Antifibrotic Drug Discovery in Acute Lung Injury

描述(申请人提供)：急性肺损伤是一种毁灭性的疾病，每年全球发病率高达数十万。它发生在严重肺炎、败血症、创伤或其他危及生命的疾病的背景下，是新出现的感染和生物恐怖袭击最可怕的表现之一。病理上，气体交换装置受到跨壁损害，临床表现为呼吸衰竭。在疾病过程中，损伤的空隙充满了增殖的成纤维细胞，导致肺僵硬和严重的低氧血症。当病人死亡时，成纤维细胞及其结缔组织产物留在肺泡空隙中。相反，当患者存活下来时，成纤维细胞的及时凋亡会导致气体交换表面的恢复。为了改善患者的预后，我们试图确定一种符合药物发现过程的细胞凋亡调控途径。在提供细胞凋亡受到翻译调控的第一个证据的研究中，我们发现成纤维细胞的活性受到真核细胞翻译起始因子4F(EIF4F)的水平和活性的调节。EIF4F的限速成分eIF4E的异位过表达可阻断成纤维细胞的凋亡，而eIF4E抑制蛋白4E-BP1的过表达在体外和体内均可引发成纤维细胞的凋亡。因此，我们认为成纤维细胞在愈合肺中的病理性持久性可能是编码关键的抗凋亡蛋白的mRNAs异常翻译激活的结果，并假设能够将帽依赖的翻译水平滴定到生理水平的治疗方法有可能恢复愈合肺中肺成纤维细胞对凋亡的敏感性。为了证明这一方法的概念，我们将提供博莱霉素肺损伤小鼠模型的初步数据，显示：1)缺乏翻译抑制子4E-BP1的小鼠在肺损伤后比野生型小鼠发生更多的纤维化；以及2)肺损伤后靶向清除成纤维细胞可以减少纤维化并提高存活率。我们计划通过两个具体目标来检验我们的假设。目的1：合成并评价与真核细胞翻译起始因子4E(EIF4E)的7-甲基鸟苷mRNA帽结合口袋(EIF4E)作用的海因类化合物，eIF4E是控制成纤维细胞凋亡关键调控因子mRNA翻译的真正分子靶点。目的2.将一种新型的基于Gone表达芯片的分子靶点发现系统应用于急性肺损伤患者肺成纤维细胞的原代培养，以筛选可作为抗纤维化药物候选分子靶点的凋亡调节蛋白。如果成功，我们的工作将证实或否定eIF4E作为抗纤维化治疗靶点，并可能导致识别新的急性肺损伤特异性分子靶点，用于抗纤维化药物的发现。

DESCRIPTION (provided by applicant): Acute lung injury is a devastating illness with an annual world-wide incidence in the hundreds of thousands. It occurs in the context of severe pneumonia, sepsis, trauma or other life-threatening illnesses, and is among the most feared manifestations of emerging infections and bioterrorist attacks. Pathologically, there is a transmural insult to the gas exchange apparatus that manifests clinically as respiratory failure. In the course of the disease process, the injured airspace fills with proliferating fibroblasts resulting in stiff lungs and profound hypoxemia. When patients die, fibroblasts and their connective tissue products persist in the alveolar airspace. In contrast, when patients survive, timely fibroblast apoptosis leads to restoration of the gas exchange surface. To improve patient outcome, we sought to identify an apoptotic regulatory pathway amenable to the drug discovery process. In studies providing the first evidence that apoptosis was subject to translational control, we discovered that fibroblast viability is regulated by the level and activity of the mRNA cap-binding apparatus, eukaryotic translation initiation factor 4F (eIF4F). Ectopic over expression of eIF4E, the rate limiting component of eIF4F, blocks fibroblast apoptosis; whereas over expression of the eIF4E repressor protein, 4E-BP1, triggers fibroblast apoptosis in vitro and in vivo. We therefore propose that pathological persistence of fibroblasts in the healing lung may result from aberrant translational activation of mRNAs encoding critical antiapoptotic proteins and hypothesize that therapies capable of titrating cap-dependent translation to physiological levels have the potential to restore sensitivity of lung fibroblasts in the healing lung to apoptosis. To provide proof of concept for this approach, we will present preliminary data in the murine bleomycin model of lung injury showing that: 1) mice lacking translational repressor 4E-BP1 develop more fibrosis than wild type mice after lung injury; and 2) targeted elimination of fibroblasts after lung injury leads to decreased fibrosis and increased survival. We plan to test our hypothesis through 2 specific aims. Aim 1: Synthesize and evaluate hydantoin-based compounds hitting the 7-methyl guanosine mRNA cap-binding pocket of eukaryotic translation initiation factor 4E (eIF4E), a bona fide molecular target controlling the translation of mRNA encoding key regulators of fibroblast apoptosis. Aim 2. Apply a novel gone expression microarray-based molecular target discovery system to primary cultures of lung fibroblasts from patients with acute lung injury to identify apoptotic regulatory proteins that are candidate molecular targets for antifibrotic drug discovery. If successful, our work will confirm or refute eIF4E as an antifibrotic therapeutic target, and may lead to the identification of new acute lung injury-specific molecular targets for antifibrotic drug discovery.