SPAK inhibitor ZT-1a for ischemic stroke therapy

项目摘要 中风仍然是第五大死亡原因， 在美国，长期的成人残疾，因为只有~15% 患者可以 从目前的标准疗法中获益（ 组织纤溶酶原激活剂与血管内 再通），因为它们的治疗窗短。因此，识别新的中风治疗靶点 神经保护药物仍然是一个未满足的迫切需求。高血压是最重要的危险因素 血管紧张素II（Ang II）诱导的神经源性高血压与脑卒中流行病的恶化有关。 缺血性脑损伤然而，临床试验研究显示，中风后血压（BP）没有任何好处- 降低缺血性卒中急性期治疗对改善死亡或残疾风险的作用， 它会阻碍脑灌注。这些发现促使我们开发一种新的策略来阻断AngII介导的 在缺血性脑中的有害作用，而不降低血压和脑灌注。 丝氨酸-苏氨酸WNK激酶家族[不含赖氨酸（K）]及其两种下游激酶SPAK（丝氨酸-苏氨酸WNK激酶家族）， STE 20/SPS 1相关的脯氨酸/富含丙氨酸的激酶）和OSR 1（氧化应激反应激酶1）激活 通过蛋白质磷酸化的多离子转运蛋白和通道。Na+-K+-2Cl-协同转运蛋白亚型1（NKCC 1） 是WNK-SPAK/OSR 1激酶的主要底物之一。刺激 的 WNK-SPAK激酶类 通过蛋白磷酸化增加脑NKCC 1活性，并通过NKCC 1- 介导的Na+和Cl-超负荷、细胞毒性水肿和兴奋性毒性。我们的初步研究表明，血管紧张素II诱导 高血压小鼠在缺血性脑损伤中WNK-SPAK-NKCC 1蛋白复合物表达增加2-5倍。 脑，这是伴随着恶化的结果，梗死，水肿，和神经功能缺损， 永久性大脑中动脉闭塞模型（pdMCAO）。卒中后给予新型非ATP 在这些小鼠中，竞争性选择性SPAK抑制剂ZT-1a显著减少了梗死和水肿， 改善神经功能恢复。然而，Ang II信号通路如何调节WNK-SPAK-NKCC 1 ZT-1a在Ang II诱导的缺血性脑损伤中的蛋白复合物表达和功效 高血压小鼠仍然未知。我们假设（1）Ang II刺激Ang II受体亚型1（AT 1 R）- NF-κB级联反应导致卒中后WNK-SPAK-NKCC 1信号复合物上调;（2）升高 WNK-SPAK-NKCC 1信号直接导致缺血性神经元损伤和神经功能损害的恶化。 （3）中风后给予新型SPAK激酶抑制剂ZT-1a可减少缺血性脑损伤 通过防止大脑SPAK-NKCC 1信号的过度激活。这些假设将在三个测试 具体目标。总之，我们研究了血管紧张素II诱导的高血压并发症的原因， 缺血性中风的结果部分地通过刺激CNS中的WNK-SPAK-NKCC 1信号传导途径。 这项研究的完成将使我们能够获得新的知识，是否靶向脑WNK-SPAK-NKCC 1 信号通路将改善缺血性卒中伴高血压患者的预后。

PROJECT ABSTRACT Stroke remains as the 5th leading cause of death and long-term adult disability in the USA because only ~15% patients can benefit from current standard therapies ( tissue plasminogen activator and endovascular recanalization) due to their short therapeutic windows. Therefore, identification of novel stroke therapeutic targets for neuroprotective drugs remains to be an unmet urgent need. Hypertension is the most significant risk factor for stroke epidemics and angiotensin II (Ang II)-induced neurogenic hypertension is associated with worsened ischemic brain damage. However, the clinical trial studies show no benefits of post-stroke blood pressure (BP)- lowering treatment in the acute stage of ischemic stroke on improvement of the risk of death or disability because it hinders cerebral perfusion. These findings challenge us to develop a novel strategy to block the AngII -mediated detrimental effects in the ischemic brains without lowering BP and cerebral perfusion. The serine-threonine WNK kinase family [with no lysine (K)], and its two downstream kinases SPAK (the STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) activate multiple ion transporters and channels via protein phosphorylation. Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) is one of the major substrates of the WNK-SPAK/OSR1 kinases. Stimulation of the WNK-SPAK kinases increased brain NKCC1 activity via protein phosphorylation and led to ischemic cell damage through NKCC1- mediated Na+ and Cl- overload, cytotoxic edema and excitotoxicity. Our pilot study reveals that Ang II-induced hypertensive mice exhibited 2-5 fold increase in the WNK-SPAK-NKCC1 protein complex expression in ischemic brains, which was accompanied with worsened outcomes in infarct, edema, and neurological deficit in the permanent middle cerebral artery occlusion model (pdMCAO). Post-stroke administration of a novel, non-ATP competitive, selective SPAK inhibitor ZT-1a in these mice significantly reduced infarction and edema, and improved neurological function recovery. However, how Ang II signaling pathway regulates WNK-SPAK-NKCC1 protein complex expression and efficacy of ZT-1a in reducing ischemic brain injury in the Ang II-induced hypertensive mice remains unknown. We hypothesize that (1) Ang II stimulates Ang II receptor subtype 1 (AT1R)- NF-κB cascade which leads to upregulation of WNK-SPAK-NKCC1 signaling complex after stroke; (2) elevated WNK-SPAK-NKCC1 signaling directly contributes to the worsened ischemic neuronal damage and neurological deficits; (3) post-stroke administration of the novel SPAK kinase inhibitor ZT-1a reduces ischemic brain damage by preventing excessive activation of brain SPAK-NKCC1 signaling. These hypotheses will be tested in three specific aims. In summary, we investigate that Ang II-induced hypertension comorbidity causes worsened ischemic stroke outcome in part via stimulating the WNK-SPAK-NKCC1 signaling pathway in the CNS. Completion of this study will enable us to gain new knowledge on whether targeting brain WNK-SPAK-NKCC1 signaling pathway will improve outcomes of ischemic stroke patients with comorbid hypertension.