Development of a therapeutic Kv7 opener to restore motor excitability in ALS

项目总结 肌萎缩侧索硬化症(ALS)是最具破坏性的神经退行性疾病之一，目前尚无治疗方法 显著延长患者的生命。因此，需要治疗肌萎缩侧索硬化症的新疗法。QurAlis正在接近这一点 需要纠正导致运动轴突神经病变和神经变性的过度兴奋。这 过度兴奋与Kv7.2/7.3传导的延迟整流钾(K+)电流的缺陷有关 电压门控离子通道。大约50%的肌萎缩侧索硬化症患者的运动有明显的过度兴奋 皮质和脊髓运动神经元，并可使用神经生理学生物标志物进行鉴定。雷替加宾，一种抗- 癫痫和Kv7.2/7.3的正向调节剂，最近被证明逆转这种过度兴奋 ALS患者的临床试验，但不幸的是，与非靶点效应有关，不再可用。 QurAlis的目标是生产一种更有效和更具体的Kv7.2/7.3激活剂。在第一阶段，目标1，我们将制定一个 基于转录重编程的iMN生产的协议。在目标2中，我们将验证iMNS细胞 与使用标准图案化系数编程的iMN的特性进行比较，并将验证 使用OptopatchTM平台的iMN的特性。在目标3中，我们将生产一个大规模的超低温储存库 对IMN的研究。在第一阶段的结论中，我们将达到iMN纯度的80%，并证明这些细胞 是电活动的，并且对雷替加宾的反应具有浓度依赖性的神经元活动减少 EC50在1到5微米之间。此外，我们将有足够规模的IMN生产来执行 化学结构-活性关系筛选(例如，至少1.5亿个细胞的冷冻批次)。在第二阶段， 目的4，我们将开发新的化学物质并测试它们对Kv7.2/7.3的调节能力 结构活性分析，在目标5中，我们将识别降低患者来源的iMN兴奋性的化合物。 在第二阶段结束时，我们将确定10-20种化合物，这些化合物符合我们的目标产品配置文件 目标和非目标效力(目标4里程碑)，我们将有两个或更多化合物改变Kv7.2/7.3 患者来源的iMN的活性在其作用大小大于或等于雷替加宾的情况下导致兴奋性降低 临床有效剂量2微米，目标5里程碑)。我们有初步数据表明我们的方法 目标是可行的：我们有3个独立的新型化学支架，它们是Kv7.2/7.3的有效激活剂 它的活性已经在异构体离子通道和患者来源的MNS(传统的 协议)使用Optopatch平台。在这项研究结束时，我们将确定几个线索 准备好进行ADME、PK和非GLP毒理学研究的化合物，我们将有一个扩大的方案 用于生产患者来源的iMN，这将促进针对ALS其他途径的未来药物开发 患者(例如，自噬)。

PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) is one of the most devastating neurodegenerative diseases and no therapies significantly extend patient lives. Thus, new therapeutics to treat ALS are needed. QurAlis is approaching this need by correcting the hyperexcitability that leads to motor axon neuropathy and neurodegeneration. This hyperexcitability is linked to deficits in the delayed-rectifier potassium (K+) current conducted by Kv7.2/7.3 voltage-gated ion channels. Approximately 50% of ALS patients have significant hyperexcitability in their motor cortex and spinal motor neurons and can be identified using neurophysiological biomarkers. Retigabine, an anti- epileptic and positive modulator of Kv7.2/7.3, has recently been shown to reverse this hyperexcitability in a clinical trial of ALS patients but unfortunately, is associated with off-target effects and is no longer available. QurAlis’s goal is to produce a more potent and specific Kv7.2/7.3 activator. In Phase I, Aim 1, we will develop a protocol for transcriptional reprogramming-based production of iMNs. In Aim 2, we will validate the iMNs cellular properties compared to iMNs programmed using standard patterning factors and will validate the electrical properties of iMNs using the OptopatchTM platform. In Aim 3, we will produce a large-scale cryopreserved bank of iMN. At Phase I conclusion, we will have achieved an iMNs purity >80% and demonstrated that these cells are electrically active and have a concentration-dependent reduction in neuronal activity in response to retigabine with an EC50 between 1 and 5 µM. In addition, we will have iMN production at a sufficient scale to perform chemical structure-activity relationship screening (e.g., frozen batches of at least 150 million cells). In Phase II, Aim 4, we will develop new chemical matter and test compounds for their ability to modulate Kv7.2/7.3 in structure-activity assays, and in Aim 5 we will identify compounds that reduce excitability in patient-derived iMNs. At conclusion of Phase II, we will have identified 10-20 compounds that meet our target product profile for on- target and off-target potency (Aim 4 milestone) and we will have two or more compounds that shift Kv7.2/7.3 activity in patient-derived iMNs toward reduced excitability at an effect size greater or equal to retigabine at its clinically efficacious dose of 2 µM. Aim 5 milestone). We have preliminary data demonstrating that our approach and goals are feasible: we have 3 independent novel chemical scaffolds that are potent activators of Kv7.2/7.3 whose activity has been validated in both heteromeric ion channels and in patient-derived MNs (traditional protocol) using the Optopatch platform. At conclusion of this study, we will have identified several lead compounds that are ready for ADME, PK, and non-GLP toxicology studies, and we will have a scaled-up protocol for producing patient-derived iMNs that will facilitate future drug development that target other pathways in ALS patients (e.g., autophagy).