Improved fluorescence assays to measure the defects associated with F508del-CFTR allow identification of new active compounds

Article abstract of DOI:10.1111/bph.13715

Background and Purpose: Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for a Clˉ/HCO₃ˉ channel. F508del, the most common CF-associated mutat

Full text of DOI:10.1111/bph.13715

1
Title: Improved fluorescence assays to measure the defects associated with F508del-
CFTR allow identification of new active compounds
Running Title: Improved fluorescence assays for monitoring CFTR
Emily Langron1, Michela I. Simone2, Clémence M.S. Delalande3, Jean-Louis Reymond3, David L.
Selwood4 and Paola Vergani1
1 Research Department of Neuroscience, Physiology and Pharmacology
University College London
Gower Street
London WC1E 6BT
UK
2 Discipline of Chemistry, School of Environmental and Life Sciences,
Priority Research Centre for Chemical Biology and Clinical Pharmacology
The University of Newcastle
University Drive
Callaghan, NSW 2308, Australia.
3 Department of Chemistry and Biochemistry
University of Bern
Freiestrasse 3, BERN 3012
Switzerland
4 Wolfson Institute for Biomedical Research
University College London
The Cruciform Building
Gower Street
London WC1E 6BT
UK
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/bph.13715
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Correspondence to: emily.langron.12@ucl.ac.uk; p.vergani@ucl.ac.uk
Word count (excluding Methods and References): 3486
Abstract
BACKGROUND AND PURPOSE
Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene, which codes for a Clˉ/HCO3ˉ channel.
F508del, the most common CF-associated mutation, causes both gating and biogenesis
defects in the CFTR protein. This paper describes the optimisation of two fluorescence
assays, capable of measuring CFTR function and cellular localisation, and their use in a pilot
drug screen.
EXPERIMENTAL APPROACH
HEK293 cells expressing YFP-F508del-CFTR, in which halide sensitive YFP is
tagged to the N-terminal of CFTR, were used to screen a small library of compounds based
on the VX-770 scaffold. Cells expressing F508del-CFTR-pHTomato, in which a pH sensor is
tagged to the fourth extracellular loop of CFTR, were used to measure CFTR plasma
membrane exposure following chronic treatment with the novel potentiators.
KEY RESULTS
Active compounds with efficacy ~50% of VX-770, micromolar potency, and
structurally distinct from VX-770 were identified in the screen. The F508del-CFTR-
pHTomato assay suggests that the hit compound MS131A, unlike VX-770, does not decrease
membrane exposure of F508del-CFTR.
CONCLUSIONS AND IMPLICATIONS
Negative influence on F508del-CFTR biogenesis/stability by most known potentiators
requires membrane exposure to be monitored early during development of drugs targeting
CFTR. Combined use of the two fluorescence assays described here provides a useful tool for
the identification of improved potentiators and correctors. The assays could also prove useful
for basic scientific investigation on F508del-CFTR, and other CF-causing mutations.
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TARGETS
Ion channels
CFTR
LIGANDS
Forskolin
VX-770, Ivacaftor
VX-809, Lumacaftor
These Tables of Links list key protein targets and ligands in this article that are hyperlinked* to
corresponding entries in http://www.guidetopharmacology.org, the common portal for data from
the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in
The Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).
Abbreviations
CF
Cystic Fibrosis
CFTR
Cystic Fibrosis Transmembrane conductance Regulator
change in fluorescence reporting on membrane-localised CFTR-
FM
pHTomato
FIN
vesicles
change in fluorescence reporting on CFTR-pHTomato in biosynthetic
EC50
half-maximal effective concentration
enhanced Green Fluorescent Protein
normalised YFP fluorescence
eGFP
F/Fmax
FpHTomato
GFP
weighted average fluorescence obtained during pHTomato assay
Green Fluorescent Protein
MES
2-(N-morpholino)ethanesulfonic acid
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nH
Hill coefficient
Po
open probability
RFU
SSMD
τib
Relative Fluorescence Units
strictly standardised mean difference
interburst duration
τb
burst duration
YFP
WT
yellow fluorescent protein
wild type
Introduction
Cystic fibrosis (CF) is the most common life-shortening hereditary disease in the
Caucasian population, affecting approximately 10,800 patients in the UK alone (UK CF
Registry annual report 2015). The disease is caused by mutations in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene, which codes for the CFTR anion
channel (Riordan et al., 1989). To date, over 2,000 mutations have been identified
(www.genet.sickkids.on.ca; www.cftr2.org). Approximately 90% of patients, however, carry
at least one copy of F508del, the most common CF-causing mutation, which results in both a
biogenesis and a gating defect in the CFTR protein (Cheng et al., 1990; Dalemans et al.,
1991). F508del prevents maturation to complex-glycosylated, plasma membrane-located
CFTR (Cheng et al., 1990). A global misfolding of the protein causes retention of F508del-
CFTR at the endoplasmic reticulum, from where it is targeted for proteasomal degradation
(Ward et al., 1995). Any F508del-CFTR which escapes degradation and reaches the plasma
membrane has a short half-life (< 4 h; Lukacs et al., 1993), due to increased degradation and
reduced recycling (Okiyoneda et al., 2010). F508del-CFTR membrane exposure can be
improved in the lab using low temperature incubation, allowing the study of ion channel
function (Denning et al., 1992). Using this technique, the open probability (Po) of F508del-
CFTR was measured as being ~15-fold smaller than that of wild type (WT)-CFTR (Miki et
al., 2010), indicating a severe gating defect.
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In 2012, VX-770 (Ivacaftor, Vertex Pharmaceuticals) became the first drug targeting
the CFTR protein, approved for treatment of CF. Acting on the root cause, VX-770 has
demonstrated a clear positive impact in the clinic (Ramsey et al., 2011; Davies et al., 2013).
