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: 10027338
• 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/10027338
• 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.

我们的目标是为人类caspase-7（C7）如何变构提供物理和化学原理 控制，特别是小分子。 caspase失调，催化和自催化， 在许多神经退行性，炎症性疾病和癌症中已隐含。由于一个 高电动的活跃部位，可药用性低和选择性问题，活动现场的发展 抑制剂是有问题的。但是，针对变构具有很大的兴趣 C7的口袋，与活动地点相距超过17Å。当前的建议包括数据 这代表了我们对小药物样分子的理解的突破性进步 用于变构“拨打” C7的活动。最初的基础已成为可能 通过基于碎片的药物发现的进步（FBDD），其中包括化学融合 信息信息，生物物理方法，例如表面等离子体共振，X射线晶体学和 分子动力学。该提案中描述的工作集中在我们发现一系列 在这个变构袋中结合的可逆变构抑制剂，这是第一个类似药物的化合物 在caspase中显示这种活动。从我们的FBDD运动中获得的变构效应是 用于阐明抑制复合物的几个高分辨率晶体结构，这表明 该酶的特定变构控制的前进方向。使用FBDD的使用，如我们两个所示 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 对于executioner caspase的可逆变构抑制剂的结构活动关系的基础 药物靶标类。这两个结构清楚地表明，变构抑制剂与 C7的远程变构袋，产生以C7的催化硫醇（CYS186）为导向 生产构象（指向P1袋而不是进入活动地点）。另一个重要 这些变构抑制复合物的特征是晶体学B因子大大增加 （相对于许多重要环区域的未抑制C7的晶体结构。这个建议 将专注于获得许多其他不同变构效应的高分辨率结构 从我们尚未与C7共结合的FBDD广告系列中，包括13个已确认 抑制剂，5个不抑制的粘合剂和一个激活剂；这项工作的完成将提供 结构性Rosetta石头（可以直接比较抑制剂，非抑制剂和激活剂的能力） 了解C7的催化能力如何受到变构袋中远程连接的影响。之上 成功完成，我们不仅会了解C7变构中的化学空间占用率 口袋会导致抑制作用，但更重要的是，我们将知道这些远程约束的物种是如何 达到C7催化能力的抑制作用。