Experimental evaluation of proposed small-molecule inhibitors of water channel aquaporin-1

Article abstract of DOI:10.1124/mol.116.103929

The aquaporin-1 (AQP1) water channel is a potentially important drug target, as AQP1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. Here, we measured the AQP1 inhibition efficacy of 12 putative small-molecule AQP1 inhibitors reported in six recent studies, and one AQP1 activator. Osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express AQP1, in hemoglobin-free membrane vesicles from rat and human erythrocytes, and in plasma membrane vesicles isolated from AQP1-transfected Chinese hamster ovary cell cultures. As a positive control, 0.3 mM HgCl₂ inhibited AQP1 water permeability by >95%. We found that none of the tested compounds at 50 μM significantly inhibited or increased AQP1 water permeability in these assays. Identification of AQP1 inhibitors remains an important priority.

Full text of DOI:10.1124/mol.116.103929

1521-0111/89/6/686–693$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.116.103929
Mol Pharmacol 89:686–693, June 2016
Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics
Experimental Evaluation of Proposed Small-Molecule Inhibitors
of Water Channel Aquaporin-1
Cristina Esteva-Font, Byung-Ju Jin, Sujin Lee, Puay-Wah Phuan, Marc O. Anderson,
and A. S. Verkman
Departments of Medicine and Physiology, University of California, San Francisco, California (C.E.-F., B.-J.J., S.L., P.-W.P., A.S.V.);
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California (M.O.A.)
Received February 19, 2016; accepted March 17, 2016
ABSTRACT
The aquaporin-1 (AQP1) water channel is a potentially important membrane vesicles from rat and human erythrocytes, and in
drug target, as AQP1 inhibition is predicted to have therapeutic plasma membrane vesicles isolated from AQP1-transfected
action in edema, tumor growth, glaucoma, and other conditions. Chinese hamster ovary cell cultures. As a positive control,
Here, we measured the AQP1 inhibition efficacy of 12 putative 0.3 mM HgCl2 inhibited AQP1 water permeability by .95%.
small-molecule AQP1 inhibitors reported in six recent studies, We found that none of the tested compounds at 50 mM
and one AQP1 activator. Osmotic water permeability was significantly inhibited or increased AQP1 water permeability in
measured by stopped-flow light scattering in human and rat these assays. Identification of AQP1 inhibitors remains an
erythrocytes that natively express AQP1, in hemoglobin-free important priority.
suggested the potential utility of AQP1 inhibitors in the
Introduction
treatment of edema (Schnermann et al., 1998), tumors
The aquaporins (AQPs) are a family of small, plasma
(Saadoun et al., 2005; Esteva-Font et al., 2014), and glaucoma
membrane proteins that transport water and/or small polar
(Zhang et al., 2002), and potentially for brain swelling (Oshio
solutes such as glycerol (Carbrey and Agre, 2009; Verkman,
et al., 2005) and other conditions.
2012). The AQPs are a potentially important class of targets
High-resolution structural data show that AQP1 monomers
for drug development (Jeyaseelan et al., 2006; Wang et al.,
consist of six transmembrane helical segments and two
2006; Frigeri et al., 2007; Verkman et al., 2014; Beitz et al.,
partially spanning segments that surround a central aqueous
2015), though there has been limited progress to date on the
pore (de Groot et al., 2001; Sui et al., 2001). AQP1 monomers
discovery and validation of AQP inhibitors. Challenges in the
further assemble in membranes as homotetramers (Verbavatz
identification of AQP inhibitors include difficulties in assaying
et al., 1993). Molecular dynamics modeling supports the
water permeability and the structural features of the AQPs,
conclusion that the single-file passage of water through the
which include a narrow pore that excludes small molecules.
central pore of an AQP1 monomer, as well as interactions with
Aquaporin-1 (AQP1), originally identified as responsible for
residues lining the pore, is responsible for AQP1 water
high water permeability in erythrocytes (Preston and Agre,
selectivity (Hub and de Groot, 2008). The narrowest section
1991), is a particularly compelling drug target. In addition to
of the pore, named the ar/R constriction, consists of aromatic
erythrocytes, AQP1 is expressed in various fluid secreting and
and arginine residues and has a diameter of 2.8 Å (Sui et al.,
absorbing epithelia in kidneys, gastrointestinal organs, the
2001). The AQP1 pore excludes passage of small molecules
central nervous system, the eye, and others as well in most
and even protons, with the latter related to lack of stabiliza-
microvascular endothelia including tumor microvessels
tion of hydronium and/or geometric constraints (Gonen and
(Hasegawa et al., 1994a; Nielsen et al., 1995; Mobasheri and
Walz, 2006; Kato et al., 2006).
