基于β-catenin/Lef1相互作用为靶标的新型抗肿瘤药物高通量筛选模型的建立
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摘要
旨在以β-catenin/Lef1相互作用为靶标,建立基于ELISA原理的适用于靶向β-catenin/Lef1相互作用小分子抑制剂筛选的高通量筛选模型。利用DNA重组技术,将构建的β-catenin-pET-30a(+)重组质粒转化大肠杆菌Escherichia coli Rosetta (DE3),经诱导培养后进行β-catenin原核表达。采用亲和层析方法分离纯化β-catenin后,以GSTPulldown实验进行生物学活性鉴定。利用ELISA原理建立β-catenin/GST-Lef1结合的分子模型,通过优选GST-Lef1最佳包被浓度和β-catenin最佳反应浓度,建立适用于靶向β-catenin/Lef1相互作用小分子抑制剂筛选的高通量筛选模型。SDS-PAGE和Western blotting实验证实β-catenin的原核表达。GST Pulldown实验证实纯化的β-catenin具有良好的生物学活性。通过对基于ELISA原理建立的β-catenin/GST-Lef1结合的分子模型优化,选用10μg/mL GST-Lef1和6μg/mLβ-catenin建立ELISA高通量筛选模型,其Z?因子为0.76。本研究成功建立了基于ELISA原理的β-catenin/GST-Lef1结合的分子模型,为靶向β-catenin/Lef1相互作用小分子抑制剂的高通量筛选奠定了基础。
To develop an enzyme-linked immunosorbent assay(ELISA)-based high throughput screening(HTS) method for β-catenin/Lef1 interaction antagonists screening, Escherichia coli Rosetta(DE3) competent cells were transformed withβ-catenin-pET-30a(+) plasmid. β-catenin protein was expressed after induction and purified using affinity chromatography.The biological activity of purified β-catenin was further analyzed by GST Pulldown assay. The β-catenin/GST-Lef1 binding model was established using ELISA principle, and the ELISA-based HTS method was further optimized through determination of an optimal coated concentration of GST-Lef1 and working concentration of β-catenin. The results showed that β-catenin protein was successfully expressed and purified. The GST Pulldown assay demonstrated a perfect biological activity for purified β-catenin. Subsequently, the ELISA-based HTS method was performed using 10 μg/mL GST-Lef1 and 6 μg/mLβ-catenin, with the Z' factor of 0.76. Taken together, we have successfully developed a simple, robust and reliable ELISA-based HTS method for screening of novel Wnt inhibitors targeting β-catenin/Lef1 interaction.
To develop an enzyme-linked immunosorbent assay(ELISA)-based high throughput screening(HTS) method for β-catenin/Lef1 interaction antagonists screening, Escherichia coli Rosetta(DE3) competent cells were transformed withβ-catenin-pET-30a(+) plasmid. β-catenin protein was expressed after induction and purified using affinity chromatography.The biological activity of purified β-catenin was further analyzed by GST Pulldown assay. The β-catenin/GST-Lef1 binding model was established using ELISA principle, and the ELISA-based HTS method was further optimized through determination of an optimal coated concentration of GST-Lef1 and working concentration of β-catenin. The results showed that β-catenin protein was successfully expressed and purified. The GST Pulldown assay demonstrated a perfect biological activity for purified β-catenin. Subsequently, the ELISA-based HTS method was performed using 10 μg/mL GST-Lef1 and 6 μg/mLβ-catenin, with the Z' factor of 0.76. Taken together, we have successfully developed a simple, robust and reliable ELISA-based HTS method for screening of novel Wnt inhibitors targeting β-catenin/Lef1 interaction.
引文
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[35]Zhang JH,Chung TDY,Oldenburg KR.A simple statistical parameter for use in evaluation and validation of high throughput screening assays.J Biomol Screen,1999,4(2):67-73.
