Caspase-6 allosteric inhibitors: activity probes and neurodegeneration treatment

Caspase-6 变构抑制剂：活性探针和神经变性治疗

• 批准号: 8507707
• 负责人: Jeanne Ann Hardy
• 金额: $ 3.74万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-15 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8507707
• 关键词: Active Sites; Address; Adverse effects; Allosteric Site; Alzheimer' s Disease; Amyloid beta-Protein Precursor; Animal Model; Apoptosis; Apoptotic; Binding; Binding Sites; Biological; Biological Assay; Caspase; Caspase-1; Cell Death; Cell model; Cells; Chemicals; Circular Dichroism; Cleaved cell; Crystallography; Development; Disease; Dissection; Dose; Eligibility Determination; Enzyme-Linked Immunosorbent Assay; Enzymes; Event; Exhibits; Family; Family member; Goals; Hereditary Disease; Huntington Disease; Individual; Inhibitory Concentration 50; Institutes; Lamin Type A; Lead; Location; Measures; Methods; Molecular; Molecular Conformation; Mutagenesis; Mutate; Nerve Degeneration; Neurodegenerative Disorders; Organism; Penetrance; Peptide Hydrolases; Pharmaceutical Preparations; Play; Proteins; Role; Signal Transduction; Site; Specificity; Structure; Thermodynamics; Time; Toxic effect; Transgenic Mice; Ursidae Family; Validation; Variant; base; caspase-3; caspase-6; drug discovery; high throughput screening; human Huntingtin protein; inhibitor/antagonist; interest; member; novel; prevent; screening; small molecule; statistics

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.

描述（由申请人提供）：Caspase-6 是多种神经退行性疾病（包括阿尔茨海默病和亨廷顿病）发展的关键因素。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 裂解的转基因小鼠可免受神经变性，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标。 Caspase-6 也是控制凋亡细胞死亡的凋亡蛋白家族的成员。迄今为止，还不可能枚举 caspase-6 在细胞中与其他家族成员相关或独特的具体作用。这一信息至关重要，因为如果长期以 caspase-6 为目标治疗亨廷顿舞蹈病和阿尔茨海默病，阻断这些额外的作用可能会导致负面副作用。不存在半胱天冬酶特异性探针，因为所有可用于细胞或动物模型的探针都在半胱天冬酶的活性位点起作用，并且所有半胱天冬酶都具有非常相似的活性位点。该筛选利用了最近在 caspase-6 中发现的变构位点，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 中这一独特变构位点的探针。对 caspase-6 具有特异性选择性的化学探针将能够区分 caspase-6 的天然生物学作用 首次发现其他凋亡半胱天冬酶。这将验证 caspase-6 作为治疗神经退行性疾病的新靶点。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退行性疾病新药物的先导化合物。该筛选使用稳健的筛选方案来鉴定我们通过晶体学和诱变鉴定的变构位点上的 caspase-6 变构抑制剂。该变构位点是 caspase-6 所独有的，在任何其他 caspase 中都不存在。在试点筛选中，观察到 2.9% 的命中率和 30% 抑制的截止值。可以通过使用更严格的截止值将命中率调整到所需的水平。还观察到 Z' 分数为 0.8，表明 caspase-6 是此类筛选的易于处理的目标。一组 61 次命中在主要测定中进行了重新测试，并进行了两次二次测定。还建立并验证了另外两种辅助测定方法。该筛选还采用了基于生物物理和光谱观察的新型三级测定。该屏幕中目标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有关探针结合的模式和位置​​的机械信息。该测定是第一个也是唯一一个可以将变构位点抑制剂与任何 caspase 中的活性位点抑制剂区分开来的测定。由于这是首次能够鉴定 caspase-6 选择性化合物的筛选，我们的筛选将允许发现控制这种重要蛋白酶的探针。