Novel Anti-Stroke Agents Targeting Toxic Protein Aggregation

针对有毒蛋白聚集的新型抗中风药物

• 批准号: 10589978
• 负责人: Bingren Hu
• 金额: --
• 依托单位: VA SAN DIEGO HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589978
• 关键词: Abate; Affect; Animal Model; Animals; Autophagocytosis; Behavioral; Biological Assay; Brain; Brain Injuries; Brain Ischemia; Categories; Cell Culture Techniques; Cessation of life; Confocal Microscopy; Dissociation; Double-Blind Method; EIF4EBP1 gene; Enzymes; Eukaryotic Initiation Factors; Event; Excision; FRAP1 gene; Future; Genes; Genetic Transcription; Goals; Hemorrhage; Hydrophobicity; Infarction; Inflammation; Injections; Injury; Ischemia; Ischemic Brain Injury; Ischemic Stroke; Laboratories; Leucine; Messenger RNA; Mission; Modeling; Molecular Chaperones; Mus; Natural regeneration; Neurologic Deficit; Neurons; Paper; Pathologic; Pathway interactions; Peptide Initiation Factors; Performance; Perfusion; Pharmaceutical Preparations; Phase; Phosphorylation; Polyribosomes; Process; Proteins; Publishing; Randomized; Recovery; Reperfusion Injury; Reperfusion Therapy; Research; Ribavirin; Ribosomes; Role; Stress; Stroke; System; TLR4 gene; Testing; Therapeutic; Time; Translations; Up-Regulation; Veterans; Western Blotting; acute stroke; cytokine; drug discovery; genetic regulatory protein; high throughput technology; improved; inhibitor; mortality; mouse model; nervous system disorder; neuron loss; neuroprotection; novel; polypeptide; post stroke; programs; protein aggregation; response; side effect; stroke model; stroke patient; systemic inflammatory response; targeted agent; therapeutic target; transcription factor CHOP

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.

该建议是研究有毒新生多肽链（NPC）的作用 中风脑损伤。中风是一种毁灭性的神经系统疾病，影响了数百万的退伍军人。治疗 重新灌注是当前治疗急性中风的金标准。但是，由于潜力 出血副作用，只有大约5％的中风患者接受再灌注疗法。 信使RNA（mRNA）转换为蛋白质也称为NPC合成。我们的实验室 最初发现，大规模的NPC聚集发生在注定死亡的缺血后神经元中。我们 已经发表了30篇有关此主题的论文。这些研究表明，NPC合成在两个中被抑制 不同的阶段。抑制的第一阶段是短暂的，在所有后再灌注0.5-1 h后完全减弱 缺血神经元；因此，与脑缺血后未来的神经元死亡无关。第一阶段 抑制后，在所有缺血后的再灌注4小时后，恢复了> 70％的NPC合成 神经元。 NPC合成抑制的第二阶段是从再灌注4小时逐渐进行的 继续前进，只有注定要死的神经元。我们的研究表明，第二阶段的抑制是 由于共同翻译的链酮保护NPC的缺血后丧失。没有这种保护，NPCS 在合成过程中暴露其粘稠的疏水片段，并在核糖体上不可逆地聚集 脑缺血后的再灌注4小时。 这些研究导致了新发现，即通过减少靶向大规模NPC聚集 具有真核启动因子（EIF）抑制剂在核糖体上的“无保护” NPC的负载可以保护大脑免受 中风损伤。通过使用高吞吐技术，几种“直接” EIF4E（相互作用）抑制剂已经 确定，例如，针对EIF4E到EIF4G和EIF4E-TO-4E-BP1相互作用（4E1RCAT）的双重抑制剂（4E1RCAT）和 利巴韦林。这些发现为了解NPC汇总的作用提供了绝佳的机会 中风脑损伤并开发出新的抗中风药物类别。我们的实验室已经研究了几个EIF 抑制剂。其中，4EGI-1在小鼠中风模型中提供了最佳的抗震动效率。因此，我们使用了 4EGI-1在双盲，随机对照动物研究中。这些研究清楚地表明了小鼠 用i.p.与45％相比，再灌注30分钟后注射4EGI-1没有中风死亡率 车辆组的死亡率。 4EGI-1处理可显着减少梗塞量，改善物理 恢复和行为表现，并改善中风后神经系统缺陷。 拟议的研究的目标是进一步研究NPC聚集在中风大脑中的作用 损伤并确定针对中风脑损伤的最佳靶标和抑制剂（AIM 1）；并调查 潜在的脑保护机制的细节（目标2和3）。我们将测试以下假设：（1） 抑制EIF4E不仅减少了核糖体上缺血后“未受保护” NPC的负载，而且还减少 开关从cap依赖性到帽子依赖性状态的交换翻译。此开关关闭帽子依赖性 NPC翻译同时优先考虑分子伴侣的帽帽非依赖性翻译；因此，保护​​后 再灌注过程中有毒聚集的缺血性“无保护” NPC。 （2）4EGI-1治疗导致鲁棒 ATF4的上调和切碎转录因子以诱导必要的自噬基因；因此，促进 脑缺血后去除有毒的NPC聚集体。 （3）抑制EIF4E减轻冲程后的抑制 炎。尽管抑制其他NPC合成倡议因素/调节因子也可能减少 核糖体上的“未保护” NPC，这些其他优势（例如，共同翻译分子的上调 EIF4E抑制作用提供的伴侣和自噬蛋白）使EIF4E具有边缘为 降低脑缺血后有毒NPC聚集的治疗靶标。 EIF4E抑制剂显示出极好的 有可能成为退伍军人的新型抗震动药物。