High affinity antagonists of the vanilloid receptor

Article abstract of DOI:10.1124/mol.62.4.947

The vanilloid receptor VR1 has attracted great interest as a sensory transducer for capsaicin, protons, and heat, and as a therapeutic target. Here we characterize two novel VR1 antagonists, KJM429 [N-(4-tert-butylbenzyl)-N′-[4-(methylsulfonylamino)benzyl]thiourea] and JYL1421 [N-(4-tertbutylbenzyl)-N′-[3-fluoro-4-(methylsulfonylamino)benzyl] -thiourea], with enhanced activity compared with capsazepine on rat VR1 expressed in Chinese hamster ovary (CHO) cells. JYL1421, the more potent of the two novel antagonists, inhibited [³H]resiniferatoxin binding to rVR1 with an affinity of 53.5 ± 6.5 nM and antagonized capsaicin-induced calcium uptake with an EC₅₀ of 9.2 ± 1.6 nM, reflecting 25- and 60-fold greater potencies than capsazepine. Both JYL1421 and KJM429 antagonized RTX as well as capsaicin and their mechanism was competitive. The responses to JYL1421 and KJM429 differed for calcium uptake by rVR1 induced by heat or pH. JYL1421 antagonized the response to both pH 6.0 and 5.5, whereas KJM429 antagonized at pH 6.0 but was an agonist at lower pH (<5.5). For heat, JYL1421 fully antagonized and KJM429 partially antagonized. Capsazepine showed only weak antagonism for both pH and heat. Responses of rVR1 to different activators could thus be differentially affected by different ligands. In cultured dorsal root ganglion neurons, JYL1421 and KJM429 likewise behaved as antagonists for capsaicin, confirming that the antagonism is not limited to heterologous expression systems. Finally, JYL1421 and KJM429 had little or no effect on ATP-induced calcium uptake in CHO cells lacking rVR1, unlike capsazepine. We conclude that JYL1421 is a competitive antagonist of rVR1, blocking response to all three of the agonists (capsaicin, heat, and protons) with enhanced potency relative to capsazepine.

Full text of DOI:10.1124/mol.62.4.947

0
026-895X/02/6204-947–956
MOLECULAR PHARMACOLOGY
U.S. Government work not protected by U.S. copyright
Mol Pharmacol 62:947–956, 2002
Vol. 62, No. 4
1729/1014115
Printed in U.S.A.
High Affinity Antagonists of the Vanilloid Receptor
1
YUN WANG, TAMAS SZABO, JACQUELINE D. WELTER, ATTILA TOTH, RICHARD TRAN, JIYOUN LEE,
SANG UK KANG, YOUNG-GER SUH, PETER M. BLUMBERG, and JEEWOO LEE
National Cancer Institute, Bethesda, Maryland (Y.W., T.S., J.D.W., A.T., R.T., P.M.B.); and College of Pharmacy, Seoul National University,
Seoul, Korea (Ji.L., S.U.K., Y.-G.S., Je.L.)
Received March 12, 2002; accepted July 16, 2002
This article is available online at http://molpharm.aspetjournals.org
ABSTRACT
The vanilloid receptor VR1 has attracted great interest as a uptake by rVR1 induced by heat or pH. JYL1421 antagonized
sensory transducer for capsaicin, protons, and heat, and as the response to both pH 6.0 and 5.5, whereas KJM429
a therapeutic target. Here we characterize two novel VR1 antagonized at pH 6.0 but was an agonist at lower pH (Ͻ5.5).
antagonists, KJM429 [N-(4-tert-butylbenzyl)-NЈ-[4-(methyl- For heat, JYL1421 fully antagonized and KJM429 partially
sulfonylamino)benzyl]thiourea] and JYL1421 [N-(4-tert- antagonized. Capsazepine showed only weak antagonism
butylbenzyl)-NЈ-[3-fluoro-4-(methylsulfonylamino)benzyl]- for both pH and heat. Responses of rVR1 to different acti-
thiourea], with enhanced activity compared with vators could thus be differentially affected by different li-
capsazepine on rat VR1 expressed in Chinese hamster ovary gands. In cultured dorsal root ganglion neurons, JYL1421
(
CHO) cells. JYL1421, the more potent of the two novel and KJM429 likewise behaved as antagonists for capsaicin,
3
antagonists, inhibited [ H]resiniferatoxin binding to rVR1 with confirming that the antagonism is not limited to heterologous
an affinity of 53.5 Ϯ 6.5 nM and antagonized capsaicin- expression systems. Finally, JYL1421 and KJM429 had little
induced calcium uptake with an EC50 of 9.2 Ϯ 1.6 nM, or no effect on ATP-induced calcium uptake in CHO cells
reflecting 25- and 60-fold greater potencies than capsa- lacking rVR1, unlike capsazepine. We conclude that
zepine. Both JYL1421 and KJM429 antagonized RTX as well JYL1421 is a competitive antagonist of rVR1, blocking re-
as capsaicin and their mechanism was competitive. The sponse to all three of the agonists (capsaicin, heat, and
responses to JYL1421 and KJM429 differed for calcium protons) with enhanced potency relative to capsazepine.