[2]Su S,Wu W.Regulation of target gene transcription by Wnt/β-catenin signaling.Sci Sin Vitae,2014,44(10):1029-1042(in Chinese).苏尚,吴畏.Wnt/β-catenin信号通路对靶基因转录的调控.中国科学:生命科学,2014,44(10):1029-1042.
[3]Barker N,Clevers H.Mining the Wnt pathway for cancer therapeutics.Nat Rev Drug Discov,2006,5(12):997-1014.
[4]Clevers H,Nusse R.Wnt/β-catenin signaling and disease.Cell,2012,149(6):1192-1205.
[5]Nguyen DX,Chiang AC,Zhang XHF,et al.WNT/TCFsignaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis.Cell,2009,138(1):51-62.
[6]Chen CY,Jan YH,Juan YH,et al.Fucosyltransferase 8as a functional regulator of nonsmall cell lung cancer.Proc Natl Acad Sci USA,2013,110(2):630-635.
[7]Iqbal W,Alkarim S,AlHejin A,et al.Targeting signal transduction pathways of cancer stem cells for therapeutic opportunities of metastasis.Oncotarget,2016,7(46):76337-76353.
[8]Steeg PS.Targeting metastasis.Nat Rev Cancer,2016,16(4):201-218.
[9]Metzeler KH,Heilmeier B,Edmaier KE,et al.High expression of lymphoid enhancer-binding factor-1(LEF1)is a novel favorable prognostic factor in cytogenetically normal acute myeloid leukemia.Blood,2012,120(10):2118-2126.
[10]Yeung J,Esposito MT,Gandillet A,et al.β-Catenin mediates the establishment and drug resistance of MLLleukemic stem cells.Cancer Cell,2010,18(6):606-618.
[11]Petropoulos K,Arseni N,Schessl C,et al.A novel role for Lef-1,a central transcription mediator of Wnt signaling,in leukemogenesis.J Exp Med,2008,205(3):515-522.
[12]Gandhirajan RK,Poll-Wolbeck SJ,Gehrke I,et al.Wnt/β-catenin/LEF-1 signaling in chronic lymphocytic leukemia(CLL):a target for current and potential therapeutic options.Curr Cancer Drug Targets,2010,10(7):716-727.
[13]Gandhirajan RK,Staib PA,Minke K,et al.Small molecule inhibitors of Wnt/β-catenin/Lef-1 signaling induces apoptosis in chronic lymphocytic leukemia cells in vitro and in vivo.Neoplasia,2010,12(4):326-335.
[14]Chen YY,Liu G,Zhang J,et al.Bacterial expression,purification and biological activity evaluation of Plk1PBD protein.J Chongqing Med Univ,2018,43(11):1453-1457(in Chinese).陈云雨,刘刚,张晶,等.Plk1 PBD蛋白原核表达、分离纯化与活性鉴定.重庆医科大学学报,2018,43(11):1453-1457.
[15]Behrens J,von Kries JP,Kühl M,et al.Functional interaction ofβ-catenin with the transcription factor LEF-1.Nature,1996,382(6592):638-642.
[16]von Kries JP,Winbeck G,Asbrand C,et al.Hot spots inβ-catenin for interactions with LEF-1,conductin and APC.Nat Struct Biol,2000,7(9):800-807.
[17]Gong SQ,Han XW,Li XH,et al.Development of a high-throughput screening strategy for upregulators of the OPG/RANKL ratio with the potential for antiosteoporosis effects.J Biomol Screen,2016,21(7):738-748.
[18]Chen YY,Miao DD,Zhang YM,et al.Development of a novel fluorescence polarization-based high-throughput screening method for PLK1 PBD inhibitors.Chin Med Biotechnol,2017,12(5):385-390(in Chinese).陈云雨,缪冬冬,张叶明,等.靶向PLK1 PBD小分子抑制剂荧光偏振高通量筛选模型的建立.中国医药生物技术,2017,12(5):385-390.
