ΔF508缺失突变型CFTR错误折叠小分子矫正剂的设计、合成及其作用机理研究

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.

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。

囊性纤维化跨膜传导调节因子(CFTR)是一种环磷酸腺苷调节的氯离子通道蛋白，基因突变可导致该蛋白的合成、翻译异常和功能丧失，使外分泌腺管上皮细胞膜对氯离子的通透性降低、钠离子重吸收增加，从而使外分泌液中钠离子、氯离子浓度异常增高，故 CFTR 基因突变主要影响外分泌腺功能，从而导致囊性纤维化的发生，其中最常见的一种突变型是 CFTR 上第 508 位的苯丙氨酸缺失。.为了考察ΔF508突变引起CFTR构象的变化，项目组采用原位限制性蛋白水解来了解CFTR各结构域的变化。经胰蛋白酶水解后，野生型和ΔF508突变型的水解产物有明显的不同。定量检测野生型和ΔF508突变型的50-kD片段，可以看到两者明显变化。这些数据可以说明 F508 突变对构象带来的变化，以及NBD1在结构上的矫正可能是重要的补救手段。.项目组比较NBD1野生型和突变型构象在热力学和动力学两个方面的不同，采用副本交换分子动力学和同源建模等技术，构建ΔF508-NBD1的结合模型，并用已知药物验证筛选模型的有效性。进一步筛选ZINC和NCI化合物数据库，找到了苗头化合物。同时通过片段对接和经典药物设计方法，设计了不同三个系列化合物，合成30多个新化合物。.经过SPR筛选后得到10个化合物,测试了这些化合物对ΔF508错误折叠的纠正能力。首先我们在稳定表达ΔF508-CFTR(HEK-ΔF)的HEK296细胞系中进行试验。在5μM浓度下，10个化合物对ΔF508-CFTR的条带B作用很小，但其中约一半的化合物可以显著增加ΔF508-CFTR的条带C。几乎所有的化合物显示出可以适度增加C/B的比值。在10µM浓度下，9个化合物都能减少了的ΔF508-CFTR的条带B。大约一半的化合物促使条带C量的增加，最终导致C/B比值急剧增加。这些数据表明大部分化合物对ΔF508- CFTR错误折叠有矫正作用，且不增加条带B的量。这个性质与目前大部分ΔF508矫正剂（增加CFTR 的条带B）有所不同纠正剂。.进一步将化合物加到CFBE-ΔF细胞中，检测氯离子外排，比较化合物氯离子调控能力。用能表达野生型的CFTR的CFBE细胞作阳性对照。项目组最终得到2-3个具有较好离子调控能力的化合物。为治疗囊性纤维化药物开发提供潜在的化合物。

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