Marples, 2004). Data largely from AQP1 knockout mice has
Heavy-metal, sulfhydryl-reactive small molecules such as
HgCl2 inhibit AQP1 water permeability by interaction with
This work was supported by the National Institutes of Health National
residue cysteine 187 near the extracellular surface of the
AQP1 aqueous pore (Preston et al., 1993; Zhang et al., 1993).
However, because of their lack of selectivity and toxicity,
heavy metals are not drug development candidates.
Institute of Diabetes and Digestive and Kidney Diseases [Grants DK101373,
DK35124, DK72517, DK99803], the National Institute of Biomedical Imaging
and Bioengineering [Grant EB00415], and the National Eye Institute [Grant
EY13574].
dx.doi.org/10.1124/mol.116.103929.
ABBREVIATIONS: AqB013, 3-butylamino-4-phenoxy-N-pyridin-4-yl-5-sulfamoyl-benzamide; AqF026, 4-chloro-2-[(2-furanylmethyl) amino]-5-
[[(phenylmethyl) amino] sulfonyl]-benzoic acid methyl ester; AQP, aquaporin; CHO, Chinese hamster ovary; DMSO, dimethylsulfoxide; HTS,
high-throughput screening; NSC164914, tributyl-(2,4,5-trichlorophenoxy) stannane; NSC168597, tributyl lead chloride; NSC301460, trichopolyn I;
NSC657298, (E)-1-[1-ethyl-4-hydroxy-4-[(E)-2-(4-methylphenyl) ethenyl] piperidin-3-yl]-3-(4-methylphenyl) prop-2-en-1-one; NSC670226, 2-[4-tert-
butyl-1-[(4-fluorophenyl) methyl] cyclohexyl] oxy-N,N-dimethylethanamine; NSC670229, 2-[4-tert-butyl-1-[(4-methylphenyl) methyl] cyclohexyl]
oxy-N,N-dimethylethanamine; PBS, phosphate-buffered saline.
686

Small-Molecule AQP1 Inhibitors
687
Biosciences (Denver, CO). All other chemicals were purchased from
Sigma-Aldrich (St. Louis, MO). Compounds 4 (AqB013 [3-butylamino-
4-phenoxy-N-pyridin-4-yl-5-sulfamoyl-benzamide]), 5 (AqF026 [4-chloro-
2-[(2-furanylmethyl) amino]-5-[[(phenylmethyl) amino] sulfonyl]-benzoic
acid methyl ester]), and 12 [1-(7-(2,4-dichlorophenyl)-5-fluoro-2,3-
dihydrobenzofuran-2-yl)-N-methylmethanamine] were synthesized. Com-
pound 5 (AqF026) was synthesized from furosemide as reported (Yool
et al., 2013), and the analytic data matched with the reported data.
Compound 4 (AqB013) was synthesized from bumetanide through
an imidazolide intermediate (Migliati et al., 2009). To a suspension of
bumetanide (100 mg, 0.27 mmol) in ethyl acetate (1 mL) was added
1,1ʹ-carbonyldiimidazole (50 mg, 0.3 mmol) under N2. The mixture
was heated for 1 hour and cooled to room temperature. The resulting
precipitate was filtered and washed with ethyl acetate and dried in
vacuo to give intermediate 3-(butylamino)-5-(1H-imidazole-1-
carbonyl)-2-phenoxybenzenesulfonamide as a white solid (105 mg,
90%). To this compound (10 mg, 0.024 mmol) in tetrahydrofuran
(1 mL) was added 4-aminopyridine (4 mg, 0.042 mmol) and
trifluoroacetic acid (catalytic amount) and stirred overnight. The
reaction mixture was evaporated under reduced pressure. Compound
4 was obtained as a yellowish white solid (6 mg, 56%) after silica gel
column chromatography. 1H-NMR (300 MHz, CD3OD): d 8.19 (brs, 1H),
7.62 (m, 2H), 7.35–7.29 (m, 2H), 7.26 (d, 1H, J 52.0 Hz), 7.15–7.13 (m, 1H),
7.08 (t, 1H, J 5 6.3 Hz), 6.95 (d, 2H, J 5 7.7 Hz), 3.08 (t, 2H, J 5
6.9 Hz), 1.46–1.36 (m, 2H), 1.17–1.09 (m, 2H), 0.79 (t, 2H, J 5 7.3 Hz);
liquid chromatography with mass spectrometry (electrospray
ionization): 441 (M1H)1.
