Diisothiocyanate derivatives as potent, insurmountable antagonists of P2Y6 nucleotide receptors

Article abstract of DOI:10.1016/j.bcp.2004.01.011

The physiological role of the P2Y₆ nucleotide receptor may involve cardiovascular, immune and digestive functions based on the receptor tissue distribution, and selective antagonists for this receptor are lacking. We have synthesized a series of symmetric aryl diisothiocyanate derivatives and examined their ability to inhibit phospholipase C (PLC) activity induced by activation of five subtypes of recombinant P2Y receptors. Several derivatives were more potent at inhibiting action of UDP at both human and rat P2Y ₆ receptors expressed in 1321N1 human astrocytes than activation of human P2Y₁, P2Y₂, P2Y₄ and P2Y₁₁ receptors. The inhibition by diisothiocyanate derivatives of 1,2-diphenylethane (MRS2567) and 1,4-di-(phenylthioureido) butane (MRS2578) was concentration-dependent and insurmountable, with IC₅₀ values of 126±15nM and 37±16nM (human) and 101±27nM and 98±11nM (rat), respectively. A derivative of 1,4-phenylendiisothiocyanate (MRS2575) inhibited only human but not rat P2Y₆ receptor activity. MRS2567 and MRS2578 at 10μM did not affect the UTP (100nM)-induced responses of cells expressing P2Y₂ and P2Y₄ receptors, nor did they affect the 2-methylthio-ADP (30nM)-induced responses at the P2Y₁ receptor or the ATP (10μM)-induced responses at the P2Y₁₁ receptor. Other antagonists displayed mixed selectivities. The selective antagonists MRS2567, MRS2575 and MRS2578 (1μM) completely blocked the protection by UDP of cells undergoing TNFα-induced apoptosis. Thus, we have identified potent, insurmountable antagonists of P2Y₆ receptors that are selective within the family of PLC-coupled P2Y receptors.

Full text of DOI:10.1016/j.bcp.2004.01.011

Biochemical Pharmacology 67 (2004) 1763–1770
Diisothiocyanate derivatives as potent, insurmountable
antagonists of P2Y6 nucleotide receptors
Liaman K. Mamedovaa, Bhalchandra V. Joshia, Zhan-Guo Gaoa,
b
Ivar von Kugelgen , Kenneth A. Jacobson
a,*
¨
aLaboratory of Bioorganic Chemistry, Molecular Recognition Section, National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, Bldg. 8A, Rm. B1A-19, Bethesda, MD 20892-0810, USA
bDepartment of Pharmacology, University of Bonn, D-53113 Bonn, Germany
Received 9 December 2003; accepted 20 January 2004
Abstract
The physiological role of the P2Y6 nucleotide receptor may involve cardiovascular, immune and digestive functions based on the
receptor tissue distribution, and selective antagonists for this receptor are lacking. We have synthesized a series of symmetric aryl
diisothiocyanate derivatives and examined their ability to inhibit phospholipase C (PLC) activity induced by activation of five subtypes of
recombinant P2Y receptors. Several derivatives were more potent at inhibiting action of UDP at both human and rat P2Y6 receptors
expressed in 1321N1 human astrocytes than activation of human P2Y1, P2Y2, P2Y4 and P2Y11 receptors. The inhibition by
diisothiocyanate derivatives of 1,2-diphenylethane (MRS2567) and 1,4-di-(phenylthioureido) butane (MRS2578) was concentration-
dependent and insurmountable, with IC50 values of 126 ꢀ 15 nM and 37 ꢀ 16 nM (human) and 101 ꢀ 27 nM and 98 ꢀ 11 nM (rat),
respectively. A derivative of 1,4-phenylendiisothiocyanate (MRS2575) inhibited only human but not rat P2Y6 receptor activity. MRS2567
and MRS2578 at 10 mM did not affect the UTP (100 nM)-induced responses of cells expressing P2Y2 and P2Y4 receptors, nor did they
affect the 2-methylthio-ADP (30 nM)-induced responses at the P2Y1 receptor or the ATP (10 mM)-induced responses at the P2Y11
receptor. Other antagonists displayed mixed selectivities. The selective antagonists MRS2567, MRS2575 and MRS2578 (1 mM)
completely blocked the protection by UDP of cells undergoing TNFa-induced apoptosis. Thus, we have identified potent, insurmountable
antagonists of P2Y6 receptors that are selective within the family of PLC-coupled P2Y receptors.
