CN103649757A - 用于鉴别β-折叠聚集的蛋白质配体的方法 - Google Patents

用于鉴别β-折叠聚集的蛋白质配体的方法 Download PDF

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CN103649757A
CN103649757A CN201280030461.2A CN201280030461A CN103649757A CN 103649757 A CN103649757 A CN 103649757A CN 201280030461 A CN201280030461 A CN 201280030461A CN 103649757 A CN103649757 A CN 103649757A
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E·博罗尼
C·采奇
A·多恩
L·戈比
V·格斯奇-迈尔
F·格吕宁格尔
D·罗特
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Abstract

本发明提供了使用噻嗪红R，鉴别β-折叠聚集的蛋白质配体的测定。

Description

用于鉴别β-折叠聚集的蛋白质配体的方法
本发明涉及用于鉴别配体的方法，所述配体结合形成β折叠的聚集的蛋白质。
在阿兹海默病患者的大脑中，Tau蛋白质聚集体与神经变性疾病的发展密切相关，可用作神经变性过程的早期成像标志物。还有其他的神经变性疾病表现出tau聚集。与淀粉样-β成像组合，可以区分tau病和阿兹海默病。这是用于诊断和患者分层的有价值的工具。Tau成像是针对tau病理学和神经变性的项目的有效生物标志物。
因此，需要用于鉴别PET（正电子成像术）成像配体的方法，所述配体用于阿兹海默病患者或其他tau病中的tau聚集体的特异性成像。
本发明的方法基于这样的发现，即，相比与形成β-折叠聚集体的单体结合的噻嗪红R(Thiazine Red R)，与β-折叠聚集体结合的噻嗪红R（CASNr.2150-33-6）表现出强烈增加的荧光。
术语“β-折叠聚集的蛋白质”在本文中使用时指形成具有β-折叠结构的聚集体的蛋白质。Tau蛋白、α-突触核蛋白和Aβ肽形成具有β-折叠结构的聚集体。
术语“蛋白质”在本文使用时指氨基酸的聚合物，不涉及特定的长度。因此，肽、寡肽和蛋白质片段都包括在多肽的定义中。术语“蛋白质”与术语“多肽”可互换的使用。
术语“tau蛋白”在本文中使用时指来自任何动物（例如哺乳动物，物种，包括人）的天然序列tau蛋白和tau变体（下文进一步定义）。可以从多种来源（包括人组织类型）分离Tau多肽，或通过重组和/或合成方法制备Tau多肽。
该测定可以使用天然或重组生产的tau蛋白。“重组蛋白”是通过在异源细胞中表达而分离、纯化或鉴别的蛋白质，所述细胞被瞬时或稳定的转导或转染经改造驱动蛋白质在宿主细胞中表达的重组表达载体。可以在原核细胞（例如大肠杆菌(E.coli)）、酵母（例如栗酒裂殖酵母（S.pombe））或真核细胞（例如HEK293、Sf9昆虫细胞）中生产重组tau。优选的，Sf9昆虫细胞用于重组tau的高表达。用于测定的tau蛋白可以是纯化的。术语“纯化的”在本文中使用时是指从其天然环境或从重组生产来源中移出的、分离或分开的多肽，所述多肽中至少60%，更优选至少80%没有它们天然相关的其他组分，例如膜和微粒体。
“天然序列tau”指不论其制备方式，具有与天然存在的tau多肽相同的氨基酸序列的多肽。天然序列tau可以是从天然分离的，或通过重组和/或合成方法制备的。术语“天然序列tau”具体涵盖了tau的天然存在的截短的或分泌的形式，天然存在的变体形式（例如可变剪接的形式）和天然存在的等位变体。NCBI数据库中人tau多肽的标识符是NP_005901（Seq.Id.No.1）。
术语“tau变体”指天然序列tau的氨基酸序列变体，在天然序列中含有一个或多个氨基酸取代和/或缺失和/或插入。氨基酸序列变体一般与天然序列tau的氨基酸序列具有至少约75%，优选至少约80%，更优选至少约85%，甚至更优选至少约90%，最优选至少约95%序列同一性。
术语“Aβ肽”在本文中使用时是指来自任何动物（例如哺乳动物，物种，包括人）的天然序列Aβ肽和Aβ肽变体（下文进一步定义）。可以从多种来源（包括人组织类型）分离Aβ肽，或通过重组和/或合成方法制备Aβ肽。
该测定可以使用天然或重组生产的Aβ肽。“重组蛋白”是通过在异源细胞中表达而分离、纯化或鉴别的蛋白质，所述细胞被瞬时或稳定的转导或转染经改造以驱动蛋白质在宿主细胞中表达的重组表达载体。可以在原核细胞（例如大肠杆菌(E.coli)）、酵母（例如栗酒裂殖酵母（S.pombe））或真核细胞（例如HEK293、Sf9昆虫细胞）中生产重组Aβ肽。用于测定的Aβ肽可以是纯化的。术语“纯化的”在本文中使用时是指从其天然环境或从重组生产来源中移出的、分离或分开的多肽，所述多肽中至少60%，更优选至少80%没有它们天然相关的其他组分，例如膜和微粒体。
“天然序列Aβ肽”指不论其制备方式，具有与天然存在的Aβ肽相同的氨基酸序列的多肽。应注意，“Aβ肽”具有若干天然存在的形式，因而，人的形式指Aβ39、Aβ40、Aβ41、Aβ42和Aβ43。最主要的形式Aβ42具有氨基酸序列（从N末端开始）：