However, it is only effective for CF caused by G551D and a number of other, less common,
gating mutations (Van Goor et al., 2009; Yu et al., 2012). The G551D mutation causes severe
gating defects, but does not affect folding or plasma membrane exposure (Gregory et al.,
1991; Bompadre et al., 2007). Consistent with the pleiotropy of the F508del mutation, VX-
770 alone was not effective on patients homozygous for F508del (Flume et al., 2012).
Treatment of F508del-CF will likely require a combination of a corrector compound, such as
VX-809 (Lumacaftor, Vertex Pharmaceuticals), to promote F508del-CFTR membrane
localisation, together with a potentiator, to increase Po (Rowe and Verkman, 2013). An
example of one such combination therapy, Orkambi (VX-770+VX-809, Vertex
Pharmaceuticals), was recently approved for the treatment of F508del homozygous patients.
A Phase III study revealed a mean absolute improvement in lung function (measured by
forced expiratory volume in one second), of 2.6 – 4% (Wainwright et al., 2015). Compared to
in vitro results using acute VX-770 treatment alongside chronic VX-809 (conductance up to
30% of WT-CFTR; Van Goor et al., 2011), clinical efficacy was low.
It has since been shown that chronic VX-770 treatment decreases F508del-CFTR
membrane exposure, regardless of the method of rescue (Cholon et al., 2014; Veit et al.,
2014). The same was true of all potentiators that were tested, except one (Veit et al., 2014).
The negative effect of chronic VX-770 on F508del-CFTR biogenesis and stability possibly
contributed to the relatively disappointing Orkambi clinical trial results (but see Matthes et
al., 2016).
This paper describes two fluorescence assays, capable of measuring the defects
associated with F508del-CFTR: inefficient gating and defective biogenesis, resulting in low
steady-state levels of CFTR protein at the plasma membrane. Yellow fluorescent protein
(YFP)-CFTR, in which halide sensitive YFP (Galietta et al., 2001a) is tagged to the N-
terminal of CFTR, provides a sensitive readout of CFTR function. CFTR-pHTomato, with a
pH sensor tagged to the fourth extracellular loop of CFTR, can quantify membrane and
internal CFTR, independently of each other, and independently of function.
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YFP-F508del-CFTR was used to screen a pilot library of compounds with some
structural similarity to VX-770. The hits from this screen were tested for long-term F508del-
CFTR destabilisation effects, using the F508del-CFTR-pHTomato assay.
Methods
YFP-CFTR
GFP-CFTR, in which enhanced GFP (eGFP) is tagged to the N-terminal of CFTR via
a 23 amino acid linker, was kindly provided by Bruce Stanton (Geisel School of Medicine,
NH), in pcDNA3.1. To obtain YFP-CFTR, eight mutations (F46L, L64F, S65G, V68L,
S72A, H148Q, I152L and T203Y) were introduced in eGFP-CFTR, using site-directed
mutagenesis (Quikchange protocol, Stratagene).
CFTR-pHTomato
CFTR-pHTomato is expressed from the pIRES2-eGFP plasmid, which contains an
internal ribosome entry site (IRES). This plasmid directs transcription of a single mRNA,
containing coding sequences for both CFTR-pHTomato and eGFP, and translation of the two
separate proteins. WT-CFTR in pIRES2-eGFP (pIRES-eGFP-CFTR) was a gift from David
Gadsby (Rockefeller University, NY). pHTomato in the pRham plasmid was provided by Dr
Li (Peking University, China) and Prof. Richard Tsien (NYU School of Medicine, NY),
while pcDNA3.1-CFTR was a gift from Luis Galietta (Istituto Gaslini, Genova, Italy).
pHTomato was inserted at a position, following Asp901, known not to interfere with cellular
processing and function (Howard et al., 1995; Schultz et al., 1997). Using a primer overlap-
extension strategy AgeI and BmtI sites were first introduced within extracellular loop 4 in
pcDNA3.1-CFTR. A fragment including the coding sequence for pHTomato, flanked by the
two restriction sites, was obtained by PCR using the pRham-pHTomato plasmid as template.
Following restriction digest and ligation to obtain pcDNA3.1-CFTR-pHTomato, the
pHTomato insertion was subcloned into the pIRES2-eGFP-CFTR plasmid, using the SalI and
BspEI restrictions sites, present in WT-CFTR.
Screening library and compound syntheses
The UCL Chemibank (a resource of diverse, mostly drug-like compounds) was
searched for compounds with similarity to VX-770. A set of compounds sharing the
quinolone scaffold of VX-770 were identified (patent WO2015189560A1). This set also
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included dissimilar intermediates which were also screened to identify MS131A and
MS134A. Similarity was calculated as Tanimoto coefficients of ECFP 4 fingerprints using
Pipeline Pilot.
http://pubs.acs.org/doi/abs/10.1021/ci100050t
MS131A, diethyl 2-(((3-(difluoromethoxy)phenyl)amino)methylene)malonate
3-(Difluoromethoxy)aniline (0.51 g, 3.21 mmol) was stirred with diethyl
ethoxymethylenemalonate (650 L, 3.21 mmol) at 85°C for two hours, at 100°C for 18 hours
and at 120°C for 3.3 hours. T.l.c. analysis (acetone : cyclohexane, 1:3) showed presence of
one product (Rf 0.13) and consumption of both starting materials (Rf 0.32 and 0.46). The
reaction solution was concentrated in
vacuo to give diethyl 2-(((3-
(difluoromethyl)phenyl)amino)methylene)malonate (1.00 g, quant.) as a dark yellow oil.
NMR Data – 1H-NMR (400 MHz, CDCl3): 11.0 (1H, d, JNH,HE 10.8 Hz, NH), 8.46 (1H, d,
4
JHE,NH 10.8 Hz, HE), 7.35 (1H, t, JHA,CHF2 6.8 Hz, HA), 6.98 (1H, ddd, J 6.4, 1.6, 0.4 Hz,
2
HC), 6.91-6.88 (2H, m, HB & HD), 6.53 (1H, t, JCH,F 58.8 Hz, CHF2), 4.31, 4.25 (2 x q,
JCH2,CH3 5.6 Hz for both q, 2 x CH2), 1.38, 1.33 (2 x t, JCH3,CH2 5.6 Hz for both t, 2 x CH3).