A vanilloid receptor (VR1) that is activated by capsaicin, neuralgia, diabetic neuropathy, cluster headache, osteoar-
low pH, and temperatures higher than 42°C has been cloned thritis, and pruritus (Rains and Bryson, 1995; Kim and
from rat dorsal root ganglia (Caterina et al., 1997; Tominaga Chancellor, 2000).
et al., 1998). It is a nonselective cation channel, with high
The exciting potential therapeutic applications for va-
permeability for divalent cations, expressed on unmyelinated nilloids have motivated efforts to identify or design novel
pain-sensing nerve fibers (C-fibers) and small A␦ fibers in the derivatives with improved properties (Walpole et al.,
dorsal root, trigeminal, and nodose ganglia. Initially, activa- 1993a,b,c; Wrigglesworth et al., 1996). An important advance
tion of VR1 by pungent agonists such as capsaicin leads to was the identification of resiniferatoxin (RTX), a diterpene
excitation of primary sensory neurons gating nociceptive in- related to the phorbol esters, as an ultrapotent capsaicin
puts to the central nervous system. Subsequently, these fi- analog (Szallasi and Blumberg, 1989). RTX demonstrated
bers may become desensitized/defunctionalized, and this de- that orders of magnitude of additional affinity for VR1 could
sensitization forms a basis for the therapeutic use of VR1 be captured through appropriate chemistry. Furthermore,
agonists (Szallasi and Blumberg, 1999). Potential therapeu- because RTX was much more potent than capsaicin for de-
tic applications include detrusor hyperreflexia, postherpetic sensitization, whereas it was only modestly more potent for
inducing acute pain, as determined in the eye wiping assay,
the behavior of RTX strongly suggested that these different
This research was partly supported by a Fund 2000 grant from the Korea biological endpoints could be dissociated, at least in part.
Research Foundation.
1
This was important, because desensitization is a therapeutic
goal, whereas the induction of acute pain is the limiting
Present address: Neuroscience Research Institute, Peking University,
00083, Beijing, People’s Republic of China.
1
ABBREVIATIONS: RTX, resiniferatoxin; HEK, human embryonic kidney; CHO, Chinese hamster ovary; rVR1, cloned rat vanilloid receptor
subtype-1; CHO/rVR1 cell, Chinese Hamster Ovary cells transfected with cloned rat vanilloid receptor subtype-1; DRG, dorsal root ganglion;
DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; JYL1421, N-(4-tert-butylbenzyl)-NЈ-[3-fluoro-4-(methylsulfonylamino)ben-
zyl]thiourea; KJM429, N-(4-tert-butylbenzyl)-NЈ-[4-(methylsulfonylamino)benzyl]thiourea; DPBS, Dulbecco’s phosphate-buffered saline; PBS,
phosphate-buffered saline; CPZ, capsazepine; MES, 2-[N-Morpholino]ethanesulfonic acid.
947

9
48
Wang et al.
toxicity for capsaicin. Because of its favorable therapeutic Lim, H.-K. Choi, Y.-G. Suh, H.-K. Park, et al., manuscript in
index, RTX is currently in clinical trials (Kim and Chancel- preparation). In the present study, we have characterized in
lor, 2000).
detail, using CHO cells heterologously expressing rVR1, two
Desensitization of C-fiber sensory neurons represents one of these antagonists, KJM429 and JYL1421. JYL1421, in
attractive therapeutic strategy. A complementary strategy is particular, is 60-fold more potent than capsazepine as a cap-
through pharmacological antagonists for VR1. Because of the saicin antagonist and blocks the action of all three VR1
long duration of desensitization after treatment with VR1 agonists—capsaicin, heat, and protons.
agonists, antagonists may be of particular utility when short-
term blockade of VR1 is desired. So far, only a single antag-
Materials and Methods
onist of VR1, capsazepine, has been studied extensively
Bevan et al., 1992). Unfortunately, capsazepine has only
modest potency and is somewhat nonspecific, also antagoniz-
(
Materials. The antagonists JYL1421 [N-(4-tert-butylbenzyl)-NЈ-
3-fluoro-4-(methylsulfonylamino)benzyl]thiourea] and KJM429
[
ing voltage sensitive calcium channels and the nicotinic cho- [N-(4-tert-butylbenzyl)-NЈ-[4-(methylsulfonylamino)benzyl]thiourea]
linergic receptor (Docherty et al., 1997; Liu and Simon, 1997; were synthesized as described below. The structures of these two
3
Wardle et al., 1997).
compounds and of capsazepine are shown in Fig. 1. [ H]RTX (37
Ci/mmol) was provided by PerkinElmer Life Science (Boston, MA).
The efforts of several groups have thus been directed at the
development of novel antagonists. Exploiting the high po-
tency of RTX, Wahl et al. (2001) have described 5-iodo-RTX
45
Ca was from ICN Biomedicals, Inc. (Irvine, CA). Nonradioactive
RTX, capsaicin, and capsazepine were purchased from Alexis Corp
(
San Diego, CA).
as a potent VR1 antagonist with a K of 4.3 nM for binding to
d
Synthesis of KJM429. A mixture of 4-aminobenzylamine (10 g,
2 mmol) and di-tert-butyl dicarbonate (20 g, 90 mmol) in THF (100
ml) was stirred for 2 h at room temperature and concentrated in
vacuo. The residue was purified by flash column chromatography on
expressing rat VR1. Compared with capsazepine in this sys- silica gel with EtOAc/hexanes (2:3) as eluant to afford tert-butyl
rat VR1 heterologously expressed in human embryonic kid-
ney (HEK) 293 cells and with an IC50 of 3.9 nM for inhibiting
the capsaicin-induced membrane current in X. laevis oocytes
8
tem, 5-iodo-RTX was thus 40-fold more potent. Although the N-(4-aminobenzyl)carbamate as a white solid (17.5 g, 96%): m.p. ϭ
1
binding of 5-iodo-RTX was reversible, its antagonism was not 75°C; H NMR (CDCl
) ␦ 7.06 (d, 2 H, J ϭ 8.3 Hz, H-2,6), 6.63 (dt, 2
3
H, J ϭ 2.7, 8.3 Hz, H-3,5), 4.73 (bs, 1 H, NHBoc), 4.18 (d, 2 H, J ϭ 5.6
Hz, CH NH), 3.60 (bs, 2 H, NH ), 1.45 (s, 9 H, C(CH ) ).
competitive with capsaicin, suggesting that further study of
its mechanism is still needed. In vivo, intrathecal adminis-
tration of 5-iodo-RTX to mice blocked the acute pain response
to injection of capsaicin. For human VR1, 5-iodo-RTX was
reported to antagonize with an ID50 of 27 nM (McDonnell et
al., 2002).
