CN113262230B - 一种靶向猪链球菌丝氨酸、苏氨酸蛋白激酶抑制剂及应用 - Google Patents
一种靶向猪链球菌丝氨酸、苏氨酸蛋白激酶抑制剂及应用 Download PDFInfo
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Abstract
本发明属于药物靶标筛选技术领域,具体涉及一种靶向猪链球菌丝氨酸、苏氨酸蛋白激酶抑制剂及应用。基于猪链球菌stk突变体在CDM培养基中生长缺陷的表型和化学发光技术,根据小分子处理时,猪链球菌SC19在CDM培养基中生长状况初筛出可能的STK抑制剂,进一步通过自磷酸化酶活反应筛选出靶向STK的猪链球菌抑制剂。利用本发明的对药库的筛选鉴定出2种化合物即抑制剂APY29和AT9283,对猪链球菌丝苏氨酸蛋白激酶STK以及猪链球菌均具有抑制作用。
Description
技术领域
本发明属于药物靶标筛选技术领域,具体涉及一种靶向猪链球菌丝氨酸、苏氨酸蛋白激酶(STK)抑制剂及应用。
背景技术
在革兰氏阳性菌厚壁门的细菌中有一种真核样的丝苏氨酸蛋白激酶STK,其蛋白的二级结构相对比较保守,是由胞外的3或4个串联PASTA结构域与胞内的激酶结构域组成,一般可以被称为PASTA激酶(Pensinger DA,Schaenzer AJ,Sauer JD.Do Shoot theMessenger: PASTA Kinases as Virulence Determinants and AntibioticTargets.Trends Microbiol. 2018Jan;26(1):56-69.doi:10.1016/j.tim.2017.06.010.Epub 2017Jul 19.PMID: 28734616;PMCID:PMC5741517.)。机制上,STK是通过自磷酸化作用而激活后磷酸化底物来实现其功能的(Zheng W,Cai X,Li S,LiZ.Autophosphorylation Mechanism of the Ser/Thr Kinase Stk1 FromStaphylococcus aureus.Front Microbiol.2018Apr 20;9:758.doi: 10.3389/fmicb.2018.00758.PMID:29731745;PMCID:PMC5920020.)。STK是一个全局性的因子,其参与调控细胞生长、分裂,细胞形态、碳代谢,致病性等过程。例如:与野生型猪链球菌SC19菌株相比,stk缺失株对小鼠的致病力显著减弱,小鼠定植试验中,各组织器官的载菌量显著较少(Zhang C,Sun W,Tan M,Dong M,Liu W,Gao T,Li L,Xu Z,Zhou R. The Eukaryote-Like Serine/Threonine Kinase STK Regulates the Growth and Metabolism ofZoonotic Streptococcus suis.Front Cell Infect Microbiol.2017Mar 7;7:66.)。 STK作为药物靶标也已被广泛研究,例如单核李斯特杆菌和结合分枝杆菌的STK抑制剂筛选(Schaenzer AJ,Wlodarchak N,Drewry DH,Zuercher WJ,Rose WE,Striker R,Sauer JD.A screen for kinase inhibitors identifies antimicrobial imidazopyridineaminofurazans as specific inhibitors of the Listeria monocytogenes PASTAkinase PrkA.J Biol Chem.2017Oct 13;292(41):17037-17045Mori M,Sammartino JC,Costantino L,Gelain A,Meneghetti F,Villa S,Chiarelli LR;An Overview on thePotential Antimycobacterial Agents Targeting Serine/Threonine Protein Kinasesfrom Mycobacterium tuberculosis.Curr Top Med Chem.2019;19(9):646-661.)。综上所述,STK 是一个潜在的抗革兰氏阳性细菌的药物靶标,关于细菌STK的抑制剂筛选已有所研究,但尚未发现有细菌的STK抑制剂作为药物进入临床应用,所以靶向STK的药物筛选仍然值得研究。