13C-NMR (100 MHz, CDCl3): 169.0, 165.5 (2 x C=O), 162.3 (Cq-CHF2), 151.4 (CHE),
2
140.9 (Cq-NH), 131.2 (CHA), 115.7 (t, JCH,F 20.7 Hz, CHF2), 115.4, 108.7 (CHB, CHD),
113.9 (CHC), 94.7 (Cq-CHE), 60.7, 60.3 (2 x CH2), 14.5, 14.3 (2 x CH3).
MS134A, diethyl 2-(((4-methoxyphenyl)amino)methylene)malonate
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para-Anisidine (4-methoxyaniline) (2.01 g, 16.32 mmol) was stirred with diethyl
ethoxymethylenemalonate (3.28 mL, 16.24 mmol) between 100 and 125°C for 18.5 hours.
T.l.c. analysis (ethyl acetate : cyclohexane, 1:3) showed presence of one product (Rf 0.41)
and consumption of both starting materials (Rf 0.32 and 0.49). The reaction solution was
concentrated in vacuo to give diethyl 2-(((4-methoxyphenyl)amino)methylene)malonate (4.76
g, quant.) as a dark liquid. NMR Data – 1H-NMR (400 MHz, CDCl3): 10.96 (1H, d, JNH,HE
13.7 Hz, NH), 8.41 (1H, d, JHE,NH 13.8 Hz, HE), 7.05 (2H, d, JHA,HB 6.8 Hz, HA), 6.88 (1H,
d, JHB,HA 7.2 Hz, HB), 4.27, 4.21 (2 x q, JCH2,CH3 5.6 Hz for both q, 2 x CH2), 3.82 (3H,
OCH3), 1.35, 1.29 (2 x t, JCH3,CH2 5.6 Hz for both t, 2 x CH3). 13C-NMR (100 MHz,
CDCl3): 169.3, 165.9 (2 x C=O), 157.2 (Cq-OCH3), 152.7 (CHE), 132.8 (Cq-NH), 118.8,
115.1 (CHA, CHB), 92.5 (Cq-CHE), 60.3, 60.0 (2 x CH2), 55.6 (OCH3), 14.5, 14.4 (2 x CH3).
LCMS (ES+) 316 [M+Na]+, 293.1 [MH]+.
Virtual screening for analogues of MS131A was performed using the multifingerprint
browser for the ZINC database (Awale and Reymond, 2014). Analogues were selected on the
basis of the available five different similarity methods by clustering of the 1000 nearest
neighbours in each case. Focusing on compounds available from Princeton Biomolecular
Research, we obtained 61 compounds for experimental screening, which were purchased as 1
mg solid samples and conditioned as 10 mM stock solutions in DMSO for testing.
Materials, Cell culture and transfections
Unless otherwise stated, chemicals were purchased from Sigma Aldrich. VX-770 and
VX-809 were purchased from Selleck Chemicals. P2 (PG-01; Pedemonte et al., 2005b), P5
(dF508act-02; Yang et al., 2003), and Corr-4a (4a; Pedemonte et al., 2005a) were obtained
from the CFTR Chemical Compound Distribution Program, sponsored by the Cystic Fibrosis
Foundation Therapeutics. HEK293 cells were maintained in DMEM, supplemented with 2
mM L-glutamine, 100 U/ml penicillin and streptomycin, and 10% FBS (all Life
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Technologies). For fluorescence imaging, cells were seeded in poly-D-lysine (PDL)-coated,
black walled 96-well plates (Costar, Fisher Scientific).
Lipofectamine transfection was used for all cells used for patch-clamp and imaging
experiments. Cells plated in 96-well plates were transiently transfected with the appropriate
YFP-CFTR- or CFTR-pHTomato- encoding plasmid using Lipofectamine 2000 (Life
Technologies), according to manufacturer’s instructions. Following transfection, cell plates
were returned to the 37°C incubator for 24 h. Where indicated, plates were further incubated
at 30°C for 24 h prior to imaging, with or without additional drug treatment.
Single channel patch-clamp recording
HEK293 cells in 35 mm dishes, expressing WT-CFTR or YFP-WT-CFTR, along with
GFP as a marker of transfection, were used for voltage-clamp recording, in the excised
inside-out patch configuration. Excised patch recordings were made using an EPC9 amplifier
and Pulse acquisition software (HEKA Instruments Inc., MA, US). Cells were mounted in 35
mm dishes above an inverted fluorescence microscope (Nikon Eclipse TE200), with mercury
light source and GFP filters (excitation/emission 450-500 nm/510-560 nm, dichroic mirror
transmitted > 505 nm). Cells were continuously perfused with bath solution (134 mM
NMDG, 134 mM HCl, 2 mM MgCl2, 5 mM HEPES, 0.5 mM EGTA, pH 7.1). Pipette
solution contained 136 mM NMDG, 136 mM HCl, 2 mM MgCl2 and 5 mM HEPES (pH 7.4).
Pipettes were pulled from borosilicate glass capillaries (Harvard Apparatus Ltd., Cambridge,
UK) using a vertical pipette puller (Narishige PP-830, Digitimer Ltd.), were coated with
Sylgard (Sigma-Aldrich), and fire polished on a microforge (Narishige MF-830, Digitimer).
When filled with pipette solution, they typically had a resistance of around 12 MΩ.
