.
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DOI: 10.1021/acs.bioconjchem.8b00137 Bioconjugate Chem. XXXX, XXX, XXX−XXX
