CN117802199A - SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法 - Google Patents
SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法 Download PDFInfo
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Abstract
本发明公开了一种SARS‑CoV‑2主要蛋白酶Mpro的活性荧光高通量筛选方法，本发明利用含有荧光基团与荧光淬灭基团(FRET效应)的多肽为底物，FRET效应可以使底物多肽的荧光效应消失，Mpro切割底物多肽的作用使FRER效应被抑制，荧光强度上升。从而将荧光强度与SARS‑CoV‑2主要蛋白酶Mpro的酶活性关联起来，因此这样的方法可应用于Mpro抑制剂的筛选，或是生物样本中的Mpro活性检测，具有微型化、自动化、快速可靠等特点，也适用于高通量的应用，也可应用于新型Mpro活性检测试剂盒的开发和开展Mpro抑制剂的筛选服务。
Description
技术领域
本发明涉及一种SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法，属于药学技术领域。
背景技术
一种新型冠状病毒的出现，对人类健康和全球卫生系统构成了巨大的威胁。
SARS-CoV-2的快速传播归因于其与人群的密切接触、患者的无症状传播以及呼吸道飞沫和气溶胶的空气传播，COVID-19患者可出现严重低氧血症、病毒性肺炎、急性呼吸窘迫综合征、胃肠道和神经系统症状.
新型冠状病毒强大的传染能力及其引发的严重并发症，迅速引起世界各国和相关组织的重视。
SARS-CoV-2是一种β冠状病毒属的有包膜、正链、单链RNA病毒；与所有冠状病毒一样，SARS-CoV-2在表面含有一个刺突蛋白(S)。S蛋白在病毒复制过程中至关重要，因为它介导病毒进入细胞并与病毒感染的能力高度相关。
S蛋白包含两个功能亚基：S1和S2。S1负责通过其C端受体结合区域(RBD)识别宿主呼吸细胞表面的受体-血管紧张素转化酶2(ACE2)，进入细胞并促进病毒感染。S2包含完成膜融合过程所需的基本元件，主要介导病毒-细胞以及细胞-细胞膜的融合。SARS-CoV-2进入宿主细胞后，病毒会释放和解锁大于30kb的基因组RNA，从而产生两个开放阅读框：ORF1a和ORF1b。这两个ORF的首轮翻译产生PP1A和PP1AB多蛋白，多蛋白随后由半胱氨酸类蛋白酶(PLpro)和3C类蛋白酶(3CLpro或Mpro)切割水解，产生多种非结构蛋白(NSPs)，如RNA依赖性RNA聚合酶(RdRp)和解旋酶(Helicase),Nsps在病毒RNA的转录、合成和复制等方面发挥重要作用。如RDRP可完成负链亚基RNA的转录和合成，生成与不同结构蛋白相关的mRNA以及参与病毒基因组RNA的复制。Helicase在SARS-CoV-2病毒RNA复制过程中的正负链RNA的展开和大量病毒RNA的复制中起关键作用。
针对这五种病毒蛋白靶标，极有希望开发出新型的抗SARS-CoV-2药物，以期克服之前开发的抗SARS-CoV-2药物存在的老药新用，毒副作用大，治疗效果不明显以及对突变型菌株无作用等问题，为新型抗SARS-CoV-2药物的开发指明了方向。
其中SARS-CoV-2主蛋白酶(Mpro)是由306个氨基酸组成的三结构域(结构域I至III)半胱氨酸蛋白酶。结构域I(残基8-101)和结构域II(残基102-184)具有反平行β-桶结构，结构域III(残基201-303)含有5个α-螺旋，排列在一个基本上反平行的球状簇中，通过长环区(残基185-200)与结构域II连接。SARS-CoV-2Mpro在结构域I和II之间的差距中具有Cys-His催化二联体(Cys 145和His 41)，可特异性地识别nsp 4～nsp 16的11个切割位点，诱导额外的冠状病毒nsp的释放。Mpro亲水解剪切释放的nsp 4-nsp 16是病毒基因组复制和转录的载体，负责蛋白质切割和修饰以及翻译后的核酸合成等过程。
因此，SARS-CoV-2的主蛋白酶(Mpro)被认为是COVID 19抗病毒药物开发的极好靶标，因为它对病毒复制至关重要，并且具有不同于人类蛋白酶的切割特异性。
目前针对SARS-CoV-2Mpro已经开发出了包括荧光素报告子、荧光素互补酶、荧光素酶生物传感器和基于FRET的荧光活性检测方法。虽然这些方法都能用检测SARS-CoV-2Mpro的活性并筛选抑制剂，但是，这些方法也存在着许多局限性，比如筛选容易产生假阳性，特异性差，信号背景比和荧光信号响应值以及酶催化效率较低等缺陷。
发明内容
本发明的目的在于，提供一种SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法。该筛选方法效果更准确，且简单易行，成本低廉，以克服现有技术的不足。
