New paradigms of CFTR regulation

描述（由申请人提供）：CFTR是盐和水转运穿过肺和肠上皮的重要介质。这种氯离子通道的合成或调节缺陷会导致几种人类疾病，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处于早期阶段。最常见的CF突变体是deltaF 508-CFTR，其被低效地递送到细胞表面。当它到达细胞表面时，这种突变体也可能表现出有缺陷的通道门控，但这种缺陷的程度和潜在的机制是未知的。我们已经鉴定了一类新的CFTR通道开放剂，其有效地激活野生型和deltaF 508-CFTR通道。我们对这些化合物的鉴定是基于我们的发现，即CFTR孔的常用阻断剂表现为对具有低活性的通道的混合激动剂（例如，磷酸化差或氧化的通道）。基于这一观察结果，我们鉴定了这种孔阻断剂的衍生物，其表现为纯CFTR激动剂。该化合物有效地（EC 50 < 1 μ/M）并且特异性地刺激切除的膜斑中的CFTR通道开放和完整上皮单层中的CFTR介导的氯电流。在野生型通道几乎完全激活的条件下，这种开放剂显著刺激膜驻留的deltaF 508-CFTR通道的活性;因此，deltaF 508突变似乎基本上破坏了通道门控。我们提出了3个具体目标：（1）确定这些化合物刺激CFTR通道开放的机制;（2）确定deltaF 508突变影响CFTR通道门控的程度和这种抑制作用的潜在机制;（3）利用这些化合物的化学性质来开发更有效的CFTR通道开放剂并鉴定天然存在的CFTR激动剂。该项目的结果将有助于阐明控制CFTR门控的正常机制，确定最常见的CF突变对CFTR门控的影响，并可能导致新CF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.