Early studies reported AQP1 inhibition by carbonic anhy-
drase inhibitors such as acetazolamide and K1 channel
blockers such as tetraethylammonium (Brooks et al., 2000;
Detmers et al., 2006), though subsequent reevaluation using
different assays did not confirm AQP1 water transport in-
hibition by these molecules (Yang et al., 2006, 2008; Søgaard
and Zeuthen, 2008; Yamaguchi et al., 2012). More recently, a
variety of approaches, including high-throughput screening
and computational chemistry, have yielded compounds with
reported AQP1 inhibition or activation activity (Migliati et al.,
2009; Mola et al., 2009; Seeliger et al., 2013; Yool et al., 2013;
To et al., 2015; Patil et al., 2016), as summarized in Fig. 1 and
Table 1. Motivated by the high potential clinical utility of
small-molecule AQP1 inhibitors, here we tested these various
proposed AQP1 inhibitors, comparing their water trans-
port inhibition activity in different cellular systems with
that of HgCl2.
Materials and Methods
Compounds. Compounds 1 [1,3-phenylenediacrylic acid], 2 [(E,Z)-
3-methyl-4-(2-quinolinylmethylene)-2-pentenedioic acid disodium salt],
and 3 [N-(1,3-benzodioxol-5-ylmethyl)-Nʹ-2,1,3-benzothiadiazol-5-yl-
thiourea] were purchased from ChemBridge (San Diego, CA). Compounds
6 (NSC168597 [tributyl lead chloride]), 7 (NSC301460 [trichopolyn I]),
8 (NSC164914 [tributyl-(2,4,5-trichlorophenoxy) stannane]), 9 (NSC670229
[2-[4-tert-butyl-1-[(4-methylphenyl) methyl] cyclohexyl] oxy-N,
N-dimethylethanamine]), 10 (NSC670226 [2-[4-tert-butyl-1-[(4-
fluorophenyl) methyl] cyclohexyl] oxy-N,N-dimethylethanamine]),
and 11 (NSC657298 [(E)-1-[1-ethyl-4-hydroxy-4-[(E)-2-(4-methylphenyl)
ethenyl] piperidin-3-yl]-3-(4-methylphenyl) prop-2-en-1-one]) were
Compound 12 was synthesized by Suzuki coupling of (7-bromo-5-
fluoro-2,3-dihydrobenzofuran-2-yl)methyl-4-methylbenzenesulfonate
and 2,4-dichlorophenylboronic acid under microwave irradiation,
followed by alkylation with methyl amine at 60°C in dimethylsulfoxide
(DMSO) overnight. 1H-NMR (300 MHz, CD3OD): d 7.56 (dd, 1H, J 5
1.7, 0.6 Hz), 7.39–7.37 (m, 2H), 7.05–7.02 (m, 1H), 6.80 (dd, 1H, J 5 9.5,
purchased from the National Cancer Institute. Compound 13 2.7 Hz), 4.99–4.96 (m, 1H), 3.42 (m, 1H), 3.06–2.83 (m, 3H), 2.45
[N-[[trans-4-[[(4-amino-2-quinazolinyl) amino] methyl] cyclohexyl] (s, 3H); 13C-NMR (75 MHz, CD3OD): d 153.0, 152.4, 134.3, 133.8, 132.4,
methyl]-1-naphthalenesulfonamide] was purchased from Tocris
128.9, 128.8, 126.8, 120.4, 120.3, 116.1, 114.7, 112.1, 81.9, 54.8, 34.4,
Fig. 1. Chemical structures of putative AQP1 inhibi-
tors. (A) Structures of inhibitors reported by Migliati
et al. (2009), Mola et al. (2009), Yool et al. (2013),
Seeliger et al. (2013), To et al. (2015), and Patil et al.
(2016) (see Table 1). (B) Osmotic water permeability in
human erythrocytes as measured from the time course
of scattered light intensity in response to a 250 mM
inwardly directed sucrose gradient at room tempera-
ture. Erythrocytes were incubated with 0, 75, 150, or
300 mM HgCl2 for 5 minutes before measurement.

688
Esteva-Font et al.
