Departments of Medicine and Physiology (P.-W.P., J.T., A.S.V.) and Department of Pathology (W.E.F.), University of California, San Francisco, California; and Groupe de Recherche Axé sur la Structure des Protéine and Departments of Physiology (G.V., A.R., G.L.L.) and Biochemistry (G.L.L.), McGill University, Montreal, Quebec, Canada.
Departments of Medicine and Physiology (P.-W.P., J.T., A.S.V.) and Department of Pathology (W.E.F.), University of California, San Francisco, California; and Groupe de Recherche Axé sur la Structure des Protéine and Departments of Physiology (G.V., A.R., G.L.L.) and Biochemistry (G.L.L.), McGill University, Montreal, Quebec, Canada
Mol Pharmacol. 2014 Jul;86(1):42-51. doi: 10.1124/mol.114.092478. Epub 2014 Apr 15.
The most prevalent cystic fibrosis transmembrane conductance regulator (CFTR) mutation causing cystic fibrosis, ΔF508, impairs folding of nucleotide binding domain (NBD) 1 and stability of the interface between NBD1 and the membrane-spanning domains. The interfacial stability defect can be partially corrected by the investigational drug VX-809 (3-[6-[[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl]amino]-3-methyl-2-pyridinyl]-benzoic acid) or the R1070W mutation. Second-generation ΔF508-CFTR correctors are needed to improve on the modest efficacy of existing cystic fibrosis correctors. We postulated that a second corrector targeting a distinct folding/interfacial defect might act in synergy with VX-809 or the R1070W suppressor mutation. A biochemical screen for ΔF508-CFTR cell surface expression was developed in a human lung epithelium-derived cell line (CFBE41o(-)) by expressing chimeric CFTRs with a horseradish peroxidase (HRP) in the fourth exofacial loop in either the presence or absence of R1070W. Using a luminescence readout of HRP activity, screening of approximately 110,000 small molecules produced nine novel corrector scaffolds that increased cell surface ∆F508-CFTR expression by up to 200% in the presence versus absence of maximal VX-809. Further screening of 1006 analogs of compounds identified from the primary screen produced 15 correctors with an EC50 < 5 µM. Eight chemical scaffolds showed synergy with VX-809 in restoring chloride permeability in ∆F508-expressing A549 cells. An aminothiazole increased chloride conductance in human bronchial epithelial cells from a ΔF508 homozygous subject beyond that of maximal VX-809. Mechanistic studies suggested that NBD2 is required for the aminothiazole rescue. Our results provide proof of concept for synergy screening to identify second-generation correctors, which, when used in combination, may overcome the "therapeutic ceiling" of first-generation correctors.
最常见的囊性纤维化跨膜电导调节因子 (CFTR) 突变导致囊性纤维化，ΔF508 ，会损害核苷酸结合域 (NBD) 1 的折叠和 NBD1 与跨膜域之间界面的稳定性。研究药物 VX-809（3-[6-[[[1-(2,2-二氟-1,3-苯并二氧杂环戊烯-5-基)环丙基]羰基]氨基]-3-甲基-2-吡啶基]-苯甲酸）或 R1070W 突变可以部分纠正界面稳定性缺陷。需要第二代 ΔF508-CFTR 校正器来改善现有囊性纤维化校正器的适度疗效。我们假设针对不同折叠/界面缺陷的第二个校正器可能与 VX-809 或 R1070W 抑制突变协同作用。通过在人肺上皮细胞系 (CFBE41o(-)) 中表达带有辣根过氧化物酶 (HRP) 的嵌合 CFTR，在存在或不存在 R1070W 的情况下，开发了一种用于 CFTR 细胞表面表达的生化筛选。使用 HRP 活性的发光读数，对大约 110000 种小分子进行筛选，产生了 9 种新型校正器支架，在存在最大 VX-809 的情况下，将细胞表面 ∆F508-CFTR 表达增加了高达 200%。对从初次筛选中鉴定出的 1006 种化合物的类似物进行进一步筛选，得到了 15 种 EC50<5µM 的校正剂。八种化学支架在恢复表达 ∆F508 的 A549 细胞中的氯离子通透性方面与 VX-809 表现出协同作用。一种氨基噻唑将囊性纤维化纯合子个体的人支气管上皮细胞中的氯离子电导率提高到超过最大 VX-809 的水平。机制研究表明，NBD2 是氨基噻唑拯救所必需的。我们的研究结果为协同筛选提供了概念验证，以鉴定第二代校正器，当与第一代校正器联合使用时，这些校正器可能会克服第一代校正器的“治疗上限”。
