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（包括阿尔茨海默氏症和亨廷顿疾病）发展的关键因素。淀粉样蛋白或亨廷汀蛋白中的caspase-6裂解位点突变以防止caspase-6裂解受到神经变性的保护，使caspase-6成为治疗阿尔茨海默氏症和亨廷顿疾病的有吸引力的目标。 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，不可能列举与其他家庭成员相关或与众不同的细胞中caspase-6的特定角色。该信息至关重要，因为如果在亨廷顿和阿尔茨海默氏症的长期治疗中caspase-6的目标是caspase-6，则阻止这些额外的角色可能会导致负面副作用。没有caspase特异性探针存在，因为在胱天蛋白酶的活性位点中使用的所有可用探针在细胞或动物模型功能中都具有非常相似的活性位点。该屏幕利用了Caspase-6中最近发现的变构位点，该位点在任何其他caspase中都不存在。这个变构位置使得第一次实现caspase-6特异性抑制。该项目的目的是开发一个专门针对Caspase-6中这个独特的变构位点的探针。特定于caspase-6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caspase是第一次。这将允许验证caspase-6作为治疗神经变性的新靶标。除了验证用于发现药物发现的变构部位外，这些探针还将用作发现新型药物神经变性的铅化合物。该屏幕使用强大的筛选方案来识别我们通过晶体学和诱变确定的变构位点上的caspase-6变构抑制剂。这个变构位点是caspase-6独有的，并且在任何其他caspase中都不存在。在试点屏幕中，观察到截止抑制作用为2.9％，命中率为2.9％。可以通过使用更严格的截止值将命中率调节到所需的水平。还观察到z'评分为0.8，这表明caspase-6是此类筛选的可拖动目标。在主要测定中重新测试了一个61个命中板，并进行了两个次要测定。还建立了两个辅助测定法。该屏幕还利用基于生物物理和光谱观测的新三级测定。在此屏幕中靶向的变构抑制构象在圆形二分色谱中表现出独特的特征。因此，使用圆形二科主义测定法提供了有关探针结合模式和位置​​的机械信息。该测定是第一个也是唯一可用的测定法，可以将变构位点抑制剂与主动部位抑制剂Inany Caspase区分开。由于这是符合caspase-6选择性化合物的第一个屏幕，因此我们的屏幕将允许发现探针控制这种重要的蛋白酶。