Department of Chemistry , University of California , Berkeley , California 94720 , United States.
Mol Pharm. 2018 Mar 5;15(3):759-767. doi: 10.1021/acs.molpharmaceut.7b00928. Epub 2018 Feb 9.
The most promising F508del-CFTR corrector, VX-809, has been unsuccessful as an effective, stand-alone treatment for CF patients, but the rescue effect in combination with other drugs may confer an acceptable level of therapeutic benefit. Targeting cellular factors that modify trafficking may act to enhance the cell surface density of F508-CFTR with VX-809 correction. Our goal is to identify druggable kinases that enhance F508del-CFTR rescue and stabilization at the cell surface beyond that achievable with the VX-809 corrector alone. To achieve this goal, we implemented a new high-throughput screening paradigm that quickly and quantitatively measures surface density and total protein in the same cells. This allowed for rapid screening for increased surface targeting and proteostatic regulation. The assay utilizes fluorogen-activating-protein (FAP) technology with cell excluded and cell permeant fluorogenic dyes in a quick, wash-free fluorescent plate reader format on live cells to first measure F508del-CFTR expressed on the surface and then the total amount of F508del-CFTR protein present. To screen for kinase targets, we used Dharmacon's ON-TARGET plus SMARTpool siRNA Kinase library (715 target kinases) with and without 10 μM VX-809 treatment in triplicate at 37 °C. We identified several targets that had a significant interaction with VX-809 treatment in enhancing surface density with siRNA knockdown. Select small-molecule inhibitors of the kinase targets demonstrated augmented surface expression with VX-809 treatment.
最有前途的 F508del-CFTR 校正剂 VX-809 作为 CF 患者的有效单一治疗方法并不成功，但与其他药物联合使用的挽救效果可能带来可接受的治疗益处。针对可改变运输的细胞因子可能通过与 VX-809 校正剂一起作用来增强 F508-CFTR 的细胞表面密度。我们的目标是确定可靶向的激酶，这些激酶可以增强 F508del-CFTR 的挽救和稳定作用，使其超过 VX-809 校正剂单独作用的效果。为了实现这一目标，我们实施了一种新的高通量筛选范式，该范式可以快速定量测量相同细胞中的表面密度和总蛋白。这使得可以快速筛选出增加的表面靶向和蛋白质稳定调节。该测定法利用氟发生蛋白（FAP）技术，在活细胞上使用细胞排除和细胞渗透的荧光染料进行快速、无洗涤的荧光板读数格式，首先测量表面表达的 F508del-CFTR，然后测量存在的 F508del-CFTR 总蛋白量。为了筛选激酶靶标，我们使用 Dharmacon 的 ON-TARGET plus SMARTpool siRNA 激酶文库（715 个靶标激酶），在 37°C 下进行三重复实验，同时或不进行 10 μM VX-809 处理。我们确定了几个靶标，它们在通过 siRNA 敲低增强表面密度方面与 VX-809 治疗有显著相互作用。选择几种激酶靶标的小分子抑制剂，与 VX-809 治疗联合使用时表现出增强的表面表达。
