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

脊髓延髓肌萎缩症（SBMA）是由雄激素受体（AR）基因CAG/谷氨酰胺束扩张引起的下运动神经元和骨骼肌退行性疾病。多聚谷氨酰胺AR（polyQ AR）经历了依赖性核转位和解折叠，这些步骤对毒性和男性进行性肌无力的发展至关重要。为了减轻这种毒性，我们将寻求一种新的策略，通过靶向基于Hsp 90/Hsp 70的伴侣机制来降解致病蛋白。我们先前已经证明，热休克蛋白70的变构调节有利于ADP结合状态促进聚Q AR泛素化和降解。本申请的目的是鉴定稳定Hsp 70的ADP结合状态并促进毒性蛋白的清除的先导小分子。我们的方法是基于初步研究，表明在ADP结合状态下，Hsp 70的热稳定性增加了13°C。我们最初使用这种方法从NIH临床收藏库中手动筛选727种小分子，并发现一种化合物埃索美拉唑，它使Hsp 70的熔化温度增加了近10°C。我们证实了这种化合物的活性是立体特异性的，并且在二级测定中它增强了Hsp 70依赖性泛素化和客户蛋白清除。由于埃索美拉唑的效力较低，我们将该测定小型化为384孔格式，并对35，840种化合物进行多重初步筛选（Z评分为0.8）。该测定在去卷积和剂量滴定后鉴定了40种具有活性的化合物。这些初步结果表明，我们已经开发出一种强大的高通量筛选，以寻找一种有效的，更有用的化合物。我们提出了以下具体目标，以确定一个足够活性的化合物，将稳定ADP结合的构象的Hsp 70和增强泛素化和降解的polyQ AR。在本申请的R21阶段，我们将从35，840种化合物开始，完成对Hsp 70稳定剂的稳健中试筛选。此外，我们将开发和执行基于ThermoFluor的计数筛选和二次筛选，以使用定量ELISA评估对泛素化和polyQ AR清除的影响。定量里程碑标志着R21/R33过渡。R33阶段旨在对另外130，000种化合物进行高通量筛选，并通过药物化学，NMR和计算方法优化活性物质。在完成这些研究时，我们期望已经鉴定出Hsp 70的小分子稳定剂的一到两个化学支架，其作用有利于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治疗网络赠款，以促进最有前途的药物的临床发展。

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.