# 2004 Elsevier Inc. All rights reserved.
Keywords: P2Y6 nucleotide receptor; GPCR; Pyrimidines; Purines; Isothiocyanate; Apoptosis
1. Introduction
these eight receptors differs greatly and includes both
adenine and uracil nucleotides [4]. The pyrimidine-selec-
tive receptors include: P2Y4, P2Y6 and P2Y14 receptors,
which are activated by UTP, UDP and UDP-glucose,
respectively. P2Y2 receptors are activated by both ATP
and UTP. The remaining subtypes are activated by adenine
nucleotides.
The physiological role of the P2Y6 receptor may involve
cardiovascular, immune, and digestive functions, based on
the receptor tissue distribution [4–8]. P2Y6 receptors are
associated with vasoconstriction [9]. Recently, we have
reported that activation by UDP of P2Y6 receptors inhib-
ited apoptosis induced by TNFa through a mechanism
dependent on PKC and Erk [10]. Also, it was reported that
extracellular nucleotides stimulated contractions of human
cerebral arteries primarily by activation of the P2Y6
receptor [11], suggesting antagonists of the P2Y6 receptor
may be useful in the treatment of vasospasm.
In addition to their intracellular functions in signaling
and genetic coding, nucleotides have an important role as
extracellular signaling molecules. The P2 receptors consist
of two superfamilies: 8 G protein-coupled 7TM receptors
(P2Y) and 7 ligand-gated ion channels (P2X) [1–3]. The
P2Y1,2,4,6,11 receptors are coupled preferentially to the
activation of phospholipase C (PLC), via Gq/11 proteins.
The P2Y12,13,14 receptors are coupled preferentially to the
inhibition of adenylate cyclase. The agonist preference of
Abbreviations: CHO, Chinese hamster ovary; DIDS, 4,40-diisothiocya-
natostilbene-2,20-disulfonic acid disodium salt; MRS2279, 2-chloro-N6-
methyl-(N)-methanocarba-20-deoxyadenosine-30,50-bisphosphate; PIA,
N6-phenylisopropyladenosine; PLC, phospholipase C; TNFa, tumor
necrosis factor a; Tris, tris(hydroxymethyl)aminomethane
* Corresponding author. Tel.: þ1-301-496-9024; fax: þ1-301-480-8422.
E-mail address: kajacobs@helix.nih.gov (K.A. Jacobson).
0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2004.01.011

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L.K. Mamedova et al. / Biochemical Pharmacology 67 (2004) 1763–1770
SCN
SO3H
SCN
SO3H
SO3H
SO3H
NCS
NCS
2 H2-DIDS
1 DIDS
SCN
SO3H
SO3H
NHCOCH3
3 SITS
Fig. 1. Structures of diisothiocyanates tested previously as inhibitors of P2Y receptors.
Selective antagonists for the P2Y6 receptor have not
previously been reported [4]. The lack of selective receptor
probes for the P2Y6 and other subtypes of nucleotide
receptors has impeded the ability to examine their phar-
macology. DIDS (1) and its dihydro derivative, H2DIDS
(2) (Fig. 1) have been shown to inhibit the PLC activity
induced by UDP at rat P2Y6 receptors [12]. It appears that
both isothiocyanate groups are needed for this activity,
since SITS (3) was inactive. In this study we have
explored the structure–activity relationship (SAR) of aryl
diisothiocyanates at P2Y receptors. We tested these com-
pounds as antagonists of human P2Y1, P2Y2, P2Y4 and
P2Y6 receptors stably expressed in 1321N1 astrocytoma
cells and the human P2Y11 receptor stably expressed in
CHO cells.
isothiocyanatophenyl)ethane (5). 1H NMR (CDCl3): d 2.87
(s, 4H), 6.92–7.22 (m, 8H). FAB-MS 297.1 (M þ 1).
