Structure activity and molecular modeling analyses of ribose- and base-modified uridine 5′-triphosphate analogues at the human P2Y 2 and P2Y4 receptors

Article abstract of DOI:10.1016/j.bcp.2005.11.010

With the long-term goal of developing receptor subtype-selective high affinity agonists for the uracil nucleotide-activated P2Y receptors we have carried out a series of structure activity and molecular modeling studies of the human P2Y₂ and P2Y₄ receptors. UTP analogues with substitutions in the 2′-position of the ribose moiety retained capacity to activate both P2Y₂ and P2Y₄ receptors. Certain of these analogues were equieffective for activation of both receptors whereas 2′-amino-2′-deoxy-UTP exhibited higher potency for the P2Y ₂ receptor and 2′-azido-UTP exhibited higher potency for the P2Y₄ receptor. 4-Thio substitution of the uracil base resulted in a UTP analogue with increased potency relative to UTP for activation of both the P2Y₂ and P2Y₄ receptors. In contrast, 2-thio substitution and halo- or alkyl substitution in the 5-position of the uracil base resulted in molecules that were 3-30-fold more potent at the P2Y₂ receptor than P2Y₄ receptor. 6-Aza-UTP was a P2Y₂ receptor agonist that exhibited no activity at the P2Y₄ receptor. Stereoisomers of UTPαS and 2′-deoxy-UTPαS were more potent at the P2Y ₂ than P2Y₄ receptor, and the R-configuration was favored at both receptors. Molecular docking studies revealed that the binding mode of UTP is similar for both the P2Y₂ and P2Y₄ receptor binding pockets with the most prominent dissimilarities of the two receptors located in the second transmembrane domain (V90 in the P2Y₂ receptor and I92 in the P2Y₄ receptor) and the second extracellular loop (T182 in the P2Y₂ receptor and L184 in the P2Y₄ receptor). In summary, this work reveals substitutions in UTP that differentially affect agonist activity at P2Y₂ versus P2Y₄ receptors and in combination with molecular modeling studies should lead to chemical synthesis of new receptor subtype-selective drugs.

Full text of DOI:10.1016/j.bcp.2005.11.010

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"
Structure activity and molecular modeling analyses of
ribose- and base-modified uridine 50-triphosphate
analogues at the human P2Y2 and P2Y4 receptors
b
a
a
Kenneth A. Jacobson a, , Stefano Costanzi , Andrei A. Ivanov , Susanna Tchilibon ,
*
Pedro Besada a, Zhan-Guo Gao a, Savitri Maddileti c, T. Kendall Harden c
a Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
b Computational Chemistry Core Laboratory, National Institute of Diabetes and Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
c Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
With the long-term goal of developing receptor subtype-selective high affinity agonists for
the uracil nucleotide-activated P2Y receptors we have carried out a series of structure
activity and molecular modeling studies of the human P2Y2 and P2Y4 receptors. UTP
analogues with substitutions in the 20-position of the ribose moiety retained capacity to
activate both P2Y2 and P2Y4 receptors. Certain of these analogues were equieffective for
activation of both receptors whereas 20-amino-20-deoxy-UTP exhibited higher potency for
the P2Y2 receptor and 20-azido-UTP exhibited higher potency for the P2Y4 receptor. 4-Thio
substitution of the uracil base resulted in a UTP analogue with increased potency relative to
UTP for activation of both the P2Y2 and P2Y4 receptors. In contrast, 2-thio substitution and
halo- or alkyl substitution in the 5-position of the uracil base resulted in molecules that were
3–30-fold more potent at the P2Y2 receptor than P2Y4 receptor. 6-Aza-UTP was a P2Y2
receptor agonist that exhibited no activity at the P2Y4 receptor. Stereoisomers of UTPaS and
20-deoxy-UTPaS were more potent at the P2Y2 than P2Y4 receptor, and the R-configuration
was favored at both receptors. Molecular docking studies revealed that the binding mode of
UTP is similar for both the P2Y2 and P2Y4 receptor binding pockets with the most prominent
dissimilarities of the two receptors located in the second transmembrane domain (V90 in
the P2Y2 receptor and I92 in the P2Y4 receptor) and the second extracellular loop (T182 in the
P2Y2 receptor and L184 in the P2Y4 receptor). In summary, this work reveals substitutions in
UTP that differentially affect agonist activity at P2Y2 versus P2Y4 receptors and in combina-
tion with molecular modeling studies should lead to chemical synthesis of new receptor
subtype-selective drugs.
