Discovering and Exploiting Caspase Regulatory, Allosteric and Exosites

发现和利用 Caspase 调节、变构和外切位点

• 批准号: 10623661
• 负责人: Jeanne Ann Hardy
• 金额: $ 38.94万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623661
• 关键词: Active Sites; Allosteric Site; Alzheimer' s Disease; Apoptosis; Apoptotic; Area; Binding; Biology; CASP6 gene; CASP9 gene; Cancerous; Caspase; Cell Death; Cells; Clinical; Development; Disease; Drug Targeting; Family; Goals; Human; Malignant Neoplasms; Marketing; Molecular Conformation; Myocardial Infarction; Nerve Degeneration; PARK7 gene; Parkinson Disease; Pharmaceutical Preparations; Pharmacologic Substance; Phosphorylation; Play; Protein Isoforms; Regulation; Reporting; Research; Research Project Grants; Role; Stroke; Work; Zinc; inhibitor; interest; nanobodies; neurofibrillary tangle formation; novel; prevent; success; tau Proteins; tau-1

PROJECT SUMMARY Caspases are cysteine proteases that control apoptotic cell death. Caspases are activated to kill cancerous cells, but inhibiting caspases can prevent deleterious cell death in diseases like heart attack and stroke. Thus, there has been significant interest in caspases as drug targets. This interest heightened further with the finding that caspase-6 plays a central role in neurodegeneration. Unfortunately, no caspase-directed therapies are on the market, primarily because research has focused on the active site, the most conserved region of the family. It is clear that each caspase is regulated in a unique manner. A comprehensive understanding of which is essential to achieve caspase-specific inhibition. Thus, our long-term project goal has been to define and exploit unique regulatory features for each apoptotic caspase. Our pursuit of that goal has been successful. Due to our understanding of the unique features of each apop- totic caspase, we developed an allosteric caspase-6 inhibitor that is more potent than any reported (33 nM) and is also by far the most selective, preferring caspase-6 500-fold or more over all other caspases. This selectivity was possible because the allosteric site we targeted is unique to caspase-6, locking it into a helical conformation not attainable by any other caspase. It is gratifying that our intense and systematic study of caspase regulation - cleavage state, conformational change, zinc binding and phosphorylation - culminated in a structural understanding that enabled us to meet our goal of caspase-selective allosteric inhibition. Thus, we propose research that will further extend our understanding of the regulation of the apoptotic caspases in new key areas - substrate selectivity by caspase isoforms, core unfolding and aggregation of caspase-9 by phosphorylation, and interactions between caspase core and prodomains to achieve substrate selection. In addition, we are currently moving this caspase-6 inhibitor forward toward clinical use. While we are thrilled at the success of developing an inhibitor that can block the function of just one of the twelve human caspases, the substrate-selective inhibitors proposed here promise to be even more invaluable. For example, preventing caspase-6 cleavage of Tau would have a major impact on preventing the formation of neurofibrillary tangles in Alzheimer’s Disease. On the other hand, cleavage of DJ-1 (PARK7) by caspase- 6 is important for preventing Parkinson’s Disease. Thus, an ideal caspase-6 inhibitor for treatment of Alz- heimer’s disease would block Tau cleavage, but would still cleave apoptotic substrates and DJ-1, without promoting cancer or Parkinson’s disease. The proposed studies identifying exosites for Tau and DJ-1 will enable us to develop substrate-selective inhibitors (nanobodies) that can block cleavage of Tau, but not of DJ-1. Together this work plan increases both our fundamental understanding of the biology and regulation of caspases and enables development of a novel highly tuned class of caspase-directed therapies.

项目总结 半胱氨酸蛋白酶是一种控制细胞凋亡的半胱氨酸蛋白酶。半胱氨酸天冬氨酸酶被激活以杀死癌症 但抑制caspase可以防止心脏病发作和中风等疾病中有害的细胞死亡。因此， 人们对半胱氨酸天冬氨酸酶作为药物靶点有很大的兴趣。这一发现进一步加剧了人们的兴趣 半胱氨酸氨基转移酶-6在神经退行性变中起核心作用。不幸的是，没有半胱氨酸天冬氨酸酶导向的疗法。 在市场上，主要是因为研究集中在活跃的地点，最保守的区域 一家人。很明显，每一种caspase都以一种独特的方式受到调控。全面了解 是实现caspase特异性抑制的关键。因此，我们的长期项目目标一直是定义和 为每个凋亡的caspase开发独特的调控功能。 我们对这一目标的追求是成功的。由于我们对每个APOP的独特功能的了解- 我们开发了一种变构caspase-6抑制剂，它比任何报道的都更有效(33 NM)。 而且也是目前为止最有选择性的，喜欢caspase-6 500倍或更多。这 选择性是可能的，因为我们针对的变构位点是caspase-6所特有的，将其锁定在螺旋结构中。 任何其他半胱氨酸天冬氨酸酶不能获得的构象。令人欣慰的是，我们对 Caspase调控-切割状态、构象变化、锌结合和磷酸化-达到顶峰 在结构上的理解，使我们能够达到caspase选择性变构抑制的目标。因此， 我们提出的研究将进一步扩大我们对凋亡caspase调控的理解。 新的关键领域-caspase异构体对底物的选择性，核心的展开和caspase-9的聚集 磷酸化，以及caspase核心和原结构域之间的相互作用，实现底物选择。在……里面 此外，我们目前正在将这种caspase-6抑制剂推向临床应用。 当我们为成功开发出一种可以阻断其中一种细胞功能的抑制剂而感到兴奋时 十二种人类半胱氨酸天冬氨酸酶，这里提出的底物选择性抑制物有望具有更大的价值。 例如，阻止Tau的caspase-6裂解将对防止形成有重大影响 阿尔茨海默病中的神经原纤维缠结。另一方面，Caspase-1对DJ-1(Park7)的切割 6对预防帕金森氏症很重要。因此，caspase-6抑制剂是治疗阿尔茨海默病的理想药物。 海默氏病会阻止Tau的切割，但仍然会切割凋亡底物和DJ-1，而不是 宣传癌症或帕金森氏症。拟议的鉴定Tau和DJ-1外显子的研究将 使我们能够开发底物选择性抑制剂(纳米体)，可以阻止Tau的切割，但不能 DJ-1。总而言之，这项工作计划既增加了我们对生物学的基本理解，也增加了对 半胱氨酸酶，并使一类新的高度可调的半胱氨酸天冬氨酸酶导向疗法的开发成为可能。