CN112831542B - 一种基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法 - Google Patents
一种基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法 Download PDFInfo
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Abstract
本发明公开了一种基于MALDI‑TOF‑MS体外筛选CDKs家族蛋白激酶抑制剂的方法，包含以下步骤：首先制备CDKs体外磷酸激酶实验缓冲液(KRB)的母液；然后加入待测药物样品进行体外磷酸激酶反应制备质谱分析样品；最后通过MALDI‑TOF‑MS检测来比较样品中磷酸化的短肽和未磷酸化的短肽离子峰的峰面积大小来判断待测药物抑制CDKs的体外活性。本发明所提供的方法是利用药物样品与细胞提取的蛋白激酶在ATP的帮助下进行体外磷酸化对应的底物短肽，然后利用质谱检测出磷酸化的短肽和未磷酸化的短肽，两者的峰面积比反映了抑制CDKs的活性大小。发明所提供的方法采用体外实验和MALDI‑TOF‑MS检测磷酸化结果，样品需求少，药物用量少，不用进行复杂的细胞实验，还可进行大规模的样品筛选。
Description
技术领域
本发明属于氨基代谢物同分异构体鉴定领域，具体涉及一种基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法。
背景技术
细胞周期蛋白依赖性激酶(cyclin-dependent kinases，CDKs)由于受周期蛋白cyclin的激活而得名，是一组丝氨酸/苏氨酸蛋白激酶。智人基因组的完整序列表明，在30000个预测基因中，有13个CDK和25个Cyclins。11个CDKS及其相关的Cyclins在人类中已被鉴定。
在众多的激酶中，CDKs由于其作用的复杂性，参与了许多重要的细胞过程。它们调节细胞分裂、凋亡、转录和分化，这涉及许多疾病，如人类的各类癌症，艾滋病等。CDKs的不同亚型在细胞分裂、凋亡、转录和分化的过程中所起的作用不尽相同，根据其在细胞周期进展和转录调控中的作用大概分为两组，第一组包括CDK亚型1、2、4、6等，主要参与细胞周期，第二组包括CDK亚型7、8、9等，与转录控制相关1。
CDKs中CDK4/6与Cyclin D结合形成CDK4/6-Cyclin D复合物，是细胞周期激活的重要组成部分，并介导细胞从G1期过渡到S期，在S期细胞生长并合成蛋白质，为合成DNA做准备2。在转录性CDKs中，CDK9引起了许多群体的关注。CDK9与Cyclin T或Cyclin K形成异源二聚体复合物，使RNA聚合酶II(RNAPII)的羧基末端结构域(CTD)磷酸化，从而控制转录进程3。CDK9和Cyclin T1组成正转录延伸因子b(P-TEFb)，它能刺激大多数蛋白编码基因的转录延伸4。
在癌症的治疗中，由于CDKs在控制细胞分裂中的重要作用，许多针对CDKs的药物被设计并在进行临床试验。近年来有几种CDK抑制剂已经通过了FDA的批准，他们基本都是选择性CDK4/6抑制剂。美国FDA于2015年2月批准了首个用于治疗HR+/her2乳腺癌的CDK抑制剂，辉瑞公司的palbociclib5。诺华的ribociclib和礼来的abemaciclib也分别于2017年3月和9月获得FDA的快速批准，用于治疗HR+/her2乳腺癌6,7。而据报道，作为CDK4/6选择性抑制剂的palbociclib和ribociclib都需要与来曲唑联合治疗乳腺癌8,9。Abemaciclib具有额外的激酶活性，如对CDK1、CDK2和CDK9的抑制活性10。
在各种癌症中都可以观察到CDK-Cyclin通路调节失调，导致无节制增殖11，但目前只有基于抑制CDK4/6的治疗乳腺癌药物获批上市，这既为用CDKs作为靶点治疗各种癌症提供了希望和动力，同时也说明寻找一个高通量的CDK抑制剂筛选方法具有重要的意义。MALDI-TOF-MS(基质辅助激光解吸电离飞行时间质谱)的主要组成为基质辅助激光解吸电离离子源(MALDI)和飞行时间质量分析器(TOF)，具有速度快、准确度高、分辨率高及灵敏度高等特点。相比其他质谱，MALDI-TOF-MS的简单、快速、准确和低成本是其最大的优势，我们将提供一种基于MALDI-TOF-MS体外筛选CDK家族的ATP竞争性抑制剂的方法。
参考文献
1.Asghar U,Witkiewicz AK,Turner NC,et al.The history and future oftargeting cyclin-dependent kinases in cancer therapy.Nat Rev Drug Discov2015；14:130–46.
