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.

本提案旨在研究有毒新生多肽链 (NPC) 错误折叠和聚集在 中风是一种破坏性的神经系统疾病，影响着数百万退伍军人。 重建灌注是目前治疗急性中风的金标准，但由于其潜在的潜力。 出血副作用，只有约5%的中风患者接受再灌注治疗。 将信使 RNA (mRNA) 翻译成蛋白质也称为 NPC 合成。 最初发现大量 NPC 聚集发生在缺血后注定死亡的神经元中。 已就此主题发表了 30 篇论文，这些研究表明 NPC 合成在两种情况下受到抑制。 第一阶段的抑制是短暂的，在所有后灌注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。 抑制后，所有缺血后再灌注 4 小时，NPC 合成恢复 > 70% NPC 合成抑制的第二阶段从再灌注 4 小时开始逐渐发生。 我们的研究表明，第二阶段的抑制是持续的，并且只发生在注定要死亡的神经元中。 由于缺血后 NPC 的共翻译伴侣保护丧失，如果没有这种保护，NPC 就会失去这种保护。 在合成过程中暴露其粘性疏水片段，并不可逆地聚集在核糖体上 脑缺血后再灌注4小时。 这些研究带来了新的发现，即通过减少 带有真核起始因子 (eIF) 抑制剂的核糖体上“未受保护”的 NPC 负载可以保护大脑免受 通过使用高通量技术，几种“直接”eIF4E（相互作用）抑制剂已被开发出来。 鉴定出例如针对 eIF4E-to-eIF4G 和 eIF4E-to-4E-BP1 相互作用的双重抑制剂 (4E1Rcat) 以及 这些发现为了解 NPC 聚集的作用提供了绝佳的机会。 我们的实验室研究了几种 eIF。 其中，4EGI-1在小鼠中风模型中具有最佳的抗中风功效。 4EGI-1在双盲、随机对照动物研究中清楚地证明了小鼠。 再灌注 30 分钟后腹腔注射 4EGI-1 的患者中风死亡率为 45%。 4EGI-1治疗组的死亡率显着减少了梗塞体积，改善了体质。 恢复和行为表现，并减少中风后的神经功能缺损。 拟议研究的目标是进一步研究 NPC 聚集在中风大脑中的作用 损伤并确定对抗中风脑损伤的最佳靶标和抑制剂（目标 1）； 我们将测试以下假设：（1） 抑制 eIF4E 不仅可以减少缺血后“未受保护”的 NPC 对核糖体的负荷，还可以 将转换从与上限相关的状态转换为与上限无关的状态。此开关会关闭与上限相关的状态。 NPC 翻译，同时优先考虑分子伴侣的帽独立翻译，从而保护后 缺血性“未受保护”的 NPC 在再灌注过程中免受毒性聚集 (2) 4EGI-1 治疗可产生强大的效果。 上调 ATF4 和 CHOP 转录因子以诱导必需的自噬基因； 脑缺血后去除有毒的 NPC 聚集物 (3) 抑制 eIF4E 可缓解中风后症状。 尽管抑制其他 NPC 合成起始因子/调节因子也可能减少炎症的负荷。 核糖体上“不受保护”的 NPC，这些其他优越的益处（例如，共翻译分子的上调） 上述 eIF4E 抑制提供的分子伴侣和自噬蛋白）使 eIF4E 具有优势，因为 eIF4E 抑制剂显示出出色的减少脑缺血后有毒 NPC 聚集的治疗靶点。 有潜力成为退伍军人的新型抗中风药物。