囊性纤维化 (CF) 是一种严重的常染色体隐性遗传病，其病因是囊性纤维化跨膜传导调节因子 (CFTR)的基因突变导致。90% CF患者的CFTR存在第508位苯丙氨酸缺失(ΔF508)，目前尚无有效治疗药物。ΔF508会导致CFTR蛋白的错误折叠。开发有效的矫正剂，用于纠正CFTR的错误折叠，有望治疗ΔF508引起的病变。根据目前最新作用机理，本课题提出崭新的药物设计策略，在动态构象中寻找合适的作用位点。课题组前期通过副本交换分子动力学模拟，初步构建适合开发矫正剂的模型，并得到了先导化合物。在本课题中将会进一步优化结构模型，运行基于片段设计的策略来设计新的矫正剂。合成得到的新化合物，将从促进蛋白成熟和增强离子调控两个指标来评价化合物的活性。选取活性佳的化合物，进一步探讨其作用机理，拟将采用脉冲追踪分析、原位限制性蛋白酶水解以及小分子和蛋白结晶等系列实验，阐明矫正剂的确切作用位点和作用机制。

Cystic Fibrosis (CF) is caused by impaired plasma membrane functional expression of the cystic fibrosis transmembrane conductance regulator (CFTR). The deletion of phenylalanine at residue 508 (ΔF508) in CFTR contributes to the development of CF in over 90% of patients. The F508 resides in the first nucleotide binding domain (NBD1) of CFTR, a domain that is critical for CFTR folding, trafficking and channel gating. The ΔF508 mutation causes a kinetic folding defect in NBD1. The alteration in NBD1 folding leads to conformational changes in the same domain, which spread to other domains of CFTR presumably through domain-domain interactions. Correcting the underlying conformational defects is at the core of ΔF508 CFTR rescue. .Using replica-exchange molecular dynamics simulation (REMD), we observed significant conformational change in NBD1 due to the ΔF508 mutation. Using computational docking, we identified a small molecule binding pocket within NBD1 that plays a pivotal role in ΔF508 misfolding. Based on computational simulation, small molecule correctors binding to the pocket prevent further conformational unfolding of NBD1. Using a novel fragment-based virtual screen strategy, we designed an initial lead DF508 corrector. We found that, of the ten initial derivatives of the lead corrector, more than half enhance ΔF508 CFTR processing. These preliminary data give us confidence that such an approach is not only feasible but also promising in the identification of novel ΔF508 correctors. We now propose to conduct a full-scale study through the following Specific Aims：.Aim 1. We propose to perform a full-scale NBD1 conformation-based virtual screen to identify novel lead correctors, synthesize them and test their ability to enhance ΔF508 processing. Both dose-response and cell-type dependence will be assessed. For a selected group of highly potent lead compounds, their ability to enhance the ΔF508 CFTR-dependent chloride conductance will be evaluated in CF airway epithelial cells..Aim 2. The efficacy, affinity and druggability of the initial lead compounds can be considerably improved by structural optimization. We propose to perform such optimization by integrating traditional medicinal chemistry, computer-based simulation and biological analyses. Side groups of the lead compounds will be optimized based on traditional medicinal chemistry, verified by computational simulation, and finally tested for their ability to enhance ΔF508 CFTR rescue. The experimental results will be fed back to direct better optimization so that highly potent correctors can be generated..Aim 3. We propose to test representative lead correctors for their ability to improve the ΔF508 CFTR synthesis, maturation, stability both in the ER and at the cell periphery, and domain conformation (NBD1 as well as other CFTR domains). Pulse-chase analysis, in situ limited proteolysis and X-ray diffraction experiments will be employed for these purposes.