Citation: ZHANG Qianqian, ZHANG Xuepei, ZHANG Hanzhi, KANG Jingwu. Inhibitor screening and selectivity assessment against multiple cellular protein kinases by capillary electrophoresis with laser-induced fluorescence detection[J]. Chinese Journal of Chromatography, ;2013, 31(7): 646-655. doi: 10.3724/SP.J.1123.2013.04032 shu

Inhibitor screening and selectivity assessment against multiple cellular protein kinases by capillary electrophoresis with laser-induced fluorescence detection

1.
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China

Corresponding author: KANG Jingwu,
Received Date: 18 April 2013

Fund Project: National Natural Science Foundations of China (90713021, 20975109, 20675086) (90713021, 20975109, 20675086)

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.
- capillary electrophoresis (CE),
- laser-induced fluorescence detection,
- protein kinase inhibitor screening,
- selectivity assessment
1. [1]
  [1] Cohen P. Nat Rev Drug Discov, 2002, 1(4): 309
2. [2]
  [2] Critchfield J W, Coligan J E, Folks T M, et al. Proc Natl Acad Sci USA, 1997, 94(12): 6110
3. [3]
  [3] Bellacosa A, Kumar C C, Cristofano A D, et al. Adv Cancer Res, 2005, 94: 29
4. [4]
  [4] Ren R. Nat Rev Cancer, 2005, 5(3): 172
5. [5]
  [5] Hanahan D, Weinberg R A. Cell, 2000, 100(1): 57
6. [6]
  [6] Sebolt-Leopold J S, English J M. Nature, 2006, 441(7092): 457
7. [7]
  [7] Zhang J, Yang P L, Gray N S. Nat Rev Cancer, 2009, 9(1): 28http://www.ncbi.nlm.nih.gov/pubmed/19104514
8. [8]
  [8] Fedorov O, Muller S, Knapp S. Nat Chem Biol, 2010, 6(3): 166
9. [9]
  [9] Wesche H, Xiao S H, Young S W. Comb Chem High Throughput Screen, 2005, 8(2): 181
10. [10]
  [10] von Ahsen O, Bmer U. ChemBioChem, 2005, 6(3): 481
11. [11]
  [11] Sadler T M, Achilleos M, Ragunathan S, et al. Anal Biochem, 2004, 326(1): 106
12. [12]
  [12] Ohml N, Wingfield J M, Yazawa H, et al. J Biomol Screen, 2000, 5(6): 463
13. [13]
  [13] Gaudet E A, Huang K S, Zhang Y, et al. J Biomol Screen, 2003, 8(2): 164
14. [14]
  [14] Seethala R, Menzel R. Anal Biochem, 1997, 253(2): 210
15. [15]
  [15] Shults M D, Kozlov I A, Nelson N, et al. ChemBioChem, 2007, 8(8): 933
16. [16]
  [16] Dasanu C A, Dutcher J, Alexandrescu D T. South Med J, 2007, 100(3): 328
17. [17]
  [17] Shults M D, Janes K A, Lauffenburger D A, et al. Nat Meth, 2005, 2(4): 277
18. [18]
  [18] Yu Y, Anjum R, Kubota K, et al. Proc Natl Acad Sci U S A, 2009, 106(28): 11606
19. [19]
  [19] Meredith G D, Sims C E, Soughayer J S, et al. Nat Biotech, 2000, 18(3): 309
20. [20]
  [20] Proctor A, Wang Q, Lawrence D S, et al. Analyst, 2012, 137(13): 3028
21. [21]
  [21] Fernandes N, Allbritton N L. Biochem Biophys Res Commun, 2009, 387(2): 414
22. [22]
  [22] Fernandes N, Bailey D E, Van Vranken D L, et al. ACS Chem Biol, 2007, 2(10): 665
23. [23]
  [23] Seethala R, Menzel R. Anal Biochem, 1998, 255(2): 257
24. [24]
  [24] Kubota K, Anjum R, Yu Y, et al. Nat Biotech, 2009, 27(10): 933
25. [25]
  [25] Bao J, Regnier F E. J Chromatogr A, 1992, 608(1/2): 217
26. [26]
  [26] Craig D B, Arriaga E A, Wong J C Y, et al. J Am Chem Soc, 1996, 118(22): 5245
27. [27]
  [27] Tang Z, Wang Z, Kang J. Electrophoresis, 2007, 28(3): 360
28. [28]
  [28] Regnier F E, Patterson D H, Harmon B J. TrAC Trend Anal Chem, 1995, 14(4): 177
29. [29]
  [29] Xue Q, Yeung E S. Nature, 1995, 373(6516): 681
30. [30]
  [30] Glatz Z. J Chromatogr B, 2006, 841(1/2): 23
31. [31]