Received 26 September 2005
Accepted 4 November 2005
Keywords:
Structure activity relationship
G protein-coupled receptor
Nucleotides
Phospholipase C
Pyrimidines
Homology modeling
Abbreviations:
PLC, phospholipase C
HEPES, N-(2-hydroxyethyl)-
piperazine-N0-2-ethanesulfonic acid
ATP, adenosine 50-triphosphate
CTP, cytidine 50-triphosphate
GTP, guanosine 50-triphosphate
TM, transmembrane domain
TBAP, tetrabutylammonium
dihydrogen phosphate
# 2005 Elsevier Inc. All rights reserved.
TEAA, triethylammonium acetate
UDP, uridine 50-diphosphate
UTP, uridine 50-triphosphate
* Corresponding author. Tel.: +1 301 496 9024; fax: +1 301 480 8422.
E-mail address: kajacobs@helix.nih.gov (K.A. Jacobson).
0006-2952/$ – see front matter # 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2005.11.010

b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 5 4 0 – 5 4 9
541
1.
Introduction
homology models of the P2Y2 and P2Y4 receptors with the goal
of extending insight gained from the empirical structure
activity studies to predict optimized structures that will be
pursued by application of new molecular syntheses. The
combination of these structure activity analyses and mole-
cular modeling studies provides resolution of key differences
in the ligand binding pockets of the P2Y2 versus P2Y4 receptor
that will be of heuristic importance for future ligand
development.
Pharmacological effects of UTP (uridine 50-triphosphate) and
other uracil nucleotides on second messenger signaling
pathways and on various tissue responses provided the initial
indications of the existence of cell surface receptors that
specifically recognize extracellular pyrimidines [1,2]. This
concept was confirmed and extended over the past decade
with the cloning of three different G protein-coupled recep-
tors, the P2Y2, P2Y4 and P2Y6 receptors [3–6] that are activated
by uracil nucleotides, and by the direct demonstration of
regulated release of UTP from a variety of cell types [7,8].
The P2Y2 receptor is activated equipotently by both UTP
and ATP (adenosine 50-triphosphate) and is distributed in a
broad range of tissues. For example, this receptor plays
important physiological roles in epithelial cells of the lung,
gastrointestinal tract, eye, and other tissues [9,10]. The human
P2Y4 receptor is selectively activated by UTP, and ATP is a
potent competitive antagonist at this receptor [11]. However,
the P2Y4 receptor of several other species is activated by both
UTP and ATP [11–13], and therefore, it has proven difficult to
differentiate the P2Y4 receptor from the P2Y2 receptor on the
basis of its cognate agonists in, for example, rat and mouse
tissues. The P2Y6 receptor is selectively activated by UDP
(uridine 50-diphosphate), and UTP is a weak agonist or inactive
at this receptor [6,14].
2.
Materials and methods
2.1.
Reagents
20-Ara-fluoro-20-deoxyuridine (25) was purchased from R.I.
Chemical, Inc. (Orange, CA). All other reagents and solvents
were purchased from Sigma–Aldrich (St. Louis, MO).
2.2.
Uracil nucleotide analogues
Most of the nucleotide analogues studied (compounds 5–10,
12–17, and 20) were purchased from TriLink Biotechnologies
(San Diego, CA). Compound 19 was custom synthesized by
TriLink Biotechnologies and was the gift of Dr. Victor Marquez,
NCI, Frederick, MD. Compounds 1–4 and 3-methyluridine (26)
were purchased from Sigma (St. Louis, MO). Compounds 21–24
were manufactured by Axxora (San Diego, CA)/Biolog Life
Science Inst. (Bremen, Germany).