2.1.2. General procedure for the synthesis of 6–11
Either 1,3- or 1,4-phenylenediisothiocyanate (14 or 15,
5 mmol) [13] was dissolved in dry acetonitrile (20 ml). To
the above solution was added alkyl diamine (1 mmol) in
acetonitrile (10 ml), and the resulting reaction mixture was
stirred at room temperature for 1 h. Solvent was removed
by evaporation, and the residue was purified by silica gel
chromatography eluting with methanol–chloroform (5:95)
to furnish as a solid (yield 55–60%). 1,2-Di-[(4-isothio-
cyanatophenyl)-thioureido] ethane (6). 1H NMR (DMSO-
d6): d 3.61–3.78 (m, 4H), 7.51 (d, J ¼ 8:7 Hz, 4H), 7.56 (d,
J ¼ 7:2 Hz, 4H), 8.00 (brs, 2H), 9.76 (brs, 2H). FAB-MS
445.1 (M þ 1). 1,3-Di-[(4-isothiocyanatophenyl)-thiour-
eido] propane (7). 1H NMR (DMSO-d6): d 1.62–1.83
(m, 2H), 3.38–3.60 (m, 4H), 7.42 (d, J ¼ 12 Hz, 4H),
7.52 (d, J ¼ 12 Hz, 4H), 7.95 (brs, 2H), 9.65 (brs, 2H).
FAB-MS 459.1 (M þ 1). 1,4-Di-[(4-isothiocyanato phe-
nyl)-thioureido] butane (8). 1H NMR (DMSO-d6): d
1.51 (brs, 4H), 3.42 (brs, 4H), 7.20–7.58 (m, 8H), 7.85
(brs, 2H), 9.62 (brs, 2H). FAB-MS 473.1 (M þ 1).
2. Materials and methods
2.1. Chemical synthesis
2.1.1. General procedure for the synthesis of 4 and 5
A mixture of the diamine (12 or 13, 1.0 mmol), sodium
bicarbonate (4.0 mmol), water (10 ml) and chloroform
(30 ml) was stirred at room temperature. After 10 min
thiophosgene (4 mmol, Aldrich Chemical Co.) was added
and the mixture stirred for 2 h at room temperature. The
phases were separated, the organic layer was dried
(Na2SO4) and evaporated. The residue was purified by
silica gel chromatography eluting with ethyl acetate-petro-
leum ether (5:95) to furnish the product as a solid (yield 65–
70%). trans-1,2-Di-(4-isothiocyanatophenyl)ethylene (4).
1H NMR (CDCl3): d 7.14 (s, 2H), 7.34 (t, J ¼ 12 Hz, 4H),
7.58 (d, J ¼ 9 Hz, 4H). FAB-MS 295.1 (M þ 1), 1,2-di-(4-
1,2-Di-[(3-isothiocyanato phenyl)-thioureido] ethane (9).
1H NMR (DMSO-d6): d 3.71 (brs, 4H), 7.08–7.20 (m, 2H),
7.28–7.42 (brs, 4H), 7.61 (brs, 2H), 8.03 (brs, 2H), 9.72 (brs,
2H). FAB-MS 445.1 (M þ 1). 1,3-Di-[(3-isothiocyanato-
1
phenyl)-thioureido] propane (10). H NMR (DMSO-d6):
d 1.76–2.01 (m, 2H), 3.32 (brs, 4H), 6.95–7.45 (m, 6H),
7.55 (brs, 2H), 8.00 (brs, 2H), 9.65 (brs, 2H). FAB-MS 459.1
(M þ 1). 1,4-Di-[(3-isothiocyanato phenyl)-thioureido]
1
butane (11). H NMR (DMSO-d6): d 1.58 (m, 4H), 3.45
(brs, 4H), 7.04–7.20 (m, 2H), 7.25–7.40 (m, 4H), 7.65 (m,
2H), 7.95 (brs, 1H), 9.62 (brs, 2H). FAB-MS 473.1 (M þ 1).

L.K. Mamedova et al. / Biochemical Pharmacology 67 (2004) 1763–1770
1765
2.2. Cell culture and membrane preparation
30 min at 4 8C in a total assay volume of 200 ml. For
adenosine A1 receptor binding, an agonist radioligand
[3H]R-PIA (2.0 nM) was incubated with membranes
(40 mg protein/tube) from CHO cells stably expressing
human adenosine A1 receptors for 60 min at 25 8C [20].
Radiolabeled ligand concentrations used in all assays
approximated the Kd values of the receptor. Binding reac-
tions were terminated by filtration through Whatman GF/B
glass-fiber filters under reduced pressure with a MT-24 cell
harvester (Brandel), and radioactivity was determined with
a 1414 liquid scintillation counter (Wallac, Win Spectral,
Perkin Elmer Life Sciences).
