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

基本信息

批准号：
10439889
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
项目类别：
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/10439889
关键词：
Active Sites Acylation Affect Apoptosis Apoptotic Binding Biophysics CASP7 gene Caspase Cells Charge Chemicals 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 insight interest molecular dynamics novel response screening simulation small molecule

项目摘要

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，特别是通过小分子的半胱天冬酶失调，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。高电荷活性位点，具有低成药性和选择性问题，活性位点的开发然而，人们对靶向变构抑制剂产生了巨大的兴趣。 C7 的口袋，距离活性位点超过 17 Å 当前的提案包括数据。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 的活性这一初步基础工作已成为可能。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 射线晶体学和本提案中描述的工作集中于我们一系列的发现。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为了显示半胱天冬酶中的这种活性，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，如我们的两个所示。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-活性关系的基础这两种结构清楚地表明变构抑制剂与药物靶点的结合。 C7 的远程变构口袋，产生 C7 的催化硫醇盐 (Cys186) 定向于非-的结构生产性构象（指向 P1 口袋而不是活性位点）。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。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 共结晶，其中包括 13 个已确认的抑制剂、5种不抑制的结合剂和一种激活剂，完成这项工作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）了解变构袋上的远程连接如何影响 C7 的催化能力。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。