The existence of three different G protein-coupled recep-
tors that recognize uracil nucleotides has made difficult the
pharmacological characterization or selective activation of
these receptors in native tissues. As has proved to be the case
with the P2Y receptors, i.e. P2Y1, P2Y11, P2Y12, and P2Y13
receptors, that are activated by adenine nucleotides, the
metabolism and interconversion of extracellular nucleotides
add complexities to the study of uracil nucleotide-activated
receptors in native tissues [15]. For example, the ectonucleo-
side triphosphate diphosphohydrolase, NTPDase2, converts
extracellular UTP to UDP [16], whereas ectonucleoside dipho-
sphokinase forms UTP from UDP with the transfer of the
g-phosphate from ATP [17].
2.3.
Chemical synthesis
2.3.1. Chemical methods
Methods used to prepare compounds 11 and 18 are depicted
schematically in Fig. 1. 1H NMR spectra were obtained with a
Varian Gemini 300 spectrometer using D2O as a solvent. The
chemical shifts are expressed as relative ppm from HOD
(4.78 ppm). 31P NMR spectra were recorded at room tempera-
ture by use of Varian XL 300 spectrometer (121.42 MHz);
orthophosphoric acid (85%) was used as an external standard.
Purity of compounds was checked using a Hewlett-Packard
1100 HPLC equipped with a Luna 5m RP-C18(2) analytical
column (250 mm ꢀ 4.6 mm; Phenomenex, Torrance, CA).
System A—linear gradient solvent system: 5 mM TBAP-CH3CN
from 80:20 to 40:60 in 20 min, then isocratic for 2 min; the flow
rate was 1 mL/min. System B—linear gradient solvent system:
10 mM TEAA-CH3CN from 100:0 to 90:10 in 20 min, then
isocratic for 2 min; the flow rate was 1 mL/min. Peaks were
detected by UV absorption with a diode array detector. All
derivatives tested for biological activity showed >99% purity in
the HPLC systems. High-resolution mass measurements were
performed on Micromass/Waters LCT Premier Electrospray
Time of Flight (TOF) mass spectrometer coupled with a Waters
HPLC system. Purification of the nucleotide analogues for
biological testing was carried out on (diethylamino)ethyl
(DEAE)-A25 Sephadex columns with a linear gradient (0.01–
0.5 M) of 0.5 M ammonium bicarbonate as the mobile phase.
Compounds 27 and 28 were additionally purified by HPLC
using system C (10 mM TEAA-CH3CN from 100:0 to 90:10 in
30 min, then isocratic for 2 min; the flow rate was 2 mL/min)
Drugs that selectively activate or block the uracil nucleo-
tide-activated P2Y receptors would provide armamentaria for
circumvention of some of the problems inherent in the study
of these physiologically important signaling proteins. How-
ever, receptor subtype-selective agonists or antagonists are
not available for the uracil nucleotide-activated P2Y receptors
[18]. Therefore, we have undertaken a series of pharmacolo-
gical studies designed to systematically evaluate the effects of
various modifications of the UTP structure on the capacity of
analogues to activate the UTP-activated P2Y2 and P2Y4
receptors. Since UTP activates the human P2Y2 and P2Y4
receptors with similar potencies, our first goal is to identify
substitutions in UTP analogues that differentially affect
activity at either of these receptors. Conversely, identification
of partial agonists at these receptors would open a path to
synthesis of selective high affinity antagonists of the UTP-
activated P2Y receptors in a manner similar to the approach
we have followed to identify
a subnanomolar affinity
antagonist for the ADP-activated P2Y1 receptor [19–21]. The
results reveal encouraging progress in the first of these goals.
We also conducted docking studies with rhodopsin-based

542
b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 5 4 0 – 5 4 9
Fig. 1 – Synthetic methods for the preparation of compounds 11 and 18.