Human 1321N1 astrocytoma cells stably transfected
with the hP2Y1–6 receptors and CHO cells stably trans-
fected with the human P2Y11 receptors [14,15] were grown
at 37 8C in a humidified incubator with 5% CO2/95% air in
Dulbecco’s modified Eagle’s medium (JRH Biosciences,
Inc.) supplemented with 10% fetal bovine serum (FBS),
100 Units/ml penicillin, 100 mg/ml streptomycin and
2 mM L-glutamine. The cells were grown to ꢁ60% con-
fluence for the experiments.
For membrane preparation, human astrocytoma cells
expressing human P2Y1 receptors were grown to approxi-
mately 80% confluence and then harvested. The cells were
homogenized and suspended and then centrifuged at
100 ꢂ g for 5 min at room temperature. The pellet was
resuspended in 50 mM tris(hydroxymethyl)aminomethane
(Tris) hydrochloride buffer (pH 7.4). The suspension was
homogenized with a polytron homogenizer (Brinkmann)
for 10 s and was then recentrifuged at 20,000 ꢂ g for
20 min at 4 8C. The resultant pellets were resuspended
in Tris buffer (pH 7.4), and the suspension was stored at
ꢃ80 8C until the binding experiments. The protein con-
centration was measured with the Bradford assay [16].
2.5. Induction of apoptosis
TNFa was used to induce apoptosis. Medium containing
5 ng/ml cycloheximide was added to the cells grown to
ꢁ60% confluence. Cycloheximide was included in all
experiments concerning TNFa-induced apoptosis. The cells
were treated with both UDP and TNFa for 4 h. Antagonists
(1 mM) were added to the incubation medium 20 min prior
to addition of UDP (100 nM) and TNFa (20 ng/ml). After
4 h the media was changed and was left for 16 h. The media
contained 5 ng/ml cycloheximide during the entire incuba-
tion. Cell death was observed 16 h later.
2.3. Determination of inositol phosphates
2.6. Degree of cell death
The quantity of inositol phosphates was measured by a
modification of the method of Kim et al. [17] and Gao et al.
[18]. Agonists were dissolved as stock solutions in Tris
buffer (pH 7.4), and antagonists were dissolved in DMSO
(5 mM) and stored at ꢃ20 8C. The antagonists were not
stable to storage in aqueous medium. The P2Y1–6-1321N1
and P2Y11-CHO cells were grown to confluence in 6-well
plates in the presence of myo-[3H]inositol (2 mCi/ml) for
24 h. Cells were then treated for 30 min at 37 8C with anta-
gonists or buffer in the presence of 20 mM LiCl, followed by
another 30 min incubation at 37 8C with the appropriate
agonist. Agonists used were: P2Y1, 2-MeSADP; hP2Y2,
UTP;hP2Y4, UTP;hP2Y6, UDP; hP2Y11, ATP. Thereaction
was terminated upon aspiration of the medium and addition
of cold formic acid (20 mM). After 30 min, supernatants
were neutralized with NH4OH, and applied to Bio-Rad
DowexAG1-X8anionexchangecolumns.Allofthecolumns
were then washed with water followed by a 60 mM sodium
formate solution containing 5 mM sodium tetraborate. Total
inositol phosphates were eluted with 1 M ammonium for-
mate containing 0.1 M formic acid, and radioactivity were
measured using a liquid scintillation counter.
After treatment, cells were washed once with PBS and
were stained with propidium iodide (PI) solution (final
concentration; 2 mg/ml). The numbers of unstained (live)
and stained (dead) cells were measured with a Cytoflour
4000 (Perseptive Biosystems) fluorescence plate reader,
with excitation at 485 nm and emission at 645 nm.
2.7. Reagents
myo-[3H]Inositol (20 Ci/mmol) was obtained from
American Radiolabeled Chemicals. Dowex AG 1-X8 resin
was purchased from Bio-Rad. DMEM and FBS were from
Life Technologies. [3H]MRS2279 was from Perkin-Elmer,
and [3H]R-PIA was from Amersham. All other reagents
were purchased from Sigma Chemical Co.
2.8. Statistical analysis
Pharmacological parameters were analyzed by Graph-
PAD Prism software. Data were expressed as mean ꢀ S:E.