2
2
3 3
A cooled solution of the above amine (1.11 g, 5 mmol) at 0°C in
pyridine (10 ml) was treated with methanesulfonyl chloride (0.464
ml, 6 mmol) and stirred for 16 h at room temperature. The reaction
mixture was cooled in an ice-bath, neutralized carefully with 1 N
hydrochloric acid solution, diluted with water, and extracted several
times with dichloromethane. The combined organic layers were
In a complementary approach, channel blockers of VR1
were prepared through combinatorial chemistry of N-alkyl washed with water and brine, dried over magnesium sulfate, and
glycine trimers (Garcia-Martinez et al., 2002). Two deriva- concentrated in vacuo. The residue was purified by flash column
tives with IC50 values of 0.7 and 2.6 ␮M were described. chromatography on silica gel with EtOAc/hexanes (1:1) as eluant to
afford tert-butyl N-[(4-methylsulfonylamino)benzyl]carbamate as a
These compounds were noncompetitive with capsaicin, as
expected, and showed selectivity for VR1 relative to several
other channels.
In our ongoing effort to design improved vanilloids, we
have identified motifs conferring significantly enhanced po-
tency for rVR1 agonists (Lee et al., 1999, 2001a, 2002). Based
on these structures, we have designed a series of derivatives
1
white solid (1.38 g, 92%): m.p. ϭ 162°C; H NMR (CDCl ) ␦ 7.28 (d,
3
2
1
H, J ϭ 8.3 Hz, H-2,6), 7.18 (dd, 2 H, J ϭ 2.2, 6.3 Hz, H-3,5), 6.76 (s,
H, NHSO ), 4.88 (bs, 1 H, NHBoc), 4.28 (d, 2 H, J ϭ 5.6 Hz,
2
CH NH), 2.99 (s, 3 H, SO CH ), 1.46 (s, 9 H, C(CH ) ).
2
2
3
3 3
A cooled solution of the above compound (0.6 g, 2 mmol) in dichlo-
romethane (10 ml) in an ice-bath was treated with trifluoroacetic
acid (2.5 ml), which was added slowly, and the mixture was stirred
that function as rVR1 antagonists (Lee et al., 2001b; J. Lee, for 1.5 h at 0°C. The mixture was concentrated in vacuo carefully to
J. Lee, M. Kang, M.-Y. Shin, J.-M. Kim, S.-U. Kang, J.-O. afford 4-(methylsulfonylamino)benzyl amine salt as a white solid in
Fig. 1. Structures of KJM429, JYL1421, and capsazepine.

Antagonists of Vanilloid Receptor
949
a quantitative yield. The salt was washed with diethyl ether and
1
used for the next step without further purification: H NMR (DMSO-
d ) ␦ 9.87 (s, 1 H, NHSO ), 8.14 (bs, 3 H, NH ), 7.40 (d, 2 H, J ϭ 8.5
6
2
3
Hz, H-2,6), 7.22 (d, 2 H, J ϭ 8.5 Hz, H-3,5), 3.97 (s, 2 H, CH ), 2.99
2
(
s, 3 H, SO CH ).
2 3
A solution of above amine salt (2 mmol) in dimethylformamide (4
ml) was treated with triethylamine (0.28 ml, 2 mmol) and stirred for
h at room temperature. To the mixture was added 4-t-butylbenzyl
1
isothiocyanate (0.41 g, 2 mmol). After being stirred for 24 h at room
temperature, the mixture was diluted with water and extracted
several times with ethyl acetate. The combined organic layers were
washed with water and brine, dried over magnesium sulfate, and
concentrated in vacuo. The residue was purified by flash column
chromatography on silica gel with EtOAc/hexanes (2:3) as eluant to
1
afford KJM429 as a white solid (0.73 g, 90%): m.p. ϭ 83°C; H NMR
(
CDCl ) ␦ 7.37 (d, 2 H, J ϭ 8.3 Hz), 7.18–7.25 (m, 4 H), 7.15 (d, 2 H,
3
J ϭ 8.3 Hz), 6.53 (s, 1 H, NHSO ), 6.12 (bs, 1 H, NHCS), 5.96 (bs, 1
2
H, NHCS), 4.66 (d, 2 H, J ϭ 5.4 Hz, NHCH ), 4.57 (d, 2 H, J ϭ 5.2 Hz,
2
NHCH ), 3.00 (s, 3 H, SO CH ), 1.31 (s, 9 H, C(CH ) ); MS m/z 405
2
2
3
3 3
ϩ
(
M ); Anal. Calcd for C20H27N O S : C, 59.23; H, 6.71; N, 10.36; S,
3 2 2
15.81. Found: C, 59.44; H, 6.73; N, 10.32; S, 15.79.
Synthesis of JYL1421. A cooled solution of 2-fluoro-4-iodoaniline
(
5 g, 21.1 mmol) in pyridine (30 ml) at 0°C was drop-wise treated
with methanesulfonyl chloride (2.45 ml, 31.6 mmol). After being
stirred for 3 h at room temperature, the mixture was diluted with
water and extracted with ethyl acetate several times. The combined
organic layers were washed with water and brine, dried over sodium
sulfate, and concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel with EtOAc/hexanes (1:2) as
eluant to afford N-(2-fluoro-4-iodophenyl)methanesulfoamide as a
1
white solid (5.32 g, 80%): m.p. ϭ 118°C; H NMR (CDCl ) ␦ 7.50 (d,
3
2
H, J ϭ 8.0 Hz), 7.33 (t, 1 H, J ϭ 8.0 Hz), 6.54 (bs, 1 H, NH), 3.04
(
s, 3 H, SO CH ).
2
3
A mixture of the above iodide (5.32 g, 17 mmol), zinc cyanide (1.2
g, 10.2 mmol), and tetrakis(triphenylphosphine) palladium (0.976 g,
.85 mmol) in dimethylformamide (15 ml) was heated at 80°C for 8 h.