with a Luna 5m RP-C18(2) semipreparative column (250 mm ꢀ
(1 mg, 1%). 1H NMR (D2O) d 8.01 (d, J = 7.8 Hz, 1H), 6.05 (d,
J = 8.1 Hz, 1H), 6.01 (d, J = 3.6 Hz, 1H), 4.41 (m, 2H), 4.27 (m, 3H),
3.31 (s, 3H); 31P NMR (D2O) d ꢁ8.68, ꢁ11.32 (d, J = 21.4 Hz); HRMS
m/z found 417.0116 (M ꢁ H+)ꢁ. C10H15N2O12P2 requires
417.0100; HPLC (System A) 14.3 min (99%), (System B)
12.4 min (99%). (20R,30R,40S,50R)-1-(30,40-dihydroxy-50-tripho-
sphoryloxymethyl-tetrahydro-furan-20-yl)-3-methyl-1H-pyri-
midine-2,4-dione, ammonium salt (18) was obtained as a
white solid (8 mg, 15%). 1H NMR (D2O) d 7.98 (d, J = 8.1 Hz, 1H),
6.02 (m, 2H), 4.44 (m, 1H), 4.40 (m, 1H), 4.28 (m, 3H), 3.29 (s, 3H);
31P NMR (D2O) d ꢁ6.64 (d, J = 20.2 Hz), ꢁ10.88 (d, J = 19.5 Hz),
ꢁ21.91 (t, J = 20.2 Hz); HRMS m/z found 496.9750 (M ꢁ H+)ꢁ.
C10H16N2O15P3 requires 496.9764; HPLC (System A) 18.3 min
(99%), (System B) 13.5 min (99%).
10.0 mm; Phenomenex, Torrance, CA).
2.3.2. General procedure for preparation of nucleoside 50-
triphosphate (compound 18)
3-Methyluridine (26) (25 mg, 0.10 mmol) and Proton Sponge
(31 mg, 0.15 mmol) were dried for several hours in high
vacuum and then dissolved in trimethyl phosphate (1 mL).
After cooling the solution at 0 8C, phosphorous oxychloride
(0.02 mL, 0.19 mmol) was added. The mixture reaction was
stirred at 0 8C for 2 h.
A solution of tributylammonium
pyrophosphate (303 mg, 0.64 mmol) and tributylamine
(0.09 mL, 0.39 mmol) in DMF (1 mL) was added and stirring
was continued at 0 8C for additional 20 min. Five milliliters of
0.2 M triethylammonium bicarbonate (TEAB) solution was
added, and the reaction mixture was stirred at room
temperature for 45 min. Analysis of the reaction mixture by
analytical HPLC (System A) indicated the formation of three
compounds, the corresponding nucleotide mono-, di-, and
triphosphates. The mixture was subsequently frozen and
lyophilized, and the residue was purified by Sephadex-DEAE
A-25 resin ion-exchange column chromatography and when
was necessary (compounds 27 and 28) by semipreparative
HPLC as described above. The corresponding nucleotide
mono-, di-, and triphosphates were collected, frozen, and
lyophilized as the triethylammonium or ammonium salts.
(20R,30R,40S,50R)-1-(30,40-dihydroxy-50-phosphoryloxymethyl-
tetrahydro-furan-20-yl)-3-methyl-1H-pyrimidine-2,4-dione,
triethylammonium salt (27) was obtained as a white solid
(6 mg, 11%). 1H NMR (D2O) d 8.06 (d, J = 8.4 Hz, 1H), 6.04 (d,
J = 8.1 Hz, 1H), 6.02 (d, J = 4.2 Hz, 1H), 4.37 (m, 2H), 4.28 (m, 1H),
4.07 (m, 2H), 3.30 (s, 3H); 31P NMR (D2O) d 1.88; HRMS m/z found
337.0404 (M ꢁ H+)ꢁ. C10H14N2O9P requires 337.0437; HPLC
(System A) 7.6 min (99%), (System B) 11.5 min (99%).
(20R,30R,40S,50R)-1-(30,40-dihydroxy-50-diphosphoryloxymethyl-
tetrahydro-furan-20-yl)-3-methyl-1H-pyrimidine-2,4-dione,
triethylammonium salt (28) was obtained as a white solid
(20R,30S,40S,50R)-1-(30-Fluoro-40-hydroxy-50-triphosphorylox-
ymethyl-tetrahydro-furan-20-yl)-1H-pyrimidine-2,4-dione,
ammonium salt (11). Compound 11 (29 mg, 26%) was obtained
as
a
white solid from 20-ara-fluoro-20-deoxyuridine (25)
following the general procedure for 18. 1H NMR (D2O)
d 7.93 (d, J = 8.1 Hz, 1H), 6.34 (d, J = 15.6 Hz, 1H), 5.94 (d,
J = 8.1 Hz, 1H), 5.24 (d, J = 51.6 Hz, 1H), 4.60 (d, J = 19.5 Hz, 1H),
4.25 (m, 3H); 31P NMR (D2O) d ꢁ9.86, ꢁ11.46 (d, J = 19.6 Hz),
ꢁ23.05; HRMS m/z found 484.9576 (M ꢁ H+)ꢁ. C9H13N2O14FP3
requires 484.9564; HPLC (System A) 19.0 min (99%), (System B)
11.6 min (99%).