TABLE 1
Reported small-molecule AQP1 modulators
Compound
Reported IC50
Reference
Identification Method
(mM)
Code
Name
Inhibitor
Compound 1
1,3-Phenylenediacrylic acid
Seeliger et al.,
2013
Seeliger et al.,
2013
Seeliger et al.,
2013
Migliati et al.,
2009
Mola et al., 2009
Mola et al., 2009
Mola et al., 2009
Mola et al., 2009
Xenopus laevis oocytes
Xenopus laevis oocytes
Xenopus laevis oocytes
8
17
18
20
Compound 2
(E,Z)-3-methyl-4-(2-quinolinylmethylene)-2-
pentenedioic acid disodium salt
N-(1,3-benzodioxol-5-ylmethyl)-Nʹ-2,1,3-
benzothiadiazol-5-yl-thiourea
3-Butylamino-4-phenoxy-N-pyridin-4-yl-5-
sulfamoyl-benzamide
Compound 3
AqB013 (Compound 4)
Virtual screen, Xenopus
laevis oocytes
Calcein cell-based assay
Calcein cell-based assay
Calcein cell-based assay
Calcein cell-based assay
NSC168597 (Compound 6)
NSC301460 (Compound 7)
NSC164914 (Compound 8)
NSC670229 (Compound 9)
Tributyl lead chloride
Trichopolyn I
49
28
40
27
Tributyl-(2,4,5-trichlorophenoxy) stannane
2-[4-Tert-butyl-1-[(4-methylphenyl) methyl]
cyclohexyl] oxy-N,N-dimethylethanamine
2-[4-Tert-butyl-1-[(4-fluorophenyl) methyl]
cyclohexyl] oxy-N,N-dimethylethanamine
(E)-1-[1-ethyl-4-hydroxy-4-[(E)-2-(4-
methylphenyl) ethenyl] piperidin-3-yl]-3-
(4-methylphenyl)
NSC670226 (Compound 10)
NSC657298 (Compound 11)
To et al., 2015
To et al., 2015
Yeast freeze-thaw assay
Yeast freeze-thaw assay
20
48
prop-2-en-1-one
Compound 12
Compound 13
1-(7-(2,4-Dichlorophenyl)-5-fluoro-2,3-
dihydrobenzofuran-2-yl)-N-
Patil et al., 2016
Patil et al., 2016
Calcein cell-based assay
Calcein cell-based assay
10
3
methylmethanamine
N-[[trans-4-[[(4-amino-2-quinazolinyl) amino]
methyl] cyclohexyl] methyl]-1-
naphthalenesulfonamide
Activator
Reported EC50
(mM)
AqF026 (Compound 5)
4-Chloro-2-[(2-furanylmethyl) amino]-5-
[[(phenylmethyl) amino] sulfonyl]-benzoic
acid methyl ester
Yool et al., 2013
Virtual screen, Xenopus
laevis oocytes
3.3
33.2; liquid chromatography with mass spectrometry (electrospray
Preparation of Plasma Membrane Vesicles from CHO
Cells. Enriched plasma membrane fractions were prepared as de-
ionization): 326 (M1H)1.
Collection of Human and Rat Blood. Human venous blood scribed elsewhere (Rossi et al., 2012). CHO cells from 10 confluent
obtained from a single donor was collected into K3EDTA Vacutainers
(Greiner, Kremsmunster, Austria). Whole rat blood was collected from
175 cm2 flasks (Thermo Scientific, Rochester, NY) were homogenized
by 20 strokes of a glass Dounce homogenizer in 250 mM sucrose,
adult Wistar rats (250–300 g) purchased from Charles River Labora- 10 mM Tris-HCl, and 1 mM EDTA containing protease inhibitors
tories (Wilmington, MA) by cardiac puncture under isoflurane anes- (Complete Mini; Roche Diagnostics, Mannheim, Germany). The
thesia. Animal protocols were approved by the University of homogenate was then centrifuged at 4000g for 15 minutes at 4°C,
California, San Francisco Committee on Animal Research. and the enriched plasma membrane fraction was obtained by
Preparation of Hemoglobin-Free Erythrocyte Ghosts. Ghost centrifugation at 17,000g for 45 minutes. The resultant pellet was
membranes were prepared by the procedure of Zeidel et al. (1992), suspended in PBS for stopped-flow measurements.
with modifications. Collected blood was washed 3 times with phosphate-
Stopped-Flow Measurements. Osmotic water permeability was
buffered saline (PBS) by centrifugation at 800g for 5 minutes at 4°C.
measured by stopped-flow light scattering (or fluorescence) using a
The erythrocyte pellet was resuspended in 0.1x PBS (hypotonic buffer), Hi-Tech Sf-51 instrument (Wiltshire, United Kingdom) as described by
and the membranes were washed twice in the same buffer by Jin et al. (2015). Intact erythrocytes (hematocrit ∼0.5%), hemoglobin-
centrifugation at 30,000g for 10 minutes at 4°C. Hypertonic (10x) PBS free erythrocyte ghost membranes (∼0.4 mg protein/ml), plasma
was added to restore isotonicity, and membranes were incubated for membrane vesicles from CHO cells (∼0.8 mg protein/ml), or calcein-
1 hour at 37°C to allow resealing. The resulting ghost membrane labeled erythrocytes were suspended in PBS and subjected to a
vesicles were resuspended at ∼0.4 mg protein/ml for stopped-flow 250 mOsm inwardly directed gradient of sucrose. Some experiments
measurements.
were performed with a 150 mOsm outwardly directed NaCl gradient
produced by mixing equal volumes of the membrane suspension in
Erythrocyte Labeling. Erythrocytes were washed 3 times with
PBS (3000g, 15 minutes) and then fluorescently labeled by incubation PBS with distilled water. The resultant kinetics of cell volume were
with 15 mM calcein-AM (Invitrogen, Carlsbad, CA) at 37°C for
1.5 hours. Erythrocytes were then washed twice with PBS (3000g,
10 minutes) to remove extracellular dye and diluted 15-fold in PBS.