0
The mixture was diluted with water and extracted with ethyl acetate
several times. The combined organic layers were washed with water
and brine, dried over sodium sulfate, and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel
with EtOAc/hexanes (1:2) as eluant to afford N-(2-fluoro-4-cyanophe-
nyl)methanesulfoamide as a white solid (3.21 g, 87%): m.p. ϭ 203°C;
1
H NMR (CDCl ) ␦ 7.73 (t, 1 H, J ϭ 8.3 Hz), 7.47 (m, 2 H), 7.00 (bs,
3
1
H, NH), 3.14 (s, 3 H, SO CH ).
2 3
A stirred suspension of the above nitrile (2.142 g, 10 mmol), 5%
3
Fig. 2. Comparison of antagonists for inhibition of specific [ H]RTX
palladium on carbon (0.2 g) and several drops of concentrated hydro-
chloric acid in MeOH (50 ml) was hydrogenated under a balloon of
hydrogen for 16 h. The reaction mixture was filtered and the filtrate
was concentrated in vacuo to afford 3-fluoro-4-(methylsulfonylami-
no)benzyl amine salt as a white solid, which was used for the next
4
5
2ϩ
binding and of Ca uptake induced by capsaicin and RTX. Ca uptake
was induced by 50 nM capsaicin or 0.26 nM RTX in the presence of the
indicated concentrations of the antagonists. Points represent mean val-
ues of quadruplicate determinations in single, representative experi-
ments; error bars indicate S.E.M. All experiments were repeated at least
two additional times with similar results.
coupling step without further purification: m.p. ϭ 256°C (decom-
1
pose); H NMR (CD OD) ␦ 7.56 (t, 1 H, J ϭ 8.3 Hz), 7.28 (dd, 2 H),
3
4
.07 (s, 2 H, CH ), 2.98 (s, 3 H, SO CH ).
ϩ
2
2
3
(M ); Anal. Calcd for C20H26FN O S : C, 56.71; H, 6.19; N, 9.92; S,
3
2 2
A solution of above amine salt (2 mmol) and triethylamine (0.28
ml, 2 mmol) in dimethylformamide (2 ml) was stirred at room tem-
perature for 30 min and treated with 4-t-butylbenzyl isothiocyanate
0.41 g, 2 mmol). After being stirred at room temperature for 20 h,
15.14. Found: C, 56.91; H, 6.22; N, 9.89; S, 15.10.
Molecular Biology. A cDNA encoding the vanilloid receptor
rVR1 was cloned from rat DRG total RNA by reverse-transcription-
polymerase chain reaction using primers base on the published nu-
(
the mixture was diluted with water and extracted with ethyl acetate cleotide sequence (Caterina et al., 1997). A 2.7-kilobase cDNA was
several times. The combined organic layers were washed with water isolated and the nucleotide sequence was verified to be identical to
and brine, dried over magnesium sulfate, and concentrated in vacuo. the published sequence. This cDNA was subcloned into pUG102–3
The residue was purified by flash column chromatography on silica (BD Biosciences Clontech, Palo Alto, CA).
gel with EtOAc/hexanes (1:1) as eluant to afford JYL-1421 as a white
Stable VR1 Expression Cell Line Preparation and Subcul-
solid (0.762 g, 90%): m.p. ϭ 59°C; H NMR (CDCl ) ␦ 7.47 (t, 1 H, J ϭ ture. The pUHG102–3 rVR1 plasmid was transfected into Chinese
1
3
8.3 Hz), 7.38 (d, 2 H, J ϭ 8.3 Hz), 7.23 (d, 2 H, J ϭ 8.3 Hz), 7.02 (m, hamster ovary (CHO) cells carrying the pTet Off regulatory express-
2
H), 6.54 (s, 1 H, NHSO ), 6.29 (bs, 1 H, NHCS), 6.01 (bs, 1 H, sion plasmid (BD Biosciences Clontech). In these cells, expression of
2
NHCS), 4.72 (d, 2 H, J ϭ 5.6 Hz, NHCH ), 4.54 (d, 2 H, J ϭ 5.4 Hz, the pUHG plasmid is repressed in the presence of tetracycline but is
2
NHCH ), 3.00 (s, 3 H, SO CH ), 1.31 (s, 9 H, C(CH ) ).; MS m/z 423 induced upon removal of the antibiotic. Stable clones were isolated in
2
2
3
3 3

9
50
Wang et al.
culture medium containing puromycin (10 ␮g/ml) and maintained in (Szallasi et al., 1992). Binding assay mixtures were set up on ice and
3
medium supplemented with tetracycline (1 ␮g/ml) (Szallasi et al., contained 40 pM [ H]RTX, various concentrations of competing li-
1
999). Cells used for assays were grown in culture medium without gands, 0.25 mg/ml bovine serum albumin (Cohn fraction V), and 5 ϫ
4
5
antibiotic for 48 h before use. For radioligand binding experiments, 10 to 5 ϫ 10 VR1-transfected cells. The final volume was adjusted
2ϩ
2ϩ
cells were seeded in T75 cell culture flasks in media without antibi- to 450 ␮l with DPBS with Ca and Mg and 0.25 mg/ml bovine
otics and grown to approximately 90% confluence. The flasks were serum albumin. Nonspecific binding was determined in the presence
then washed with PBS and harvested in PBS containing 5 mM of 100 nM nonradioactive RTX. The binding reaction was initiated by
EDTA. The cells were pelleted by gentle centrifugation and stored at transferring the assay mixtures to a 37°C water bath and was
45
Ϫ20°C until assayed. For assay of Ca uptake, cells were seeded into terminated after a 60-min incubation period by cooling the tubes on
2
4-well plates and grown to 70 to 90% confluence. For calcium
ice. Membrane-bound RTX was separated from the free by pelleting
the membranes in a Beckman 12 (Beckman Coulter, Fullerton, CA)
imaging, cells were grown on glass coverslips (25 mm).
