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

脊髓和球性肌萎缩症（SBMA）是一种由雄激素受体（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。聚谷氨酰胺AR （polyQ AR）经历激素依赖的核易位和展开，这是毒性和男性进行性肌肉无力发展所必需的步骤。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Hsp90/ hsp70的伴侣蛋白机制来降解致病蛋白。我们之前已经证明，Hsp70的变构调节有利于ADP结合状态，促进polyQ AR泛素化和降解。本应用的目的是鉴定一种先导小分子，稳定Hsp70的adp结合状态，促进有毒蛋白的清除。我们的方法是基于初步研究表明，当处于adp结合状态时，Hsp70的热稳定性提高了13°C。我们最初使用该方法人工筛选来自NIH临床收集文库的727个小分子，发现一种化合物，埃索美拉唑，可以将Hsp70的熔化温度提高近10°C。我们证实了该化合物的活性是立体特异性的，并且在二次分析中，它增强了hsp70依赖的泛素化和客户蛋白清除。由于埃索美拉唑的效价较低，我们将该试验缩小到384孔格式，并对35,840种化合物进行了多路主筛选（z评分为0.8）。经反褶积和剂量滴定，鉴定出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方法来寻找有效的、更有用的化合物。我们提出了以下具体目标，以确定一种足够活跃的化合物，以稳定Hsp70的adp结合构象，并增强polyQ AR的泛素化和降解。在该申请的R21阶段，我们将从35,840个化合物开始完成Hsp70稳定剂的稳健中试筛选。此外，我们将开发并执行基于热荧光的反筛选和二次筛选，以使用定量elisa评估泛素化和polyQ AR清除的影响。定量里程碑标志着R21/R33的过渡。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、核磁共振和计算方法优化活性。在这些研究完成后，我们希望能够鉴定出1 - 2种Hsp70小分子稳定剂的化学支架，它们有利于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Blueprint神经治疗网络资助，以促进最有希望的药物的临床开发。

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.