[19]Lepourcelet M,Chen YNP,France DS,et al.Small-molecule antagonists of the oncogenic Tcf/β-catenin protein complex.Cancer Cell,2004,5(1):91-102.
[20]Fang L,Zhu QH,Neuenschwander M,et al.ASmall-molecule antagonist of theβ-catenin/TCF4interaction blocks the self-renewal of cancer stem cells and suppresses tumorigenesis.Cancer Res,2016,76(4):891-901.
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[23]Wei W,Chua MS,Grepper S,et al.Small molecule antagonists of Tcf4/β-catenin complex inhibit the growth of HCC cells in vitro and in vivo.Int J Cancer,2010,126(10):2426-2436.
[24]Kim JY,Lee HY,Park KK,et al.CWP232228 targets liver cancer stem cells through Wnt/β-catenin signaling:a novel therapeutic approach for liver cancer treatment.Oncotarget,2016,7(15):20395-20409.
[25]Sukhdeo K,Mani M,Zhang YY,et al.Targeting theβ-catenin/TCF transcriptional complex in the treatment of multiple myeloma.Proc Natl Acad Sci USA,2007,104(18):7516-7521.
[26]Hsieh TH,Hsu CY,Tsai CF,et al.A novel cell-penetrating peptide suppresses breast tumorigenesis by inhibitingβ-catenin/LEF-1 signaling.Sci Rep,2016,6:19156.
[27]Ivanov AA,Khuri FR,Fu HA.Targeting protein-protein interactions as an anticancer strategy.Trends Pharmacol Sci,2013,34(7):393-400.
[28]Baeshen MN,Al-Hejin AM,Bora RS,et al.Production of biopharmaceuticals in E.coli:current scenario and future perspectives.J Microbiol Biotechnol,2015,25(7):953-962.
[29]Li YJ,Chen YY,Bi LJ.Fusion tags technology and their applications.Chin J Biotech,2006,22(4):523-527(in Chinese).李永进,陈媛媛,毕利军.融合标签技术及其应用.生物工程学报,2006,22(4):523-527.
[30]Yin HL,Yuan L.Prokaryotic expression,purification,and identification of GST-β-catenin-His double labeled fusion protein.J Grad Univ Chin Acad Sci,2012,29(6):847-852(in Chinese).尹会龙,袁莉.GST-β-catenin-His双标签融合蛋白的原核表达、纯化及鉴定.中国科学院研究生院学报,2012,29(6):847-852.
[31]Chai ZB,Zhang GL,Han JX.Application of GST-pull down technique in study on protein-protein interaction.Chin J Biol,2014,27(10):1354-1358(in Chinese).柴政斌,张更林,韩金祥.GST-pull down技术在蛋白质相互作用中的应用.中国生物制品学杂志,2014,27(10):1354-1358.
[32]Wang L,Bao Y,Yang Y,et al.Discovery of antagonists for human scavenger receptor CD36 via an ELISA-like high-throughput screening assay.J Biomol Screen,2010,15(3):239-250.
[33]Zhang XL,Nie YZ,Lan XG,et al.Preparation of anti-HBsAg epitope monoclonal antibodies and establishment of a sandwich ELISA for HBV serotype detection.Chin J Cell Mol Immunol,2014,30(2):171-175(in Chinese).张晓磊,聂玉哲,蓝兴国,等.抗HBsAg表位单克隆抗体的制备及HBV血清型夹心ELISA的建立.细胞与分子免疫学杂志,2014,30(2):171-175.
[34]Siarot L,Chutiwitoonchai N,Sato H,et al.Identification of human immunodeficiency virus type-1 Gag-TSG101interaction inhibitors by high-throughput screening.Biochem Biophys Res Commun,2018,503(4):2970-2976.
[35]Zhang JH,Chung TDY,Oldenburg KR.A simple statistical parameter for use in evaluation and validation of high throughput screening assays.J Biomol Screen,1999,4(2):67-73.