88
Yangthara et al.
Danvers, MA), washed, and detected by enhanced chemilumines- swab. Compound 1 (135 mg, 0.614 mmol) was added drop-wise in
cence (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK).
dioxane (5 ml) and stirred overnight at 100°C. The 2-pyrrolone was
YFP Fluorescence Measurement of I؊ Influx. Transfected purified by column chromatography and recrystallized to give
FRT cells were plated in black-walled, 96-well plates with transpar- V2Rinh-02 (153 mg, 60% yield). 1H NMR (400 MHz, CD3OD): ␦ 7.72
ent plastic bottom (Corning-Costar, Acton, MA), cultured overnight (d, J ϭ 7.6 Hz, 2H), 7.59 (t, J ϭ 7.2 Hz, 1H), 7.48 (d, 8.0 ϭ Hz, 2H),
to confluence, washed three times with PBS, and treated with spec- 7.24 (m, 2H), 7.10 (t, J ϭ 8.8 Hz, 1H), 7.03 (d, J ϭ 8.6 Hz, 2H), 6.81
ified compounds in a final volume of 60 l. YFP-H148Q/I152L fluo- (d, J ϭ 8.6 Hz, 2H), 5.26 (s, 1H), 3.91 (m, 1H), 3.36 (s, 1H), 2.97 to
2.68 (m, 2H), 2.71 (m, 1H). Liquid chromatography-mass spectrom-
etry: m/z 418.1 [Mϩ H]ϩ (C18 column, 99%, 200–400 nm; Nova-Pak,
Eatontown, NJ).
cAMP Measurement. Cells were grown in 24-well plates, treated
with test compounds for 30 min, lysed by sonication, centrifuged to
remove cell debris, and assayed for cAMP according to manufacturer’s
instructions (R&D Systems, Minneapolis, MN). For CHO-K1 cells, 24 h
after transfection with 2-adrenergic receptors, cells were trypsinized
and plated onto 24-well plates overnight before cAMP assay.
Receptor Binding Assay. Radiolabeled vasopressin binding was
measured in intact FRT cells stably expressing human V2R or V1aR.
Confluent cells in 24-well plates were washed twice with ice-cold
binding buffer (PBS containing 0.1% glucose and 0.2% bovine serum
albumin). Cells were incubated for 2 h at 4°C with binding buffer
containing 1 nM [3H]AVP (PerkinElmer Life and Analytical Sci-
ences, Boston, MA) and specified concentrations of V2Rinh-02,
washed twice with ice-cold PBS, and lysed in 0.1 N NaOH containing
0.2% SDS. Radioactivity was measured with a scintillation counter.
Nonspecific binding, determined by radioactivity with dDAVP (for
V2R) or SR 49059 (for V1aR) incubation, was subtracted. dDAVP and
SR 49059 (Sanofi Aventis, Montpellier, France) bind selectively to
V2R and V1a receptor, respectively (Serradeil-Le Gal et al., 1993).
rescence was measured using a commercial plate reader (FluoStar
Optima; BMG LabTechnologies, Offenburg, Germany) equipped with
custom excitation and emission filters (500 nm and 544 nm, respec-
tively; Chroma, Brattleboro, VT). Fluorescence intensity in each well
was measured for a total of 14 s. In each well, 100 l of PBS/IϪ (PBS
with 100 mM ClϪ replaced by IϪ) was injected by a syringe pump at
2 s after the start of data collection.
CFTR Cl؊ Current Measurement. Cells were cultured on Snap-
well filters (Costar 3801) until confluence (transepithelial resistance,
Ͼ500 ⍀). Apical membrane current was measured in an Ussing
chamber (Vertical diffusion chamber; Costar) with Ringer’s solution
bathing the basolateral surface and half-Ringer’s bathing the apical
surface. The composition of Ringer’s solution was 130 mM NaCl, 2.7
mM KCl, 1.5 mM KH2PO4, 1 mM CaCl2, 0.5 mM MgCl2, 10 mM
sodium HEPES, and 10 mM glucose, pH 7.3. Half-Ringer’s solution
was the same, except that 65 mM NaCl was replaced with sodium
gluconate, and CaCl2 was increased to 2 mM. Chambers were bub-
bled continuously with air. Apical membrane current was measured
using a DVC-1000 voltage-clamp apparatus (World Precision Instru-
ments, Sarasota, FL).