现有的STK抑制剂的筛选多利用STK蛋白的激酶活性测定来实现,酶活反应中的三个基本组分别为STK激酶、ATP、磷酸化的底物。STK激酶活性测定的核心问题是测量激酶将多少含量的ATP的γ-P转移至底物或者其自身磷酸化位点上。
关于激酶活性测定方法目前主要有以下几种:
放射性同位素法:使用放射性同位素标记的ATP作底物,反应一段时间后通过放射自显影检测激酶本身或者底物条带的灰度判断激酶的活性。
酶联免疫吸附法:用发生反应后的激酶体系包被至固相载体上,然后用专一性抗磷酸化底物的抗体孵育,再用酶标二抗孵育,显色,测量光吸收值,然后根据结果判断激酶的活性 (Bhagwat SV,Kahler J,Yao Y,Maresca P,Brooks M,Crew A,Boisclair M,Pachter JA. High-throughput screening for mTORC1/mTORC2 kinase inhibitorsusing a chemiluminescence-based ELISA assay.Assay Drug Dev Technol.2009Oct;7(5):471-8.)。
免疫印迹法:将反应后的激酶体系制样,然后进行SDS-PAGE电泳,将电泳结束后的SDS-PAGE转至硝酸纤维素膜,膜经BSA封闭后,用特异性的磷酸化抗体孵育,再用酶标二抗孵育,放射自显影后,通过条带的灰度,判断激酶的活性(Hirano S.Western blotanalysis. Methods Mol Biol.2012;926:87-97.)。
荧光共振能量转移:在多肽的底物的C和N分别连上荧光共振能量转移的供体与配体,当多肽底物被激酶磷酸化后,其不能被蛋白水解酶水解,荧光共振能量转移现象依然存在,若多肽底物未被磷酸化,则其容易被蛋白酶水解,荧光共振能量转移现象消失,最终,根据激发光和发射光的强度来判断激酶的活性(Rodems SM,Hamman BD,Lin C,Zhao J,ShahS, Heidary D,Makings L,Stack JH,Pollok BA.A FRET-based assay platform forultra-high density drug screening of protein kinases and phosphatases.AssayDrug Dev Technol. 2002Nov;1(1Pt 1):9-19.)。
荧光素酶间接测量法:基于荧光素酶测量激酶反应体系中剩余ATP含量的分析方法.在一个反应体系中,激酶会消耗一部分ATP,荧光素酶会利用体系中的剩余ATP来催化荧光素发光,发光值可以通过酶标仪检测,所以激酶的活性与荧光素发光值成反比(Baki A,Bielik A, Molnár L,Szendrei G,KeserüGM.A high throughput luminescent assayfor glycogen synthase kinase-3beta inhibitors.Assay Drug Dev Technol.2007Feb;5(1):75-83.)。
在蛋白水平上进行药物筛选的弊端就是抑制剂往往只在体外抑制STK的激酶活性,对细菌并无抑制作用。
发明内容
本发明的目的在于提供一种两轮的,靶向猪链球菌丝氨酸、苏氨酸蛋白激酶(STK)抑制剂的高通量筛选方法和该方法所得的作为药物靶标的抑制剂。
本发明还在于对上述高通量筛选方法和作为药物靶标的抑制剂的应用,本发明利用所述方法筛选出抑制猪链球菌STK激酶活性,同时也能抑制猪链球菌的抑制剂。
本发明基于在人工CDM培养基(van de Rijn I,Kessler RE.Growthcharacteristics of group A streptococci in a new chemically definedmedium.Infect Immun.1980 Feb;27(2):444-8.)中猪链球菌stk突变体(由SC19菌株缺失stk基因获得)生长缺陷的表型和化学发光技术,通过测量加有药物的野生型猪链球菌SC19(申请人的农业微生物学国家重点实验室于2005年临床分离,参见Li W,Liu L,Qiu D,etal.Identification of Streptococcus suis serotype 2genes preferentiallyexpressed in the natural host.[J]. International Journal of MedicalMicrobiology,2010,300(7):482-488.)在CDM培养基中生长8h后的OD600nm值,初始筛选出可能靶向STK的猪链球菌抑制剂,针对初始筛选出的抑制剂,通过化学发光技术,测量STK自磷酸化反应体系中剩余ATP的含量来反应猪链球菌STK自磷酸化活性。经过两轮筛选,筛选出对STK和猪链球菌均有抑制作用的抑制剂。在后续的筛选过程中,具有高通量筛选的优势,对1240种小分子化合物进行筛选,初筛出2种对STK与猪链球菌均有抑制作用的抑制剂。