2.Harbour JW,Luo RX,Dei Santi A,et al.Cdk phosphorylation triggerssequential intramolecular interactions that progressively block Rb functionsas cells move through G1.Cell 1999；98:859–69.
3.Romano G.Deregulations in the cyclin-dependent kinase-9-relatedpathway in cancer:implications for drug discovery and development.ISRN Oncol2013；2013:1–14.
4.Morales F,Giordano A.Overview of CDK9 as a target in cancerresearch.Cell Cycle 2016；15:519–27.
5.DiPippo AJ,Patel NK,Barnett CM.Cyclin-dependent kinase inhibitorsfor the treatment of breast cancer:past,present,and future.Pharmacotherapy2016；36:652–67.
6.Shah A,Bloomquist E,Tang S,et al.FDA approval:ribociclib for thetreatment of postmenopausal women with hormone receptor-positive,HER2-negative advanced or metastatic breast cancer.Clin Cancer Res 2018；24:2999–3004.
7.FDA OKs abemaciclib for ER+,HER2-breast cancer.Cancer Discov 2017；7:OF1.
8.Im SA,Mukai H,Park IH,et al.Palbociclib plus letrozole as first-line therapy in postmenopausal Asian women with metastatic breast cancer:results from the phase III,randomized PALOMA-2study.J Glob Oncol 2019；5:1–19.
9.Meattini I,Desideri I,Scotti V,et al.Ribociclib plus letrozole andconcomitant palliative radiotherapy for metastatic breast cancer.Breast 2018；42:1–2.
10.Hafner M,Mills CE,Subramanian K,et al.Multiomics profilingestablishes the polypharmacology of FDA-approved CDK4/6inhibitors and thepotential for differential clinical activity.Cell Chem Biol 2019；26:1067–80.
11.Sarosiek T.Inhibitors of cyclin-dependent kinases(CDK)–a new groupof medicines in therapy of advanced breast cancer.Polski Merkuriusz Lekarski:organ Polskiego Towarzystwa Lekarskiego 2018；44:5–9.
发明内容
针对上述现有技术存在的不足之处，本发明目的在于提供一种基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法。为了达成上述目的，本发明的解决方案是：