High-Throughput Screening. The compound library for screen-
ing contained 50,000 chemically diverse, drug-like small molecules
(ChemDiv, San Diego, CA). Stock compounds were stored in 96-well
plates at 2.5 mM in dimethyl sulfoxide. Compounds occupied 80
wells, with the remaining 16 wells containing only dimethyl sulfox-
ide (for positive and negative controls). Screening was done using an
automated apparatus (Beckman Coulter, Fullerton, CA) containing a
CO2 incubator, carousels for compound plates and pipette tip boxes,
plate washer (Elx405; Bio-Tek Instruments, Winooski, VT), liquid
handling station (Biomek FX; Beckman Coulter), and two plate
readers (FluoStar Optima; BMG LabTechnologies). Robotic opera-
tions were controlled by SAMI software (version 3.3; Beckman
Coulter).
For high-throughput screening, cells expressing human wild-type
V2R were plated in 96-well plates using a LabSystems Multidrop
Dispenser. After overnight growth to confluence, cells were washed
with PBS, and dDAVP (1 nM; Ferring Pharmaceuticals, Suffern, NY)
was added together with test compounds (20 M). The first and last
columns of each plate were used for positive (PBS) and negative
(dDAVP, no test compound) controls. IϪ influx was assayed as de-
scribed above after a 30-min incubation at 37°C in a CO2 incubator.
Data Analysis. IϪ influx (d[IϪ]/dt at t ϭ 0) was computed from
fluorescence time course data as described previously (Muanprasat
et al., 2004). Percentage of inhibition was computed using the fol-
lowing equation: % inhibition ϭ 100 ϫ (negative control Ϫ com-
pound)/(negative control Ϫ positive control). Positive and negative
control values denote d[IϪ]/dt obtained from the first and last col-
umns of each plate. Primary screening data were subjected to histo-
gram analysis for “hit” selection.
Results
Expression of the Wild-Type and the Mutant V2Rs in
FRT Cells. Stably transfected FRT cell lines were generated
that coexpress human wild-type CFTR, YFP-H148Q/I152L,
and c-myc-tagged wild-type V2R or the mutant V2R-W164S.
The c-myc-tag was inserted at the external-facing V2R N
terminus. Wild-type V2R showed a plasma membrane distri-
bution by c-myc staining (Fig. 1B), whereas no membrane
staining was seen in nontransfected cells or cells expressing
V2R-W164S that has a defect in cellular processing with
retention at the endoplasmic reticulum (Oksche et al., 1996).
Immunoblot analysis with c-myc antibody showed bands at
ϳ40 and 64 kDa, corresponding to nonglycosylated and gly-
cosylated V2R, respectively (Innamorati et al., 1996; Sadeghi
et al., 1997). These results indicate stable surface expression
of wild-type V2R in FRT cells.
Forskolin, an adenylyl cyclase activator, and dDAVP, a
V2R-selective vasopressin receptor agonist (Chang et al.,
2005), increase cytoplasmic cAMP concentration and hence
activate CFTR. CFTR activity was assayed from the kinetics
of decreasing YFP-H148Q/I152L fluorescence after external
IϪ addition. Forskolin treatment increased CFTR activity in
wild-type, mutant, and nontransfected cells, whereas dDAVP
Synthesis Procedures. To synthesize 2,4-dioxo-4-phenyl-ethyl- increased CFTR activity only in cells expressing wild-type
butylate 1, a solution of anhydrous benzene (50 ml) and acetophe-
none (1.2 g, 0.010 mol) was added to a suspension of NaH in oil (60%;
0.8 g, 0.020 mol), and the mixture was stirred for 30 min. To a
solution of diethyl oxalate (2.19 g, 0.015 mol), benzene (10 ml) was
added drop-wise, and the reaction mixture was stirred for 6 h. The
mixture was filtered over Celite and purified by chromatography to
give 2,4-dioxo-4-phenyl-ethylbutylate 1 (1.76 g; 85% yield). To syn-
thesize 4-benzoyl-5-(4-fluorophenyl)-3-hydroxy-1-(4-hydroxyphenyl-
ethyl)-2,5-dihydro-2-pyrrolone (V2Rinh-02), tyramine (93 mg, 0.675
mmol) and 4-fluorobenzaldehyde (84 mg, 0.675 mmol) were heated to
V2R (Fig. 2A). Increased CFTR activity in response to fors-
kolin or dDAVP was inhibited by the CFTR blocker CFTRinh
-
172 (Ma et al., 2002). Inhibition of dDAVP-stimulated IϪ
influx was found with the partial V2R agonist [1--mercapto-
, -cyclopentamethylenepropionyl1, O-ET-TYR2, VAL4,
ARG8]-vasopressin (Sigma) and the V2R antagonist SR
121463B (Sanofi Pharmaceutical) (Serradeil-Le Gal et al.,
1996; Manning et al., 1997). Concentration-activation data
are summarized in Fig. 2B. The IC50 value for CFTR activa-
110°C for 10 min. Water was removed during reflux with a cotton tion by dDAVP of ϳ0.1 nM is less than that observed in V2R