Antifibrotic Drug Discovery in Acute Lung Injury

描述（由申请人提供）：急性肺损伤是一种毁灭性的疾病，每年全球范围内的发病率为数十万。 它发生在严重肺炎、败血症、创伤或其他危及生命的疾病的情况下，是新出现的感染和生物恐怖袭击最令人恐惧的表现之一。 在病理学上，存在对气体交换装置的透壁损伤，其在临床上表现为呼吸衰竭。在疾病过程中，受伤的空气充满了增生的成纤维细胞，导致僵硬的肺和严重的低氧血症。当患者死亡时，成纤维细胞及其结缔组织产物持续存在于肺泡气隙中。 相反，当患者存活时，及时的成纤维细胞凋亡导致气体交换表面的恢复。 为了改善患者的预后，我们试图确定一种适合药物发现过程的凋亡调控途径。 在提供细胞凋亡受翻译控制的第一个证据的研究中，我们发现成纤维细胞活力受mRNA帽结合装置，真核翻译起始因子4F（eIF 4F）的水平和活性的调节。 异位过度表达eIF 4 E，eIF 4F的限速成分，阻断成纤维细胞凋亡;而过度表达eIF 4 E阻遏蛋白，4 E-BP 1，在体外和体内触发成纤维细胞凋亡。 因此，我们提出，在愈合的肺成纤维细胞的病理持久性可能会导致异常的翻译激活的mRNA编码的关键抗凋亡蛋白，并假设能够滴定帽依赖性翻译的生理水平的治疗有可能恢复肺成纤维细胞的敏感性愈合肺细胞凋亡。为了提供这种方法的概念证明，我们将在肺损伤的鼠博来霉素模型中提供初步数据，显示：1）缺乏翻译阻遏物4 E-BP 1的小鼠在肺损伤后比野生型小鼠发展更多的纤维化;和2）肺损伤后靶向消除成纤维细胞导致纤维化减少和存活增加。 我们计划通过两个具体目标来验证我们的假设。 目标1：合成并评估基于乙内酰脲的化合物，其作用于真核生物翻译起始因子4 E（eIF 4 E）的7-甲基鸟苷mRNA帽结合口袋，eIF 4 E是一种真正的分子靶标，控制编码成纤维细胞凋亡关键调节因子的mRNA的翻译。 目标二。将一种新的基于基因表达微阵列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，以鉴定作为抗纤维化药物发现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反驳eIF 4 E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.