The cytosolic face of the patch was continuously perfused using a gravity-driven
microperfusion system. CFTR was activated by addition of 300 nM PKA (~100 U/ml
catalytic subunit from bovine heart, Sigma-Aldrich P-2645) and 2 mM ATP for
approximately 2 minutes in order to reach steady state phosphorylation. After this time, PKA
was removed and recording continued in 2 mM ATP. No significant difference was found in
the value of kinetic parameters obtained before and after PKA removal, consistent with a
very slow dephosphorylation rate in this system. Continuous recordings were acquired at 5
kHz and pre-filtered at 1 kHz, at room temperature with the pipette potential held at + 60 mV
(- 60 mV membrane potential).
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Analysis of patch data
Records were exported to ClampFit (Molecular Devices), and filtered digitally at 50
Hz. Programs developed by László Csanády (Semmelweis University, Budapest) were used
to analyse traces (Csanády, 2000). Briefly, the distributions of dwell-times at all conductance
levels were jointly fit assuming a three-state gating model (C1 ↔ O ↔ C2), in which C1 ↔ O
represents a simplified opening and closing (entry and exit from bursts), and O ↔ C2
represents ATP-independent fast, flickery closures. Maximum Likelihood fitting provided
estimates for opening rate (rCO), closing rate (rOC) and flicker rates (rOF and rFO: on and off
rates of flicker closures, respectively) of the channel, and thus interburst duration (τib,
1/rCO), and open burst duration (τb, = (1/rOC)*(1+rOF/rFO)).
=
Both rCO and rOC can be reliably estimated when the number of channels in the patch
is known. In most recordings the number of channels in the patch could not be estimated with
sufficient confidence (see Vergani et al., 2003 for criteria used to determine whether number
of channels in the patch was unknown). In these circumstances, noise analysis was used to
estimate Po. τb can still be estimated from dwell time distributions, as it is not steeply
dependent on the number of channels. Therefore, when the number of channels in a patch
could not be determined, Po was obtained from noise analysis, τb from Maximum Likelihood
fitting of dwell-time histograms, and τib as
Equation [1]
YFP-CFTR wide-field imaging
All imaging was carried out using ImageXpress (ImageXpress Micro XLS, Molecular
Devices); an image-acquisition system equipped with wide-field inverted fluorescence
microscope, CMOS camera and fluidics robotics. 96-well cell plates were contained in an
environmental chamber, at 37°C or 30°C. YFP-CFTR was imaged using a 20X objective, and
excitation/emission filters 472 ± 30 nm and 520 ± 35 nm. For each plate, the laser intensity
and exposure were optimised to achieve the highest possible fluorescence whilst avoiding
both photobleaching and saturation (illumination intensity 100-150/225 cd, and exposure 0.1
– 0.2 s)
Before imaging, cells were washed twice with 100 µl standard buffer (140 mM NaCl,
4.7 mM KCl, 1.2 mM MgCl2, 5 mM HEPES, 2.5 mM CaCl2, 1 mM Glucose, pH 7.4). Images
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were taken for 150 s at a frequency of 0.5 Hz. 50 µl extracellular Iˉ (as standard buffer with
140 mM NaCl replaced with 300 mM NaI; resulting in 100 mM final [Iˉ]) was added at 20 s,
and activating compounds were added at 60 s (in 100 mM Iˉ) at the concentrations indicated.
For experiments in which extracellular [Iˉ] was lower (Figure 1), the sum of [NaCl] and [NaI]
was maintained at 140 mM.
YFP-CFTR confocal imaging
Confocal images were acquired using the Opera High Content Screening System
(PerkinElmer Life and Analytical Sciences, Bucks., UK). Cells were washed as above,
incubated with Hoechst nuclear stain, and imaged with a 40X objective at 37°C. YFP-CFTR
imaging was carried out using a triple bandpass excitation filter (488/561/640 nm) and
emission filter 540 ± 37.5 nm. Hoechst was excited by UV light and a 425 nm dichroic
longpass mirror; emission filter was at 450 ± 25 nm.
CFTR-pHTomato imaging
Before imaging, cells were washed twice with 100 µl standard buffer (as above).
During imaging, extracellular pH was changed using addition of 50 µl pH 6 buffer (as
standard buffer, with 5 mM HEPES replaced with 10 mM MES: final [MES] 3.3 mM, final
value ~ pH 6.5), and 50 µl pH 9 buffer (as standard buffer, with 5 mM HEPES replaced with
100 mM Tris: final [Tris] 25 mM, ~ pH 8.8). Two pHTomato images (acquisition frequency
0.5 Hz) were taken in each condition, using bandpass excitation/emission filters 531 ± 20 nm
and 592 ± 20 nm. Single eGFP and Hoechst nuclear stain images were also acquired for each
well, using excitation/emission filters 472 ± 30 nm and 520 ± 35 nm, and 377 ± 25 nm and
447 ± 30 nm, respectively.
Data analysis
Images were analysed using ImageJ (http://rsbweb.nih.gov/ij/). For each well, data
were exported as a stack, with each time point represented by an image in the stack. For YFP-
CFTR, image areas corresponding to transfected cells were selected, before addition of Iˉ,
using a fluorescence threshold. Fluorescence was normalised to this maximal value. Very
high extracellular [Iˉ] allows accumulation of [Iˉ]in which completely quenches YFP-CFTR
fluorescence intensity to background levels (Figure 1), confirming that anion binding
abolishes fluorescence in our system. Thus fluorescence quenching can be described by an
equation including a Hill-Langmuir component:
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-
-
Equation [2]
Where
is the normalised fluorescence, KI is the dissociation constant for Iˉ binding to
YFP (1.9 mM; Galietta et al., 2001a) and [Iˉ]in is the concentration of Iˉ at the chromophore.
Experimentally, we can alter [Iˉ]in by increasing extracellular Iˉ concentration, [Iˉ]out. The fact
that we can describe the observed changes in fluorescence as a function of [I-]out with
equation [2] (Figure 1), suggests that membrane potential is not greatly affected by changes
in [Iˉ]out.
-
Equation [2] can be rearranged to express [I ]in as a function of
:
-
Equation [3]
A plot of [I-]in against time was used to calculate the maximal rate of Iˉ entry into cells
( , mM s-1), which was then used to quantify CFTR activation (Figure 2).
