Caspase-6 allosteric inhibitors: activity probes and neurodegeneration treatment

描述（由申请人提供）：Caspase-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的关键因素。在淀粉样蛋白前体蛋白或亨廷顿蛋白中caspase-6切割位点发生突变以阻止caspase-6切割的转基因小鼠免受神经变性，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也是控制凋亡细胞死亡的凋亡蛋白家族的一员。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caspase-6长期用于治疗亨廷顿舞蹈症和阿尔茨海默氏症，阻断这些额外的作用可能会导致负面的副作用。没有caspase特异性探针存在，因为所有可用的用于细胞或动物模型的探针都在caspase的活性位点起作用，所有的caspase都有非常相似的活性位点。这个筛选利用了最近在caspase-6中发现的一个在其他caspase中不存在的变构位点。这个变构位点使得首次实现caspase-6特异性抑制成为可能。该项目的目标是开发一种专门针对caspase-6中这种独特的变构位点的探针。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caspase-6区分开来

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.