Dialing down caspase-7 through allosteric control: An integrated approach

通过变构控制降低 caspase-7：一种综合方法

• 批准号: 10259744
• 负责人: Michael Ashley Spies
• 金额: $ 30.9万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10259744
• 关键词: Active Sites; Acylation; Affect; Apoptosis; Apoptotic; Binding; Biophysics; CASP7 gene; Caspase; Cells; Charge; Chemicals; Cleaved cell; Complex; Crystallization; Data; Deposition; Development; Disease; Drug Targeting; Enzyme Precursors; Enzymes; Family; Foundations; Goals; Human; Hydrolysis; Inflammation; Inflammatory; Kinetics; Knowledge; Learning; Ligation; Malignant Neoplasms; Measurable; Methods; Modeling; Molecular Conformation; Nerve Degeneration; Pathway interactions; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Positioning Attribute; Post-Translational Protein Processing; Process; Property; Resolution; Role; Scheme; Series; Source; Specificity; Stimulus; Structural Models; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; System; Testing; Work; X-Ray Crystallography; base; biophysical techniques; cheminformatics; deacylation; drug discovery; flexibility; genetic regulatory protein; inhibitor/antagonist; insight; interest; molecular dynamics; novel; response; screening; simulation; small molecule

Our goal is to provide a physical and chemical rationale for how human caspase-7 (C7) is allosterically controlled, particularly by small molecules. Caspase dysregulation, both catalytic and autocatalytic, has been implicated in numerous neurodegenerative, inflammatory diseases and cancers. Due to a highly charged active site, with low druggability and selectivity problems, development of active site inhibitors has been problematic. However, there has been enormous interest in targeting an allosteric pocket of C7, which is located more than 17 Å from the active site. The current proposal includes data that represents a breakthrough advance in our understanding of how small drug-like molecules may be used to allosterically “dial down” the activity of C7. This initial groundwork has been made possible by advances in Fragment Based Drug Discovery (FBDD), which includes a confluence of chemical informatics, biophysical methods such as Surface Plasmon Resonance, X-ray crystallography and molecular dynamics. The work described in this proposal centers on our discovery of a series of reversible allosteric inhibitors that bind in this allosteric pocket, which are the first drug-like compounds to show such activity in caspases. The allosteric effectors obtained from our FBDD campaign were used to elucidate several high resolution crystal structures of the inhibited complex, which revealed a way forward for specific allosteric control for this enzyme. The use of FBDD, as illustrated by our two inhibited high resolution crystal structures, PDB-ID 5V6U and 5V6Z, provide us with the first rational basis for structure-activity relationships for reversible allosteric inhibitors for the executioner caspase class of drug targets. These two structures clearly show that binding of the allosteric inhibitor to the remote allosteric pocket of C7, yields structures with C7’s catalytic thiolate (Cys186) oriented in a non- productive conformation (pointing into the P1 pocket instead of into the active site). Another important feature of these allosterically inhibited complexes is a large increase in crystallographic B-factors (relative to crystal structures of uninhibited C7) of a number of important loop regions. This proposal will focus on obtaining high resolution structures of the many other distinct allosteric effectors resulting from our FBDD campaign, which have not been co-crystallized with C7, including 13 confirmed inhibitors, 5 binders that do not inhibit, and one activator; completion of this work will provide a structural Rosetta Stone (an ability to directly compare inhibitor, non-inhibitor and activator) for understanding how C7’s catalytic power is affected by remote ligation at the allosteric pocket. Upon successful completion, not only will we learn what chemical space occupancy in the C7 allosteric pocket results in inhibition, but more importantly, we will know how these remotely bound species are achieving their dampening of C7’s catalytic power.

我们的目标是提供一个物理和化学的理由，如何人胱天蛋白酶-7（C7）是变构的， 控制，特别是小分子。半胱天冬酶失调，包括催化和自催化， 与许多神经变性、炎症性疾病和癌症有关。由于 高电荷活性位点，具有低可药用性和选择性问题，活性位点的开发 抑制剂是有问题的。然而，人们对靶向变构药物 C7的口袋，其位于距离活性位点超过17 μ m处。目前的提案包括数据 这代表了我们对小药物样分子如何 用于变构“下调”C7的活性。这一初步的基础工作已经成为可能， 基于片段的药物发现（FBDD）的进展，其中包括化学物质的融合， 信息学、生物物理学方法，如表面等离子体共振、X射线晶体学和 分子动力学本提案中所描述的工作集中在我们发现的一系列 可逆的变构抑制剂，结合在这个变构口袋里，这是第一个药物样化合物 在半胱天冬酶中显示出这种活性。从我们的FBDD活动中获得的变构效应物是 用于阐明抑制复合物的几种高分辨率晶体结构，其揭示了 对这种酶进行特异性变构控制的方法。FBDD的使用，如我们的两个例子所示， 抑制高分辨率晶体结构，PDB-ID 5V 6 U和5V 6 Z，为我们提供了第一个合理的 可逆性胱天蛋白酶变构抑制剂构效关系的基础 一类药物靶点。这两种结构清楚地表明，变构抑制剂与蛋白质的结合是不稳定的。 C7的远程变构口袋，产生C7的催化硫醇盐（Cys 186）以非- 生产构象（指向P1口袋而不是活性位点）。另一个重要 这些变构抑制复合物的特征是晶体学B因子的大量增加 （相对于未抑制的C7的晶体结构）的一些重要的环区域。这项建议 将专注于获得许多其他不同的变构效应物的高分辨率结构， 来自我们的FBDD活动，尚未与C7共结晶，包括13个已确认的 抑制剂、5种不抑制的粘合剂和1种活化剂;完成这项工作将提供 结构Rosetta Stone（直接比较抑制剂、非抑制剂和活化剂能力） 了解C7的催化能力如何受到变构口袋的远程连接的影响。后 成功完成，我们不仅会了解到C7化学空间占据的变构 口袋会导致抑制，但更重要的是，我们将知道这些远程绑定的物种是如何 从而抑制C7的催化能力