一种基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法，其特征在于，具体步骤如下：
(1)制备CDKs体外磷酸激酶实验缓冲液(KRB)的母液：所述母液由Hepes(pH 7.6)溶液，KCl溶液，MgCl2溶液，DTT溶液，β-glycerol phosphate溶液和Na3VO4溶液组成；
(2)进行体外磷酸激酶反应：将待测药物样品与步骤(1)中所制备的CDKs体外磷酸激酶实验缓冲液(KRB)的母液，2R底物肽段，CDKs家族蛋白激酶和ATP一起进行水浴孵育，终止反应后将所得产物除盐后即可制备得到质谱分析样品；
(3)MALDI-TOF-MS检测：将步骤(2)中制得的样品与基质混合后点样，等完全干燥后即可进行MALDI-TOF-MS测试，MALDI-TOF-MS模式选择适合生物大分子(蛋白质、核酸、多聚物)的线性模式和正电荷模式，分子量扫描范围设为500～3000kDa，激光器能量为12-36％，通过比较样品中磷酸化的短肽和未磷酸化的短肽离子峰的峰面积来即可判断待测药物抑制CDKs的体外活性大小。
优选地，步骤(2)中所述的水浴孵育条件为：水浴温度30℃，水浴时间10-60min。
优选地，步骤(3)中所述的基质选自SA,DHB或HCCA中的一种。
优选地，步骤(2)中所述的CDKs家族蛋白激酶包括CDK9,DYRK1A，CDK1,CDK4,CDK6及CDK7等蛋白激酶。
本发明的具体原理如下：
本发明所提供的基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法是利用药物样品与细胞提取的蛋白激酶在ATP的帮助下进行体外磷酸化对应的底物短肽，然后再用质谱检测出磷酸化的短肽和未磷酸化的短肽，两者的峰面积之比反映了CDKs的活性大小。具体说来，本发明所提供的方法需要先寻找CDKs对应的底物肽，以及反应对应的体外磷酸激酶实验缓冲液，确保在ATP的帮助下，CDKs可以磷酸化该底物肽，且已发现的CDKs抑制剂可以抑制该磷酸化过程，再利用该方法大量筛选CDKs抑制剂。具体的例子在实施例部分会再详细说明。
本发明的优点：
(1)本发明所提供的基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法是一种可以自动化及高通量检测的MALDI-TOF-MS方法，可以快速地获得每组实验中CDKs的活性，从而快速判断出药物对CDKs的抑制作用。
(2)本发明所提供的基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法不用进行复杂的细胞实验进行激酶活性的评估，终止反应后样品可以长期保存，因此可以用于大规模样品的快速筛选。
(3)本发明所提供的基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法还具有操作简单、精确度高及重复性好的优点。
附图说明
图1为加药前后CDK9磷酸化底物肽PSYSPTSPSYSPT的作用模式图。
图2为P-TEFb的银染定量结果图。
图3为阳性药FLP以不同浓度作用于CDK9的峰面积变化图。
图4为1-8号药物筛选结果图。
具体实施方式
下面进一步结合实施例以详细说明本发明。同样应理解，以下实施例只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限制，示例中具体的质量、反应时间和温度、工艺参数等也仅是合适范围中的一个示例，本领域的技术人员根据本发明的上述内容做出的一些非本质的改进和调整均属于本发明的保护范围。实施例中未注明具体技术或条件者，均为按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市场购买的常规产品。
本实用所使用的蛋白激酶CDKs和Cyclins复合体为商业化试剂，也可通过以下方法制备：
实验选用表达蛋白量高的293T贴壁细胞进行蛋白表达。
1.将100mm的培养皿中的细胞转移至150mm培养皿中用DMEM培养基(有血清无抗性)培养1天至细胞密度达80％-90％；
