SPAK inhibitor ZT-1a for ischemic stroke therapy

项目摘要 中风仍然是第五大死亡原因， 在美国长期的成年人残疾，因为只有15% 患者可以 受益于当前的标准疗法( 组织型纤溶酶原激活剂与血管内膜 再通)，因为它们的治疗窗口很短。因此，寻找新的卒中治疗靶点 对神经保护药物的需求仍然是一个尚未得到满足的迫切需求。高血压是最重要的危险因素 对于中风流行和血管紧张素II(Ang II)诱导的神经源性高血压与病情恶化有关 缺血性脑损伤。然而，临床试验研究表明，中风后血压(BP)没有好处- 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 它会阻碍大脑的灌流。这些发现挑战我们开发一种新的策略来阻止血管紧张素转换酶介导的 在不降低血压和脑灌注量的情况下，对脑缺血有不利影响。 丝氨酸-苏氨酸WNK激酶家族[不含赖氨酸(K)]及其下游的两个蛋白Spak( STE20/SPS1相关的富含脯氨酸/丙氨酸的激酶)和氧化应激反应蛋白1(OSR1)被激活 通过蛋白质磷酸化的多个离子转运体和通道。Na+-K+-2Cl-共转运蛋白亚型1(NKCC1) 是WNK-Spak/OSR1激酶的主要底物之一。刺激 这个 WNK-SPAK激酶 通过蛋白磷酸化增加脑内NKCC1活性，并通过NKCC1-1导致缺血细胞损伤 介导Na+、Cl-超载、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 高血压小鼠脑缺血时WNK-Spak-NKCC1蛋白复合体表达增加2-5倍 大脑，伴随着脑梗塞、水肿和神经功能障碍的恶化结局 永久性大脑中动脉闭塞模型(PdMCAO)。一种新型非三磷酸腺苷的卒中后给药 在这些小鼠中，竞争性的选择性Spak抑制剂ZT-1a显著减少了脑梗塞和水肿，并 改善神经功能恢复。然而，Ang II信号通路如何调控WNK-Spak-NKCC1 ZT-1a蛋白复合体的表达及对血管紧张素Ⅱ诱导的缺血性脑损伤的保护作用 高血压小鼠仍不清楚。我们假设(1)Ang II刺激Ang II受体亚型1(AT1R)- 核因子-κB级联导致卒中后WNK-SPAK-NKCC1信号复合体上调；(2)升高 WNK-SPAK-NKCC1信号直接参与缺血性神经元损伤加重和神经功能损害 (3)卒中后应用新型Spak激酶抑制剂ZT-1a可减少缺血性脑损伤 通过防止大脑Spak-NKCC1信号的过度激活。这些假说将在三个方面进行检验 明确的目标。综上所述，我们调查了血管紧张素转换酶II引起的高血压共病恶化的原因。 缺血性卒中的结果部分是通过刺激中枢神经系统中的WNK-SPAK-NKCC1信号通路实现的。 这项研究的完成将使我们能够获得新的知识，即靶向大脑WNK-Spak-NKCC1 信号通路将改善合并高血压的缺血性卒中患者的预后。

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.