Caspase-6 allosteric inhibitors: activity probes and neurodegeneration treatment

描述(由申请人提供)：Caspase-6是几种神经退行性疾病发展的关键因素，包括阿尔茨海默氏症和亨廷顿病。在转基因小鼠中，淀粉样前体蛋白或亨廷顿蛋白中的caspase-6裂解位点发生突变，以防止caspase-6裂解，从而防止神经退化，使caspase-6成为治疗阿尔茨海默病和亨廷顿病的有吸引力的靶点。Caspase-6也是控制细胞死亡的凋亡蛋白家族的成员。到目前为止，还不可能列举caspase-6在细胞中与其他家庭成员相关或独特的具体角色。这一信息具有核心重要性，因为如果将caspase-6作为亨廷顿病和阿尔茨海默病长期治疗的靶点，阻断这些额外的作用可能会导致负面副作用。不存在caspase特异的探针，因为所有用于细胞或动物模型的探针都是在caspase的活性部位起作用的，而且所有的caspase都有非常相似的活性部位。这个筛选利用了最近在caspase-6中发现的一个变构位点，该位点在任何其他caspase中都不存在。这个变构位点使首次实现对caspase-6的特异性抑制成为可能。这个项目的目标是开发一种探针，专门针对caspase-6中的这个独特的变构位点。一种对caspase-6具有特异性和选择性的化学探针将使caspase-6的天然生物学作用与 其他凋亡的半胱氨酸天冬氨酸酶首次出现。这将使caspase-6成为治疗神经退行性变的新靶点。除了验证药物发现的变构部位外，这些探针还将作为发现神经退行性变新药的先导化合物。这一筛选使用了一种强大的筛选方案，用于鉴定我们通过结晶学和突变鉴定的变构位点上的caspase-6变构抑制物。这个变构位点是caspase-6所独有的，在任何其他caspase中都不存在。在引导屏幕上，观察到了2.9%的命中率和30%的抑制截止值。通过使用更严格的截止线，命中率可以调整到所需的水平。Z‘值也达到了0.8，这表明caspase-6是这类筛查的一个容易处理的靶点。在第一次测试中重新测试了61次命中结果，并进行了两次二次测试。还建立并验证了另外两个二级检测方法。这一筛选还利用了一种基于生物物理和光谱观察的新的第三次化验。这个屏幕中目标的变构抑制构象在圆二色谱中显示了一个独特的特征。因此，使用圆二色谱分析提供了关于探针结合模式和位置的机械信息。该方法是第一种也是唯一一种可用于区分半胱氨酸天冬氨酸氨基转移酶中的变构位点抑制物和活性位点抑制物的方法。由于这是第一个能够鉴定caspase-6选择性化合物的筛查，我们的筛查将允许发现控制这一重要蛋白酶的探针。

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.