Novel Anti-Stroke Agents Targeting Toxic Protein Aggregation

本研究旨在研究有毒新生多肽链（NPC）的错误折叠和聚集在 中风脑损伤中风是一种毁灭性的神经系统疾病，影响数百万退伍军人。疗法来 重建灌注是目前治疗急性卒中的金标准。然而，由于潜在的 出血性副作用，只有约5%的中风患者接受再灌注治疗。 信使RNA（mRNA）翻译成蛋白质也称为NPC合成。本实验室 最初发现大规模的NPC聚集发生在缺血后注定死亡的神经元中。我们 发表了30篇关于这个主题的论文。这些研究表明，NPC的合成在两种情况下受到抑制， 不同的阶段抑制的第一阶段是短暂的，在所有的后灌注中，在再灌注0.5-1小时后完全减弱。 缺血性神经元;因此，与脑缺血后的未来神经元死亡无关。的第一阶段 在所有的缺血后再灌注中，在再灌注4小时抑制之后恢复>70%的NPC合成。 神经元NPC合成抑制的第二阶段从再灌注4 h开始逐渐发生 而且只存在于注定死亡的神经元中。我们的研究表明，抑制的第二阶段是 这是由于缺血后NPC的共翻译伴侣保护的丧失。如果没有这些保护，NPC 在合成过程中暴露出它们的粘性疏水片段，并不可逆地聚集在核糖体上， 脑缺血后再灌注4 h。 这些研究已经导致了新的发现，通过减少大规模NPC聚集， 核糖体上的“未保护的”NPC负载与真核起始因子（eIF）抑制剂可以保护大脑免受 中风损伤通过使用高通量技术，已经研究了几种“直接”eIF 4 E（相互作用）抑制剂。 识别，例如，针对eIF 4 E与eIF 4G和eIF 4 E与4 E-BP 1相互作用的双重抑制剂（4 E1 Rcat），和 利巴韦林。这些发现为理解NPC聚集在 中风脑损伤和开发一类新的抗中风药物。我们的实验室研究了几种eIF 抑制剂的其中，4 EGI-1在小鼠中风模型中提供了最好的抗中风功效。因此，我们使用 4 EGI-1在双盲、随机对照动物研究中。这些研究清楚地表明， 再灌注30分钟后腹腔注射4 EGI-1治疗没有中风死亡率，相比之下， 媒介物组的死亡率。4 EGI-1治疗显著减少了梗死体积，改善了物理 恢复和行为表现，并减少中风后的神经功能缺损。 本研究的目的是进一步探讨NPC聚集在脑卒中中的作用 损伤，并确定最佳的靶点和抑制剂对中风脑损伤（目标1）;并调查 潜在大脑保护机制的详细信息（目标2和3）。我们将检验以下假设：（1） 抑制eIF 4 E不仅减少了缺血后“未保护”的NPC在核糖体上的负荷，而且 将转换从cap-dependent状态切换到cap-independent状态。此开关关闭cap-dependent NPC翻译，同时优先考虑分子伴侣的帽非依赖性翻译;因此， 缺血性“未保护的”NPC在再灌注期间免于毒性聚集。(2)4 EGI-1治疗导致稳健的 上调ATF 4和CHOP转录因子以诱导必需的自噬基因;因此， 清除脑缺血后的有毒NPC聚集体。(3)抑制eIF 4 E可缓解卒中后 炎症虽然抑制其他NPC合成起始因子/调节因子也可以降低NPC的负荷， 核糖体上的“未受保护的”NPC，这些其他上级益处（例如，共翻译分子的上调 通过上述eIF 4 E抑制提供的蛋白质（分子伴侣和自噬蛋白）给予eIF 4 E优势， 治疗目标是减少脑缺血后毒性NPC聚集。eIF 4 E抑制剂显示出优异的生物相容性。 有可能成为退伍军人的一类新的抗中风药物。

This proposal is to study the role of toxic Nascent Polypeptide Chain (NPC) misfolding and aggregation in stroke brain injury. Stroke is a devastating neurological disease, affecting millions of Veterans. Therapies to reestablish perfusion are the current gold standard for treating acute stroke. However, because of the potential hemorrhagic side effect, only about 5% of stroke patients receive reperfusion therapy. Translation of messenger RNA (mRNA) into protein is also known as NPC synthesis. Our laboratory originally discovered that massive NPC aggregation occurs in post-ischemic neurons destined for death. We have published 30 papers on this subject. These studies have shown that NPC synthesis is inhibited in two distinct phases. The first phase of inhibition is transient, abating entirely after 0.5-1 h of reperfusion in all post- ischemic neurons; thus, not correlating with future neuronal death after brain ischemia. This first phase of inhibition is followed by a recovery of >70% of NPC synthesis at 4 h of reperfusion among all post-ischemic neurons. The second phase of NPC synthesis inhibition takes place progressively from 4 h of reperfusion onward and is only in neurons destined to die. Our studies have shown that this second phase of inhibition is due to the post-ischemic loss of co-translational chaperone protection of NPCs. Without this protection, NPCs expose their sticky hydrophobic segments during synthesis and are irreversibly aggregated on ribosomes from 4 h of reperfusion onward after brain ischemia. These studies have led to the novel discovery that targeting massive NPC aggregation by reducing the load of “unprotected” NPCs on ribosomes with eukaryotic initiation factor (eIF) inhibitors can protect brain from stroke injury. By using high throughput technologies, several “direct” eIF4E (interaction) inhibitors have been identified, e.g., a dual inhibitor against eIF4E-to-eIF4G and eIF4E-to-4E-BP1 interactions (4E1Rcat) and ribavirin. These discoveries provide an outstanding opportunity to understand the role of NPC aggregation in stroke brain injury and to develop a novel category of anti-stroke agents. Our laboratory has studied several eIF inhibitors. Among them, 4EGI-1 offers the best anti-stroke efficacy in mouse stroke models. Therefore, we used 4EGI-1 in double-blind, randomized controlled animal studies. These studies clearly demonstrated that mice treated with i.p. injection of 4EGI-1 after 30 min of reperfusion had no stroke mortality compared to the 45% mortality in the vehicle group. 4EGI-1 treatment significantly reduced the infarct volume, improved physical recovery and behavioral performance, and decreased neurological deficits after stroke. The goals of the proposed research are to investigate further the role of NPC aggregation in stroke brain injury and to identify the best targets and inhibitors against stroke brain injury (Aim 1); and to investigate the details of the underlying brain protection mechanisms (Aims 2 and 3). We will test the following hypotheses: (1) Inhibition of eIF4E not only reduces the load of post-ischemic “unprotected” NPCs on ribosomes, but also switches translation from a cap-dependent to a cap-independent state. This switch shuts down cap-dependent NPC translation while prioritizing cap-independent translation of molecular chaperones; thus, protecting post- ischemic “unprotected” NPCs from toxic aggregation during reperfusion. (2) 4EGI-1 treatment leads to a robust upregulation of ATF4 and CHOP transcription factors to induce essential autophagic genes; thus, facilitating the removal of toxic NPC aggregates after brain ischemia. (3) Inhibition of eIF4E mitigates post-stroke inflammation. Although inhibition of other NPC synthesis initiation factors/regulators may also reduce the load of “unprotected” NPCs on ribosomes, these other superior benefits (e.g., upregulation of co-translational molecular chaperones and autophagic proteins) offered by eIF4E inhibition described above give eIF4E an edge as the therapeutic target to reduce toxic NPC aggregation after brain ischemia. The eIF4E inhibitors show an excellent potential to become a new class of anti-stroke drugs for Veterans.