Cell Culture. Chinese hamster ovary (CHO) cells stably express-
ing human AQP1 or AQP4-M23, generated as reported (Ma et al.,
measured from the time course of 90° scattered light intensity at
530 nm (or calcein fluorescence) in which increasing scattered light
intensity corresponds to decreasing cell volume.
For the testing of putative AQP1 modulators, compounds in DMSO
(0.5% final DMSO concentration) were incubated with cell or mem-
1993, Phuan et al., 2012), and nontransfected CHO cells were grown in brane suspensions for .10 minutes at 50 mM before stopped-flow
Ham’s F-12 nutrient medium supplemented with 10% fetal bovine measurement. Relative osmotic water permeability was determined
serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. Geneticin by exponential regression using Prism software (GraphPad Software,
(500 mg/ml) was used as a selection marker for the cells expressing
AQP1 and AQP4-M23. Cells were grown at 37°C in 5% CO2
and 95% air.
San Diego, CA).
Erythrocyte Hemoglobin Release. Compounds (50 mM, 0.5%
final DMSO concentration) were incubated with a freshly prepared

Small-Molecule AQP1 Inhibitors
689
erythrocyte suspension for 15 minutes and centrifuged at 3000 rpm for
10 minutes. Hemoglobin released in the supernatant was measured by
absorbance at 540 nm. In some studies, erythrocyte morphology was
assessed by phase-contrast microscopy at 1000Â magnification.
Statistical Analysis. Data are presented as the mean standard
error of the mean (S.E.M.) of at least four independent experiments,
and were analyzed with paired Student’s t test or one-way analysis of
variance (ANOVA).
Results
Figure 1A shows chemical structures of the 12 putative
AQP1 inhibitors and one AQP1 activator studied here.
HgCl2 was used as a positive control for inhibition. Figure 1B
shows HgCl2 concentration-dependent inhibition of water
permeability in human erythrocytes, which natively express
AQP1. Osmotic water permeability was measured by the
established stopped-flow light-scattering method in which a
dilute erythrocyte suspension was mixed rapidly with an
anisosmolar solution to impose a 250 mM inwardly directed
sucrose gradient. The sucrose gradient causes osmotic
water efflux and cell shrinkage, seen as increasing scat-
tered light intensity at 530 nm wavelength. The IC50 for
HgCl2 inhibition of erythrocyte AQP1 water permeability
was ∼85 mM.
Similar studies were performed for the putative AQP1
modulators at 50 mM, a concentration predicted from pub-
lished data to strongly inhibit (or weakly activate) AQP1
water permeability (published IC50 values listed in Table 1).
Compounds were incubated with the erythrocyte suspension
for at least 15 minutes before stopped-flow measurements.
Representative light-scattering curves are shown for human
Fig. 2. Osmotic water permeability in human erythrocytes and hemoglobin-
free ghost membranes derived therefrom. Osmotic water permeability was
measured from the time course of scattered-light intensity in response to a
250 mM inwardly directed sucrose gradient. (A) Representative time
course data for negative control (0.5% DMSO vehicle alone), 0.3 mM HgCl2
(positive control), and indicated compounds (each 50 mM). Cells and ghosts
were incubated with test compounds for 15 minutes before measurements.
(B) Relative osmotic water permeability (S.E., n = 4). *P , 0.05 compared
with control.
To investigate the possible reasons for the effects of
erythrocytes in Fig. 2A (left) with averaged data (6 S.E.M.) compounds 9, 10, and 13 in the light-scattering assay of
summarized in Fig. 2B (left). Whereas HgCl2 strongly in- osmotic swelling, we evaluated erythrocyte toxicity by use
hibited osmotic water permeability in human erythrocytes, no of a hemoglobin-release assay and by cell morphology. Com-
significant effect was seen for any of the 13 test compounds.
pounds 6 and 8 to 13 caused significantly greater hemoglobin
Reasoning that the lack of inhibition might be due to the release than the vehicle control. Direct examination of
presence of hemoglobin in the erythrocyte cytoplasm, which erythrocyte morphology at high magnification showed marked
potentially could bind compounds, we performed similar abnormalities for compounds 6, 9, 10, 12, and 13, with cell
studies in sealed, hemoglobin-free ghost membranes prepared crenation and variable aggregation (Fig. 5B). These abnor-
from human erythrocytes. Similar to the results in Fig. 2A, no malities may account for the apparent artifacts in the light-
significant effect on osmotic water permeability by the test scattering assay of cell swelling.
compounds was seen in ghost membranes, with HgCl2 show-
ing strong inhibition as positive control.