DRG Neuron Isolation and Culture. Two- to 3-week-old rats bench-top centrifuge (15 min, maximal velocity), the tips of the tubes
were killed by decapitation under CO2 anesthesia. The spinal col- containing the pellets were cut off, and the radioactivity was deter-
umns were removed aseptically and dorsal root ganglia from all mined by scintillation counting. Equilibrium binding parameters (Ki,
levels were dissected out and collected in DMEM containing 0.5% Bmax, and cooperativity) were determined by fitting the Hill equation
heat inactivated FBS (Invitrogen, Carlsbad, CA), 1 mM Na-pyru- to the measured values with the aid of the program Origin 6.0
vate, 25 mM HEPES, pH 7.55, and antibiotics. Ganglia were digested (OriginLab Corp., Northampton, MA).
4
5
with 1 mg/ml collagenase (Sigma-Aldrich, St. Louis, MO) and 0.125
Ca Uptake. CHO/rVR1 cells were incubated for 5 min at 37°C or
45
mg/ml trypsin (Sigma-Aldrich) for 30 min at 37°C. The digestion was 44°C with 0.2 ␮Ci of Ca in the presence of serum-free DMEM, 0.25
terminated by addition of 10% FBS. The ganglia were triturated with mg/ml bovine serum albumin, and various concentrations of the
45
a flame-polished Pasteur pipette to form a single cell suspension. different compounds. To determine the pH dependence of Ca up-
45
Cells were then washed three times with DMEM, they were resus- take, cells were incubated for 5 min at 22°C with 1 ␮Ci Ca in the
2ϩ
2ϩ
pended in the same medium, and the number of viable cells was presence of Dulbecco’s PBS (DPBS) with Ca and Mg , supple-
determined. Cells were plated onto coverslips coated with 10 ␮g/ml mented with 0.25 mg/ml bovine serum albumin, adjusted to the
polylysine and were incubated for 24 h in DMEM containing 10% indicated pH with 1 M MES (Sigma). After incubation, cells were
2ϩ
2ϩ
FBS, antibiotics, 200 ng/ml mouse submaxillary gland 2.5 S NGF washed three times with DPBS (with Ca , Mg ), and lysed in 400
Ϫ5
(
Sigma), and 10 M cytosine arabinoside (Sigma). The medium was ␮l/well of radioimmunoprecipitation assay buffer (50 mM Tris-HCl,
then changed to fresh medium lacking the cytosine arabinoside and pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, and 0.1%
the cells incubated for a further 2 days before being used for exper-
iments.
Competition Binding Assay. Binding studies with [ H]RTX
SDS) for 20 min. Aliquots of the solubilized cell extracts were
counted in a liquid scintillation counter.
Imaging of Intracellular Calcium Levels [Ca ]i. Cells grown
3
2؉
were carried out as described previously with minor modifications on coverslips were loaded with Fura-2 acetoxymethyl ester (10 ␮M)
4
5
Fig. 3. Shift in the dose dependence of the capsaicin- and RTX-induced Ca uptake in CHO/rVR1 cells by KJM429 and JYL1421. a, b, d, e,
dose-response curves were determined for agonists alone and in the presence of the indicated doses of antagonists. Points represent mean values of
quadruplicate determinations in single, representative experiments; error bars indicate S.E.M. All experiments were repeated at least two additional
times with similar results. c, f, plot of Kapp versus the concentration of KJM429 or JYL1421.

Antagonists of Vanilloid Receptor
951
for 10 min at 37°C and an additional 50 min at room temperature (Szallasi et al., 1999). We have therefore routinely used that
CHO/rVR1 cells) or 30 min at 37°C (DRG neurons), washed and measure for determination of ligand structure activity rela-
(
then incubated at room temperature for at least an additional hour.
Coverslips were placed in a chamber at room temperature. Images of
Fura-2 loaded cells with the excitation wavelength alternating be-
tween 340 nM and 380 nM were captured using a Cohu 4915 low-
light charge-coupled device camera on an InCyt Dual-wavelength
tions. KJM429 and JYL1421, a derivative of KJM429 substi-
tuted with a fluorine in the m-position of the phenyl ring in
3
the A region, inhibited [ H]RTX binding to rVR1 with K
i
values of 62.6 Ϯ 10.1 (four experiments) and 53.5 Ϯ 6.5 nM
Fluorescence Imaging and Photometry System (Intracellular Imag- (three experiments), respectively (Fig. 2a). Capsazepine, in
ing Inc., OH). After subtraction of background fluorescence, the ratio contrast, inhibited with a K of only 1310 Ϯ 150 nM (three
i
of fluorescence intensity at the two wavelengths was calculated.
experiments, current studies) (6600 nM; Szallasi et al., 1999)
(
Fig. 2a). We conclude that KJM429 and JYL1421 are 10 to
Results
25-fold more potent than capsazepine for binding to rVR1.
We evaluated antagonism by the ability of the compounds
to inhibit the Ca uptake induced by 50 nM capsaicin.
JYL1421 and KJM429 Bind to the Vanilloid Receptor
and Antagonize Its Response to Ligands. We have pre-
45
viously demonstrated that ligand binding to rVR1 heterolo- KJM429 and JYL1421 inhibited the response of CHO/rVR1
gously expressed in CHO cells or HEK293 cells closely re- cells with K values of 53.9 Ϯ 8.7 nM (four experiments) and
i
sembles that characterized in dorsal root ganglion neurons 9.2 Ϯ 1.6 nM (three experiments), respectively (Fig. 2b).
Fig. 4. Antagonism of capsaicin evoked calcium mobilization in CHO/rVR1 cells and cultured rat DRG neurons as determined by calcium imaging.
CHO/rVR1 cells or rat DRG neurons were treated with 100 nM capsaicin as indicated for 120 s before application of buffer, KJM429, or JYL1421
(
indicated by horizontal line) in the continued presence of capsaicin. Points represent the averaged signal from a minimum of 16 cells imaged
simultaneously. Each experiment was repeated at least an additional two times with similar results on independently cultured cells.

9
52
Wang et al.
