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娈丰鲁
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1 Cdk5 / p Cdk5 /p35 Cdk5 /p35 Histone H1 ATP ATP ph Z' Cdk5 /p35 ATP 3 μmol L - 1 Cdk5 /p35 30 nmol L - 1 Histone H1 70 μmol L min ph 7 Z' Cdk5 /p35 Cdk5 /p35 Cdk5 /p35 Cdk5 /p35 AD Cdk5 /p35 tau R A Establishment and application of a high-throughput screening assay for Cdk5 / p35 inhibitors YANG Ran-yao 1 FANG Jian-song 1 ZHOU Dan 1 LIU Ai-lin DU Guan-hua Institute of Materia Medica Chinese Academy of Medical Sciences and Peking Union Medical College Beijing China 2 Beijing Key Laboratory of Drug Target and Screening Research Beijing China 3 State Key Laboratory of Natural Active Substance and Function Beijing China Abstract Objective To establish a high-throughput screening assay for Cdk5 /p35 inhibitors. Methods ATP is necessary in the reaction system between Cdk5 / p35 and its substrate Histone H1. Through detecting the residue of ATP in the system based on Kinase-Glo Luminescent Kinase Assay we can easily estimate the activity of Cdk5 /p35. In this experiment the effects of some factors in the enzyme reaction system were observed such as ATP concentration enzyme concentration substrate concentration incubation temperature incubation time and ph then the optical condition of this enzymatic reaction was determined. Also the assay was evaluated using Z' factor. At last the assay was used to screen and discover active compounds. Results The high-throughput screening assay for Cdk5 /p35 inhibitors was optimized with ATP concentration 3 μmol L - 1 enzyme concentration 30 nmol L - 1 substrate concentration 70 μmol L - 1 incubation temperature 37 incubation time 60 min and ph was 7. Z 'factor was which meant the assay could be used for high-throughput screening of Cdk5 /p35 inhibitors. Finally 13 inhibitors out of 222 compounds selected by virtual screening were discovered by high-throughput 国家重大新药创制科技重大专项 ( 2014ZX ) ; 国际合作项目 ( 2011DFR31240) 杨然耀, 男, 硕士研究生, 主要从事新药发现与药学信息学的研究联系电话 : ( 010) , yangranyao@ imm. ac. cn 刘艾林, 女, 博士生导师, 主要从事新药发现与药学信息学的研究联系电话 : ( 010) , liuailin@ imm. ac. cn 杜冠华, 男, 博士生导师, 主要从事药物筛选与新药发现的研究, 联系电话 : ( 010) , dugh@ imm. ac. cn 2 781

2 screening assay. Conclusion This assay has the features of high stability high sensibility and high efficiency which can be used for high-throughput screening of Cdk5 / p35 inhibitors. The establishment of the assay laid the basis for screening of Cdk5 / p35 inhibitors and mechanism research of Alzheimer's disease. Key words Alzheimer's disease Cdk5 /p35 inhibitors hyper-phosphorylation of Tau protein high-throughput screening Alzheimer's disease AD Cdk5 /p35 AD 68 AD AD Kinase-Glo Luminescent Kinase Assay Cdk5 / p35 Promega ATP R -rosco- vitine Sigma 10 16% 23% 8% AD Histone H1 PKTPKKAKKL 68% 1 AD 40 mmol L - 1 Tris-HCl 20 mmol L - 1 MgCl 2 AD FDA 0. 1 μg ml - 1 BSA ph 7 AD NMDA 2 Costar 384 Corning Eppendorf M5 Molecular 460 AD Devices AD Tau AD 1 tau Kinase-glo Cdk5 /p35 Kinase-glo Cdk5 /p35 Histone H1 tau ATP Mg Tau ATP ATP ATP 5 cyclin-dependent ph kinase 5 Cdk5 p35 tau Z'- 6-7 Lee 8 tau 2 ATP Cdk5 /p35 ATP tau Cdk5 /p μmol L nmol L - 1 Cdk5 / Roscovitine tau p35 70 μmol L - 1 Cdk5 /p35 AD Cdk5 / p35 AD Kinase-Glo Reagent h

3 10 min 4 interation time 500 ns ATP ATP ATP 37 1 h Kinase-Glo Reagent ATP 1 10 min interation ATP 3 μmol L Histone H1 ATP 30 nmol L - 1 Cdk5 /p35 time 500 ns Histone H1 3 1 ATP nmol L Cdk5 /p35 μmol L nmol L μmol L - 1 ATP 70 μmol L h Kinase-Glo Reagent 10 min interation time 500 ns Cdk5 /p35 2 μmol L μmol L μmol L Histone H1 ATP 30 nmol L - 1 Cdk5 /p h Kinase-Glo Reagent 10 min interation time 500 ns ~ 45 nmol L ~ nmol L