Stopped-flow light-scattering measurements were also per-
formed using rat erythrocytes, which express rat AQP1, and
As it is possible, though unlikely, that inhibition efficacy sealed, hemoglobin-free ghost membranes prepared from rat
could depend on the direction of water flow, compounds were erythrocytes (Fig. 6). As found with human erythrocytes and
also tested in human erythrocytes using a stopped-flow light- ghost membranes, no significant effect on osmotic water
scattering assay of osmotic swelling in which cells were permeability was seen for any of the test compounds, with
exposed to 50% hypotonic saline. Compounds 9, 10, and 13 HgCl2 showing strong inhibition as positive control.
showed apparent weak inhibition of water permeability (30%–
40% at 50 mM) whereas the other compounds had no effect on AQP1-enriched plasma membrane vesicles were isolated from
water permeability (Fig. 3). AQP1-transfected (and control) CHO cells by homogenization
To test for AQP1 inhibition in a different cellular context,
Because potential artifacts in light-scattering assays could and differential centrifugation. Osmotic water permeability
be produced, for example, by erythrocyte crenation or aggre- was measured by stopped-flow light scattering as done for the
gation, compounds were also tested using a calcein-quenching erythrocytes and erythrocyte ghost membranes. Figure 7A
assay of osmotic swelling. This assay is based on volume- shows that membrane vesicle shrinkage was ∼5-fold more
dependent calcein quenching, which is insensitive to factors rapid in vesicles prepared from AQP1-transfected CHO cells
such as cell shape that can affect light scattering. Figure 4 than control (nontransfected) CHO cells. Osmotic water
shows that none of the 13 compounds significantly affected permeability was inhibited by ∼85% by 0.3 mM HgCl2.
erythrocyte water permeability. The reduced fluores-
cence signal intensity for compound 12 suggests partial pared from CHO cells expressing AQP4, a water-selective
erythrocyte lysis. channel that was originally named MIWC (mercurial-insensitive
As another control, plasma membrane vesicles were pre-

690
Esteva-Font et al.
Fig. 4. Osmotic swelling of calcein-labeled human erythrocytes. Osmotic
water permeability was measured from the time course of intracellular
calcein fluorescence in response to a 150 mOsm outwardly directed osmotic
gradient. (A) Representative time course data for negative control (0.5%
DMSO vehicle alone), 0.3 mM HgCl2 (positive control), and indicated
compounds (each 50 mM). (B) Relative osmotic water permeability (S.E., n = 4).
*P , 0.05 compared with negative control (0.5% DMSO vehicle alone).
Fig. 3. Osmotic swelling of human erythrocytes. Osmotic water perme-
ability was measured from the time course of scattered-light intensity
in response to
a 150 mOsm outwardly directed osmotic gradient.
(A) Representative time course data for negative control (0.5% DMSO
vehicle alone), 0.3 mM HgCl2 (positive control), and indicated compounds
(each 50 mM). (B) Relative osmotic water permeability (S.E., n = 4). *P ,
0.05 compared with negative control (0.5% DMSO vehicle alone). Cells and
ghosts were incubated with test compounds for ∼15 minutes before
measurements.
as these compounds were found to cause cell crenation and
aggregation, and inhibition was not seen in the calcein
fluorescence quenching assay. We conclude that, other than
nonselective, sulfhydryl-reactive, heavy metal-containing
compounds, no confirmed small-molecule AQP1 inhibitors
have been reported to date. Albeit a negative study, the work
here underscores the need to test putative AQP inhibitors
using robust, sensitive assays, and, given the major potential
clinical applications of AQP1 inhibitors, the need for contin-
ued screening and computational work to identify useful
inhibitors.
water channel) (Hasegawa et al., 1994b), whose water perme-
ability is not inhibited by HgCl2 because of absence of a critical
cysteine residue (Shi and Verkman, 1996). Osmotic water
permeability in the AQP4-containing membrane vesicles was
∼5-fold more rapid than in control vesicles, and, in contrast to
the AQP1-containing vesicles, it is not inhibited by HgCl2.
Figure 7B shows representative light-scattering data and
averaged results for measurements of osmotic water perme-
ability in the AQP1-containing plasma membrane vesicles. No
significant effect was found for any of the test compounds.