The analysis as described above does not take into account the presence of cytosolic
Clˉ ions, their binding to the chromophore at rest, and their displacement by Iˉ entering the
cytosol. However, the discrepancy between the [I-]in we calculate as above, assuming no
competing anion, and that obtained assuming up to 20 mM cytosolic Clˉ (Bregestovski et al.,
2009), is small (see S1, Supporting Information). To avoid introducing additional arbitrarily
chosen parameters to describe the more complex system, we therefore analyse our data
assuming no Clˉ competition, as likely done in previous publications exploiting the anion-
sensitive YFP (Galietta et al., 2001b; Pedemonte et al., 2005b).
For CFTR-pHTomato, regions corresponding to transfected cells were selected based
on eGFP fluorescence. For each selected region, the mean eGFP fluorescence (Fgreen) was
used to normalise the mean pHTomato fluorescence (Fred), to allow for differences in
transfection efficiency. A weighted average was then obtained, with each region weighted by
cell count:
Equation [4]
Dose-response curves were fitted using the Hill equation
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where nH is the Hill coefficient, EC50 is the half maximal effective concentration, y0 is the
response measured in the absence of ligand, L is ligand and a is the amplitude of the curve.
Where a value is not indicated, nH is set to 1.
Statistical Analysis
All average plots including error bars represent mean ± SEM. In cases where two
groups were compared, this was carried out using paired or unpaired t-test, as appropriate, or
Mann-Whitney U test in cases where data was not normally distributed. Significance
indicates p < 0.05. All statistical analysis was carried out using SigmaPlot (Systat Software).
Hit compounds from the pilot screen were identified using the strictly standardised
mean difference (SSMD) score (Zhang et al., 2007; Zhang, 2011). The mean and standard
deviation of the difference between the test and the negative reference was used to calculate
one SSMD value which incorporated all replicates:
Equation [5]
Where is the mean of the differences between the test compound and negative reference,
is the standard deviation of the same and is the median of all (Zhang, 2011). A
combination of a common factor ( ), and the individual sample variance ( ) was
used to stabilise the estimate of standard deviation due to the small n number (Zhang, 2011).
An SSMD value > 2 indicates a positive effect, consistently greater than the negative
control (Zhang, 2011). Compounds with weak but very consistent effects will also have high
SSMD values. To avoid selecting these compounds as hits, data were plotted on a dual-
flashlight plot, in which SSMD is plotted against average percentage activation. Chosen hit
compounds had SSMD > 2 and percentage activation > 50% of VX-770, and were therefore
both statistically and biologically favourable (Zhang, 2011).
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Data accessibility statement
Data associated with this paper will be made available in the Figshare repository.
Results
The YFP-CFTR assay
Following the development of an anion sensitive YFP (H148Q/I152L) with optimal
characteristics for CFTR research (Galietta et al., 2001a), a number of high-throughput
screening projects have utilised cell-based assays in which soluble YFP is expressed in the
cytosol (for examples see Galietta et al. 2001b; Pedemonte, et al. 2005b). Upon binding of
halides, YFP fluorescence decreases (Jayaraman et al., 2000). Exploiting a high affinity for Iˉ
compared to Clˉ (1.9 mM KI vs. 85 mM KCl; Galietta et al. 2001a) Clˉ/Iˉ exchange protocols
can report on CFTR function and, indirectly, on its membrane exposure.
We have engineered a YFP-CFTR fusion protein, in which YFP (H184Q/I152L) is
fused to the N-terminal of CFTR. Expressed in HEK293 cells it forms the basis of an
improved cell-based assay. Like in cytosolic YFP assays, in the presence of high extracellular
[Iˉ], and only if CFTR is present at the membrane and gating, Iˉ can enter the cell and quench
YFP fluorescence. Normalised fluorescence values (Figure 2A) are converted to [I-]in (Figure
2B), and CFTR activity is expressed in terms of the maximal rate of Iˉ entry into the cells
(obtained from plots such as Figure 2C). Due to the direct correlation between YFP
fluorescence intensity and levels of CFTR expression, normalising to fluorescence before
addition of Iˉ (i.e. maximal fluorescence) accounts for variability in cellular expression of the
probe, and reduces readout dispersion.
Using a high-content fluorescence microscope with fluidics capabilities (ImageXpress
Micro XLS, Molecular Devices), we can identify and separately measure image areas
corresponding to cells expressing YFP-CFTR as opposed to areas corresponding to
“background”. This allows sensitive, low-noise measurements of small, localised
fluorescence changes triggered by fluid additions. Such measurements were used to construct
a concentration-response curve to forskolin (Figure 2D). The forskolin EC50 obtained for
YFP-WT-CFTR was similar to that obtained for WT-CFTR in a mouse mammary epithelial
cell line (500 nM; Haws et al., 1996).
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WT-CFTR vs. YFP-WT-CFTR
Published studies suggest that a GFP tag on the N-terminal of CFTR is not
detrimental to either trafficking or function of the channel (Moyer et al., 1998; Vais et al.,
2004; Ban et al., 2007). The similar forskolin EC50 of tagged and untagged CFTR confirms
that an N-terminal YFP tag does not severely affect sensitivity to PKA-mediated
phosphorylation. However, critical domain interactions have been ascribed to the N-tail
(Naren et al., 1999; Chan et al., 2000; Fu et al., 2001).
To assess the effects of the YFP tag on CFTR gating kinetics, single-channel
recording in the inside-out excised patch configuration was used to compare YFP-tagged and
untagged WT-CFTR. Po was reduced by the YFP tag to 64% that of WT-CFTR, mainly due
to an increase in interburst duration (Figure 3). Thus the N-terminal YFP tag affects gating in
a way comparable to a FLAG tag (Chan et al., 2000).