4 10 ~ ~ ph ph nmol L - 1 Cdk5 /p35 ph Histone H1 ATP 37 1 h Kinase-Glo Reagent 10 min interation 6 time 500 ns ph 8 DMSO 5 DMSO 10 mg ml μg ml - 1 DMSO 500 DMSO 1 /500 DMSO 7 1 /500 DMSO DMSO 1 /500 5 ph ph 7 ph 7 7 Histone H1 ATP 30 nmol L - 1 Cdk5 /p min Kinase-Glo Reagent 7 DMSO 10 min 9 interation time 500 ns 9. 1 Z' Z' Z' > 0. 5 Z' 1 6 Z' = 1-3 SD + SD / M - M SD SD M M ATP ATP + 60 min

5 Z' Cdk5 /p35 Z' R -roscovitine Cdk5 / p35 IC μmol L - 1 R -roscovitine Cdk5 /p35 /% = R - R / R - R 100 R R R mol L - 1 Cdk5 /p Chinese Journal of New Drugs 2014,23(7) IC 50 IC 50 /μmol L - 1 J ± J ± J ± J ± J ± J ± J ± J ± J ± J ± J ± J ± J ± IC 50 R -roscovitine Cdk5 /p35 IC ± μmol L AD tau 11 Tau / Cdk5 /p35 Cdk5 /p35 tau 4 Cdk5 /p35 AD 20 8 R -roscovitine Cdk5 /p35 12 Cdk5 /p35 ATP 10 ph Cdk5 /p μg ml - 1 Cdk5 /p35 > 50% μg ml - 1 ATP 9 IC 50 ADP 13 Kinase-glo ATP Cdk5 /p ATP 13 Cdk5 /p35 50% IC

6 5 ALONSO A ZAIDI T NOVAK M et al. Hyperphosphorylation induces self-assembly of tau into tangles of paired helical fila- ments /straight filaments J eration J ph ph ph 15 ATP 3 μmol L - 1 Cdk5 /p35 30 nmol L - 1 Histone H1 70 μmol L min ph = 7 Z' Cdk5 /p THIES W BLEILER L Alzheimer's disease facts and figures J. Alzheimers Dement AMADORO G CORSETTI V CIOTTI MT et al. Endogenous Abeta causes cell death via early tau hyperphosphorylation J. Neurobiol Aging AVILA J. Tau kinases and phosphatases J. J Cell Mol Med KIMURA T TSUTSUMI K TAOKA M et al. Isomerase Pin1 stimulates dephosphorylation of tau protein at cyclin-dependent kinase Cdk5 -dependent Alzheimer phosphorylation sites J. J Biol Chem Proc Natl Acad Sci USA PATRICK GN ZUKERBERG L NIKOLIC M et al. Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegen-. Nature AUGUSTINACK JC SANDERS JL TSAI LH et al. Colocalization and fluorescence resonance energy transfer between cdk5 and AT8 suggests a close association in pre-neurofibrillary tangles and neurofibrillary tangles J. J Neuropathol Exp Neurol LEE S HALL GF SHEA TB. Potentiation of tau aggregation by cdk5 and GSK3beta J. J Alzheimers Dis KORESAWA M OKABE T. High-throughput screening with quantitation of ATP consumption a universal non-radioisotope homogeneous assay for protein kinase J. Assay Drug Dev Technol MAPELLI M MASSIMILIANO L CROVACE C et al. Mechanism of CDK5 /p25 binding by CDK inhibitors J. J Med Chem IQBAL K ALONSO A GONG C et al. Molecular pathology of Alzheimer neurofibrillary degeneration J. Acta Neurobiol Exp Wars J ZEGZOUTI H ZDANOVSKAIA M HSIAO K et al. ADP-Glo A Bioluminescent and homogeneous ADP monitoring assay for kinases J. Assay Drug Dev Technol Kinase-Glo A J RADZICKA A WOLFENDEN R. A proficient enzyme J. Science / 檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴檴 http / /pudf. alcedu. org 786

1449 1 相对校正因子的相关方法 1.1 内标多控法研究发现, 在中药各成分间存在着一定的比例关系, 只要各成分的量符合该比例关系, 中药的多 [6] 指标质量控制可以依此法

1449 1 相对校正因子的相关方法 1.1 内标多控法研究发现, 在中药各成分间存在着一定的比例关系, 只要各成分的量符合该比例关系, 中药的多 [6] 指标质量控制可以依此法 1448 相对校正因子在中药多指标测定中的应用研究进展 1, 罗祖良 2, 仇峰 1 1,, 韦日伟 2 1,, 覃禹 2, 张坤 1, 覃洁萍 2, 杨美华 1. 北京协和医学院药用植物研究所, 中草药物质基础与资源利用教育部重点实验室, 北京 100193 2. 广