2.4.
Assay of P2Y2 and P2Y4 receptor-stimulated
phospholipase C activity
Stable cell lines expressing the human P2Y2 receptor or the
human P2Y4 receptor in 1321N1 human astrocytoma cells
were generated as previously described in detail [14]. Agonist-
induced [3H]inositol phosphate production was measured in
1321N1 cells grown to confluence on 48-well plates. Twelve
hours before the assay, the inositol lipid pool of the cells was
radiolabeled by incubation in 200 mL of serum-free inositol-
free Dulbecco’s modified Eagle’s medium, containing 0.4 mCi

b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 5 4 0 – 5 4 9
543
of myo-[3H]inositol. No changes of medium were made
subsequent to the addition of [3H]inositol. On the day of the
assay, cells were challenged with 50 mL of the five-fold
concentrated solution of receptor agonists in 200 mM Hepes,
pH 7.3, containing 50 mM LiCl for 20 min at 37 8C. Incubations
were terminated by aspiration of the drug-containing medium
and addition of 450 mL of ice-cold 50 mM formic acid. After
15 min at 4 8C, samples were neutralized with 150 mL of
150 mM NH4OH. [3H]Inositol phosphates were isolated by ion
exchange chromatography on Dowex AG 1-X8 columns as
previously described [22].
were subjected to Monte Carlo driven rotations and transla-
tions during the conformational search. The search was
˚
performed on the ligand and the residues located within 5 A of
the ligand, while the remaining residues were conformation-
ally frozen. The calculations were conducted with the MMFFs
force field [26], using water as implicit solvent (GB/SA model
[27] as implemented in MacroModel [23]) and a molecular
dielectric constant of 1. The Polak-Ribier Conjugate Gradient
was used for the energy minimizations with a convergence
˚
threshold of 0.05 kJ/mol/A.
2.5.
Data analyses
3.
Results
Agonist potencies (EC50 values) were obtained from concen-
tration–response curves by non-linear regression analysis
using the GraphPad software package Prism (GraphPad, San
Diego, CA). All experiments were performed in triplicate
assays and repeated at least three times. The results are
presented as mean ꢂ S.E.M. from multiple experiments or in
3.1.
Chemical synthesis
Synthetic methods for the preparation of nucleoside 50-
triphosphate derivatives 11 and 18 from the nucleosides of
25 and 26, respectively, are shown in Fig. 1. The classical
phosphorous oxychloride method was used for the synthesis
of both derivatives [28,29]. The 50-mono (27) and diphosphate
(28) derivatives of 26 were also isolated as side products.
the case of concentration effect curves from
a single
experiment carried out with triplicate assays that were
representative of results from multiple experiments.
3.2.
Pharmacological assays and structure activity
2.6.
Reconstruction of the binding pocket model
relationships
The reconstruction of the P2Y2 and P2Y4 receptors around UTP
was performed with the InducedFit module of the Prime 1.2
The human P2Y2 and P2Y4 receptors were stably expressed in
1321N1 human astrocytoma cells using retroviral vectors as
previously described [14]. Agonist-promoted activation of
phospholipase C (PLC) was assessed in these two stable cell
lines by quantification of the accumulation of [3H]inositol
phosphates as described in Section 2. UTP markedly stimu-
lated [3H]inositol phosphate accumulation to similar levels in
P2Y2 versus P2Y4 receptor-expressing cells. Similar EC50 values
also were observed for UTP (1) for activation of the two
receptors (Fig. 2 and Table 1). ATP (2) was two-fold less potent
than UTP at the human P2Y2 receptor, and CTP (3a) and GTP
(3b) were only weakly active at this subtype. Both nucleotides
were previously shown to be antagonists at the human P2Y4
receptor [11].
homology modeling program (Schrodinger, LLC). The calcula-
¨
˚
tion was restricted to the residues located within 5 A of the
ligand.