To investigate whether cellular distribution was affected, confocal images were
obtained of HEK293 cells expressing YFP-WT-CFTR or YFP-F508del-CFTR. Images show
mainly membrane localisation of YFP-WT-CFTR (Figure 4, white arrows), and internal, ER-
like retention of YFP-F508del-CFTR (Figure 4, green arrows), as observed for untagged WT-
CFTR and F508del-CFTR.
Overall, the mild functional alteration and expected cellular distribution confirmed
suitability of the fusion protein for CFTR monitoring.
Known potentiators
Concentration-response curves to known potentiators were constructed using the
YFP-F508del-CFTR assay (Figure 5), after temperature correction (24 h incubation at 30°C).
P2 and P5 show the expected potency (Yang et al., 2003; Pedemonte et al., 2005b). The VX-
770 EC50 we obtained (441 ± 144 nM, for F508del-CFTR) is higher than some of the
published values, with EC50 values ranging from low nanomolar (Van Goor et al., 2009; Veit
et al., 2014) to 129 ± 38 nM (Van Goor et al., 2014).
Figure 5 confirms that the YFP-F508del-CFTR assay is suitable for detecting
potentiation. As the potentiator with the highest efficacy, VX-770 was chosen as the positive
control for a pilot compound screen.
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Pilot screen
A small library of 138 compounds was selected from UCL ChemiBank: a resource of
diverse, drug-like compounds for screening (The Wolfson Institute for Biomedical Research,
UCL, London). Compounds were selected on the basis of structural resemblance to VX-770,
but also included some dissimilar chemical intermediates.
Test compounds were screened at 10 µM in triplicate, in the presence of 50 µM
forskolin. Three compounds with SSMD > 2 and mean activity > 50% that of 10 µM VX-770
were identified (Figure 6A and 6B); the activity of two could be confirmed on YFP-F508del-
CFTR and YFP-G551D-CFTR (Figures 6C). Concentration-response curves were obtained
after all solutions (10 mM stock in DMSO and dilutions) were made freshly from solid stock
to exclude the possibility of sample contamination. The third hit compound was not further
investigated, as insolubility became a major obstacle. The confirmed active compounds,
MS131A
(chemical
name,
diethyl
2-(((3-
(difluoromethoxy)phenyl)amino)methylene)malonate) and MS134A (chemical name, diethyl
2-(((4-methoxyphenyl)amino)methylene)malonate), were in fact chemical intermediates en
route to cyclic structures and are quite dissimilar from VX-770 (Tanimoto similarity = 0.28).
F508del
Differences in compound efficacy when tested on F508del-CFTR vs. G551D-CFTR
confirmed that the response detected in the screen was mediated by potentiation of CFTR,
and not an alternative anion channel. Given the high structural similarity of the two hit
compounds, further work was carried out only on one. MS131A was chosen due to
marginally higher efficacy on F508del-CFTR compared to MS134A (Figure 6C).
The CFTR-pHTomato assay
The CFTR-pHTomato localisation assay exploits a pH sensitive, red fluorescent
protein. Both the excitation and the emission spectra of pHTomato are strongly pH-dependent
(pKa ~7.8). As pH increases fluorescence increases, since only the deprotonated form is
fluorescent (Li and Tsien, 2012).
In our assay, pHTomato is inserted within an extracellular loop of CFTR. This
insertional fusion does not alter the pH-dependence of pHTomato (see Supporting
Information Figure S2D). We chose a site following position 901 in extracellular loop 4,
known to be a permissive site for tag insertion on CFTR (Howard et al., 1995; Schultz et al.,
1997; Sharma et al., 2001; Hildebrandt et al., 2015). Even a relatively large tag (308 amino
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acid Horseradish Peroxidase; HRP) at this position in F508del-CFTR has been shown not to
alter drug responses or cellular localisation (Phuan et al., 2014).
CFTR-pHTomato is expressed in HEK293 cells. Transient transfection of cells has
variable efficiency, resulting in variable levels of protein expression in individual cells. To
reduce such variability, CFTR-pHTomato and eGFP are encoded on a pIRES2 plasmid: a
single, bicistronic mRNA is transcribed in cells, from which translation of both proteins
occurs. Assay readout is obtained as fluorescence on the red channel divided by that on the
green channel. Therefore, such readouts reflect steady state levels of CFTR-pHTomato, a
complex function of biosynthetic, trafficking and degradation rates, but independent of
transfection efficiency (Supporting Information, S2).
Image acquisition begins in pH 6 buffer (Figure 7A (i)). The fluorescence of
membrane localised CFTR-pHTomato, exposed to pH 6, is only ~5% of maximal, and,
because the pH of biosynthetic vesicle lumen is low (varies from neutral to pH 5; Wu et al.,
2001), fluorescence from immature CFTR-pHTomato is also low. The extracellular pH is
then changed to pH 9, using a non-permeant buffer. The increase in fluorescence intensity
corresponding to this extracellular buffer change (FM) represents membrane-localised
CFTR-pHTomato, as only CFTR-pHTomato located at the plasma membrane is exposed to
the basic extracellular environment (Figure 7A (ii)). The final step adds 40 mM NH4Cl, still
maintaining extracellular pH 9 (Figure 7A (iii)). NH3 permeates membranes and raises the pH
of internal vesicles, increasing fluorescence of internal CFTR (Li and Tsien, 2012; Xu et al.,
2014). The change in fluorescence at this final step (FIN) is used to estimate the level of
immature CFTR-pHTomato localised along the biosynthetic pathway. FM and FIN are then
used to estimate membrane and internal pools of WT-CFTR and F508del-CFTR (Figure 7B).
