Mechanism of Prostone Activation During CFTR Modulation

项目摘要/摘要 囊性纤维化是一种常染色体隐性遗传病，由囊性纤维化跨膜突变引起。 电导调节器(CFTR)基因，它破坏了CFTR的功能。大约90%的慢性萎缩性胃炎患者 至少有一份F508del CFTR的副本。F508del CFTR功能可以通过以下方式在很大程度上修复 美国市场上的双联和三联用药。另外10%的慢性纤维性心脏病患者没有 有F508del，他们中的许多突变没有恢复CFTR的药物。这笔赠款的目的是 是为了探索一种新型的CFTR调节剂-前石-激活野生型和 突变的CFTR。我们的假设是，路比前列酮和相关的前石激活了许多形式的CFTR- 正常的cftr、F508del cftr或其他罕见的突变，特别是那些已被挽救到细胞中的突变 通过市面上销售的调制器。这些原蛋白被开发为氯离子通道2(CLCN2)激活剂 但与存在于呼吸道和胃肠上皮中的受体介导的通路相互作用，以促进CFTR- 介导的氯化物分泌。可能选择的前驱蛋白是CFTR和CLCN2的双重调节剂。 CFTR介导的氯离子转运是一种有意义的、可量化的评估通道有效性的指标 校正器、激活器、增强器和放大器。校正器、稳定器和放大器通过增加 细胞表面的CFTR蛋白水平和增强剂增加CFTR通道的开放概率 以增加阴离子的渗透性。目前在体外提高cAMP的激活剂Forsklin和IBMX还没有临床应用 安全或可用，这意味着市场上的双重和三重组合调制器依赖于 内源性cAMP水平。具体目标1：量化卢比前列酮和其他新型CFTRs的作用 激活剂对非CF型、F508del CFTR型和非F508del突变型CFTR型呼吸道离子转运的影响 上皮细胞培养。目的1检验急性鲁比前列酮刺激CFTR介导的氯离子的假设 运输。目的1B检验的假设是，长期接触CFTR激活剂会招募CFTR功能并可以 刺激CLCN2介导的氯离子分泌。Aim 1C验证CFTR功能增强导致 以增加呼吸道表面液体深度。特定目的2：研究激活过程中的PGE2信号通路 加了卢比前列酮。假设一个或多个EP受体亚型是鲁比前列酮的靶点(S)。 野生型和突变型cftr激活氯离子分泌。目标2A解决来自 Lubiprostone对CFTR的作用，并不同于Lubiprostone对CLCN2的激活。Aim 2B测试的假设是 EP2和EP4受体与CFTR在同一细胞中表达。

PROJECT SUMMARY/ABSTRACT Cystic fibrosis (CF) is an autosomal recessive disease resulting from mutations in the CF transmembrane conductance regulator (CFTR) gene that disrupts the functions of CFTR. Approximately 90% of people with CF have at least one copy of F508del CFTR. F508del CFTR function can be repaired to a significant extent by double and triple combination medications on the market in the USA. The other 10% of people with CF do not have F508del and many of their mutations do not have a medication to restore CFTR. The GOAL of this grant is to explore the efficacy of a new class of CFTR modulator—a prostone—for the ability to activate wild-type and mutant CFTR. Our HYPOTHESIS is that lubiprostone and related prostones activate many forms of CFTR— normal CFTR, F508del CFTR, or other rare mutations, especially those that have been rescued to the cell surface by the marketed modulators. The prostones were developed as chloride channel 2 (CLCN2) activators but interact with receptor-mediated pathways present in airway and gastrointestinal epithelia to boost CFTR- mediated chloride secretion. It is likely that select prostones are dual modulators of both CFTR and CLCN2. CFTR-mediated chloride transport is a meaningful and quantifiable measure to assess efficacy of channel correctors, activators, potentiators, and amplifiers. Correctors, stabilizers, and amplifiers function by increasing CFTR protein levels on the cellular surface, and potentiators increase the open probability of the CFTR channel to increase anion permeability. Current activators that raise cAMP in vitro, forskolin and IBMX, are not clinically safe or available, which means that the double and triple combination modulators on the market are relying on endogenous levels of cAMP. Specific Aim 1: To quantify the effects of lubiprostone and other novel CFTR activators on ion transport in non-CF, F508del CFTR, and non-F508del mutant CFTR-expressing airway epithelial cell cultures. Aim 1A tests the hypothesis that acute lubiprostone stimulates CFTR-mediated chloride transport. Aim 1B tests the hypothesis that chronic exposure to CFTR activators recruits CFTR function and can stimulate CLCN2-mediated chloride secretion. Aim 1C tests the hypothesis that increased CFTR function leads to increased airway surface liquid depth. Specific Aim 2: To study the PGE2 signaling pathway during activation with lubiprostone. The hypothesis is that one or more EP receptor subtypes are target(s) of lubiprostone during activation of chloride secretion by wild-type and mutant CFTR. Aim 2A addresses GPCR coupling from lubiprostone to CFTR and differentiates from lubiprostone activation of CLCN2. Aim 2B tests the hypothesis that EP2 and EP4 receptors are expressed in the same cells as CFTR.