Ambrosetti Adam D, Hagedorn Zachary J, Bono Taylor R, Wen Hui, Nguyen Rodney, Rodriguez-Cruz Kevin, Ali Judge, Palacio Hayes, Phillips Aubrey J, Gilliland Stephanie D, Freeman Alana J, Thompson Jake, Fu Lianwu, McNicholas Carmel M, Rowe Steven M, Wang X Robert
McWhorter School of Pharmacy, Department of Pharmaceutical, Social and Administrative Sciences, Samford University, Birmingham, Alabama, United States.
Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, United States.
Am J Physiol Lung Cell Mol Physiol. 2025 Jun 1;328(6):L832-L843. doi: 10.1152/ajplung.00287.2024. Epub 2025 May 7.
Lumacaftor, the corrector of Orkambi, enhances the processing of F508del cystic fibrosis transmembrane conductance regulator (CFTR), but its impact on the channel activity of rescued F508del CFTR (rF508del) is unclear. Using an electrode-based, real-time iodide efflux assay performed at room temperature, acute exposure to lumacaftor was shown to increase the processing of F508del CFTR without a proportional increase in channel activity in a CFBE41o-cell line stably expressing F508del CFTR (CFBE-DF). A similar effect was not observed on wild-type CFTR in a HEK293 cell line. At 37°C, rF508del channel activity is significantly inhibited in CFBE-DF cells by acute exposure to 5 µM lumacaftor, but not to 5 µM tezacaftor or 1 µM elexacaftor, the two correctors of Trikafta. Lumacaftor's inhibitory effect was characterized by a major left shift of the peak channel activity relative to the peak CFTR processing in the dose-response chart, which is absent for tezacaftor or elexacaftor. Ussing chamber analysis on polarized CFBE-DF cells reveals an inhibitory effect for lumacaftor on the forskolin- and ivacaftor-induced change in short-circuit current. Single channel patch clamp on HEK-DF cells shows that acute application of cytosolic lumacaftor significantly decreases rF508del channel open probability. Taken together, despite its strong corrector activity, lumacaftor inhibits rF508del channel activity, compromising the degree of functional rescue. This effect may contribute to the limited clinical efficacy of Orkambi. Small-molecule correctors bind to F508del cystic fibrosis transmembrane conductance regulator (CFTR) and restore its trafficking to the plasma membrane to function as an anion channel. Despite its high efficacy as a corrector, lumacaftor inhibits the channel opening of rescued F508del CFTR, making it a weak CFTR modulator. The current work highlights the impact of CFTR correctors on the channel activity of rescued F508del CFTR as an important variable in the efficacy of modulator therapy.
鲁马卡托（Orkambi中的校正剂）可增强F508del囊性纤维化跨膜传导调节因子（CFTR）的加工过程，但其对挽救的F508del CFTR（rF508del）通道活性的影响尚不清楚。在室温下使用基于电极的实时碘外流试验，结果显示在稳定表达F508del CFTR的CFBE41o细胞系（CFBE-DF）中，急性暴露于鲁马卡托可增加F508del CFTR的加工过程，但通道活性并未成比例增加。在HEK293细胞系中，野生型CFTR未观察到类似效应。在37°C时，CFBE-DF细胞中急性暴露于5 μM鲁马卡托会显著抑制rF508del通道活性，但暴露于5 μM泰扎卡托或1 μM依列卡托（Trikafta的两种校正剂）则不会。鲁马卡托的抑制作用表现为剂量反应图中通道活性峰值相对于CFTR加工峰值大幅左移，而泰扎卡托或依列卡托则不存在这种情况。对极化的CFBE-DF细胞进行尤斯灌流室分析显示鲁马卡托对福斯可林和依伐卡托诱导的短路电流变化有抑制作用。对HEK-DF细胞进行单通道膜片钳实验表明，胞质内急性应用鲁马卡托会显著降低rF508del通道开放概率。综上所述，尽管鲁马卡托具有很强的校正活性，但它会抑制rF508del通道活性，从而影响功能挽救的程度。这种效应可能导致Orkambi的临床疗效有限。小分子校正剂与F508del囊性纤维化跨膜传导调节因子（CFTR）结合，使其转运至质膜，发挥阴离子通道的功能。尽管鲁马卡托作为校正剂具有很高的疗效，但它会抑制挽救的F508del CFTR的通道开放，使其成为一种较弱的CFTR调节剂。目前的研究突出了CFTR校正剂对挽救的F508del CFTR通道活性的影响，这是调节剂治疗疗效的一个重要变量。