2.每盘培养皿需：(1)将10-15μg PRK5M-CDKs-flag和10-15μg对应的PRK5M-Cyclins-HA稀释至750μL的DMEM培养基(无血清无抗性)；(2)将20μL 2*PEI稀释到750μL的DMEM培养基(无血清无抗性)；
3.混合含质粒和含PEI的DMEM培养基(无血清无抗性)，室温孵育25min，形成DNA-PEI复合物；每盘细胞加入1.5mL复合物溶液，轻微晃动使其均匀分布；
4.培养24小时后，将每盘培养皿的培养基换成20mL新鲜的DMEM培养基(有血清有抗性)，再培养24小时后收细胞。
5.将细胞用细胞刮刮下来，收集到离心管中，400g，4℃离心5min，再用1*PBS洗两次，每次400g，4℃离心5min；
6.每个150mm培养皿加入1mL全细胞裂解液(50mM Hepes-KOH(pH7.9)，350mMNaCl,5mM EDTA)，再加入1mM DTT，0.5mM PMSF，之后用Vortex将细胞震散裂解细胞；
7.用接触式探针超声，30％功率，超声10s，停10s，重复12次，之后14000g，4℃，离心20min。收集管内液体至新的离心管，将flag-beads加入离心管，4℃翻转孵育过夜；
8.将过夜溶液用buffer D0.3(20mM Hepes-KOH(pH7.9)，300mM KCl,0.2mM EDTA,10％甘油)洗3次，3000g，4℃，再用buffer D0.1洗2次，3000g，4℃，离心0.5min，降低盐浓度，在此过程中将beads转移到1.5mL EP管中，最后一次用buffer D0.1(20mM Hepes-KOH(pH7.9)，100mM KCl,0.2mM EDTA,10％甘油)洗过后，要用扁枪头吸走所有液体，此时beads应为白色。
9.加入适量的1*Flag-peptide洗脱液，室温翻转洗脱30min；用针管在1.5mL EP管管盖和管底扎孔，放在一个新的1.5mL EP管之上，用掌上离心机离心1分钟，让洗脱后的液体充分流入新的1.5mL EP管中(洗脱后的液体的成分即为CDKs和Cyclins复合体)，取部分液体做银染实验测定复合体含量，其余-80℃保存。
CDKs蛋白激酶体外实验方法
配CDKs体外磷酸激酶实验缓冲液(KRB)的母液5*KRB，含250mM Hepes(pH 7.6)，170mM KCl，35mM MgCl2，12.5mM DTT，25mMβ-glycerol phosphate，2.5mM Na3VO4。
1.体外磷酸激酶实验(10μL体系)
30℃水浴孵育30min，加1μL 10％TFA终止反应；
2.除盐
(1)将整块C8膜用针孔扎出一小团C8膜，放入50μL进口枪头中，压实在枪头口，加入适量C18填料(很少即可)；
(2)用约40μLACN活化C18填料，填料充分润湿，装填紧实即可，可通过控制离心力控制离心速率，转速大约为0.3g，1min；
(3)用约80μL 0.1％TFA平衡C18填料，将ACN替换掉，转速大约为0.5g，2min/次；
(4)将样品转移至stage-tip中，转速大约为0.1g，5min；
(5)用约80μL 0.1％TFA冲洗C18填料，除盐，转速大约为0.5g，2min/次；用15μLACN将肽段洗脱下来。
3.MALDI-TOF-MS检测分析
各取1μL样品和1μL基质(SA,DHB,HCCA)混合，点样，等完全干燥后测试，进行质谱测试判断肽段是否磷酸化。MALDI-TOF-MS模式选择适合生物大分子(蛋白质、核酸、多聚物)的线性模式和正电荷模式，分子量扫描范围设为500～3000kDa，若检测信号较弱，可适当提高检测器增益(控制在2倍以内)，激光器能量为12-36％。
4.结果处理
通过质谱处理软件可以直接获得MALDI-TOF-MS所得的质谱图中各个峰的峰面积，将所有磷酸化底物肽峰的质谱峰强度值求和，包括加氢峰、加钠离子峰、加钾离子，二钠峰和二钾峰等加和峰，除以对应的所有未磷酸化底物肽峰的峰面积和，可以得到一个相对的数值。以未加药处理的样品的数值为1，可以推算出各个加药组两类峰面积之比的变化百分比，以面积比减少为正值，值越大则药物抑制效果越强。同时，根据不同药物浓度处理条件下磷酸化肽与未磷酸化肽的比值进行拟合作图，从而计算药物的EC50.
