deCarvalho Ana C V, Ndi Chi P, Tsopmo Apollinaire, Tane Pierre, Ayafor Johnson, Connolly Joseph D, Teem John L
Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA.
Mol Med. 2002 Feb;8(2):75-87.
Cystic fibrosis (CF) results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel localized at the plasma membrane of diverse epithelia. The most common mutation leading to CF, Delta F508, occurs in the first nucleotide-binding domain (NBD1) of CFTR. The Delta F508 mutation disrupts protein processing, leading to a decreased level of mutant channels at the plasma membrane and reduced transepithelial chloride permeability. Partial correction of the Delta F508 molecular defect in vitro is achieved by incubation of cells with several classes of chemical chaperones, indicating that further investigation of novel small molecules is warranted as a means for producing new therapies for CF.
The yeast two-hybrid assay was used to study the effect of CF-causing mutations on the ability of NBD1 to self-associate and form dimers. A yeast strain demonstrating defective growth as a result of impaired NBD1 dimerization due to Delta F508 was used as a drug discovery bioassay for the identification of plant natural product compounds restoring mutant NBD1 interaction. Active compounds were purified and the chemical structures determined. The purified compounds were tested in epithelial cells expressing CFTR Delta F508 and the resulting effect on transepithelial chloride permeability was assessed using short-circuit chloride current measurements.
Wild-type NBD1 of CFTR forms homodimers in a yeast two-hybrid assay. CF-causing mutations within NBD1 that result in defective processing of CFTR (Delta F508, Delta I507, and S549R) disrupted NBD1 interaction in yeast. In contrast, a CF-causing mutation that does not impair CFTR processing (G551D) had no effect on NBD1 dimerization. Using the yeast-based assay, we identified a novel limonoid compound (TS3) that corrected the Delta F508 NBD1 dimerization defect in yeast and also increased the chloride permeability of Fisher Rat Thyroid (FRT) cells stably expressing CFTR Delta F508.
The establishment of a phenotype for the Delta F508 mutation in the yeast two-hybrid system yielded a simple assay for the identification of small molecules that interact with the mutant NBD1 and restore dimerization. The natural product compound identified using the system (TS3) was found to increase chloride conductance in epithelial cells to an extent comparable to genistein, a known CFTR activator. The yeast system will thus be useful for further identification of compounds with potential for CF drug therapy.
囊性纤维化（CF）由囊性纤维化跨膜传导调节因子（CFTR）基因突变引起，该基因编码一种位于多种上皮细胞膜上的氯离子通道。导致CF的最常见突变Delta F508发生在CFTR的第一个核苷酸结合结构域（NBD1）中。Delta F508突变破坏蛋白质加工过程，导致质膜上突变通道水平降低以及跨上皮氯离子通透性降低。通过用几类化学伴侣孵育细胞，可在体外部分纠正Delta F508分子缺陷，这表明有必要进一步研究新型小分子，作为开发CF新疗法的一种手段。
采用酵母双杂交试验研究导致CF的突变对NBD1自我缔合和形成二聚体能力的影响。由于Delta F508导致NBD1二聚化受损而表现出生长缺陷的酵母菌株被用作药物发现生物测定法，以鉴定恢复突变型NBD1相互作用的植物天然产物化合物。对活性化合物进行纯化并确定其化学结构。在表达CFTR Delta F508的上皮细胞中测试纯化的化合物，并使用短路氯离子电流测量评估其对跨上皮氯离子通透性的影响。
在酵母双杂交试验中，CFTR的野生型NBD1形成同源二聚体。NBD1内导致CFTR加工缺陷的致CF突变（Delta F508、Delta I507和S549R）破坏了酵母中的NBD1相互作用。相比之下，不损害CFTR加工的致CF突变（G551D）对NBD1二聚化没有影响。利用基于酵母的试验，我们鉴定出一种新型柠檬苦素化合物（TS3），它纠正了酵母中Delta F508 NBD1二聚化缺陷，还增加了稳定表达CFTR Delta F508的Fisher大鼠甲状腺（FRT）细胞的氯离子通透性。
在酵母双杂交系统中建立Delta F508突变的表型，为鉴定与突变型NBD1相互作用并恢复二聚化的小分子提供了一种简单的试验方法。使用该系统鉴定出的天然产物化合物（TS3）被发现可将上皮细胞中的氯离子传导率提高到与已知CFTR激活剂染料木黄酮相当的程度。因此，酵母系统将有助于进一步鉴定具有CF药物治疗潜力的化合物。
