Identification of Small Molecule Correctors of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Delta508 Mutation Function in Human Bronchial Epithelial Cells. Measured in Cell-Based System Using Plate Reader - 7017-01_Other_SinglePoint_HTS_Activity
Assay Overview: Halide-sensitive YFP quenching assay in CFBE cells expressing a mutated form of CFTR. ..more
BioActive Compounds: 1253
Keywords: Cystic Fibrosis, CFTR and corrector
Assay Overview: Halide-sensitive YFP quenching assay in CFBE cells expressing a mutated form of CFTR.
Cystic fibrosis (CF) is an autosomal recessive disease, caused by mutations in the gene coding for the Cystic Fibrosis Transmembrane conductance Regulator (CFTR). About 90% of people with CF have at least one copy of the "F508del" mutation (deletion of phenylalanine at position 508, or CFTR-F508del), and over 70% of CF patients are homozygous for this mutation. CFTR-F508del proteins are improperly folded, defective in trafficking to the plasma membrane, which results in the impairment of the channel functions. The mutated channel is still functional but is not present at the cell surface to achieve its normal functions. This assay aims to evaluate the correcting activity of small molecules on the function of CFTR-F508del. The correcting activity is determined by examining the fluorescence quenching ability of human bronchial epithelial CFBE cells expressing CFTR-F508del as well as a halide-sensitive Yellow Fluorescent Protein (YFP). Compound(s) restoring CFTR-F508del function will allow additional mutated CFTR channels to be expressed at the cell surface resulting in an enhanced fluorescent quenching ability of these CFBE cells.
Expected outcome: Identify small molecules decreasing the fluorescence mediated by a halide-sensitive YFP present in CFBE cells expressing a mutated form of the human CFTR protein (F508del) by the quencher sodium iodide. Compounds reducing the fluorescence further down compared to the neutral control by at least 8% in duplicate will be considered as active hits.
Cystic Fibrosis airway epithelial cells (CFBE) have been transiently transfected (electroporation) with vectors containing an Yellow Fluoresencent Protein (YFP) halide-sensitive mutant and the human Cystic Fibrosis Transmembrane conductance Regulator with the deletion of the amino acid phenylalanine 508 (CFTR-F508del) provided by Dr. Jinliang Sui from the Flatley Discovery laboratory (Charlestown, MA).
Day 1 (Cell plating and compound pinning in assay plates)
Prior to perform the luciferase reporter assay experiments, the CFBE cells are thawed and reconstituted in MEM medium (Invitrogen, 11965) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS) (Gibco, 16140), 1X antibiotic (Penicillin/Streptamycin/Glutamine) (Gibco, Ref. no. 10378-016). CFBE cells are then plated in 384-well format cell culture treated solid black wall clear bottom assay plates (Corning, Cat. # 3712) at a density of 6,000 cells/well using the multidrop dispenser (standard cassette)(Thermo Scientific) in a final volume of 50 ul. The assay plates are placed in 5% CO2 incubator where they are incubated for 2h at 37oC. After 2h of incubation, the assay plates are pulled out of the incubator and are placed side by side on a pinning table adjacent to compound plates containing the MLPCN library and a sentinel plate containing 32 wells with the positive control C18. 100 nl of the compounds and the positive control C18 (final concentration 10 uM) are then collected on metal pins from these compound plates and transferred to the assay plate. The pins are washed with DMSO and methanol between each transfer. The assay plates are then moved back to the 5% CO2 incubator and incubated for an additional 24h.
Day 2 (Cell wash, CFTR channel activation, quenching and reading on the Flipr)
The assay plates are coming out of the incubator and washed once with PBS (50 ul) using a Biotek plate washer and 20ul of 20 uM Forskolin (Cayman Chemicals) and 3uM potentiator P3 (final concentration) in DPBS is added to the assay plate for 1h at 37oC. Then, the assay plates are cooled down for 30 minutes at room temperature and 25 ul of YFP quencher sodium iodide (72.5 mM final concentration) in DPBS is added and the fluorescence is measured every second for one minute on the Flipr tetra (Molecular Devices).
DPBS (Dulbecco's Phosphate buffered saline)
2.7 mM KCL
8.1 mM Na2PO4
1.5 mM KH2PO4
0.7 mM CaCl2*2H2O
1.1 mM MgCl2
137 mM NaCl
145 mM NaI
PRESENCE OF CONTROLS: Neutral control wells (NC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 8.
PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T
Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T
Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T
Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)