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.

我们的目标是为人类 caspase-7 (C7) 的变构提供物理和化学原理 控制，特别是由小分子控制。 Caspase 失调，催化和自催化， 与许多神经退行性疾病、炎症性疾病和癌症有关。由于一个 高电荷活性位点，具有低成药性和选择性问题，活性位点的开发 抑制剂一直是个问题。然而，人们对靶向变构 C7 的口袋，距离活性位点超过 17 Å。当前提案包含数据 这代表着我们对药物样小分子如何发挥作用的理解取得了突破性进展 用于变构“降低”C7 的活性。初步的基础工作已经成为可能 基于片段的药物发现 (FBDD) 的进步，其中包括化学物质的融合 信息学、生物物理方法，如表面等离子共振、X 射线晶体学和 分子动力学。本提案中描述的工作集中于我们发现的一系列 结合在这个变构袋中的可逆变构抑制剂，这是第一个类药物化合物 在半胱天冬酶中显示这种活性。从我们的 FBDD 活动中获得的变构效应器是 用于阐明受抑制复合物的几种高分辨率晶体结构，揭示了 该酶的特异性变构控制的前进方向。 FBDD 的使用，如我们两个所示 抑制高分辨率晶体结构 PDB-ID 5V6U 和 5V6Z 为我们提供了第一个理性的 刽子手半胱天冬酶可逆变构抑制剂的结构-活性关系的基础 药物靶标的类别。这两个结构清楚地表明变构抑制剂与 C7 的远程变构口袋，产生具有 C7 催化硫醇盐 (Cys186) 取向的结构 生产性构象（指向 P1 口袋而不是活性位点）。另一个重要的 这些变构抑制复合物的特点是晶体 B 因子大幅增加 （相对于不受抑制的 C7 的晶体结构）许多重要的环区域。这个提议 将重点关注获得许多其他不同变构效应子的高分辨率结构 来自我们的 FBDD 活动，尚未与 C7 共同结晶，其中包括 13 个已确认的活动 抑制剂、5 种不抑制的结合剂和 1 种激活剂；这项工作的完成将提供 结构罗塞塔石碑（直接比较抑制剂、非抑制剂和激活剂的能力） 了解变构袋处的远程连接如何影响 C7 的催化能力。之上 成功完成，我们不仅将了解 C7 变构中的化学空间占用情况 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 从而抑制 C7 的催化能力。