本发明的技术方案：SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法，以含有荧光基团与荧光淬灭基团的多肽为底物，将底物与SARS-CoV-2主要蛋白酶Mpro孵育，使荧光强度与SARS-CoV-2主要蛋白酶Mpro关联起来。
前述的SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法中，以含有5-(2-氨基乙氨基)-1-萘磺酸荧光基团与4-[4-(二甲基氨基)苯偶氮]苯甲酸荧光淬灭基团的多肽为底物，5-(2-氨基乙氨基)-1-萘磺酸荧光基团与4-[4-(二甲基氨基)苯偶氮]苯甲酸荧光淬灭基团作为荧光对分别连接到多肽的N端和C端，多肽序列如下：
[EDANS-Glu]-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Ala-[Lys-DABCYL]-Gly。
前述的SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法中，以含有荧光基团与荧光淬灭基团的多肽为底物具有如下结构式：
式中，多肽为通过肽键相连的3个以上的氨基酸。
前述的SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法中，具体筛选方法为，在孔板格式中，将反应缓冲液中不同浓度的SARS-CoV-2主要蛋白酶Mpro加入到每个孔中，然后加入25μM的底物，底物与SARS-CoV-2主要蛋白酶Mpro的体积比为1：1，在25℃下共孵育60min后，检测其荧光信号值。
前述的SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法中，孵育过程中所使用的反应缓冲液成分包括：pH 7.0的Bis–Tris、NaCl、MgCl2、DTT和BSA。
本发明的有益效果：与现有技术相比，本发明作为底物的多肽所含的两个基团，属于荧光供体-荧光淬灭受体的关系，根据荧光能量共振转移(fluorescence resonanceenergy transfer,FRET)原理，当荧光供体-荧光淬灭受体的这两个基团通过共价键(肽键)连接保持一定的距离内，荧光基团发出的荧光会被荧光淬灭基团吸收，导致底物的荧光强度非常弱，当蛋白水解酶经裂解液处理后，多肽的肽键断裂，荧光基团和荧光淬灭基团失去了FRET的有效距离，荧光基团的荧光强度大大增强，荧光强度和底物多肽关联起来，底物多肽又和Mpro的活性相关，因此，FRET信号降低，荧光信号增强，与Mpro活性具有相关性。
由于采用了上述技术方案，本发明利用含有荧光基团与荧光淬灭基团(FRET效应)、多肽为底物，将荧光强度与Mpro的酶活性关联起来。这样的方法可应用于Mpro抑制剂的筛选，具有微型化、自动化、快速可靠等特点，适用于高通量的应用，也可应用于新型Mpro活性检测试剂盒的开发和开展Mpro抑制剂的筛选服务。且本发明的筛选效果准确，简单易行，成本低廉。
附图说明
图1：SARS-CoV-2Mpro底物FRET多肽的合成；
图2：基于FRET的SARS-CoV-2Mpro荧光活性筛选方法示意图；
图3：固定底物浓度(25μM)进行Mpro滴定的浓度-荧光响应曲线；
图4：不同孵育时间，线性范围内的四种酶浓度的荧光信号值变化；
图5：酶动力学测定；
图6：已知的Mpro抑制剂和其它化合物对开发的Mpro测定方法的验证；
图7：已知的Mpro抑制剂Ensitrelvir IC50的测定。
具体实施方式
下面结合附图和实施例对本发明作进一步的说明，但并不作为对本发明限制的依据。
本发明的实施例1：SARS-CoV-2主要蛋白酶(Mpro)的活性荧光筛选方法，包括有以下内容：
试剂和仪器
台式大摇床(美国Thermo)；超净工作台(江苏苏净集团有限公司)；台式小摇床(ZQTY-70S)；化学发光凝胶成像系统(英国Syngene)；紫外切胶仪(天跟生化有限公司)；台式冷冻离心机(美国Thermo)；恒温培养箱(DH6000BⅡ)；智能恒流泵(BT1-1000V-LCD)；高压破碎仪(ATS Engineering Inc)；酶标仪(BIO-RAD)；Milli-Q超纯水仪(法国MilliporePharmacia)；台式冷冻离心机(美国Thermo)；涡旋仪(美国Thermo)；电泳系统(美国BIO-RAD)；STS-2水平摇床(上海琪特分析仪器有限公司)；化合物均购自毕得医药公司或安耐吉。
SARS-CoV-2FRET多肽的合成与纯化