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA（Seq.Id.No.3）。在Aβ41、Aβ40、Aβ39中，分别缺失C末端氨基酸A、IA和VIA。在Aβ43形式中，在上述序列（Seq.Id.No.3）的C末端包括额外的苏氨酸残基。用于本测定的优选的Aβ肽是具有Seq.Id.No.4中给出的氨基酸序列Aβ40。
天然序列Aβ肽可以是从天然分离的，或通过重组和/或合成方法制备的。术语“天然序列Aβ肽”具体涵盖了Aβ肽的天然存在的截短的或分泌的形式，天然存在的变体形式和天然存在的等位变体。术语“Aβ肽变体”指天然序列Aβ肽的氨基酸序列变体，其在天然序列中含有一个或多个氨基酸取代和/或缺失和/或插入。氨基酸序列变体一般与天然序列Aβ肽的氨基酸序列具有至少约75%，优选至少约80%，更优选至少约85%，甚至更优选至少约90%，最优选至少约95%序列同一性。
术语“α-突触核蛋白”在本文中使用时是指来自任何动物（例如哺乳动物，物种，包括人）的天然序列α-突触核蛋白和α-突触核蛋白变体（下文进一步定义）。可以从多种来源（包括人组织类型）分离α-突触核蛋白多肽，或通过重组和/或合成方法制备α-突触核蛋白多肽。
该测定可以使用天然或重组生产的α-突触核蛋白蛋白质。“重组蛋白”是通过在异源细胞中表达而分离、纯化或鉴别的蛋白质，所述细胞被瞬时或稳定的转导或转染经改造驱动蛋白质在宿主细胞中表达的重组表达载体。可以在原核细胞（例如大肠杆菌(E.coli)）、酵母（例如栗酒裂殖酵母（S.pombe））或真核细胞（例如HEK293、Sf9昆虫细胞）中生产重组α-突触核蛋白。优选的，Sf9昆虫细胞用于重组α-突触核蛋白的高表达。用于测定的α-突触核蛋白可以是纯化的。术语“纯化的”在本文中使用时是指从其天然环境或从重组生产来源中移出的、分离或分开的多肽，所述多肽中至少60%，更优选至少80%没有它们天然相关的其他组分，例如膜和微粒体。
“天然序列α-突触核蛋白”指不论其制备方式，具有与天然存在的α-突触核蛋白多肽相同的氨基酸序列的多肽。天然序列α-突触核蛋白可以是从天然分离的，或通过重组和/或合成方法制备的。术语“天然序列α-突触核蛋白”具体涵盖了α-突触核蛋白的天然存在的截短的或分泌的形式，天然存在的变体形式（例如可变剪接的形式）和天然存在的等位变体。人α-突触核蛋白多肽的氨基酸序列在Seq.Id.No.2中给出。
术语“α-突触核蛋白变体”指天然序列α-突触核蛋白的氨基酸序列变体，在天然序列中含有一个或多个氨基酸取代和/或缺失和/或插入。氨基酸序列变体一般与天然序列α-突触核蛋白的氨基酸序列具有至少约75%，优选至少约80%，更优选至少约85%，甚至更优选至少约90%，最优选至少约95%序列同一性。
术语“化合物”在本文中用于与本发明的测定相关时描述的“测试化合物”或“示踪候选化合物”的背景中。由此，这些化合物包括有机或无机化合物，其来自合成或天然来源。化合物包括无机或有机化合物，例如但不限于特征是相对低分子量的多核苷酸、脂类或激素类似物。
附图简介
图1显示了用于聚集的和单体蛋白质的噻嗪红R的饱和分析。每个聚集的蛋白质上有2个不同的针对噻嗪红（Thiazin-red）的结合位点。确定了两个结合位点的Kd（Kd1、Kd2）A:Tau B:Aβ，C：Α-突触核蛋白。
实验部分
在Tris10mM pH8，24h，37℃的条件下，从大肠杆菌纯化的重组人微管相关蛋白Tau在5μM浓度下用花生四烯酸（100μM）聚集。用花生四烯酸（100μM）聚集合成的Aβ40，在Tris10mM pH8中，37℃进行3天，在150rpm振荡下。
用花生四烯酸（100μM）聚集从大肠杆菌纯化的人重组-Α-突触核蛋白，在Tris10mM pH8中，37℃进行5天，在150rpm振荡下。
进行针对聚集的蛋白质的噻嗪红饱和分析，确定噻嗪红与聚集的蛋白质的亲和力（Kd）。表1显示了噻嗪红与聚集的tau、Aβ和α-突触核蛋白的亲和力常数。结果显示，每个聚集的蛋白质上有2个针对噻嗪红的具有不同亲和力的结合位点。
所添加的噻嗪红的浓度对应于各个聚集的蛋白质结合位点的Kd，以诱导可以被添加替换剂化合物抑制的荧光信号。
为了确定替换剂化合物与聚集的蛋白质的噻嗪红结合位点的亲和力，测定中添加不同浓度的化合物，范围从0.3nM至10000nM。
与之平行的，在不含噻嗪红的条件下，同时测量化合物与聚集的蛋白质的自发荧光。使用配体和聚集的蛋白质作为阴性对照，使用噻嗪红、具有已知活性的参照化合物和聚集的蛋白质作为阳性对照。
在Perkin Elmer OptiPlate384中实施测定，黑暗中，45ul测定体积，测定缓冲液是不含CaCl2和MgCl2的DPBS（GIBCO N.14020）。在DMSO中稀释测试化合物，加入2μl至测定中（5%DMSO终浓度）。通过添加聚集的蛋白质（竞争条件）开始测定。短暂的摇动平板（用Sterico variomagteleshake摇动1min），在室温孵育30min。在Excitation531nm/Emission595nm下，用En:Vision（Perkin Elmer）进行测量
表1显示了不同化合物与聚集的tau、Aβ和α-突触核蛋白针对高亲和力结合位点的亲和力常数。
虽然出于清楚理解的目的，通过示例和实施例详细的描述了上述发明，但说明书和实施例不应被视为限制了本发明的范围。本文引用的所有专利和科学文献的公开内容都通过引用全文明确整合到本文中。
Figure IDA0000442539560000021
Figure IDA0000442539560000031