具体的本发明的技术方案如下所述:
一种基于在CDM培养基中猪链球菌stk突变体生长缺陷表型和化学发光技术的靶向丝氨酸、苏氨酸蛋白激酶STK的抑制剂的高通量筛选方法,所述的方法如下所述:
本发明筛选出了两种小分子抑制剂APY29和AT9283,其化学结构式如下所示:
结构式APY29所示的化合物为N2-1H-苯并咪唑-6-基-N4-(5-环丙基-1H-吡唑-3-基)-2,4- 嘧啶二胺,分子式为C17H16N8;分子量为332.36;
结构式AT9283所示的化合物为1-环丙基-3-(3-(5-(吗啉甲基)-1H-苯并[d]咪唑-2- 基)-1H-吡唑-4-基)脲,分子式为C19H23N7O2;分子量为381.432。
本发明的筛选方法为:
(1)在透明的96孔板中加入200μLCDM培养基稀释的初始OD600为0.01的SC19菌液,用多通道移液器加1μLDMSO或DMSO溶解的母液浓度为10mM小分子化合物,设置不加药物的SC19阳性与不加药物的Δstk阴性对照各4个重复,放入37℃温箱静置培养8小时后,用酶标仪测量最终的 OD600nm值;
(2)采用如下公式计算药物生长的抑制率:
药物生长抑制率=(阳性组OD600nm值平均值-待测药物组OD600nm值)/(阳性组OD600nm 值平均值–阴性组OD600nm值平均值)×100%;
(3)在黑色的平型底96孔板中加入44μL包含终浓度为10mM的MgCl2,10μM STK(1-300),pH 7.0的激酶缓冲液(50mM HEPES,1mM DTT和0.01%Brij35)的混合体系,用多通道移液器加 1μLDMSO溶解的母液浓度为10mM的(1)中筛选出的生长抑制率大于85%的药物,设置阳性对照与阴性对照各3个重复;
(4)将所述96孔板置于4℃孵育30min,然后加5μL 1mM ATP,置于37℃温箱反应30min后取出在室温下冷却5min,加入50μLReagent,反应10min后,用酶标仪测量相对发光值RLU;
(5)采用如下公式计算药物酶活的抑制率:
药物酶活抑制率=(待测药物化学发光值–阳性组化学发光平均值)/(阴性组化学发光平均值–阳性组化学发光平均值)×100%;
(6)按照上述步骤对MedChemExpress公司的激酶抑制剂药库HY-L001进行筛选,得到对STK (1-300)的酶活抑制率为的抑制剂为APY29和AT9283。
上述方法可在猪链球菌真核样丝苏氨酸蛋白激酶STK抑制剂APY29和AT9283筛选中应用。
应用本发明所述方法筛选可以得到猪链球菌丝氨酸、苏氨酸蛋白激酶STK的抑制剂APY29 和AT9283。
附图说明
图1:本发明的原核表达重组质粒pET28a-STK(1-300)构建流程图,
图2:CDM培养基中的SC19与Δstk生长曲线图。附图标记说明:SC19与Δstk在人工培养基CDM 中37℃静置培养的生长曲线,Δstk生长显著慢于SC19。
图3:小分子对SC19的生长抑制率散点图。图3是1240种小分子对SC19在CDM培养基的生长抑制率散点图;
图4:抑制剂APY29与AT9283小分子与抑制剂APY29与AT9283对STK IC50的测量结果。图4中的A 图是抑制剂APY29与AT9283小分子的结构式;图4中的B图是APY29和AT9283对STK酶活IC50的测量,APY29和AT9283抑制STK的IC50分别为4.12μM和9.406μM。
图5:抑制剂APY29与AT9283处理条件下SC19与Δstk生长曲线图。在200μL包含终浓度100μM 的APY29与AT9283的TSB培养基中,SC19与Δstk生长曲线图,在对数生长中后期,与未经处理的SC19和Δstk相比,在APY29作用下SC19和Δstk生长变慢,在AT9283作用下SC19生长变慢,Δstk未见明显变化。
具体实施方式
实施例1靶向真核样丝苏氨酸蛋白激酶STK的猪链球菌抑制剂APY29和AT9283的筛选
1.STK(1-300)重组表达质粒的构建与蛋白纯化
将原核表达载体pET28a经由NdeI与XhoI双酶切处理后,用1%琼脂糖凝胶电泳和纯化回收后,用oligo7软件设计引物(表1),以猪链球菌SC19基因组(NZ_CP020863)为模板,扩增 STK的胞内结构域片段,用同源重组酶连接酶切载体和PCR片段(图1),转化至DH5α感受态大肠杆菌中,若干个小时后,用菌落PCR鉴定阳性克隆,摇菌提取质粒送商业测序公司进行测序。
表1 原核表达重组质粒pET28a-STK(1-300)引物序列
