SPLUNC1-Derived Peptides as ENaC Antagonists

描述（由申请人提供）：和的细胞外环？ENaC可被弗林蛋白酶型转化酶、丝氨酸蛋白酶和中性粒细胞在多个位点上进行蛋白水解切割。 弹性蛋白酶，导致通道激活并增加Na+吸收。在囊性纤维化（CF）气道中，ENaC部分由于过度的蛋白水解裂解而异常活跃，这有助于气道表面液体（ASL）体积的消耗。这反过来可能导致粘液淤滞和气道感染的发生率增加，经常导致患者死亡。我们最近已经鉴定出SPLUNC 1是ENaC的有效抑制剂，其结合ENaC的细胞外侧并降低质膜ENaC水平。我们还表明，当SPLUNC 1被shRNA敲低时，正常人支气管上皮细胞培养物不再能调节ENaC活性。我们假设SPLUNC 1通过降低ENaC表面密度，防止暴露于细胞外蛋白酶来限制Na+吸收。我们已经确定了SPLUNC 1的活性位点，并基于该位点合成了一个18个氨基酸的肽，称为S18，它特异性结合到ENaC。尽管S18强烈抑制NL和CF气道上皮中的ENaC，但SPLUNC 1不能调节CF气道上皮中的ENaC。由于缺乏CFTR相关的HCO 3分泌，CF ASL呈酸性。分子模拟表明SPLUNC 1可能经历pH诱导的构象变化，其“掩埋”其活性位点，从而阻止与酸性CF ASL中的ENaC结合。因此，我们建议（i）解决SPLUNC 1的晶体结构，以进一步完善该模型，（ii）确定细胞外SPLUNC 1可以降低ENaC表面密度的机制，以及（iii）确定SPLUNC 1为什么不能在CF气道中发挥作用。S18样肽在CF肺病的治疗中可能是治疗有益的。此外，我们的数据表明，吸入高渗HCO 3-可以提供显着的优势，高渗盐水恢复CF粘液清除。 公共卫生相关性：CF粘液由于遗传引起的肺中盐和水运输的不平衡而脱水，使气道易于粘液堵塞和复发性感染。在本申请中，我们建议了解CF气道如何调节盐和水的运输以维持粘液清除。我们还提出开发可以抑制CF气道中的过度盐吸收以改善CF粘液水合的新型肽。

DESCRIPTION (provided by applicant): The extracellular loops of ¿ and ? ENaC can be roteolytically cleaved at multiple sites by furin-type convertases, serine proteases and neutrophil elastase, leading to activation of the channel and increased Na+ absorption. In cystic fibrosis (CF) airways, ENaC is abnormally hyperactive due in part to excessive proteolytic cleavage, which contributes to a depletion of airway surface liquid (ASL) volume. This in turn may lead to mucus stasis and increased incidence of airway infections that frequently lead to the death of the patient. We have recently identified SPLUNC1 as a potent inhibitor of ENaC that binds to the extracellular side of ENaC and diminishes plasma membrane ENaC levels. We have also shown that when SPLUNC1 is knocked down by shRNA, normal human bronchial epithelial cultures can no longer regulate ENaC activity. We hypothesize that SPLUNC1 limits Na+ absorption by reducing ENaC surface densities, preventing exposure to extracellular proteases. We have identified the active site of SPLUNC1, and have synthesized an 18 amino acid peptide based on this site, called S18, which specifically binds to ¿ENaC. Although S18 robustly inhibits ENaC in NL and CF airway epithelia, SPLUNC1 fails to regulate ENaC in CF airway epithelia. CF ASL is acidic due to the lack of CFTR-associated HCO3- secretion. Molecular modeling indicates that SPLUNC1 may undergo a pH- induced conformational change that "buries" its active site, preventing binding to ENaC in the acidic CF ASL. Thus, we propose to (i) solve the crystal structure of SPLUNC1 to further refine this model, (ii) determine the mechanism whereby extracellular SPLUNC1 can reduce ENaC surface density and (iii) determine why SPLUNC1 fails to function in CF airways. S18-like peptides may be therapeutically beneficial in the treatment of CF lung disease. Furthermore, our data suggest that inhaled hypertonic HCO3- may offer significant advantages over hypertonic saline for restoring CF mucus clearance. PUBLIC HEALTH RELEVANCE: CF mucus is dehydrated due to a genetically induced imbalance in salt and water transport in the lungs, leaving the airways prone to mucus plugging and recurrent infections. In this application, we propose to understand how CF airways can regulate salt and water transport to maintain mucus clearance. We also propose to develop novel peptides that can inhibit excessive salt absorption in CF airways to improve CF mucus hydration.