It is not known why AQP1 appears to be refractory to
identification of small-molecule inhibitors. Part of the reason
may be its unique structure, in which small, relatively rigid
monomers containing water-only pores are assembled in
Discussion
We tested 12 putative AQP1 inhibitors and one putative membranes as homotetramers. The narrow AQP1 water pore
activator for their efficacy in reducing or increasing osmotic excludes small molecules; even if a small molecule could bind
water permeability in rat and human erythrocytes and ghost to the vestibule adjacent to the pore, there may remain many
membranes, and plasma membrane vesicles from AQP1- paths for water flow around the small molecule, as if trying to
transfected CHO cells. At 50 mM, a concentration well above cork a wine bottle with a randomly shaped stone. However,
reported IC50 values for each of the compounds, no significant though direct pore blockade may be difficult to achieve,
water transport inhibition or activation was found using allosteric closure of the pore from a distant site would seem
stopped-flow assays with sensitivity to detect as small as a possible. Perhaps the tight, relatively rigid structure of
5%–10% change in water permeability. The well-studied, the AQP1 monomer, as well as its narrow pore region
albeit nonselective AQP1 inhibitor HgCl2 showed strong and small extracellular footprint, resists allosteric pore
inhibition in all assay systems. The small apparent inhibition closure by externally bound molecules. In unpublished work
by compounds 9, 10, and 13 seen in the light-scattering (M.O. Anderson, C. Esteva-Font, and A.S. Verkman) we did
swelling assay in human erythrocytes was likely an artifact, not identify any useful AQP1 inhibitors from a screen

Small-Molecule AQP1 Inhibitors
691
Fig. 5. Compound effects on hemoglobin release and
erythrocyte morphology. (A) Hemoglobin release from
human erythrocytes after 15 minutes of incubation with
test compounds at 50 mM (S.E., n = 4). *P , 0.05
compared with control. (B) Representative phase-
contrast photomicrographs of human erythrocytes after
15-minute incubations with test compounds.
of ∼150,000 synthetic small molecules with an erythrocyte largely relied on the Xenopus oocyte expression system and
lysis assay that was used successfully to identify nanomolar- calcein-loaded cell cultures, both of which are subject to
potency urea transport inhibitors (Levin et al., 2007). We also artifacts. For example, compounds that affect cell size or shape,
did not identify any useful AQP1 inhibitors in a computational cell volume regulation, nonaquaporin ion or solute trans-
docking study of 106 commercially available compounds, porters, or calcein fluorescence quenching could appear to
followed by a water transport assay of 2000 compounds with inhibit water permeability in these systems. Inhibitors of
the highest docking scores.
cellular proteins involved in major transport functions, such
It is also unclear why many putative AQP1 modulators have as bumetanide, acetazolamide, and tetraethylammonium, may
been reported in the literature, but water transport inhibi- affect resting cell volume and volume regulation. Artifacts in
tion (or activation) cannot be confirmed, including the 13 water transport measurements using the Xenopus oocyte
small molecules studied here, and previously acetazolamide, expression system have also led to the conclusion that a wide
tetraethylammonium, and dimethylsulfoxide (Yang et al., 2006; variety of drugs, including many common, chemically unrelated
Tanimura et al., 2009). As discussed elsewhere (Verkman et al., antiepileptics and carbonic anhydrase inhibitors, inhibit brain
2014), part of the reason may be that the functional assays water channel aquaporin-4 (AQP4) (Huber et al., 2009), with
Fig. 6. Osmotic water permeability in rat erythrocytes and
hemoglobin-free ghost membranes derived therefrom. Os-
motic water permeability was measured from the time
course of scattered light intensity in response to a 250 mM
inwardly directed sucrose gradient. (A) Representative time
course data for negative control (0.5% DMSO vehicle alone),
0.3 mM HgCl2 (positive control), and indicated compounds
(each 50 mM). Cells and ghosts were incubated with test
compounds for ∼15 minutes before measurements. (B)
Relative osmotic water permeability (S.E., n = 4). *P ,
0.05 compared with control.

692
Esteva-Font et al.
loop diuretic inhibitors of the NKCC cotransporter, reporting
inhibition by bumetanide. Of 45 bumetanide scaffolds syn-
thesized, compound 4 here (AqB013) was identified in oocyte
assays as an inhibitor of AQP1 and AQP4 with IC50 ∼20 mM.
In a follow-on study by the same investigators, compound 4
was tested in a brain injury model for reducing edema,
though no beneficial effect was found (Oliva et al., 2011). The
same group later found that an analog of the loop diuretic
furosemide, compound 5 here (AqF026), activated AQP1,
increasing its water permeability by ∼20% in the oocyte
assay (Yool et al., 2013). The possibility that off-target
actions of these compounds might be responsible for the
apparent effects on oocyte water permeability, such as
actions of the many NKCC-related ion transporters, was
not considered.
Mola et al. (2009) screened approximately 3500 compounds
using a calcein fluorescence assay in AQP1 and AQP4-
expressing cells in a plate-reader assay in which cells were
exposed to a 200 mM inwardly directed gradient of NaCl.