Known Correctors
Concentration-response curves were obtained for VX-809 and Corr-4a, after
temperature correction at 30°C to enhance the F508del-CFTR-pHTomato signal. Both
correctors increased membrane-localised CFTR in a concentration-dependent manner (Figure
8A). The EC50 values we obtained were similar to published values (Pedemonte et al., 2005a;
Van Goor et al., 2011). Maximal correction by VX-809 is ~2-fold greater than that which is
achieved by Corr-4a, also consistent with published data (Farinha et al., 2013; He et al., 2013;
Okiyoneda et al., 2013). In addition, while we could not detect a relationship between the
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concentration of Corr-4a and internal F508del-CFTR, VX-809 incubation caused a
concentration-dependent increase in the levels of immature protein (Figure 8B), as well as
well as of levels of plasma-membrane exposed protein.
Effects of chronic potentiator treatment on F508del-CFTR
CFTR-pHTomato was used to measure membrane destabilisation of F508del-CFTR
caused by chronic treatment with potentiators. Membrane-localised F508del-CFTR, rescued
by 10 µM VX-809 and 30°C incubation, was decreased by ~20% following 24 h incubation
with 10 µM VX-770. There was no significant change in internal CFTR (Figure 9A). In
contrast, MS131A had no significant effect on F508del-CFTR levels, after rescue by 10 µM
VX-809 and low temperature (Figure 9B). In addition, at the end of the 24 h incubation,
bound MS131A still potentiates gating (Figure 9C), ruling out the possibility that the lack of
F508del-CFTR destabilisation might be caused by compound degradation during chronic
incubation.
10 µM MS131A did not cause significant cell death during chronic incubation
(DMSO control 0.9% ± 0.2% vs. 10 µM MS131A 1.7% ± 0.5%, n = 12, measured using
NucGreen Dead 488 (Molecular Probes)), suggesting cell toxicity of the compound is low.
Discussion
YFP-CFTR functional assay can be used to identify F508del-CFTR potentiators
Effective pharmacological treatment of F508del-CF requires novel potentiators,
devoid of the negative effects on biogenesis and stability that have been reported for almost
all known F508del-CFTR potentiators (Cholon et al., 2014; Veit et al., 2014). This paper
describes a pilot screen using the YFP-CFTR assay, capable of sensitive CFTR functional
measurements. The presence of the fused N-terminal YFP has relatively minor effects on the
gating function and cellular localisation of CFTR (Figure 3 and Figure 4), similar to what has
been previously described for GFP-CFTR (Moyer et al., 1998; Vais et al., 2004; Ban et al.,
2007). Our estimates of P2 and P5 potency confirmed published EC50 values (Yang et al.,
2003; Pedemonte et al., 2005b), and in our assay VX-770 appeared to be only slightly less
potent than previously reported (Van Goor et al., 2009; Veit et al., 2014, Van Goor et al.,
2014). We cannot rule out that the YFP tag could alter the binding of VX-770, slightly
reducing the apparent affinity, but minor cell-specific differences in expression of CFTR-
interacting proteins and/or lipids are also possibilities.
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A pilot screen of 138 compounds identified two novel potentiator compounds, whose
activity was confirmed on F508del-CFTR and G551D-CFTR (Figure 6C). Our original
intention was to screen compounds similar to VX-770, however, serendipitously, we
observed potentiation with chemical intermediates MS131A and MS134A with dissimilar
structures i.e. lacking the quinolone sub-structure of VX-770. It is interesting to note that the
time course of activation by VX-770 and by the novel compounds also differs. After addition
of forskolin (adenylate cyclase activator) and VX-770 quenching occurs only after a delay,
consistent with activation requiring a build-up of cAMP levels followed by PKA-catalysed
phosphorylation. However, upon addition of forskolin + MS131A quenching is immediate.
More research will be necessary to determine whether this is due to the new compound not
requiring prior phosphorylation, or an alternative mechanism.
CFTR-pHTomato assay
We also introduce the CFTR-pHTomato assay, a novel membrane-localisation assay
capable of estimating membrane and internal CFTR, independent of channel function, in a
rapid and inexpensive manner (Figure 7). Recently published assays have overcome the
immediate need for techniques to quantify F508del-CFTR localisation without relying on
channel function (Carlile et al., 2007; Botelho et al., 2015; Larsen et al., 2016). Like CFTR-
pHTomato, each of these assays has advantages and limitations. One advantage of CFTR-
pHTomato is that it does not require incubation with antibodies or other relatively expensive
compounds. Used alongside a protein synthesis inhibitor, CFTR-pHTomato assays could be
used – as a rapid and cheap alternative to pulse-chase experiments – to investigate altered
stability at the plasma membrane of mutant CFTR. A reduced membrane half-life is one of
the consequences of the F508del mutation (Lukacs et al., 1993), so rapid quantification of
this parameter could be valuable in drug discovery projects. However, due to the inefficient
folding and low peripheral stability, the levels of uncorrected F508del-CFTR protein at the
plasma membrane are extremely low. This results in a very low signal for the pHTomato
assay, and a signal:noise ratio that is not as high as with chemiluminescence.
The assay was validated using concentration-response curves to known corrector
compounds (Figure 8). To enhance the fluorescence signal, these analyses were carried out
on temperature-corrected F508del-CFTR. Consistent with our data (Figure 8B), VX-809 has
been shown to stabilise F508del-CFTR during folding, to increase both the internal and
membrane-localised pools of protein, whereas Corr-4a is understood to stabilise only
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F508del-CFTR located at the membrane (Farinha et al., 2015, but also see Bali et al., 2016).
The similarity of our observations to published data suggests that the pHTomato tag, like
other tags at this location (Howard et al., 1995; Schultz et al., 1997; Phuan et al., 2014), does
not severely alter cellular trafficking or drug mechanisms.
F508del-CFTR destabilisation by chronic potentiator treatment
Destabilisation of membrane-localised F508del-CFTR by chronic VX-770 treatment
was confirmed using the CFTR-pHTomato assay (Figure 9A). Chronic treatment with 10 µM
novel potentiator MS131A did not decrease membrane or internal F508del-CFTR in the
CFTR-pHTomato assay (Figure 9B) and we could exclude that the activity was lost due to
compound degradation during incubation (Figure 9C). Thus MS131A, like compounds based
on potentiator P5 (Phuan et al., 2015), can potentiate F508del-CFTR without causing
membrane destabilisation.