2.4. Radioligand binding assays
3. Results
P2Y1 receptor binding experiments were performed as
previously described [19]. Briefly, membranes (40 mg pro-
tein) from astrocytoma cells stably expressing human P2Y1
receptors were incubated with [3H]MRS2279 (8 nM) for
3.1. Chemistry
Symmetric aryl diisothiocyanate derivatives (4–11,
Table 1) were synthesized as shown in Fig. 1. Diaryl-4,40-

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Table 1
Inhibition by aryl diisothiocyanate derivatives of the activation of PLC induced by P2Y receptor agonists
Compound (MRS number)
Position of NCS
Percent inhibitiona
SCN
R
hP2Y1
hP2Y2
hP2Y4
hP2Y6
<5
hP2Y11
NCS
R =
4 (2568)
5 (2567)
6 (2564)
7 (2575)
8 (2576)
9 (2570)
10 (2577)
11 (2578)
trans-CH=CH–
–CH2–CH2–
4
4
4
4
4
3
3
3
<5
<5
<5
<5
<5
<5
<5
95 ꢀ 7
<5
–NHCSNH(CH2)2NHCSNH–
–NHCSNH(CH2)3NHCSNH–
–NHCSNH(CH2)4NHCSNH–
–NHCSNH(CH2)2NHCSNH–
–NHCSNH(CH2)3NHCSNH–
–NHCSNH(CH2)4NHCSNH–
<5
<5
<5
<5
<5
<5
77 ꢀ 4
96 ꢀ 6
78 ꢀ 2
71 ꢀ 10
86 ꢀ 6
100 ꢀ 6
62 ꢀ 4
<5
90 ꢀ 5
80 ꢀ 4
<5
92 ꢀ 4
40
77 ꢀ 4
<5
<5
<5
<5
<5
98 ꢀ 2
<5
<5
<5
20 ꢀ 3
a Concentration of antagonists was 10 mM. Specific subtypes of human P2Y receptors were expressed in 1321N astrocytoma cells (hP2Y1, hP2Y2, hP2Y4,
and hP2Y6) or in CHO cells (hP2Y11) [13]. Agonists used were: P2Y1, 2-MeSADP (30 nM); hP2Y2, UTP (100 nM); hP2Y4, UTP (100 nM); hP2Y6, UDP
(300 nM); hP2Y11, ATP (10 mM). The concentrations of agonists used were roughly equivalent to their EC50 values. The potency (IC50 values) of MRS2567,
MRS2575 and MRS2578 at both human and rat P2Y6 receptors were listed in the text.
diisothiocyanates (4 and 5) were prepared in good yields
from their corresponding amines 12 and 13, respectively,
by a standard procedure using thiophosgene and sodium
bicarbonate [13]. The symmetric diisothiocyanates (6–11)
were synthesized in good yield by a different route, i.e.
from the interaction of alkyl diamines and large excess of
either 1,3- or 1,4-phenylenediisothiocyanates (Fig. 2).
induced responses of cells expressing human P2Y2 or P2Y4
receptors, nor did they affect the 2-MeSADP (30 nM)-
induced responses of cells expressing the P2Y1 receptor. In
addition, they did not affect the ATP (10 mM)-induced
responses of cells expressing the P2Y11 receptor. Thus,
MRS2567 and MRS2578 selectively blocked P2Y6 recep-
tor activity versus activity at P2Y1, P2Y2, P2Y4 or P2Y11
receptors. However, MRS2577 blocked activity at both
P2Y4 and P2Y6 receptors, without affecting other subtypes.
It was interesting that another derivative of 1,4-phenyl-
endiisothiocyanate (MRS2575) inhibited only human
P2Y6 receptor activity. This compound at 10 mM concen-
tration had no effect on rat P2Y6 or human P2Y1, P2Y2,
P2Y4 and P2Y11 receptors (Table 1). This antagonist
inhibited in an insurmountable manner UDP activity at
the P2Y6 receptor. Inhibition of human P2Y6 receptor
activity by MRS2575 occurred in a concentration-depen-
dent manner with an IC50 value of 155 ꢀ 49 nM (Fig. 5A
and B).
3.2. Inhibition of agonist-induced production of inositol
phosphates by aryl diisothiocyanate derivatives
The ability of the diisothiocyanate derivatives to inhibit
agonist-induced accumulation of inositol phosphates in
1321N1 human astrocytes expressing P2Y6 receptors
and other P2Y subtypes was studied. Fig. 3 indicates that
the concentration–response curve for UDP acting at the
P2Y6 receptor (EC50 230 ꢀ 83 nM) was inhibited by the
compounds 5 and 11, but in an insurmountable manner.
