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.

项目摘要 半胱天冬酶是控制凋亡性细胞死亡的半胱氨酸蛋白酶。半胱天冬酶被激活以杀死癌细胞 细胞，但抑制半胱天冬酶可以防止有害的细胞死亡的疾病，如心脏病发作和中风。因此，本发明的目的是， 人们对半胱天冬酶作为药物靶点产生了极大的兴趣。随着研究结果的公布， caspase-6在神经变性中起着重要作用。不幸的是，没有半胱天冬酶导向疗法 在市场上，主要是因为研究集中在活性位点，最保守的区域， 家人很明显，每一种caspase都以独特的方式受到调控。全面了解其中 是实现胱天蛋白酶特异性抑制所必需的。因此，我们的长期项目目标是定义和 利用每种凋亡半胱天冬酶的独特调控特征。 我们对这一目标的追求是成功的。由于我们对每个apop的独特功能的理解- 我们开发了一种变构caspase-6抑制剂，比任何报道的（33 nM）更有效。 并且也是迄今为止最具选择性的，优选caspase-6是所有其它caspase的500倍或更多。这 选择性是可能的，因为我们靶向的变构位点是caspase-6所独有的， 其他半胱天冬酶所不能达到的构象。令人欣慰的是，我们对这些问题的深入和系统的研究， 半胱天冬酶调节-裂解状态，构象变化，锌结合和磷酸化-达到顶峰 在结构上的理解，使我们能够满足我们的目标，半胱天冬酶选择性变构抑制。因此，在本发明中， 我们提出的研究将进一步扩展我们对细胞凋亡半胱天冬酶调控的理解， 新的关键领域-通过半胱天冬酶亚型的底物选择性，通过半胱天冬酶-9的核心解折叠和聚集， 磷酸化以及胱天蛋白酶核心和前结构域之间的相互作用以实现底物选择。在 此外，我们目前正在将这种半胱天冬酶-6抑制剂推向临床应用。 虽然我们对成功开发出一种抑制剂感到兴奋，这种抑制剂可以阻断其中一种功能， 12人半胱天冬酶，底物选择性抑制剂在这里提出的承诺是更加宝贵的。 例如，阻止Tau的半胱天冬酶-6裂解将对防止Tau形成具有重大影响。 阿尔茨海默氏症的神经系统缠结。另一方面，通过半胱天冬酶对DJ-1（PARK7）的裂解， 6对预防帕金森病很重要。因此，理想的caspase-6抑制剂用于治疗Alz- 海默氏病将阻断Tau切割，但仍将切割凋亡底物和DJ-1，而不 促进癌症或帕金森氏病。确定Tau和DJ-1的外来位点的拟议研究将 使我们能够开发底物选择性抑制剂（纳米抗体），可以阻断Tau的切割，但不能阻断Tau的切割。 DJ-1总之，这项工作计划增加了我们对生物学的基本理解， 半胱天冬酶，并能够开发一种新的高度调整的一类半胱天冬酶导向疗法。