2.7.
Conformational analysis of UTP inside the putative
binding pocket
A conformational analysis of UTP in the binding pockets of the
P2Y2 and P2Y4 receptors was performed by means of the mixed
MCMM/LMCS sampling method as implemented in Macro-
Model 9.0 [23] (Schrodinger, LLC). The approach combines the
¨
Monte Carlo Multiple Minimum (MCMM) [24] and the low-
mode conformational search (LMCS) [25]. All of the rotatable
bonds of the ligand, as well as the ligand itself as a single body,
Given the similar activities of UTP at the P2Y2 versus P2Y4
receptor, we compared the activities of a series of ribose-
Fig. 2 – Activation of the human P2Y2 (left panel), and P2Y4 (right panel) receptors by ribose-modified uracil 20-
deoxynucleotide analogues containing 20-amino (7) (!) and 20-azido (8) (~) functionality. PLC activity was measured as
described in Section 3.2 in 1321N1 human astrocytoma cells stably expressing the human P2Y receptors. The data are the
means of triplicate determinations and are representative of results obtained in at least three separate experiments with
each analog.

544
b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 5 4 0 – 5 4 9
Table 1 – In vitro pharmacological data for UTP (1) and its analogues in the stimulation of PLC at recombinant human P2Y2
and P2Y4 receptors expressed in astrocytoma cells
Compound
Modification
Structure
EC50 at hP2Y2
Relative
to UTP
EC50 at hP2Y4
Relative
to UTP
receptor (mM)a
receptor (mM)a
Native ribonucleoside-50-triphosphates
1 UTP
2 ATP
0.049 ꢂ 0.012
0.085 ꢂ 0.012
5.63 ꢂ 0.30
1
1.7
110
54
0.073 ꢂ 0.02
Antagonistb
Antagonistb
6.59b
1
3a CTP
3b GTP
2.64 ꢂ 0.30
90
Ribose-modified UTP analogues
4
5
20-Deoxy
RII = H
1.08 ꢂ 0.28
14.3 ꢂ 7.7
NE
22
290
>1000
1.3
1.9 ꢂ 0.5
8.2 ꢂ 2.1
NE
26
110
>1000
16
20-Deoxy-20-methoxy
30-Deoxy-30-methoxy
20-Amino-20-deoxy
20-Azido-20-deoxy
20-Deoxy-20-fluoro
Arabino
RII = OCH3
RIII = OCH3
RII = NH2
6
7
0.062 ꢂ 0.008
5.0 ꢂ 2.1
1.2 ꢂ 0.3
1.1 ꢂ 0.1
0.54 ꢂ 0.14
0.71 ꢂ 0.08
0.52 ꢂ 0.08
8
RII = N3
RII = F
RII = H, RARA = OH
RII = H, RARA = F
100
16
15
9
0.78 ꢂ 0.11
0.087 ꢂ 0.010
0.52 ꢂ 0.15
7.4
10
11
1.8
9.7
20-Deoxy-arabino-20-fluoro-
11
7.1
Uracil-modified UTP analogues
12
13
14
15
16
17
18
19
20
5-Bromo
R5 = Br
R5 = I
R5 = CH3
R2 = S
R4 = S
0.75 ꢂ 0.1
0.83 ꢂ 0.1
0.48 ꢂ 0.1
0.035 ꢂ 0.004
0.026 ꢂ 0.01
8.6 ꢂ 3.7
15
17
2.1 ꢂ 0.7
4.0 ꢂ 1.7
3.9 ꢂ 1.6
0.35 ꢂ 0.01
0.023 ꢂ 0.005
NE
29
55
5-Iodo
5-Methyl
2-Thio
9.8
0.71
0.53
180
24
53
4.8
4-Thio
0.32
>1000
47
6-Aza
X = N
R3 = CH3
3-Methyl
Zebularine analogue
Pseudouridine
1.20 ꢂ 0.20
8.9 ꢂ 0.5
3.4 ꢂ 0.8
NE
R
c
4 = H, H
180
16
>1000
41
0.78 ꢂ 0.5
3.0 ꢂ 0.3
Phosphate-modified UTP analogues
21
22
23
24
Rp-a-thio
RR = S
5.4 ꢂ 1.5
14 ꢂ 5.5
12.5 ꢂ 6.7
NE
110
290
27 ꢂ 5
81 ꢂ 8
NE
370
1100
Sp-a-thio
RS = S
20-Deoxy-Rp-a-thio-triphosphate
20-Deoxy-Sp-a-thio triphosphate
RR = S, RII = H
RS = S RII = H
260
>1000
>1000
>1000
NE
NE: no effect at 10 mM.
