New paradigms of CFTR regulation

描述（由申请人提供）：CFTR是盐和水通过肺和肠上皮运输的重要介质。这种氯离子通道的合成或调节缺陷导致多种人类疾病，包括囊性纤维化（CF）、男性不育和腹泻。治疗这些cftr相关疾病的药物开发尚处于早期阶段。最常见的CF突变体是deltaF508-CFTR，它不能有效地传递到细胞表面。这种突变体在到达细胞表面时也可能表现出缺陷的通道门控，但这种缺陷的程度和潜在的机制尚不清楚。我们已经确定了一类新的CFTR通道开启器，它可以有效地激活野生型和deltaF508-CFTR通道。我们对这些化合物的鉴定是基于我们的发现，一种常用的CFTR孔阻滞剂对活性低的通道（例如，低磷酸化或氧化通道）具有混合激动剂的作用。基于这一观察结果，我们确定了这种孔阻滞剂的衍生物，其表现为纯CFTR激动剂。该化合物有效（EC50 < 1 mu/M）并特异性刺激切除膜斑块中的CFTR通道开放和完整上皮单层中CFTR介导的氯离子电流。在野生型通道几乎完全活跃的情况下，该开启剂显著刺激膜上驻留的deltaF508-CFTR通道的活性；因此，deltaF508突变似乎实质上破坏了通道门控。我们提出了三个具体目标：(1)确定这些化合物刺激CFTR通道打开的机制；(2)确定deltaF508突变对CFTR通道门控的影响程度以及这种抑制作用的潜在机制；(3)利用这些化合物的化学性质开发更有效的CFTR通道打开剂，并鉴定天然存在的CFTR激动剂。本项目的结果应有助于阐明控制CFTR门控的正常机制，明确最常见的CF突变对CFTR门控的影响，并可能导致CF新药物的开发。

DESCRIPTION (provided by applicant): CFTR is an essential mediator of salt and water transport across lung and gut epithelia. Defects in the synthesis or regulation of this chloride channel cause several human disorders including cystic fibrosis (CF), male infertility and diarrhea. The development of drugs to treat these CFTR-related disorders is at an early stage. The most common CF mutant is deltaF508-CFTR, which is inefficiently delivered to the cell surface. This mutant may also exhibit defective channel gating when it reaches the cell surface, but the extent of this defect and the underlying mechanisms are unknown. We have identified a new class of CFTR channel opener that potently activates wild type and deltaF508-CFTR channels. Our identification of these compounds was based on our discovery that a commonly used blocker of the CFTR pore behaves as a mixed agonist toward channels that have low activity (e.g., poorly phosphorylated or oxidized channels). Based on this observation, we identified a derivative of this pore blocker that behaves as a pure CFTR agonist. This compound potently (EC50 < 1 mu/M) and specifically stimulates CFTR channel opening in excised membrane patches and CFTR-mediated chloride currents in intact epithelial monolayers. This opener dramatically stimulates the activities of membrane-resident deltaF508-CFTR channels under conditions when the wild type channel is nearly fully active; thus, the deltaF508 mutation appears to substantially disrupt channel gating. We propose 3 specific aims: (1) to define the mechanism by which these compounds stimulate CFTR channel opening; (2) to determine the extent to which the deltaF508 mutation affects CFTR channel gating and the underlying mechanism for this inhibitory effect; and (3) to exploit the chemistry of these compounds to develop more potent CFTR channel openers and identify naturally occurring CFTR agonists. The results of this project should help clarify the normal mechanisms that control CFTR gating, define the effect of the most common CF mutation on CFTR gating, and possibly lead to the development of new CF drugs.