Capsazepine was once again appreciably less potent, with a observed, however, that the DRGs were somewhat less sen-
K of 520 Ϯ 12 nM (three experiments) (Fig. 2b). The levels of sitive to the antagonists than were the CHO/rVR1 cells. We
i
inhibition under these assay conditions were Ͼ98% for all assume that this reflects the effect of spare receptors, and
three compounds. We conclude that both KJM429 and DRGs have likewise been found to be somewhat less sensitive
JYL1421 function as full antagonists under these conditions. to other vanilloids for calcium uptake than are heterologous
JYL1421 was thus 60-fold more potent than capsazepine; rVR1 overexpressors.
KJM429 was somewhat less so. We further evaluated antag-
JYL1421 and KJM429 Antagonized Heat-Induced
45
onism by inhibition of the Ca uptake induced by 0.26 nM (44°C) Calcium Uptake. Heat represents a second and
RTX. Results were similar to those with capsaicin, with Ki physiologically relevant class of agonists for activation of
values of 30.4 Ϯ 5.7 nM (three experiments), 12.4 Ϯ 1.6 nM VR1. It has been reported that capsazepine was less effective
(
three experiments), and 315 Ϯ 24 nM (three experiments), for inhibiting response to heat than response to capsaicin
for KJM429, JYL1421, and capsazepine, respectively (Fig. (Nagy and Rang, 1999; Savidge et al., 2001). Using calcium
c). The antagonism was thus not dependent on our choice of uptake, we examined the ability of KJM429, JYL1421, and
2
45
capsaicin as the agonist for induction of Ca uptake.
capsazepine to block heat-induced (44°C) calcium uptake in
4
5
KJM429 and JYL1421 Antagonize Ca Uptake In- CHO/rVR1 cells. Unlike vanilloid stimulated calcium uptake,
duced by Capsaicin and RTX through a Competitive which was fully antagonized by all three compounds under
Mechanism. To test whether KJM429 and JYL1421 were our assay conditions, heat-induced calcium uptake was dif-
antagonizing vanilloid action through a competitive mecha- ferentially antagonized by the three compounds (Fig. 5).
nism, we determined the dose response curves for induction JYL1421 was an almost complete antagonist at 300 nM,
45
of Ca uptake by capsaicin and RTX as a function of the
concentration of KJM429 or JYL1421. Cells were incubated
with fixed concentrations of antagonist together with varied
concentrations of capsaicin for 5 min (Fig. 3, a, b, d, e). For
both antagonists, the dose-response curves for induction of
4
5
Ca uptake in CHO/rVR1 cells by both agonists were shifted
to the right with no depression of the maximal response.
Using the relationship Kapp ϭ K ϩ (K /K ) (I), where K is
d
d
i
app
the measured ED50 in the presence of the antagonist, K is
d
the ED50 in the absence of antagonist, K is the dissociation
i
constant for the antagonist, and I is the concentration of
antagonist, we obtained a linear fit between Kapp and I (Fig.
3
, c and f) (Matthews, 1993). We derived Ki values for
KJM429 of 31.2 Ϯ 0.8 nM (three experiments) as an antag-
onist of capsaicin and of 35.2 Ϯ 0.3 nM (three experiments) as
an antagonist of RTX. For JYL1421, we obtained K values of
i
8
.0 Ϯ 0.3 nM (three experiments) as an antagonist of capsa-
icin and of 9.6 Ϯ 0.1 nM (three experiments) as an antagonist
of RTX. For capsazepine, we obtained K values of 430 Ϯ 10
i
nM (three experiments) as an antagonist against capsaicin
and of 460 Ϯ 10 nM (three experiments) as an antagonist
against RTX. These values agree well with those obtained
from the curves for antagonism at a fixed concentration of
agonist as described previously. We conclude that KJM429
and JYL1421 indeed antagonized capsaicin and RTX action
through a competitive mechanism.
Antagonism of Capsaicin Action by KJM429 and
JYL1421 as Determined by Calcium Imaging Analysis
of Both CHO/rVR1 Cells and Cultured Rat DRG Neu-
rons. Using visualization of intracellular calcium with
Fura-2 and imaging of individual cells, we determined the
ability of KJM429 and JYL1421 to block the calcium uptake
induced by capsaicin, both on the cloned rVR1 heterologously
expressed in CHO cells (Fig. 4, b and c) as well as of the rVR1
endogenously present in rat DRG neurons (Fig. 4, e and f).
Cells were preincubated with capsaicin for 2 min before ad-
dition of each antagonist in the continued presence of capsa-
icin. KJM429 and JYL1421 antagonized the elevation in
intracellular calcium in response to capsaicin in both sys-
45
Fig. 5. Inhibition by different antagonists of heat (44°C) induced Ca
uptake by CHO/rVR1 cells. Points represent mean values of quadrupli-
45
tems. These results confirm our findings with Ca uptake. cate determinations in single, representative experiments; error bars
indicate S.E.M. All data were subtracted from baseline (cells incubated at
Moreover, they show that the antagonism is not an artifact of
the heterologous expression system but is also observed for
the rVR1 endogenously present in the rat DRGs. It should be additional times with similar results.
3
(
7°C just with medium) and normalized by the percentage of control
without antagonists group). All experiments were repeated at least two

Antagonists of Vanilloid Receptor
953
whereas KJM429 inhibited by only 65.4 Ϯ 5.1% (three exper- we also compared the ability of KJM429, JYL1421, and cap-
iments) and capsazepine inhibited the response by only sazepine to antagonize the activation of VR1 by acidification
6
4.4 Ϯ 4.5% (three experiments). Secondly, JYL1421 was to pH 5.5 (Fig. 6, d-f). At this pH, JYL1421 remained potent
more potent than KJM429, as previously observed for antag- but the antagonism became partial, with 63.9 Ϯ 4.6% (three
onism of vanilloid-induced calcium uptake, and was mark- experiments) inhibition of the proton induced response.
edly more potent than capsazepine.