Active compounds from the screen were retested using
erythrocytes and vesicles derived from AQP4-expressing
cells. Compounds 6, 7, 8, and 9 were reported to inhibit
AQP1 with IC50 values of 25–50 mM. In our hands these
compounds showed marked toxicity at 100 mM (data not
shown). Compounds 6, 7, and 8 are non-drug-like; compounds
6 and 8 are organolead and organotin molecules, respec-
tively, which as a general class of molecules are considered
to be neurotoxins (Chang, 1990) and cause erythrocyte lysis
ꢀ
(Kleszcynska et al., 1997). Compound 7 (trichopolyn I) is a
Fig. 7. Osmotic water permeability in plasma membrane vesicles from
CHO cells. Osmotic water permeability was measured from the time
course of scattered-light intensity in response to a 250 mM inwardly
directed sucrose gradient. (A) Representative time course data for negative
control (0.5% DMSO vehicle alone) and 0.3 mM HgCl2 in vesicles from
nontransfected CHO cells expressing AQP1- and AQP4-M23. (B) Repre-
sentative time course data for indicated compounds (each 50 mM). Vesicles
were incubated with test compounds for ∼15 minutes before measure-
ments. (C) Relative osmotic water permeability (S.E., n = 4). *P , 0.05
compared with control.
structurally complex 10-residue lipopeptide isolated from
the fungus Trichoderma polysporum that belongs to the
trichogin class of lipopeptaibols antibiotics whose mecha-
nism of action is thought to be pore formation in bacterial cell
membranes (de Zotti et al., 2009).
Recently, To et al. (2015) reported two compounds, 10 (an
analog of 9) and 11, as AQP1 inhibitors using a yeast freeze-
thaw assay performed in Escherichia coli expressing AQP1, in
which cell viability was measured after two-cycles of freeze-
thaw. The stated, though nonvalidated, rationale is that AQP1
subsequent measurements failing to confirm inhibition (Yang permits water efflux during thawing and prevents cell burst-
et al., 2008). Given the apparent very low probability of ing. Their study included light-scattering measurements on
identifying AQP inhibitors, it seems a priori unlikely that erythrocytes, though interpretation of possible inhibitory
testing common drugs such as bumetanide, acetazolamide, effects was confounded by the multiexponential kinetics of
antimigraines, and antiepileptics without large-scale screening the light-scattering data. Very recently, Patil et al. (2016)
would yield AQP inhibitors. It also seems unlikely that small reported compounds 12 and 13 as AQP1 inhibitors in a small
molecules could activate AQP1, as its water permeability is screen. Apparent compound activities were quite variable in
constitutively high and not subject to regulation.
Xenopus oocyte, erythrocyte ghost, and AQP1 proteoliposome
Compounds 1, 2, and 3 were identified by virtual (computa- assays. Here, we found both compounds to be toxic to erythro-
tional) screening of ∼106 compounds of the ZINC database and cytes and vesicles.
testing 14 compounds for inhibition of osmotic swelling in
In conclusion, we could not demonstrate modulation of
AQP1-expressing Xenopus laevis oocytes (Seeliger et al., 2013). AQP1 function by 12 reported inhibitors and one reported
The compounds were identified by molecular docking compu- activator using several direct assays of osmotic water perme-
tations to a part of the extracellular surface of human AQP1. ability and different assay conditions. In view of the multiple
Compounds 1, 2, and 3 showed ∼80% reduced osmotic swelling potential clinical applications of AQP1 inhibitors, the identi-
of the oocytes with an IC50 of 8–20 mM. However, as found here, fication of AQP1 inhibitors remains a high priority.
no inhibition was seen in light-scattering measurements on
erythrocytes, suggesting an oocyte-specific action. A bona fide
Authorship Contributions
AQP1 inhibitor would be expected to produce inhibition in
different assays and different cell systems.
Migliati et al. (2009) screened known channel blockers using
the oocyte swelling assay. Although the identities and num-
Participated in research design: Esteva-Font, Phuan, Anderson,
Verkman.
Conducted experiments: Esteva-Font, Jin, Lee.
Wrote or contributed to the writing of the manuscript: Esteva-Font,
bers of tested blockers were not mentioned, they focused on Lee, Phuan, Anderson, Verkman.

Small-Molecule AQP1 Inhibitors
693
Patil RV, Xu S, van Hoek AN, Rusinko A, Feng Z, May J, Hellberg M, Sharif NA, Wax
MB, and Irigoyen M et al. (2016) Rapid identification of novel inhibitors of the
human aquaporin-1 water channel. Chem Biol Drug Des DOI: 10.1111/cbdd.12713
[published ahead of print].
Phuan PW, Ratelade J, Rossi A, Tradtrantip L, and Verkman AS (2012) Complement-
dependent cytotoxicity in neuromyelitis optica requires aquaporin-4 protein as-
sembly in orthogonal arrays. J Biol Chem 287:13829–13839.
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