Optimizing brain penetrance of caspase-6 inhibitors to treat neurodegenerative diseases

项目总结/摘要 半胱氨酸蛋白酶caspase-6（Casp 6）与神经退行性疾病有关 已经超过十五年了在阿尔茨海默病（AD）中，Casp 6与淀粉样蛋白斑块共定位， 人类大脑中的tau缠结，以及人类和动物模型数据都表明激活的Casp 6 导致神经元炎症、神经变性和蛋白质裂解成毒性形式。 缺乏Casp 6的人类和小鼠是健康的，对炎症有抵抗力;此外，Casp 6 在携带早老素1和淀粉样蛋白β前体的家族性AD突变的5xFAD小鼠中的敲除 蛋白（APP）在神经元变性和炎症中显示出降低的水平。因此，选择性 抑制Casp 6治疗AD安全有效。为此，我们建议 设计、合成和分析活化形式的Casp 6的有效和高选择性抑制剂 （aCasp 6）基于我们为SU 110和SU 134建立的初始SAR。这些新化合物 通过共价结合在其他成员中没有发现的独特半胱氨酸残基来获得选择性， caspase家族虽然SU 110和SU 134都具有有希望的药物样性质，包括 在生物化学和基于细胞的测定中对aCasp 6的低纳摩尔抑制，低毒性， 蛋白质组研究中的靶点，血清暴露比细胞IC 50高100倍，50-77%口服 生物利用度和高达23%的脑/血浆暴露，我们将进一步探索SAR，以确定 具有改善的PK和脑渗透性质的化合物（目的1）。我们将按照标准 小鼠PK实验，以确定口服生物利用度并定义结构-脑暴露 关系。对于Aim 2，我们开发了一种基于点击的aCasp 6结合探针， 证明了足够的效力和选择性，可用作体外活性探针， aCasp6。我们将使用这种探针，结合aCasp 6蛋白半衰期测定， 测量aCasp 6底物tau，以确定靶抑制的寿命， 细胞中优化的aCasp 6抑制剂;这些数据将用于该项目的II期，以评估 在动物中的目标参与。根据这些SAR、PK和组织分布研究，我们将确定 最适合进行PD试验的化合物和在试验期间运行的疾病模型 项目的第二阶段。在随后的二期申请中，我们将开发生物标志物检测方法 并评估在用优化的先导化合物治疗后这些生物标志物的变化。 5xFAD模型和其他AD动物模型。II期研究的结论将是 目标候选人概况的细化，并将预测发展的选择 可作为IND赋能研究的候选人。

PROJECT SUMMARY/ABSTRACT The cysteine protease caspase-6 (Casp6) has been associated with neurodegenerative diseases for over fifteen years. In Alzheimer’s disease (AD), Casp6 is colocalized with amyloid plaques and tau tangles in human brain, and both human and animal model data indicate that activated Casp6 contributes to neuronal inflammation, neurodegeneration, and cleavage of proteins to toxic forms. Humans and mice lacking Casp6 are healthy and resistant to inflammation; furthermore, Casp6 knockout in 5xFAD mice bearing familial AD mutations in presenilin1 and amyloid beta precursor protein (APP) show reduced levels in neuronal degeneration and inflammation. Thus, selective inhibition of Casp6 could be safe and effective in treatment of AD. To this end, we propose to design, synthesize and profile potent and highly selective inhibitors of the activated form of Casp6 (aCasp6) based on our initial SAR established for SU110 and SU134. These novel compounds gain selectivity by binding covalently to a unique cysteine residue not found in other members of the caspase family. While both SU110 and SU134 have promising drug-like properties, including low nanomolar inhibition of aCasp6 in biochemical and cell-based assays, low toxicity, few off- targets in proteome-wide studies, serum exposures 100-fold higher than cellular IC50, 50-77% oral bioavailability, and up to 23% brain/plasma exposure, we will further explore the SAR to identify compounds with improved PK and brain penetration properties (Aim 1). We will conduct standard mouse PK experiments to determine oral bioavailability and to define structure–brain exposure relationships. For Aim 2, we have developed a click-based probe of aCasp6 binding that demonstrates sufficient potency and selectivity to use as an ex vivo activity-based probe of aCasp6. We will use this probe, in conjunction with aCasp6 protein half-life determination and measurement of the aCasp6 substrate tau, to determine the lifetime of target inhibition by optimized aCasp6 inhibitors in cells; these data will be used in Phase II of the project to assess target engagement in animals. From these SAR, PK and tissue distribution studies, we will identify the most appropriate compounds to conduct PD assays and disease models to be run during Phase II of the project. In the subsequent Phase II application, we will develop biomarker assays and evaluate changes in these biomarkers after treatment with optimized lead compound in the 5xFAD model and additional animal models of AD. The conclusion of Phase II studies will be refinement of the Target Candidate Profile and will anticipate the selection of a development candidate for IND-enabling studies.