KJM429, in contrast to JYL1421, showed agonist activity at
The Effect of KJM429 and JYL1421 on Proton-In- this pH, and capsazepine remained as a weak, partial antag-
duced Calcium Uptake. Protons represent the third class onist.
of well-characterized agonists for VR1 and are of particular
These results demonstrate that compounds cannot be re-
interest because of the physiological role that acidosis is garded simply as antagonists or agonists for rVR1 but rather
believed to play in inflammatory pain. Mutational analysis that their actions can vary depending on the specific stimulus
suggests, moreover, that this response is complex, with mul- for activation of rVR1. Thus, JYL1421 was a complete antag-
tiple residues involved in the proton-coupled activation of the onist for stimulation of rVR1 by vanilloids, heat, and pH 6.0,
channel. We first compared the ability of KJM429, JYL1421, whereas KJM421 was only a partial antagonist for the latter
and capsazepine to antagonize the activation of VR1 by acid- two stimuli, although it was a complete antagonist for va-
45
ification to pH 6.0, as assayed by Ca uptake (Fig. 6, a-c). As nilloids under our assay conditions.
with activation by heat, JYL1421 functioned as a complete
Evaluation of Specificity of Antagonists for rVR1.
antagonist, whereas KJM429 blocked uptake by 57.8 Ϯ 2.6% One of the major deficiencies of capsazepine, in addition to its
(
7
three experiments) and capsazepine reduced uptake by weak potency, is its inhibitory activity on other channels
1.1 Ϯ 5.3% (three experiments) at concentrations of 10 ␮M. [e.g., voltage gated calcium channels (Docherty et al., 1997)
In addition, JYL1421 seemed modestly more potent as an and the nicotinic cholinergic receptor (Liu and Simon, 1997)].
antagonist for pH 6.0-induced activation than it was in the Using normal CHO cells not transfected with rVR1, we have
4
5
other assays.
observed that capsazepine is also able to inhibit Ca uptake
Because of the suggested complexity of the pH response, induced by ATP (Fig. 7). Its potency (K of 1090 Ϯ 32 nM;
i
4
5
Fig. 6. Effect of different antagonists on proton (pH 6.0 and pH 5.5) induced Ca uptake in CHO/rVR1 cells. Points represent mean values of triplicate
determinations in single, representative experiments; error bars indicate SEM. All data were relative to baseline (cells incubated at pH 7.0 without
antagonist) and normalized by the percentage of control (without antagonists). All experiments were repeated at least two additional times with
similar results.

9
54
Wang et al.
three experiments) was similar to that observed for antago- pene, binds to rVR1 with some 4 orders of magnitude higher
nism of rVR1. In contrast, JYL1421 gave no significant inhi- affinity than does capsaicin (Szallasi et al., 1999). These
bition up to 300 nM (p Ͼ 0.12, Student’s t test) and KJM429 opportunities had not previously been fully revealed through
gave only 43.2 Ϯ 6.7% (three experiments) inhibition up to 1 fairly extensive medicinal chemistry (Walpole et al.,
␮
M, a concentration 20 times its K for antagonism of capsa- 1996a,b,c). Using a strategy seeking to incorporate and con-
i
icin. Similar results were obtained by calcium imaging (Fig. strain some of the functional groups contributed by the diter-
). Cells were preincubated with each of the antagonists for 2 pene moiety of RTX, we have been able to produce novel
8
min before application, in the continued presence of the an- vanilloid agonists with several hundred-fold enhancement of
tagonist, of ␣,␤-methyleneadenosine 5Ј-diphosphate, a selec- binding potencies (Lee et al., 1999, 2001a,b; 2002).
tive agonist of the ATP gated P X receptor (Fig. 8). Whereas
An important finding is that medicinal chemistry can at
2
3
1
0 ␮M capsazepine partially inhibited the response, no inhi- least in part dissect the different biological endpoints asso-
bition was observed for 100 nM JYL1421 or KJM429.
ciated with vanilloid action. RTX is much more potent for
desensitization than for induction of acute pain in the eye-
wiping assay (Szallasi and Blumberg, 1989). Piperine has
been reported to be pungent but not to desensitize (Liu and
Discussion
The pharmacology of VR1 affords great opportunities for Simon, 1996). Several capsaicin analogs with a phorbol ester
exploitation through medicinal chemistry. Recognition has as a C region were inactive for modulating thermoregulation,
emerged over the past few years that the receptor affords a although they were potent for neurogenic inflammation (Sza-
binding surface that can generate much higher binding af- llasi et al., 1989; Appendino et al., 1996). The loss of the
finities than found for capsaicin. As we have described, the range of vanilloid responses in mice null for VR1 argues
natural product RTX, which represents a standard capsaicin against the otherwise attractive model that multiple genes
analog with the C-region substituted with a tricyclic diter- for the vanilloid receptor exist (Caterina et al., 2000; Davis et
al., 2000). Nevertheless, the vanilloid pharmacology unam-
biguously demonstrates that there must be mechanisms that
can generate from VR1 heterogeneity of response and that
can be distinguished by ligand chemistry. Plausible mecha-
nisms include association of VR1 with splice variants (Xue et
al., 2001) or other proteins, different subcellular localizations
for VR1 (Olah et al., 2001), and modification of VR1 by
phosphorylation (Premkumar and Ahern, 2000) or other
mechanisms (Kwak et al., 2000). Regardless of mechanism,
the whole animal behavior has amply documented the diver-
sity of behavior from a small assortment of compounds. Much
opportunity should be afforded by a vigorous medicinal chem-
ical approach.
Building on our previous efforts to design high-affinity
vanilloid agonists, we have also been able to develop an
extensive series of antagonists for rVR1 (Lee et al., 2001b; J.
Lee, J. Lee, M. Kang, M.-Y. Shin, J.-M. Kim, S.-U. Kang,
J.-O. Lim, H.-K. Choi, Y.-G. Suh, H.-K. Pork, et al., manu-
script in preparation). Here, we have characterized in detail
two of these antagonists at the cellular level and compared
their behavior with that of capsazepine.