a
Agonist potencies were calculated using a four-parameter logistic equation and the GraphPad software package (GraphPad, San Diego, CA). EC50 values
(mean ꢂ S.E.) represent the concentration at which 50% of the maximal effect is achieved. EC50 is also expressed relative to the value for UTP at each
receptor. Relative efficacies (%) were determined by comparison with the effect produced by a maximal effective concentration of reference agonist
(UTP) in the same experiment. For all of the nucleotides, except 22 and 23, for which an EC50 is reported in this table, ꢃ100% efficacy was achieved.
ATP antagonized the human P2Y4 receptor with a KB of 0.708 mM. CTP (100 mM) inhibited the response to an EC50 concentration of UTP by ꢃ40% [11].
Compound 20 has the following structure:
b
c
.

b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 5 4 0 – 5 4 9
545
Fig. 3 – Activation of the human P2Y2 (left panel), and P2Y4 (right panel) receptors by UTP (1) and its base-modified uracil
nucleotide analogues having 5-methyl (14), 2-thio (15), and 4-thio (16) substitution. PLC activity was measured as described
in Section 3.2 in 1321N1 human astrocytoma cells stably expressing the human P2Y receptors. The data are the means of
triplicate determinations and are representative of results obtained in at least three separate experiments with each
analog.
modified UTP analogues (4–11) for activation of the two
receptor types. Although less potent than UTP in most cases,
all 20-substituted molecules retained capacity to maximally
stimulate both receptors in the test system used. Whereas
similar potencies were observed for the 20-O-methyl-substi-
tuted (5) and 20-fluoro-substituted (9) molecules at the P2Y2
versus P2Y4 receptors (Table 1), differential effects on
potencies at the two receptors were observed with 20-amino
(7) and 20-azido (8) substitutions (Fig. 2 and Table 1). That is, the
potency of 20-amino-20-deoxyuridine-50-triphosphate (7) was
unchanged from UTP at the P2Y2 receptor but was 16-fold less
at the P2Y4 receptor. Conversely, 20-azido-substitution in 8
resulted in a greater decrease in potency at the P2Y2 receptor
than at the P2Y4 receptor. Interestingly, existence of the 20-
hydroxyl of UTP in the arabino configuration in 10 resulted in a
molecule that exhibits similar potency to UTP at the P2Y2
receptor but 10-fold reduced potency at the P2Y4 receptor.
The only 30-substituted molecule, 30-O-methyluridine-50-tri-
phosphate (6), tested was inactive at both P2Y2 and P2Y4
receptors.
(21–24). These phosphate side chain modifications decreased
agonist potency by approximately two orders of magnitude.
Both stereoisomers of UTPaS (21, 22) were approximately five-
fold more potent at the P2Y2 receptor than the P2Y4 receptor
(Fig. 4 and Table 1). The R-configuration was favored by both
receptors by approximately three-fold over the S-isomer. Rp-
20-deoxy-UTPaS (23), although displaying a relatively large
standard error, was a full agonist at the P2Y2 receptor and
inactive at the P2Y4 receptor. Sp-20-deoxy-UTPaS (24) was
inactive at both the P2Y2 and P2Y4 receptors.
3.3.
Flexible docking of UTP into putative binding pockets
of the P2Y2 and P2Y4 receptors
We recently proposed, supported by experimental data, a
hypothetical binding mode of nucleotides within P2Y recep-
tors of subgroup A (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) [31,32].