  [31] Křenková J, Foret F. Electrophoresis, 2004, 25(21/22): 3550http://onlinelibrary.wiley.com/doi/10.1002/elps.200406096/abstract;jsessionid=85EAF4320CEA6C84570C3AD82B3C24AF.d02t02?deniedAccessCustomisedMessage=&userIsAuthenticated=false
32. [32]
  [32] He Y, Yeung E S. Electrophoresis, 2003, 24(1/2): 101http://124.16.154.79/gochem/searchAction!showArticle.action?article.id=10034
33. [33]
  [33] Sano M, Ueno K, Kamimori H. J Chromatogr B, 2003, 794(1): 149
34. [34]
  [34] Janes K A, Albeck J G, Peng L X, et al. Mol Cell Proteomics, 2003, 2(7): 463
35. [35]
  [35] Obata T, Yaffe M B, Leparc G G, et al. J Biol Chem, 2000, 275(46): 36108
36. [36]
  [36] Tiganis T, House C M, Kemp B E. Arch Biochem Biophys, 1996, 325(2): 289
37. [37]
  [37] Knight Z A, Shokat K M. Chem Biol, 2005, 12(6): 621
38. [38]
  [38] Zelada A, Passeron S, Lopes Gomes S, et al. Eur J Biochem, 1998, 252(2): 245
39. [39]
  [39] Kübler D, Pyerin W, Bill O, et al. J Biol Chem, 1989, 264(24): 14549
40. [40]
  [40] Pfister T D, Storey K B. Insect Biochem Mol Biol, 2002, 32(5): 505
41. [41]
  [41] Copeland R A. Anal Biochem, 2003, 320(1): 1
42. [42]
  [42] Davies S P, Reddy H, Caivano M, et al. Biochem J, 2000, 351(Pt 1): 95
43. [43]
  [43] Ross D, Kralj J G. Anal Chem, 2008, 80(24): 9467
44. [44]
  [44] Viht K, Vaasa A, Raidaru G, et al. Anal Biochem, 2005, 340(1): 165
45. [45]
  [45] Meggio F, Deana A D, Ruzzene M, et al. Eur J Biochem, 1995, 234(1): 317
46. [46]
  [46] Hill M M, Andjelkovic M, Brazil D P, et al. J Biol Chem, 2001, 276(28): 25643
47. [47]
  [47] Sarno S, Reddy H, Meggio F, et al. FEBS Lett, 2001, 496(1): 44
48. [48]
  [48] Cheng Y C, Prusoff W H. Biochem Pharmacol, 1973, 22(23): 3099
49. [49]
  [49] Traut T W. Mol Cell Biochem, 1994, 140(1): 1
50. [50]
  [50] Gribble F M, Loussouarn G, Tucker S J, et al. J Biol Chem, 2000, 275(39): 30046
1. [1]
  Jiakun BAI , Ting XU , Lu ZHANG , Jiang PENG , Yuqiang LI , Junhui JIA . A red-emitting fluorescent probe with a large Stokes shift for selective detection of hypochlorous acid. Chinese Journal of Inorganic Chemistry, 2024, 40(6): 1095-1104. doi: 10.11862/CJIC.20240002
2. [2]
  Peiran ZHAO , Yuqian LIU , Cheng HE , Chunying DUAN . A functionalized Eu³⁺ metal-organic framework for selective fluorescent detection of pyrene. Chinese Journal of Inorganic Chemistry, 2024, 40(4): 713-724. doi: 10.11862/CJIC.20230355
3. [3]
  Jun LUO , Baoshu LIU , Yunchang ZHANG , Bingkai WANG , Beibei GUO , Lan SHE , Tianheng CHEN . Europium(Ⅲ) metal-organic framework as a fluorescent probe for selectively and sensitively sensing Pb²⁺ in aqueous solution. Chinese Journal of Inorganic Chemistry, 2024, 40(12): 2438-2444. doi: 10.11862/CJIC.20240240
4. [4]
  Yihui Song , Shangshang Qin , Kai Wu , Chengyun Jin , Bin Yu . 生物化学在高水平创新型药学人才培养中的交叉融合应用——以去甲基化酶LSD1抑制剂的活性评价为例. University Chemistry, 2025, 40(6): 341-352. doi: 10.12461/PKU.DXHX202406018
5. [5]
  Dingwen CHEN , Siheng YANG , Haiyan FU , Hua CHEN , Xueli ZHENG , Weichao XUE , Jiaqi XU , Ruixiang LI . NiOOH-mediated synthesis of gold nanoaggregates for electrocatalytic performance for selective oxidation of glycerol to glycolate. Chinese Journal of Inorganic Chemistry, 2025, 41(11): 2317-2326. doi: 10.11862/CJIC.20250053
6. [6]
  Junjie Zhang , Yue Wang , Qiuhan Wu , Ruquan Shen , Han Liu , Xinhua Duan . Preparation and Selective Separation of Lightweight Magnetic Molecularly Imprinted Polymers for Trace Tetracycline Detection in Milk. University Chemistry, 2024, 39(5): 251-257. doi: 10.3866/PKU.DXHX202311084
7. [7]
