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: 10649449
• 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 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10649449
• 关键词: Active Sites; Acylation; Affect; Apoptosis; Apoptotic; Binding; Biophysics; CASP7 gene; Caspase; Cells; Charge; Chemicals; Complex; Crystallization; Crystallography; 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; Proliferating; Property; Resolution; Role; Scheme; Series; Source; Specificity; Stimulus; Structural Models; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; System; Testing; Work; X-Ray Crystallography; biophysical techniques; cheminformatics; deacylation; drug discovery; drug-like compound; flexibility; genetic regulatory protein; inhibitor; 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.

我们的目标是为人类caspase-7(C7)如何变构提供物理和化学基础 受控制的，尤指受小分子控制的。半胱氨酸天冬氨酸酶的失调，包括催化和自身催化， 与许多神经退行性、炎症性疾病和癌症有关。由于一项 高电荷活性部位，具有较低的可药性和选择性问题，开发活性部位 抑制剂一直是个问题。然而，人们一直对以变构为目标的兴趣巨大 C7的口袋，它位于离活动地点超过17ä的地方。目前的提案包括数据 这代表着我们在理解类药物小分子如何可能 用来变构“降低”C7的活性。这一初步的基础工作已经成为可能。 通过基于片段的药物发现(FBDD)的进展，其中包括化学合成 信息学、生物物理方法，如表面等离子体共振、X射线结晶学和 分子动力学。这项提案中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，这是第一个类似药物的化合物 以显示半胱氨酸天冬氨酸酶的这种活性。从我们的FBDD活动中获得的变构效应器是 用于阐明被抑制的络合物的几个高分辨晶体结构，这揭示了一个 这种酶的特定变构控制的前进方向。FBDD的使用，如我们的两个例子所示 被抑制的高分辨率晶体结构，PDB-ID 5V6U和5V6Z，为我们提供了第一个合理的 执行者半胱氨酸天冬氨酸酶可逆变构抑制剂的构效关系基础 毒品靶子的类别。这两种结构清楚地表明变构抑制剂与 C7的远程变构口袋，生成具有C7‘S催化硫代(Cys186)定向的结构 生产性构象(指向P1口袋而不是活性部位)。另一项重要的 这些变构抑制络合物的特征是结晶学B因子大幅增加 (相对于未被抑制的C7的晶体结构)一些重要的环区。这项建议 将专注于获得许多其他不同的变构效应器的高分辨率结构 来自我们的FBDD活动，尚未与C7共结晶，包括13个已确认的 缓蚀剂、5种不抑制的粘结剂和1种活化剂；完成这项工作将提供 结构性Rosetta Stone(直接比较抑制剂、非抑制剂和激活剂的能力) 了解远程结扎变构口袋对C7‘S催化能力的影响。vt.在.的基础上 成功完成后，我们不仅可以了解到C7变构中占据了哪些化学空间 口袋会导致抑制，但更重要的是，我们将知道这些远程结合的物种是如何 实现了对C7‘S催化能力的抑制。