Further studies will be required to determine whether the two ester bonds in MS131A
(Figure 6B), susceptible to hydrolysis catalysed by esterases present in the plasma, the
intestine and the liver (Oda et al., 2015), can be replaced without affecting activity. A
preliminary screening of 61 analogues of MS131A selected by virtual screening of
commercial catalogues using substructure and pharmacophore similarity searches (Awale et
al., 2015; Reymond, 2015) identified seven active compounds featuring variation of MS131A
with different substituents on the aromatic ring but conserving the problematic malonic
diester moiety.
Conclusions
YFP-CFTR and CFTR-pHTomato are novel fluorescence assays, capable of
quantifying the defects associated with F508del-CFTR: reduced open probability and reduced
membrane exposure. Using these assays, we could identify a novel compound with
potentiator action, which did not cause destabilisation of F508del-CFTR following chronic
incubation. Further structure-activity relationship studies would be required to improve
potency and drug-like qualities. In addition, although the insertion of relatively large tags
both at the N-terminal end and in the 4th extracellular loop has been shown to affect
processing and function of CFTR and F508del-CFTR only to a small extent (see Moyer et al.,
1998; Vais et al., 2004; Ban et al., 2007, and Howard et al., 1995; Schultz et al., 1997;
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Hildebrandt et al., 2015, and Figures 2D, 3 and 4), mutation and/or drug effects identified
with our assays would need to be confirmed using unmodified CFTR targets.
The pHTomato assay could be exploited for other protein trafficking diseases, the
only requirement being a suitable extracellular domain to which the pHTomato can be
tagged. One obvious protein, related to CFTR, whose cellular localisation is an important
determinant of disease severity is ABCA4, an ABC transporter mutated in Stargardt disease
(Sabirzhanova et al., 2015).
One of the weaknesses of the assays presented here is the cell line (HEK293) in which
the fusion proteins are expressed. While, in vivo, CFTR is expressed apically in polarised
epithelial cells, HEK293 cells do not form polarised monolayers, with high transepithelial
resistance. In addition, CFTR folding and trafficking are complex processes, dependent on
many accessory proteins, some of which are likely to be cell-type specific. Indeed, corrector
mechanisms have shown cell-type specificity (Pedemonte et al., 2010; Farinha et al., 2015).
For many drug-discovery projects, a cell line that more accurately represents the in vivo
environment might be preferable. On the other hand, the flexibility and ease-of-use, high
levels of expression, and low background signal obtained with our HEK293-based assays are
advantages that might prove preferable if rapid and cheap testing of hypotheses is required
(e.g. for the initial phases of projects investigating cell biology processes and/or drug
mechanisms).
Author Contributions
Experiments were conceived and designed by EL, DLS and PV. EL carried out the molecular
biology, ran the fluorescence assay acquisition and image analysis, and performed the patch-
clamp experiments. DS provided the compound library for the pilot screen, MIS prepared
MS131A and MS134A. JLR and CMSD performed the virtual screening to identify MS131A
analogues. Manuscript was written by EL and PV. All authors read and commented on the
final draft of the manuscript.
Acknowledgments
EL was supported by a Cystic Fibrosis Trust studentship [CFT Project number RS31]. We
thank Dr. Robin Ketteler and Dr. Kriston-Vizi, MRC Laboratory for Molecular Cell Biology
UCL with support from the UK Medical Research Council, for help with confocal image
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acquisition and analysis, and Dr László Csanády, Semmelweis University, for very
interesting discussions.
Conflict of interest declaration
The authors declare no conflicts of interest.
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Figure Legends
Figure 1. Steady state fluorescence measured at increasing [Iˉ]out. Solid line is a fit to
equation [2], but using [Iˉ]out as the independent variable. The apparent KI obtained is 41.4 ±
1.3 mM. At a membrane potential of -83 mV, [Iˉ]out = 41.4 mM is in equilibrium with [Iˉ]in =
1.9 mM. Alternatively, at a membrane potential of -77.5 mV, [Iˉ]out = 41.4 mM is in
equilibrium with [Iˉ]in = 2.35 mM, effective half-maximal [I-]in, assuming a [Cl-]in = 20 mM.
n = 3 wells from 1 experiment.
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Figure 2. Quantification of CFTR ion channel function using YFP-CFTR. A) YFP-WT-
CFTR fluorescence quenching in response to addition of 100 mM extracellular Iˉ (red bar)
and of 20 µM forskolin (adenylate cyclase activator, black bar). B) [I-]in increase. C) The
gradient of the trace in B) gives the rate of Iˉ entry at each time point. D) Forskolin
concentration-response curve of WT-CFTR using maximal observed Iˉ entry rate (d[Iˉ]/dt,
mM s-1) to quantify CFTR activation. EC50 = 2.2 µM ± 0.3 µM. n = 3 wells from 1
experiment.
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Figure 3. Gating of WT-CFTR and YFP-WT-CFTR expressed in HEK293 cells. A)
Representative excised patch recordings of WT-CFTR and YFP-WT-CFTR containing two
gating channels in the presence of 2 mM ATP, following exposure to 300 nM PKA. Dashed
lines: current levels corresponding to 0, 1 or 2 open channels. B) Open probability C)
Interburst duration and D) Burst duration, derived from noise analysis and dwell-time
distribution analysis. n = 11 and 15 for tagged and untagged, respectively. * p < 0.05 from
Mann-Whitney U test.
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Figure 4. YFP-WT-CFTR and YFP-F508del-CFTR localisation. Confocal images of
HEK293 cells expressing YFP-WT-CFTR (top panel) or YFP-F508del-CFTR (bottom panel).
White arrows indicate membrane localisation, green arrows indicate intracellular retention.
Yellow = YFP-CFTR, blue = Hoechst nuclear stain. Scale bar = 10 µm.
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