Caspase-6 allosteric inhibitors: activity probes and neurodegeneration treatment

描述（由申请人提供）：胱天蛋白酶-6是包括阿尔茨海默病和亨廷顿病在内的几种神经退行性疾病发展中的关键因子。其中淀粉样前体蛋白或亨廷顿蛋白中的胱天蛋白酶-6切割位点突变以防止胱天蛋白酶-6切割的转基因小鼠被保护免于神经变性，使得胱天蛋白酶-6成为治疗阿尔茨海默病和亨廷顿病的有吸引力的靶标。Caspase-6也是控制凋亡性细胞死亡的凋亡蛋白家族的成员。迄今为止，还不可能列举出caspase-6在细胞中与其他家族成员相关或独特的特定作用。这一信息至关重要，因为如果胱天蛋白酶-6被用于亨廷顿病和阿尔茨海默病的长期治疗，阻断这些额外的作用可能会导致负面的副作用。不存在半胱天冬酶特异性探针，因为用于细胞或动物模型的所有可用探针在半胱天冬酶的活性位点处起作用，并且所有半胱天冬酶具有非常相似的活性位点。该筛选利用了最近发现的caspase-6中不存在于任何其他caspase中的变构位点。这种变构位点使得首次实现胱天蛋白酶-6特异性抑制成为可能。该项目的目标是开发一种探针，专门针对caspase-6中这个独特的变构位点。对胱天蛋白酶-6具有特异性和选择性的化学探针将允许区分胱天蛋白酶-6的天然生物学作用与 其他凋亡的半胱天冬酶。这将允许验证半胱天冬酶-6作为治疗神经变性的新靶点。除了验证药物发现的变构位点外，这些探针还将作为发现神经变性新药的先导化合物。该筛选使用稳健的筛选方案，用于在我们通过晶体学和诱变鉴定的变构位点鉴定胱天蛋白酶-6变构抑制剂。这种变构位点是胱天蛋白酶-6所特有的，并且不存在于任何其他胱天蛋白酶中。在中试筛选中，观察到命中率为2.9%，抑制截止值为30%。命中率可以通过使用更严格的截止值调整到所需的水平。还观察到0.8的Z'评分，表明半胱天冬酶-6是这种类型筛选的易处理的靶标。在初步试验中重新检测了一组61个匹配结果，并进行了两次二次试验。还建立并验证了另外两项二级试验。该筛选还利用了基于生物物理和光谱观察的新型三级测定。在该筛选中靶向的变构抑制构象在圆二色光谱中表现出独特的特征。因此，使用圆二色性测定提供了关于探针结合的模式和位置的机制信息。该试验是第一个也是唯一一个可以将变构位点抑制剂与任何半胱天冬酶活性位点抑制剂区分开来的试验。由于这是第一个筛选，使识别半胱天冬酶-6选择性化合物，我们的屏幕将允许发现的探针来控制这个重要的蛋白酶。

DESCRIPTION (provided by applicant): Caspase-6 is a critical factor in the development of several neurodegenerative disorders including Alzheimer's and Huntington's Diseases. Transgenic mice in which the caspase-6 cleavage sites in Amyloid Precursor Protein or Huntingtin Protein were mutated to prevent caspase-6 cleavage are protected from neurodegeneration, making caspase-6 an attractive target for treatment of Alzheimer's and Huntington's Diseases. Caspase-6 is also a member of the family of apoptotic proteins that control apoptotic cell death. To date it has been impossible to enumerate the specific roles caspase-6 plays in the cell that are related to or unique from other family members. This information is of central importance because blocking these additional roles could potentially lead to negative side-effects if caspase-6 were targeted in treatments for Huntington's and Alzheimer's long term. No caspase-specific probes exist because all available probes for use in cell or animal models function at the active sites of caspases and all caspases have very similar active sites. This screen makes use of a recently discovered allosteric site in caspase-6 that is not present in any other caspase. This allosteric site makes it possible to achieve caspase-6 specific inhibition for the very first time. The goal of this project is to develop a probe that specifically targets this unique allosteric site in caspase-6. A chemical probe that is specific an selective for caspase-6 will allow the native biological role of caspase-6 to be distinguished from other apoptotic caspases for the first time. This will allow validation of caspase-6 as a novel target for treatment of neurodegeneration. In addition to validating the allosteric site for drug discovery, the probes will also serve as lead compounds for discovery of novel drugs for neurodegeneration. This screen uses a robust screening protocol for identifying caspase-6 allosteric inhibitors at an allosteric site we have identified by crystallography and mutagenesis. This allosteric site is unique to caspase-6 and is not present in any other caspase. In the pilot screen, a hit rate of 2.9% with a cutoff of 30% inhibition was observed. The hit-rate can be adjusted to the desired level by using more stringent cutoffs. A Z' score of 0.8 was also observed, suggesting that caspase-6 is a tractable target for this type of screening. A panel of 61 hits was retested in the primary assay and subjected to two secondary assays. Two additional secondary assays have also been established and validated. This screen also utilizes a novel tertiary assay based on biophysical and spectroscopic observations. The allosterically inhibited conformation targeted in this screen exhibits a unique signature in the circular dichroism spectra. Thus using the circular dichroism assay provides mechanistic information about both the mode and location of probe binding. This assay is the first and only assay available that allows allosteric site inhibitors to be distinguished from active site inhibitors inany caspase. As this is the first screen enabling identification of caspase-6 selective compounds, our screen will allow discovery of probes to control this important protease.