High-Content Surface and Total Expression siRNA Kinase Library Screen with VX-809 Treatment Reveals Kinase Targets that Enhance F508del-CFTR Rescue

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-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.

Fluorogen activating protein toolset for protein trafficking measurements

“…Labeling all tagged protein (inside and out) can be a useful tactic for relating the amount of protein at the cell surface with total amount of protein, distinguishing proteostatic or biosynthetic effects from trafficking effects (Figure B). Either a matched or a different color cell‐permeable dye can be used sequentially after surface labeling to give a surface/total POI measurement that increases overall mechanistic insight in a variety of multi‐cell or single‐cell assays (Figure E) …”

“…The second dye, MGnBu, is a new MG dye derivative that has the same brightness as MG‐B‐Tau allowing for quantitative collection of the combined signal of MG‐B‐Tau labeling on the surface plus MGnBu intracellular labeling with the same excitation and emission wavelengths, providing a direct measurement of the surface fraction and total protein levels on a sample‐by‐sample or cell‐by‐cell basis. This FAP approach was used to investigate all kinase targets knocked down by siRNA that could enhance CFTR corrector, VX‐809, mediated rescue of ΔF508‐CFTR . The versatility of FAP across microscopy, plate reader and flow‐cytometry instruments has allowed for diverse strategies to measure CFTR trafficking and rescue to the plasma membrane at a drug‐discovery and systems‐biology experimental scale, and for detailed mechanistic investigations.…”

“…This FAP approach was used to investigate all kinase targets knocked down by siRNA that could enhance CFTR corrector, VX-809, mediated rescue of ΔF508-CFTR. 25 The versatility of FAP across microscopy, plate reader and flow-cytometry instruments has allowed for diverse strategies to measure CFTR trafficking and rescue to the plasma membrane at a drug-discovery and systems-biology experimental scale, and for detailed mechanistic investigations.…”

“…Either a matched or a different color cell-permeable dye can be used sequentially after surface labeling to give a surface/total POI measurement that increases overall mechanistic insight in a variety of multi-cell or single-cell assays ( Figure 2E). [25][26][27] The FAP platform was developed by screening a yeast display library of single chain variable fragment antibodies (scFv) to find proteins that bind and activate two distinct cell-excluded fluorogenic dye derivatives: a malachite green with an amido-diethylene glycol amine linker (MG-2p) and a sulfonated thiazole orange with a similar linker (TO1-2p). The dL5** and AM2-2 FAP tags noncovalently bind MG and TO1 dye derivatives respectively with high affinities (Figures 3 and 4).…”

“…• High-content surface and total expression siRNA kinase library screen with VX-809 treatment reveals kinase targets that enhance F508del-CFTR rescue. (Figure 6E, dL5**:MG-bTau, and dL5**:MGnBu) 25 • Select α-arrestins control cell-surface abundance of the mammalian Kir2.1 potassium channel in a yeast model. (Figure 6C, D, dL5**:MG-11p and dL5**: MG-Ester) 74 range of conditions and diseases.…”