实施例1
以CDK9为例：
图1为加药前后CDK9磷酸化底物肽PSYSPTSPSYSPT的作用模式图，结果表明：实验的核心在于利用细胞提取的蛋白激酶在ATP的帮助下体外磷酸化对应的短肽(2Rpeptide,PSYSPTSPSYSPT)，可以用质谱检测出磷酸化的短肽和未磷酸化的短肽，两者的峰面积比反映了CDK9的活性大小。利用MALDI-TOF-MS可以自动化和高通量检测的特点，可以快速的获得每组实验中CDK9的活性，从而快速判断出待筛选的药物对CDK9的抑制作用。本实验选择CDK9抑制剂flavopiridol(FLP)做阳性药物进行研究，FLP结构如下：
实验分为两步，第一步利用质粒PRK5M-CDK9-flag和PRK5M-Cyclin T1-HA制备蛋白激酶CDKs和Cyclins的复合体P-TEFb，具体步骤如上所述。图2为获取的P-TEFb的银染结果图，结果表明：提取物中含有CDK9-flag和Cyclin T1-HA，且对蛋白进行了定量，含量约为50ng/μL。
第二步为CDK9蛋白激酶体外激酶实验：
30℃水浴孵育30min，加1μL 10％TFA终止反应；
再经过除盐，MALDI-TOF-MS检测及结果处理，所得结果阳性药FLP以不同浓度作用于CDK9的峰面积变化如图3所示：1374.386Da处是未磷酸化的峰，1454.446Da处是磷酸化的峰，当阳性药FLP以不同浓度作用于CDK9时，两个峰面积的比随之变化，反映了不同药物浓度下CDK9的活性的变化，由于320nM时磷酸化的峰峰面积过小，无法获取，去掉前9组数据算得FLP的EC50为89.4nM。
实施例2
使用实例1所得的CDK9蛋白激酶，检测了重楼皂苷Ⅱ(药物1)，吴茱萸碱(药物2)，去氢吴茱萸碱(药物3)，薯蓣皂苷(药物4)，吴茱萸次碱(药物5)，Aloperine(药物6)，Chelerythrine chloride(药物7)，Cytisine(药物8)抑制CDK9蛋白激酶的活性，具体实验步骤与实施1相似：
Aloperine(药物6)，Chelerythrine chloride(药物7)和Cytisine(药物8)的化学结构如下面所示：
CDK9蛋白激酶体外激酶实验：
30℃水浴孵育30min，加1μL 10％TFA终止反应。
(除8个药物处理组还包括加入1μLDMSO的空白对照组和工作浓度为160nM FLP的阳性对照组)；
处理数据后，图4为使用本发明所提供的方法筛选这些天然产物的结果：相对于空白对照组，阳性对照组抑制效果高达89％，1-8号药物分子均不具备抑制效果或抑制效果极其微弱。
Claims (3)
1.一种基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法，其特征在于，具体步骤如下：
(1)制备CDKs体外磷酸激酶实验缓冲液(KRB)的母液：所述母液由pH值＝7.6的Hepes溶液，KCl溶液，MgCl2溶液，DTT溶液，β-glycerol phosphate溶液和Na3VO4溶液组成；
(2)进行体外磷酸激酶反应：将待测药物样品与步骤(1)中所制备的CDKs体外磷酸激酶实验缓冲液(KRB)的母液，2R底物肽段，CDKs家族蛋白激酶和ATP一起进行水浴孵育，终止反应后将所得产物除盐后即可制备得到质谱分析样品；其中，所述的CDKs家族蛋白激酶为CDK9；所述2R底物肽段为PSYSPTSPSYSPT；
(3)MALDI-TOF-MS检测：将步骤(2)中制得的样品与基质混合后点样，等完全干燥后即可进行MALDI-TOF-MS测试，MALDI-TOF-MS模式选择适合生物大分子的线性模式和正电荷模式，分子量扫描范围设为500～3000kDa，激光器能量为12-36％，通过比较样品中磷酸化的短肽和未磷酸化的短肽离子峰的峰面积来即可判断待测药物抑制CDKs的体外活性大小。
2.根据权利要求1所述的基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法，其特征在于，步骤(2)中所述的水浴孵育条件为：水浴温度30℃，水浴时间10-60min。
3.根据权利要求1所述的基于MALDI-TOF-MS体外筛选CDKs家族蛋白激酶抑制剂的方法，其特征在于，步骤(3)中所述的基质选自SA,DHB或HCCA中的一种。
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