如图1所示，我们利用Wang树脂和Fmoc保护的甘氨酸缩合，脱Fmoc保护，得到甘氨酸缩合的Wang树脂后，进一步和Fmoc保护且被EDANS修饰的谷氨酸[Fmoc-Glu(EDANS)-OH]缩合，使用标准的FMOC/tert-Butyl固相合成策略，再根据所设计的氨基酸序列依次连接上不同的氨基酸，其中包括含有最优酰化修饰的赖氨酸，最后在氮端接上4-[4-(二甲基氨基)苯偶氮]苯甲酸(DABCYL)，经过水解树脂和脱除所有氨基酸保护基团的三氟醋酸/乙二硫醇/苯酚/茴香硫醚(TFA/EDT/Phenol/Thioanisole)混合溶液处理，通过反相高效液相色谱法分离纯化，高分辩质谱鉴定，冻干机冻干，得到相应的FRET荧光多肽。
[EDANS-Glu]-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Ala-[Lys-DABCYL]-Gly。
SARS-CoV-2主要蛋白酶(Mpro)的克隆、表达和纯化
使用TOPO和GATEWAY克隆技术(Invitrogen Corp.,Carlsbad,CA)将PCR产物克隆到pET 28b载体(Novagen)中，在大肠杆菌BL21(DE3)中进行表达，将大肠杆菌细胞转化的质粒在37℃培养，直至A600达到0.6-0.8，然后用0.5mM硫代异丙基-β-D-半乳苷苷(IPTG)诱导表达重组蛋白3CLpro，诱导时间为3小时。收集的细胞在包含缓冲液A(20mM三羟甲基甲烷磺酸盐(Tris-HCl)，pH8.0，500mM氯化钠和5mM咪唑)的条件下裂解，细胞裂解液被施加到HisTrapTM HP色谱柱中(GE Healthacare,US)进行纯化。然后使用缓冲液B(20mM三羟甲基甲烷磺酸盐(Tris-HCl)，pH 8.0，500mM氯化钠和250mM咪唑)在缓冲液A中呈12-100％线性梯度洗脱3CLpro蛋白。含有3CLpro的分数被聚集，并用储存缓冲液(20mM三羟甲基甲烷磺酸盐(Tris-HCl)，pH 8.0，50mM氯化钠和1mM乙二胺四乙酸)进行透析。样品被浓缩并在包含50％甘油的储存缓冲液中储存在-80℃。通过SDS-PAGE检验，蛋白的纯度高于95％。
SARS-CoV-2Mpro酶测定实验中酶浓度和孵育时间的优化
为了进行SARS-CoV-23CLpro抑制剂的筛选，使用并优化了荧光蛋白酶测定法。如图2所示，肽底物的C-末端与荧光团(Edans)连接，N-末端具有荧光猝灭剂(Dabcyl)，其猝灭Edans的荧光信号。当Mpro水解底物以产生两个片段时，Dabcyl与Edans分离，这减轻了荧光猝灭效应，导致荧光信号增加。为了使该筛选方法适用于高通量筛选，对酶活性测定中的酶和底物浓度进行优化。
首先在384孔板中以25μM多肽底物浓度检查不同的酶浓度对荧光强度的影响。简而言之在384孔板格式中，将反应缓冲液中的10μL酶(不同浓度)加入到每个孔中，然后加入10μL(25μM)底物，共孵育60min后，检测荧光信号值。实验原理如图2所示，实验结果如图3所示。发现在固定底物浓度(25μM)下，荧光强度随Mpro的酶浓度的增加而增加。在低酶浓度下观察到线性响应。
与上述步骤类似，接下来考察低酶浓度Mpro对荧光值的影响，并对孵育时间进行优化，以确认最佳酶浓度和孵育时间，实验结果如图4所示。对于测试的所有4酶浓度，荧光信号值随着孵育时间而增加。60min孵育导致酶浓度为25、50、75和100nM的荧光信号值分别为34520、53250、87956、94562，因此选择75nM酶浓度和60min孵育时间作为后续实验的优化条件。
反应缓冲液成分包括为Bis–Tris(pH 7.0)，NaC，MgCl2，DTT和BSA。
SARS-CoV-2Mpro酶测定实验中酶动力学和底物浓度的确定
然后进行酶动力学研究以确定该病毒蛋白酶的Km,Vmax和Kcat。实验步骤与上述类似，本实验中使用的底物浓度范围为12.5至150μM，酶浓度为75nM。实验结果如图5所示。该重组SARS-CoV-2Mpro的Km为59.13μM，Vmax为869.8nM/min，Kcat＝11.6min-1。由于抑制剂通常与底物竞争结合游离酶，因此高底物浓度可降低酶试验中测定的抑制剂效价(IC50)。因此，选择75nM Mpro和25μM底物作为筛选条件。
使用已知的Mpro抑制剂进行Mpro荧光测定法的验证
为了验证本发明的Mpro测定方法，在1536孔板形式中测定已知的SARS-CoV-2Mpro抑制剂Ensitrelvir和对Mpro无抑制作用的人类免疫缺陷病毒蛋白酶抑制剂(HIV PI)Saquinavir,Ritonavir,Indinavir,Amprenavir,Lopinavir,Telaprevir的荧光猝灭效果。实验步骤与上述类似，本实验中底物浓度为25μM，在添加Mpro(75nM)或不添加酶以及添加酶不加入化合物情况下，测定荧光强度。实验原理如图2所示，实验结果如图6所示，所有测定化合物中，只有已知的Mpro抑制剂Ensitrelvir表现出了荧光猝灭的效果，其它化合物无明显效果，体现了该酶测定法的可行性。
