Novel Anti-Stroke Agents Targeting Toxic Protein Aggregation

本研究旨在研究毒性新生多肽链(NPC)的错误折叠和聚集在脑出血中的作用。 中风脑损伤。中风是一种毁灭性的神经疾病，影响着数百万退伍军人。治疗方法 重建血流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，只有大约5%的中风患者接受再灌注治疗。 信使RNA(信使RNA)转化为蛋白质也称为NPC合成。我们的实验室 最初发现在注定要死亡的缺血后神经元中发生大量的NPC聚集。我们 已经发表了30篇关于这个主题的论文。这些研究表明，鼻咽癌的合成在两个方面受到抑制 截然不同的阶段。第一阶段的抑制是短暂的，在再灌流0.5-1h后，所有后遗症的抑制完全消失。 缺血神经元；因此，与脑缺血后未来的神经元死亡无关。这是第一阶段 在所有的缺血后再灌注4h，鼻咽癌的合成在抑制之后恢复了70% 神经元。鼻咽癌合成抑制的第二阶段从再灌流4h开始逐渐发生。 而且只存在于注定要死亡的神经元中。我们的研究表明，抑制的第二阶段是 这是由于缺血后失去了对NPC的共翻译伴侣保护。如果没有这种保护，NPC 在合成过程中暴露其粘稠的疏水片段，并不可逆转地聚集在来自 脑缺血后再灌流4h。 这些研究导致了一个新的发现，即通过减少 携带真核细胞起始因子(EIF)抑制剂的核糖体上“未受保护的”NPC可保护大脑 中风伤。通过使用高通量技术，已经开发出了几种“直接”eIF4E(相互作用)抑制剂 识别例如针对eIF4E到eIF4G和eIF4E到4E-BP1相互作用的双重抑制物(4E1Rcat)和 利巴韦林。这些发现提供了一个极好的机会来理解鼻咽癌聚集在 并开发了一类新型的抗中风药物。我们实验室已经研究了几个EIF 抑制剂。其中，4EGI-1在小鼠卒中模型中提供了最好的抗中风效果。因此，我们使用了 4EGI-1的双盲、随机对照动物研究。这些研究清楚地表明，老鼠 接受了ip治疗。再灌流30min后注射4EGI-1无卒中死亡率，而45% 赋形剂组的死亡率。4EGI-1治疗显著缩小脑梗塞体积，改善体格检查 恢复和行为表现，并减少中风后的神经功能障碍。 这项拟议研究的目标是进一步研究鼻咽癌聚集在中风脑中的作用。 并确定抗卒中脑损伤的最佳靶点和抑制剂(目标1)； 基本脑保护机制的细节(目标2和3)。我们将检验以下假设：(1) 抑制eIF4E不仅可以减少缺血后“无保护”的神经前体细胞在核糖体上的负荷，而且还可以 将转换从封口依赖状态切换到封口无关状态。此开关关闭与大小写相关的 NPC翻译，同时优先考虑分子伴侣的帽子非依赖性翻译；因此，保护后 缺血“未保护”的神经前体细胞在再灌流期间不会出现毒性聚集。(2)4EGI-1治疗导致 上调ATF4和CHOP转录因子以诱导必要的自噬基因；因此，促进 脑缺血后毒性鼻咽癌聚集体的清除。(3)抑制eIF4E可缓解卒中后 发炎。虽然抑制其他鼻咽癌合成启动因子/调节因子也可以减少 核糖体上的不受保护的NPC，这些其他优越的好处(例如，上调共翻译分子 如上所述的eIF4E抑制提供的伴侣和自噬蛋白)使eIF4E具有优势 减少脑缺血后中毒性鼻咽癌聚集的治疗靶点。EIF4E抑制剂显示出极好的 有可能成为一类新型的退伍军人抗中风药物。

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.