Table 1 indicates that similar effects were obtained for
other compounds in the series and at other subtypes of P2Y
receptors. Typically, the effect was inhibited with the
diisothiocyanate in a micromolar concentration range. Full
concentration–response curves for the inhibition of the
effects of UDP at P2Y6 receptors were measured for
compounds 5 (MRS2567) and 11 (MRS2578) (Fig. 4),
derivatives of 1,4- and 1,3-phenylendiisothiocyanate,
respectively. Results indicated that the production of ino-
sitol phosphates in human P2Y6-transfected 1321N1 astro-
cytoma cells in response to 300 nM UDP was inhibited in a
concentration-dependent manner with IC50 values of
126 ꢀ 15 nM and 37 ꢀ 16 nM, respectively. Also, these
two selective inhibitors of the hP2Y6 receptor activation
had approximately identical effects on rat P2Y6 receptors.
MRS2567 and MRS2578 inhibited the response to 300 nM
UDP at the rat P2Y6 receptor expressed in 1321N1 cells in
a concentration-dependent manner, with IC50 values of
101 ꢀ 27 nM and 98 ꢀ 11 nM, respectively. MRS2567 and
MRS2578 at 10 mM did not affect the UTP (100 nM)-
3.3. Aryl diisothiocyanate derivatives in receptor
binding experiments
Binding experiments at the human A1 AR expressed in
CHO cell membranes indicated that compounds MRS2567
(5), MRS2575 (7) and MRS2578 (11) at 10 mM concen-
tration displayed no significant inhibition of radioligand
binding (data not shown).
The diisothiocyanate derivatives MRS2576 (8) and
MRS2570 (9) inhibited agonist effects at hP2Y1 receptors
as well as at P2Y6 receptors, and consequently we examined
the ability to interferewith radioligand binding at the former
subtype. The high affinity antagonist [3H]MRS2279 was
used as a P2Y1 receptor radioligand in binding to mem-
branes from astrocytoma cells stably expressing human
P2Y1 receptors. Specific binding of [3H]MRS2279 was
defined in these experiments using the antagonist
MRS2179 (10 mM). Essentially no inhibition of binding

L.K. Mamedova et al. / Biochemical Pharmacology 67 (2004) 1763–1770
1767
Thiophosgene,
NaHCO3, CHCl3
H2N
( R )
NH2
12 R = -CH2-CH2-
13 R = -CH=CH-
(trans)
5 R = -CH2-CH2-
SCN
( R )
NCS
4 R = -CH=CH-(trans)
R1
H2N (CH2)n NH2, CH3CN
R2
NCS
14 R =
NCS
R2= H
NCS
1
15 R1= H
R2=
R1
NHCSNH (CH2)n
NHCSNH
R1
R2
R2
R = H
6 n = 2, R1= NCS
7 n = 3, R1= NCS
8 n = 4, R1= NCS
2
R = H
2
R = H
2
R1= H R2= NCS
9 n = 2,
R2= NCS
R1= H
10 n = 3,
R2= NCS
11 n = 4, R1= H
Fig. 2. Synthetic routes for the preparation of diisothiocyanate derivatives.
hP2Y6
Control
10000
7500
5000
2500
0
+MRS2567 [10
+MRS2578 [10
-11 -10
-9
-8
-7
-6
-5
-4
Log [UDP], M
Fig. 3. Inositol phosphate production in human P2Y6-transfected 1321N1 human astrocytes. After labeling with myo-[3H]inositol (1 mCi/106 cells) for 24 h,
the cells were treated for 30 min at 37 8C with antagonists in the presence of LiCl, followed by addition of the agonist, UDP, for another 30 min. The quantity
of inositol phosphates was analyzed after extraction though Dowex AG 1-X8 columns (see Section 2). Data shown are from the combined results of three
independent experiments in triplicate. The EC50 value is listed in Section 3.

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L.K. Mamedova et al. / Biochemical Pharmacology 67 (2004) 1763–1770
of [3H]MRS2279 was detected in the presence of 10 mM
hP2Y6
MRS2576 and MRS2570 (data not shown), both of
which antagonized the P2Y1 receptor-mediated effects of
2-MeSADP.
125
100
75
50
25
0
MRS2567
MRS2578
3.4. Inhibition of the anti-apoptotic effects of
a P2Y6 receptor agonist
The study of P2Y receptors has been a challenge, largely
due to the paucity of pharmacological tools for identifica-
tion of receptor subtypes. Since truly selective antagonists
for most of the subtypes have not yet been developed, the
characterization of P2Y receptors has mainly consisted of
monitoring agonist responses, such as activation of PLC.