另外，如图7所示Ensitrelvir抑制SARS-CoV-2Mpro的酶活性，IC50值为13.31nM。192nM的Ensitrelvir显示出完全抑制作用，其中荧光强度等于不存在酶时的背景底物。该酶测定法中Ensitrelvir的IC50值与文献报告值相当，表明该酶测定法的可靠性。
根据图1得知，Mpro底物FRET多肽的合成途径。
根据图2得知，上述多肽[EDANS-Glu]-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Le u-Ala-[Lys-DABCYL]-Gly能够产生FRET效应。Mpro存在情况下，FRET效应被抑制，荧光强度增加，当潜在Mpro抑制剂加入时，使FRET效应恢复，荧光强度下降。
根据图3得知，在Mpro存在的情况下，荧光强度随Mpro的酶浓度的增加而增加。在低酶浓度下观察到线性响应。
根据图4得知，对于测试的所有4种酶浓度，荧光信号值随着孵育时间而增加。60min孵育导致酶浓度为25、50、75和100nM的荧光信号值分别为34520、53250、87956、94562。
根据图5得知，该重组SARS-CoV-2Mpro的Km为59.13μM，Vmax为869.8nM/min，Kcat＝11.6min-1。
根据图6得知，在所有评价的化合物中，只有已知的Mpro抑制剂Ensitrelvir表现出了荧光猝灭的效果，其它化合物无明显效果，体现了该酶测定法的可行性。
根据图7得知，Ensitrelvir抑制SARS-CoV-2Mpro的酶活性，其IC50值为13.31nM与文献报道相当，证明测定方法的可靠性。
结论：
本发明成功设计和开发了含有荧光基团与荧光淬灭基团的多肽为底物，利用FRET荧光效应，将荧光强度与Mpro的酶活性关联起来，建立了Mpro活性检测方法。
本发明的检测方法可应用于生物样本中的Mpro活性检测，或者应用于Mpro抑制剂的初步筛选及IC50的测定。
本发明对Mpro的活性检测及其调节剂的筛选效果准确，简单易行，快速高效，成本低廉。
序列表信息:
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生成日期:2023-12-14
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申请人姓名或名称:贵州医科大学
申请人姓名或名称/语言:zh
申请人姓名或名称/拉丁名称:guizhoumedical University
发明名称:SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法(zh)
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Claims (5)
1.SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法，其特征在于：以含有荧光基团与荧光淬灭基团的多肽为底物，将底物与SARS-CoV-2主要蛋白酶Mpro孵育，使荧光强度与SARS-CoV-2主要蛋白酶Mpro关联起来。
2.根据权利要求1所述的SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法，其特征在于：以含有5-(2-氨基乙氨基)-1-萘磺酸荧光基团与4-[4-(二甲基氨基)苯偶氮]苯甲酸荧光淬灭基团的多肽为底物，5-(2-氨基乙氨基)-1-萘磺酸荧光基团与4-[4-(二甲基氨基)苯偶氮]苯甲酸荧光淬灭基团作为荧光对分别连接到多肽的N端和C端，多肽序列如下：
[EDANS-Glu]-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Ala-[Lys-DABCYL]-Gly。
3.根据权利要求1所述的SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法，其特征在于：以含有荧光基团与荧光淬灭基团的多肽为底物具有如下结构式：
式中，多肽为通过肽键相连的3个以上的氨基酸。
4.根据权利要求1所述的SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法，其特征在于：具体筛选方法为，在孔板格式中，将反应缓冲液中不同浓度的SARS-CoV-2主要蛋白酶Mpro加入到每个孔中，然后加入25μM的底物，底物与SARS-CoV-2主要蛋白酶Mpro的体积比为1：1，在25℃下共孵育60min后，检测其荧光信号值。
5.根据权利要求4所述的SARS-CoV-2主要蛋白酶Mpro的活性荧光筛选方法，其特征在于：所述反应缓冲液成分包括：pH 7.0的Bis–Tris、NaCl、MgCl2、DTT和BSA。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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