Small molecule stabilizers of Hsp70 for treatment of spinal and bulbar muscular atrophy

Hsp70小分子稳定剂治疗脊髓和延髓性肌萎缩症

基本信息

批准号：
9812011
负责人：
ANDREW P LIEBERMAN
金额：
$ 38.91万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-12-01 至 2020-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9812011
关键词：
Allosteric Regulation Androgen Receptor Binding Biological Biological Assay Biological Models Cell model Chemicals Client Clinic Clinical Collection Degenerative Disorder Development Disease Dose Drosophila genus Dyes Enzyme-Linked Immunosorbent Assay Esomeprazole Future Glutamine Grant Hip region structure Hormones Human Lead Length Libraries Manuals Measures Mediator of activation protein Metabolic Molecular Chaperones Molecular Conformation Motor Neurons Muscle Weakness Neurodegenerative Disorders Nuclear Translocation Oranges Pathway interactions Patients Pharmaceutical Chemistry Phase Proteins Public Health Receptor Gene Role Skeletal Muscle Stabilizing Agents Structure Supportive care Temperature Testing Therapeutic Titrations Toxic effect Ubiquitination United States National Institutes of Health Work assay development base chaperone machinery counterscreen disease-causing mutation drug discovery high throughput screening improved melting men miniaturize mouse model novel strategies novel therapeutics overexpression polyglutamine protein aggregation protein degradation receptor scaffold small molecule spinal and bulbar muscular atrophy therapeutic target thermostability ubiquitin-protein ligase

项目摘要

Spinal and bulbar muscular atrophy (SBMA) is a degenerative disorder of lower motor neurons and skeletal muscle caused by a CAG/glutamine tract expansion in the androgen receptor (AR) gene. The polyglutamine AR (polyQ AR) undergoes hormone-dependent nuclear translocation and unfolding, steps that are essential to toxicity and to the development of progressive muscle weakness in men. To alleviate this toxicity, we will pursue a novel strategy to degrade the disease-causing protein by targeting the Hsp90/Hsp70-based chaperone machinery. We have previously demonstrated that allosteric regulation of Hsp70 to favor the ADP- bound state promotes polyQ AR ubiquitination and degradation. The objective of this application is to identify a lead small molecule that stabilizes the ADP-bound state of Hsp70 and promotes clearance of the toxic protein. Our approach is based on preliminary studies demonstrating that the thermostability of Hsp70 increases by 13°C when in the ADP-bound state. We initially used this assay to manually screen 727 small molecules from the NIH Clinical Collection library and found one compound, esomeprazole, that increases the melting temperature of Hsp70 by nearly 10°C. We confirmed that activity of this compound is stereospecific, and that in secondary assays it enhances Hsp70-dependent ubiquitination and client protein clearance. As esomeprazole has low potency, we miniaturized this assay to 384-well format and performed a multiplexed, primary screen of 35,840 compounds (Z-score of 0.8). The assay identified 40 compounds with activity after deconvolution and dose titration. These initial results indicate that we have developed a robust high-throughput screen to search for a potent, more useful compound. We propose the following specific aims to identify a sufficiently active compound that will stabilize the ADP-bound conformation of Hsp70 and enhance ubiquitination and degradation of the polyQ AR. In the R21 phase of this application, we will complete a robust pilot screen for stabilizers of Hsp70 starting with a collection of 35,840 compounds. Additionally, we will develop and perform a ThermoFluor-based counter-screen and secondary screens to assess effects on ubiquitination and polyQ AR clearance using quantitative ELISAs. Quantitative milestones mark the R21/R33 transition. The R33 phase is aimed at performing the high-throughput screen of an additional 130,000 compounds, and optimizing actives by medicinal chemistry, NMR and computational approaches. At the completion of these studies, we expect to have identified one-two chemical scaffolds of small molecule stabilizers of Hsp70 that act to favor the ADP conformation and enhance ubiquitination and degradation of polyQ AR. Our long-term plans are to apply for a Blueprint Neurotherapeutics Network grant to facilitate development of the most promising agents to the clinic.

脊柱和鳞茎肌肉萎缩（SBMA）是由雄激素受体（AR）基因中CAG/谷氨酰胺的扩张引起的下运动神经元和骨骼肌的退化性疾病。聚谷氨酰胺AR（Polyq AR）经历了马酮依赖性的核易位和展开，这对于毒性至关重要，对男性进行性肌肉无力的发展。为了减轻这种毒性，我们将采取一种新的策略，以靶向基于HSP90/HSP70的伴侣机械来降解引起疾病的蛋白质。我们先前已经证明，HSP70的变构调节有利于ADP结合状态，促进了PolyQ AR泛素化和降解。该应用的目的是确定铅的小分子，该分子稳定HSP70的ADP结合状态并促进毒性蛋白质的清除。我们的方法基于初步研究，表明在ADP结合状态下，HSP70的热稳定性增加了13°C。我们最初使用该测定法从NIH临床收集文库手动筛选了727个小分子，并发现了一种化合物Enameprazole，将HSP70的熔化温度提高了近10°C。我们证实了该化合物的活性是立体特异性的，并且在次要测定中，它增强了HSP70依赖性的泛素化和客户蛋白清除率。由于埃索美拉唑的效力较低，因此我们将该测定法至384孔格式，并执行了35,840种化合物的多路复用，主筛选（z得分为0.8）。该测定法确定了40种反卷积和剂量滴定后活性的化合物。这些初始结果表明，我们已经开发了强大的高通量屏幕，以搜索潜在的，更有用的化合物。我们提出了以下特定目的，以识别足够活跃的化合物，该化合物将稳定HSP70的ADP结合构象并增强Polyq AR的泛素化和降解。在此应用程序的R21阶段中，我们将完成一个稳定的试点屏幕，以启用HSP70的稳定剂，从35,840种化合物的集合开始。此外，我们将开发和执行基于热氟的反屏幕和二级屏幕，以评估使用定量ELISAS对泛素化和PolyQ AR清除率的影响。里程碑标志着R21/R33过渡。 R33阶段旨在执行另外130,000种化合物的高通量屏幕，并通过药物化学，NMR和计算方法优化活性。这些研究完成后，我们希望已经确定了HSP70的小分子稳定剂的一两个化学支架，这些支架有利于ADP构象并增强PolyQ AR的泛素化和降解。我们的长期计划是申请蓝图神经疗法网络赠款，以支持诊所最有前途的代理商的开发。