One functional effect of P2Y6 nucleotide receptor activa-
tion is its cellular protective role. Activation of the rat P2Y6
receptor expressed in 1321N1 astrocytoma cells by UDP
has been shown to protect the cells from undergoing
apoptosis induced by TNFa [10,21]. The P2Y6 receptor
is a GPCR that is coupled to PLC, and the subsequent
activation of protein kinase C (PKC) by the receptor has
been shown to relate to this protective effect. We studied
-11
-10
-9
-8
-7
-6
-5
Log, M
Fig. 4. Concentration dependence of inhibition by compounds 5 and 11 of
inositol phosphate production induced by UDP (300 nM) in human P2Y6-
receptor transfected 1321N1 human astrocytes. After labeling with myo-
[3H]inositol (1 mCi/106 cells) for 24 h, the cells were treated for 30 min at
37 8C with the antagonist or buffer in the presence of LiCl, followed by
addition of agonist, UDP, for another 30 min. The quantity of inositol
phosphates was analyzed after extraction though Dowex AG 1-X8
columns (see Section 2). Data shown represent the mean ꢀ S:E:M: of
three independent experiments in duplicate. IC50 values are listed in
Section 3.
(A)
hP2Y6
Control
+ MRS2575 [10
7000
6000
5000
4000
3000
2000
1000
0
-11 -10
-9
-8
-7
-6
-5
-4
Log [UDP], M
(B)
hP2Y6
125
100
75
50
25
0
-10
-9
-8
-7
-6
-5
-4
Log [MRS2575]
Fig. 5. (A) Inositol phosphate production in human P2Y6-transfected 1321N1 human astrocytes. (B) Concentration dependence of inhibition by compound
MRS2575 (7) of inositol phosphate production induced by UDP (300 nM) in human P2Y6-receptor transfected 1321N1 human astrocytes. After labeling with
myo-[3H]inositol (1 mCi/106 cells) for 24 h, the cells were treated for 30 min at 37 8C with the antagonist or buffer in the presence of LiCl, followed by
addition of agonist, UDP, for another 30 min. The quantity of inositol phosphates was analyzed after extraction though Dowex AG 1-X8 columns (see Section 2).
Data shown represent the mean ꢀ S:E:M: of three independent experiments in triplicate.

L.K. Mamedova et al. / Biochemical Pharmacology 67 (2004) 1763–1770
1769
insurmountable. Previous studies have shown that DIDS
and H2DIDS block P2Y6 receptors at concentrations of 10–
100 mM and are clearly less potent than MRS2567,
MRS2578 and MRS2575 [12]. Compounds MRS2567
and MRS2578 can block UDP-stimulated activity at con-
centrations less than 1 mM (for human P2Y6 at IC50 values
126 ꢀ 15 nM and 37 ꢀ 16 nM, for rat P2Y6 at IC50 values
101 ꢀ 27 nM and 98 ꢀ 11 nM, respectively). Interestingly,
MRS2575 is a selective antagonist for human P2Y6 recep-
tors with an IC50 value of 155 ꢀ 49 nM, while it has no
effect on rat P2Y6 receptors. Since other compounds in this
series inhibited other P2Y receptor subtypes, a panel of
such diisothiocyanates may be useful in characterizing a
given pharmacological response to extracellular nucleo-
tides. For example, the following mixed selectivities were
observed: MRS 2564 (P2Y6, P2Y11), MRS 2576 (P2Y1,
P2Y2, P2Y4, P2Y6), and MRS 2577 (P2Y4, P2Y6).
It is likely that the isothiocyanate groups are involved in
the antagonistic effects at P2Y6 receptors. All of the
derivatives 4–11 evaluated as P2Y receptor antagonists
included two isothiocyanate groups. Isothiocyanate groups
are potent electrophiles that are sufficiently stable in
aqueous medium for biochemical experiments, yet may
be chemically reactive towards certain nucleophilic groups
on biopolymers [22], such as Lys and Cys side chains. The
possible covalent reaction of these antagonists with the
P2Y6 receptor protein has not been demonstrated. Future
studies will be needed to establish the location(s) of cross-
linking, if any, with the receptor. The degree of inhibition
and specificity of the antagonists depended on the chain
length and position of ring substitution. Thus, these inhi-
bitory effects follow a specific SAR pattern and are not the
result of general reactivity of the electrophilic group. The
selectivity of these derivatives was further indicated by
their inability to block binding at the human A1AR, which
is know to be sensitive to other electrophilic reagents [13].
