Abstract
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-TARGETplus 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.
Highlights
The cystic fibrosis transmembrane conductance regulator (CFTR) is finely controlled in its bioavailability at the apical surface of epithelial cells, where it plays a significant role as a chloride channel
The fluorogenic malachite green (MG) analog dyes become fluorescent only once bound to the dL5**FAP, which eliminates the need for wash
We have developed a new assay that selectively and quantitatively assesses the cell surface expression and overall protein content of membrane proteins in the same population of cells, and applied this assay to identify potential kinase targets that may enhance F508del- CFTR rescue to the plasma membrane in combination with VX-809 treatment
Summary
The cystic fibrosis transmembrane conductance regulator (CFTR) is finely controlled in its bioavailability at the apical surface of epithelial cells, where it plays a significant role as a chloride channel. Aberrant cellular trafficking and dysfunction of CFTR in the lung leads to decreased chloride transport, resulting in cystic fibrosis (CF) symptoms. These arise from a thickened mucus layer in lung epithelia, which causes airway obstruction, inflammation, chronic infection, and severe reduction in life expectancy. The disease is most frequently caused by the deletion of phenylalanine at position 508 of the CFTR gene (F508delCFTR).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