At the practical level, a contribution of this study is that we
have identified a rVR1 antagonist, JYL1421, which is 25- to
60-fold more potent than capsazepine, the antagonist cur-
rently in common use. It thus falls in a similar potency range
to 5-iodo-RTX. JYL1421 blocks the activation of rVR1 by both
temperature and pH as well as by capsaicin, thus showing a
broader range of antagonistic activity than capsazepine. Fi-
nally, we might expect that the enhanced potency of JYL1421
would be associated with correspondingly enhanced specific-
ity. We in fact showed that this was true for one non–VR1-
mediated response, inhibition of ATP-induced calcium up-
take. ATP-induced calcium uptake was blocked by
capsazepine at a dose similar to that active on rVR1 but was
not blocked by JYL1421 at a dose 30 times that antagonizing
rVR1. Naturally, whether JYL1421 has improved specificity
4
5
Fig. 7. Effect of different antagonists on Ca uptake evoked by treatment
with 100 ␮M ATP of control CHO cells not expressing rVR1. Points
represent mean values of quadruplicate determinations in single, repre- relative to either the other secondary targets already char-
sentative experiments; error bars indicate S.E.M. All data were relative
acterized for capsazepine or other, as-yet-unidentified tar-
gets remains unknown.
At the conceptual level, a finding of our study is that
to baseline (cells incubated without ATP and antagonists) and normal-
ized by the percentage of control (without antagonists group). All exper-
iments were repeated at least two additional times with similar results.

Antagonists of Vanilloid Receptor
955
antagonism for different activators of VR1 is associated with on the ability of the bound compound to suppress the open
different structure activity requirements. Thus, whereas all channel conformation. It is, moreover, not surprising that
three compounds, JYL1421, KJM429, and capsazepine, an- different pH values may behave differently in response to
tagonized capsaicin-induced rVR1 activation, only JYL1421 antagonists. Careful molecular analysis of residues involved
was a complete antagonist at pH 6.0. Likewise, whereas both in proton regulation of VR1 suggests several residues with
JYL1421 and KJM429 antagonized at least partially at pH different effects: an extracellular Glu residue (E600) serves
6
.0, at pH 5.5, KJM429 acted as a partial agonist rather than as an important regulatory site for proton potentiation of
as an antagonist. This issue is important because it is not yet vanilloid receptor activity over a physiologically relevant
clear which class(es) of agonist is most relevant from the range (pH 6–8), whereas mutations at a second extracellular
perspective of therapeutic development. Endogenous ligands site (E648) significantly reduce proton activated currents
for VR1 include anandamide or lipoxygenase products without altering heat- or capsaicin-evoked responses or with-
(
Hwang et al., 2000; Smart and Jerman, 2000). Likewise, out eliminating the ability of protons to potentiate responses
tissue acidification associated with inflammation could rep- to these stimuli (Jordt et al., 2000).
resent a relevant activation mechanism (Olah et al., 2001;
Consistent with the finding that VR1 channel opening in
Voilley et al., 2001). Although antagonism of VR1 activation response to different agonists, namely capsaicin, heat, and
by capsaicin may be a convenient initial screening approach, pH, can be associated with different dependence on antago-
its evaluation against the range of activators for VR1 is nist structure is our previous observation that minor changes
needed for appropriate assessment of its utility.
in structure can shift the activity of a ligand from agonist to
In principle, the divergence in structure activity for differ- antagonist (Lee et al., 2001b). For example, the replacement
ent classes of activators is not unexpected. Antagonism of of benzoate with 3,4-dimethylbenzoate in the antagonist
capsaicin action will result from binding to the capsaicin- 2-benzyl-3-{[(6,7-dihydroxy-3,4-dihydro-2(1H)-isoquinolinyl)
binding site as long as the compound does not induce the carbothioyl]amino}propyl benzoate converts the compound
conformational change associated with channel opening. An- into an agonist (Lee et al., 2001b). Similarly, 2-iodo-RTX was
tagonism against temperature or pH, in contrast, will depend described as an agonist for human VR1 with 75% of the
Fig. 8. Effect of different antagonists on calcium mobilization evoked by 100 ␮M ␣, ␤-Me-ATP in control CHO cells, not expressing rVR1, and detected
using calcium imaging. Cells were pretreated with buffer or antagonist for 120 s before application of ␣,␤-methyleneadenosine 5Ј-diphosphate
(
␣,␤-Me-ATP) in the continued presence of the buffer or antagonist. Points represent the averaged signal from a minimum of 16 cells imaged
simultaneously. Each experiment was repeated at least an additional two times with similar results on independently cultured cells.

9
56
Wang et al.
quinoline thiourea analogues as vanilloid receptor ligands. Bioorg Med Chem
:1713–1720.
Lee J, Park SU, Kim JY, Kim JK, Lee J, Oh U, Marquez VE, Beheshti M, Wang QJ,
Modarres S, and Blumberg PM (1999) 3-Acyloxy-2-phenalkylpropyl amides and
esters of homovanillic acid as novel vanilloid receptor agonists. Bioorg Med Chem
Lett 9:2909–2914.
efficacy of capsaicin, whereas 5-iodo-RTX was an antagonist
McDonnell et al., 2002). In the present study, the difference
in behavior of JYL1421 compared with KJM429 reflects
merely the m-fluoro substitution on the phenyl ring of the
A-region.
RTX has provided an attractive tool for the assessment of
the therapeutic utility of C-fiber desensitization. Together
with 5-iodo-RTX, JYL1421 represents a step in the develop-
ment of potent, specific VR1 antagonists. Capsazepine has
been reported to inhibit induction of fos in the spinal cord in
response to peripheral inflammation (Kwak et al., 1998),
consistent with a role for endogenous activators of VR1 in
response to inflammation. The N-alkylglycyl trimers active
as VR1 channel blockers attenuated response of mice to nox-
ious heat, thermal hyperalgesia in response to mustard oil,
and neurogenic inflammation in response to capsaicin (Gar-
cia-Martinez et al., 2002). Compounds such as JYL1421 or
9
(
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