将测序正确的重组质粒转化至BL21(DE3)感受态大肠杆菌中,将转化子细菌接种至LB液体养基上,置37℃,180rpm摇床条件下生长,待菌液生长至OD600为0.6-0.8之间时,加入1mM IPTG, 置于18℃,180rpm摇床中诱导10h,之后用离心机离心去上清,用磷酸盐缓冲液(PBS,常规) 溶液重悬菌体,用商购的高压破碎仪破碎细菌,将破碎后的裂解菌体溶液离心,上清用0.45μm 滤器过滤后,与镍柱充分反应,然后使用商购的蛋白纯化仪用含有咪唑的缓冲液(常规)洗脱镍柱,根据280nm的吸收峰,将收集到的成分进行取样,跑SDS-PAGE蛋白胶,评价蛋白纯度,同时用超滤管将蛋白浓缩。
2.猪链球菌SC19和Δstk在人工培养基CDM中生长曲线测定
使用人工CDM培养基将对数中期的猪链球菌SC19和Δstk菌液OD600nm稀释为0.01,然后分装至无菌的透明平底96孔中,设置猪链球菌SC19和Δstk各40个重复和空白对照8个重复,放置37℃温箱静置生长10h后,用酶标仪测量OD600nm值。SC19与Δstk的生长曲线如图2,Δstk 在CDM培养基中几乎不生长,生长速率显著低于SC19。
3.两轮药物筛选
(1)基于生长表型的药物筛选
对MedChemExpress公司的激酶抑制剂药库HY-L001的1240种药物进行筛选应用验证,具体步骤如下:
1)在透明的96孔板中加入200μLCDM培养基稀释的初始OD600为0.01的SC19菌液,用多通道移液器加1μLDMSO或DMSO溶解的母液浓度为10mM小分子,设置不加药物的SC19阳性与不加药物的Δstk阴性对照各4个重复,放入37℃温箱静置培养8小时后,用酶标仪测量最终的OD600nm 值;
2)采用如下公式计算药物生长的抑制率:
药物生长抑制率=(阳性组OD600nm值平均值-待测药物OD600nm值)/(阳性组OD600nm值平均值–阴性组OD600nm值平均值)×100%。药物的生长抑制率散点图见图3所示,
(2)基于激酶活性的药物筛选
1)在黑色的平型底96孔板中加入44μL包含终浓度为10mM的MgCl2,10μM STK(1-300), pH 7.0的激酶缓冲液(50mM HEPES,1mM DTT和0.01%Brij35)的混合体系,用多通道移液器加1μLDMSO溶解的母液浓度为10mM的(1)中筛选的生长抑制率大于85%的药物,设置阳性对照与阴性对照各3个重复。
2)将所述96孔板置于4℃孵育30min,然后加5μL 1mM ATP,置于37℃温箱反应30min后取出在室温下冷却5min,加入50μL Reagent,反应10min后,用酶标仪测量相对发光值RLU。
3)采用如下公式计算药物酶活的抑制率:
药物酶活抑制率=(待测药物化学发光值–阳性组化学发光平均值)/(阴性组化学发光平均值–阳性组化学发光平均值)×100%。
按照上述步骤从MedChemExpress公司的激酶抑制剂化合物库HY-L001中筛选出获得了抑制剂APY29和AT9283,对STK自磷酸化活性活抑制率分别为98.23%和86.50%。
3.筛选药物IC50的测定
1)对筛选出的抑制剂APY29和AT9283(图4)进行IC50测量,在96孔板黑色不透明板子中加入44μL包含终浓度为10mM的MgCl2,10μM STK(1-300),pH 7.0的激酶缓冲液(50mMHEPES、 1mM DTT和0.01%Brij35)的混合体系,设置一组阳性对照(加STK(1-300))、一组阴性对照(不加STK(1-300))以及药物组。
2)设立对照(不加STK(1-300),加药物),设置一系列浓度梯度抑制剂组,加入1μLDMSO 溶解的药物,置于4℃孵育30min,然后加入5μL1mM ATP.置于37℃反应30min后取出室温冷却 5min,加入50μL Reagent,反应10min后,用酶标仪测量相对发光值RLU。根据计算公式计算的抑制率进行IC50,
3)采用以下计算公式计算酶活的抑制率:
药物酶活抑制率=(待测药物化学发光值–阳性组化学发光平均值)/(阴性组化学发光平均值–阳性组化学发光平均值)×100%。结果见图4所示,APY29和AT9283抑制STK的IC50 分别为4.12μM和9.406μM。
4)生长曲线的测量
将199μL初始OD600为0.01的革兰氏阳性菌猪链球菌(Streptococcus suis)菌液SC19 和Δstk加入到与生长曲线仪配套的100孔板中,加入一系列浓度梯度的1μL DMSO溶解10mM 母液APY29和AT9283,同时设置加入等量DMSO的空白对照与阳性对照,每个组别设置5个重复。置于生长曲线仪中,设置37℃,中等振幅下进行测量,然后对每个时间点的差异性进行 Student’s-t检验统计学分析。结果如图5所述。
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