PIKFYVE antagonism as a therapy for C9ORF72-ALS/FTD

PIKfyve拮抗剂治疗C9ORF72-ALS/FTD的优化与验证 项目摘要/摘要 C9ORF72重复扩增突变是肌萎缩侧索硬化症(ALS)和 额颞性痴呆(FTD)，占北欧ALS病例的50%以上，占病例的10% 使其成为治疗干预的关键目标。使用特定于患者的干细胞 疾病模型、动物模型和死后组织分析，我们已经确定了一个新的治疗靶点 C9ORF72 ALS/FTD，脂蛋白激酶PIKfyve。我们发现抑制PIKfyve可以挽救内体 C9ORF72运动神经元的运输缺陷，并恢复正常的运动神经元存活。PIKfyve函数 以与图4相反的方式，图4是一种功能丧失突变导致肌萎缩侧索硬化症的磷酸酶。反义 寡核苷酸介导的PIKfyve基因敲除挽救了C9-ALS运动神经元的存活，没有表现出 任何对控制运动神经元的毒性。这一结合的功能和遗传证据有力地表明 PIKfyve激酶的小分子抑制是C9-ALS/FTD的一个可行的治疗靶点。 我们发现Apilimod可以逆转生存和其他功能缺陷，是一种有效的PIKfyve 小分子抑制剂。阿普利莫德已经在没有患者参与的情况下进行了临床测试 观察到了毒性，我们已经确定它在小鼠身上耐受性很好。我们已经开始执行一项 双管齐下的策略，以产生新的PIKfyve抑制剂。第一种方法侧重于重新搭建房屋。 阿普利莫德创造一种可以跨越血脑屏障的小分子并可以申请专利。作为后备，我们的第二个 方法使用虚拟筛选来识别抑制PIKfyve的新的、可申请专利的化学类型。对这件事 最后，我们构建了人类PIKfyve的3D同源蛋白质模型，并在 重新构筑以产生几个阿普利莫德类似物。我们采用了PIKfyve同调模型和 薛定谔的小分子药物发现套件，筛选出800多万种可用的化合物 虚拟来自ICAGEN和E-分子电子化合物系列。开展结构性活动 关系(SAR)对于这个项目，我们建立了一种生化PIKfyve激酶测定作为主要的 所有化合物评估的化验。 这个快速通道项目的目标是确定一种有效的PIKfyve抑制剂，它是血脑屏障 很有穿透力。在第一阶段，我们将使用Apilimod，表1中的6个类似物和3个最有希望的线索 在硅胶屏幕上测试我们的整个漏斗，包括活体测试。在第二阶段，我们将使用这些化验来 优化和验证开发候选对象。我们的具体目标是(第一阶段)1)验证初级和 二次体外检测；2)三级检测的验证；3)概念验证检测；(第二阶段)1) 一次和二次分析中的化合物优化；2)化合物安全性和给药的优化 3)建立C9ORF72 ALS/FTD的体内概念验证。

Optimization and validation of PIKFYVE antagonism as a therapy for C9ORF72-ALS/FTD Project Summary / Abstract The C9ORF72 repeat expansion mutation is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), accounting for over 50% of ALS cases in northern Europe and 10% of cases worldwide, making it a critical target for therapeutic intervention. Using patient-specific stem cell-based disease models, animal models, and postmortem tissue analysis, we have identified a new therapeutic target for C9ORF72 ALS/FTD, the lipid kinase PIKFYVE. We find that inhibition of PIKFYVE rescues the endosomal trafficking defects in C9ORF72 motor neurons and restores normal motor neuron survival. PIKFYVE functions in a manner that opposes FIG4, a phosphatase for which a loss-of-function mutation causes ALS. Antisense oligonucleotide-mediated knockdown of PIKFYVE rescues C9-ALS motor neuron survival, without exhibiting any toxicity toward control motor neurons. This combined functional and genetic evidence strongly indicate that small molecule inhibition of PIKFYVE kinase is a viable therapeutic target for C9-ALS/FTD. We have found that Apilimod reverses survival and other functional defects and is an effective PIKFYVE small molecule inhibitor. Apilimod has been tested in the clinic where target engagement without patient toxicity was observed, and we’ve determined it to be well tolerated in mice. We have begun execution of a two-pronged strategy to generate novel inhibitors of PIKFYVE. The first approach focuses on rescaffolding Apilimod to create a small molecule that can cross the BBB and can be patented. As a backup, our second approach employs virtual screening to identify new, patentable chemotypes which inhibit PIKFYVE. To this end, we have constructed 3D homology protein models for human PIKFYVE which we’ve used productively in rescaffolding to generate several Apilimod analogs. We employed the PIKFYVE homology models and the Small Molecule Drug Discovery Suite from Schrodinger and screened over 8 million compounds available virtually from the Icagen and E-molecule electronic compound collections. To develop structure activity relationships (SAR) for this program, we have established a biochemical PIKFYVE kinase assay as the primary assay for all compound evaluations. The goal of this Fast Track project is to identify a potent PIKFYVE inhibitor that is blood-brain-barrier penetrating. In phase I, we will use Apilimod, the 6 analogs from Table 1 and the 3 most promising leads from the in silico screen to test our entire funnel, including in vivo assays. In Phase II we will use these assays to optimize and validate a development candidate. Our specific aims are (Phase I) 1) Validation of primary and secondary in vitro assays; 2) Validation of tertiary assays; 3) Validation of proof of concept assays; (Phase II) 1) Compound optimization in primary and secondary assays; 2) Optimize of compound safety and administration through tertiary assays; 3) Establish in vivo proof of concept in C9ORF72 ALS/FTD.