  Zhi Chai , Huashan Huang , Xukai Shi , Yujing Lan , Zhentao Yuan , Hong Yan . Wittig反应的立体选择性. University Chemistry, 2025, 40(8): 192-201. doi: 10.12461/PKU.DXHX202410046
8. [8]
  Tianlong Zhang , Jiajun Zhou , Hongsheng Tang , Xiaohui Ning , Yan Li , Hua Li . Virtual Simulation Experiment for Laser-Induced Breakdown Spectroscopy (LIBS) Analysis. University Chemistry, 2024, 39(6): 295-302. doi: 10.3866/PKU.DXHX202312049
9. [9]
  Junjian Wang , Qingquan Yu , Shunyao Liu , Yuke Chen , Xiaoyu Liu , Guodong Li , Xiaoyan Liu , Hong Liu , Weijia Zhou . Laser-Induced Carbonization of Hydroxyapatite Sandwich Paper for Inkless Printing. Acta Physico-Chimica Sinica, 2024, 40(4): 2304024-0. doi: 10.3866/PKU.WHXB202304024
10. [10]
  Shihui Shi , Haoyu Li , Shaojie Han , Yifan Yao , Siqi Liu . Regioselectively Synthesis of Halogenated Arenes via Self-Assembly and Synergistic Catalysis Strategy. University Chemistry, 2024, 39(5): 336-344. doi: 10.3866/PKU.DXHX202312002
11. [11]
  Yunhao Zhang , Yinuo Wang , Siran Wang , Dazhen Xu . Progress in Selective Construction of Functional Aromatics from Nitrogenous Cycloalkanes. University Chemistry, 2024, 39(11): 136-145. doi: 10.3866/PKU.DXHX202401083
12. [12]
  Xilin Zhao , Xingyu Tu , Zongxuan Li , Rui Dong , Bo Jiang , Zhiwei Miao . Research Progress in Enantioselective Synthesis of Axial Chiral Compounds. University Chemistry, 2024, 39(11): 158-173. doi: 10.12461/PKU.DXHX202403106
13. [13]
  .
  CCS Chemistry | 超分子活化底物为自由基促进高效选择性光催化氧化
  . CCS Chemistry, 2025, 7(10.31635/ccschem.025.202405229): -.
14. [14]
  Feifei Yang , Wei Zhou , Chaoran Yang , Tianyu Zhang , Yanqiang Huang . Enhanced Methanol Selectivity in CO₂ Hydrogenation by Decoration of K on MoS₂ Catalyst. Acta Physico-Chimica Sinica, 2024, 40(7): 2308017-0. doi: 10.3866/PKU.WHXB202308017
15. [15]
  Shuhong Xiang , Lv Yang , Yingsheng Xu , Guoxin Cao , Hongjian Zhou . Selective electrosorption of Cs(Ⅰ) from high-salinity radioactive wastewater using CNT-interspersed potassium zinc ferrocyanide electrodes. Acta Physico-Chimica Sinica, 2025, 41(9): 100097-0. doi: 10.1016/j.actphy.2025.100097
16. [16]
  Xinyu Xu , Jiale Lu , Bo Su , Jiayi Chen , Xiong Chen , Sibo Wang . Steering charge dynamics and surface reactivity for photocatalytic selective methane oxidation to ethane over Au/Ti-CeO₂. Acta Physico-Chimica Sinica, 2025, 41(11): 100153-0. doi: 10.1016/j.actphy.2025.100153
17. [17]
  Jie ZHAO , Sen LIU , Qikang YIN , Xiaoqing LU , Zhaojie WANG . Theoretical calculation of selective adsorption and separation of CO₂ by alkali metal modified naphthalene/naphthalenediyne. Chinese Journal of Inorganic Chemistry, 2024, 40(3): 515-522. doi: 10.11862/CJIC.20230385
18. [18]
  Peng YUE , Liyao SHI , Jinglei CUI , Huirong ZHANG , Yanxia GUO . Effects of Ce and Mn promoters on the selective oxidation of ammonia over V₂O₅/TiO₂ catalyst. Chinese Journal of Inorganic Chemistry, 2025, 41(2): 293-307. doi: 10.11862/CJIC.20240210
19. [19]
  Baitong Wei , Jinxin Guo , Xigong Liu , Rongxiu Zhu , Lei Liu . Theoretical Study on the Structure, Stability of Hydrocarbon Free Radicals and Selectivity of Alkane Chlorination Reaction. University Chemistry, 2025, 40(3): 402-407. doi: 10.12461/PKU.DXHX202406003
20. [20]
  Jun Huang , Pengfei Nie , Yongchao Lu , Jiayang Li , Yiwen Wang , Jianyun Liu . 丝光沸石负载自支撑氮掺杂多孔碳纳米纤维电容器及高效选择性去除硬度离子. Acta Physico-Chimica Sinica, 2025, 41(7): 100066-0. doi: 10.1016/j.actphy.2025.100066

Get Citation

PDF

XML

Metrics

PDF Downloads(0)
Abstract views(491)
HTML views(39)

通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142