Claims (8)

1.用于鉴别β-折叠聚集的蛋白质配体的方法，包括：
a）使β-折叠聚集的蛋白质与噻嗪红R和化合物接触，
b）测量步骤a）的混合物中的噻嗪红R荧光，其中步骤a）的混合物中的噻嗪红R荧光相比空白减少，指示了β-折叠聚集的蛋白质配体。
2.权利要求1的方法，其中β-折叠聚集的蛋白质选自tau蛋白、Aβ肽和α-突触核蛋白。
3.权利要求1或2的方法，其中β-折叠聚集的蛋白质是tau蛋白或Aβ40肽，优选人tau蛋白或人Aβ40肽。
4.权利要求1至3的方法，其中噻嗪红R荧光是在Excitation531nm/Emission595nm测量的。
5.权利要求1至4的方法，其中β-折叠聚集的蛋白质是重组生产的β-折叠聚集的蛋白质。
6.权利要求1至5的方法，其中空白包括β-折叠聚集的蛋白质和噻嗪红R。
7.权利要求1至6的方法，其中化合物是以增加的浓度添加到步骤a）的混合物中的，从而确定测试化合物的IC50。
8.噻嗪红R用于鉴别β-折叠聚集的蛋白质配体的用途。
CN201280030461.2A 2011-06-22 2012-06-19 用于鉴别β-折叠聚集的蛋白质配体的方法 Pending CN103649757A (zh)

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