The inability of these aryl diisothiocyanate derivatives to
produce a parallel rightward shift of the concentration–
response curves suggests that they are insurmountable
P2YR antagonists [23]. Moreover, the failure of selected
derivatives to displace radioligand binding at the human
P2Y1R suggests that they are non-competitive antagonists
at the P2Y1 subtype. Since there is no radioligand for
P2Y6 receptors, it was not possible to carry out similar
binding experiments at this subtype. An additional func-
tional experiment using the antagonists has supported the
finding [10,21] that activation of the human P2Y6 receptor
by the agonist UDP prevented TNFa-induced apoptosis,
since selective antagonists MRS2575, MRS2567, and
MRS2578 at a concentration of 1 mM completely blocked
agonist-induced activation of the P2Y6 receptor.
Fig. 6. Effects of P2Y6 receptor antagonists on cell death induced by the
treatment of hP2Y6 receptor-expressing 1321N1 astrocytoma cells with
TNFa. Cells were treated with both UDP and TNFa for 4 h. Antagonists
(1 mM) were added to the incubation medium 20 min prior to addition of
UDP (100 nM) and TNFa (20 ng/ml). The media contained 5 ng/ml
cycloheximide during the entire incubation. After 16 h of continuous
treatment (as described in Section 2) the cell culture medium was replaced
by medium containing PI (2 mg/ml). Since PI transverses only leaky or
lysed cell membranes, DNA of dead cells can be stained and fluorescence
emission measured, while living cells will not be stained. Fluorescence
was measured using the Cytofluor 4000 microplate reader (excitation
485 nm/emission 645 nm), giving a direct number of dead cells. Results
represent the mean value of two experiments, each performed in triplicate.
the present set of P2Y6 receptor antagonists in a model of
the anti-apoptotic effects of P2 receptor agonists.
We have extended previous results with the rat P2Y6
receptor [10,21] to the human P2Y6 receptor. The activa-
tion of the human P2Y6 receptor by UDP (100 nM) in the
presence of TNFa (20 ng/ml) and cycloheximide (5 ng/
ml), which sensitizes the TNFa response, significantly
reduced cell death (Fig. 6). Four antagonists of P2Y6
receptors were used in the experiment, including the
selective aryl diisothiocyanate derivatives MRS2567 (5),
MRS2575 (7) and MRS2578 (11) and the non-selective
derivative MRS2564 (6) (1 mM). Measurements of intra-
cellular PI staining, a measure of cell death, indicated
that MRS2567, MRS2575 and MRS2578 completely
blocked the protection by UDP (100 nM). Therefore, it
was demonstrated using antagonists that P2Y6 receptor
activation by UDP played a crucial role in preventing
1321N1 astrocytoma cells from undergoing TNFa-induced
apoptosis.
4. Discussion
The present study demonstrated that MRS2567 and
MRS2578 block agonist effects at both human and rat
P2Y6 receptors, and MRS2575 selectively blocks effects
at human P2Y6 but not rat P2Y6 receptors. This is the first
report of selective antagonists for P2Y6 receptors, albeit
In summary, we have identified potent, insurmountable
antagonists of P2Y6 receptors that are selective within the
family of PLC-coupled P2Y receptors. These selective
antagonists could be used as pharmacological tools for
defining the role of P2Y6 and other P2Y receptors.

1770
L.K. Mamedova et al. / Biochemical Pharmacology 67 (2004) 1763–1770
[11] Nicholas RA, Watt WC, Lazarowski ER, Li Q, Harden K. Uridine
Acknowledgments
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ceptors: identification of a UDP-selective, a UTP-selective and an
ATP- and UTP-specific receptor. Mol Pharmacol 1996;50:224–9.
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diisothiocyanatostilbene-disulfonic acid (DIDS) and analogues. Nau-
nyn-Schmiedeberg’s Arch Pharmacol 2001;363:R34.
L.K. Mamedova and B.V. Joshi thank Gilead Sciences
(Foster City, CA) for financial support. We thank Prof.
Robert Nicholas and Prof. T. Kendall Harden of the
University of North Carolina (Chapel Hill, NC, USA)
for providing the P2Y6-receptor-transfected cells. We
thank Dr. Neli Melman (NIDDK) for technical assistance.
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