Briefly, the cognate agonist nucleotide of each of these
receptors is accommodated in the cavity defined by the third,
sixth, and seventh transmembrane domains (TM) and the
second extracellular loop (EL2). The ribose moiety of agonists
is located between TM3 and TM7, with the negatively charged
phosphate groups pointing toward TM6 and the nucleobase
pointing toward TM1 and TM2.
Uracil-substituted UTP analogues (12–20) also were exam-
ined. Modification of UTP with a thio moiety in the 4 position in
16 resulted in an analogue that was two-fold and three-fold
more potent than UTP at the P2Y2 and P2Y4 receptors,
respectively (Fig. 3 and Table 1). In contrast, 2-thio-substitu-
tion in 15 resulted in a molecule that was at least as potent as
UTP at the P2Y2 receptor and 5-fold less potent at the P2Y4
receptor. Halo- or alkyl substitution in the 5-position of the
base (12–14) resulted in molecules 3–10-fold more potent at
the P2Y2 receptor than the P2Y4 receptor. Aza-substitution in
the 6-position in 17 decreased potency at the P2Y2 receptor
and resulted in an analogue that was inactive at the P2Y4
receptor. A decrease in P2Y2 receptor potency and complete
loss of activity at the P2Y4 receptor also occurred with
replacement of uracil with a pyrimidinone ring moiety, i.e.
zebularine-50-triphosphate [30]. Conversely, attenuated ago-
nist activity was observed at the P2Y2 and P2Y4 receptors with
pseudouridine-50-triphosphate (20), in which the base moiety
is replaced with a reoriented uracil ring.
Our previous success in identification of new receptor-
selective high affinity agonists and antagonists of the P2Y1
receptor [19–21,33] was driven in part by a close interplay of
structure activity analyses, rational new drug syntheses, and
molecular modeling of the P2Y1 receptor binding site. Thus, we
have carried out new modeling studies as described in Section
2 to further refine insight into the binding pocket of the P2Y2
and P2Y4 receptors. Our previously published binding mode
[31,32] was utilized to position UTP in the putative binding
pockets of the unoccupied P2Y2 and P2Y4 receptors. These
respective binding pockets then were reconstructed around
docked UTP by means of homology modeling under conditions
that allowed the receptors to adapt to the presence of ligand.
We subsequently performed
a concerted conformational
analysis of UTP and the surrounding residues, exploring
simultaneously the flexibility of the ligand and the receptors.
To facilitate the comparison among receptors, throughout this
Phosphate side chain modification as phosphothioates was
examined in the stereoisomers of UTPaS and 20-deoxy-UTPaS

546
b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 5 4 0 – 5 4 9
Fig. 4 – Activation of the human P2Y2 (left panel), and P2Y4 (right panel) receptors by chiral a-phosphothioate uracil
nucleotide analogues 21 (~), 22 (^), and 23 (!). PLC activity was measured as described in Section 3.2 in 1321N1 human
astrocytoma cells stably expressing the human P2Y receptors. The data are the means of triplicate determinations and are
representative of results obtained in three separate experiments.
paper we use the GPCR residue indexing system, as explained
in detail elsewhere [31].
side chain modified UTP, i.e. stereoisomers of UTPaS and 20-
deoxy-UTPaS, also was examined. Thio-substitution at the 4-
position increases potency at both P2Y2 and P2Y4 receptors,
whereas other base- or ribose-modifications were identified
that differentially affect the capacity of analogues to activate
the P2Y2 versus P2Y4 receptor. These results lay the ground-
work for rational drug synthesis directed at generation of high
affinity agonists that selectively activate either the P2Y2
receptor or P2Y4 receptor.
The putative nucleotide binding pockets of the P2Y2 and
P2Y4 receptors are highly conserved and, not surprisingly, the
results of our docking experiments indicated very similar
binding modes of UTP within both receptors (Fig. 5). The most
prominent dissimilarities between the putative UTP binding
pockets in the P2Y2 and P2Y4 receptors were found in residues
located in TM2 (V90(2.61) in the P2Y2 receptor corresponding to
I92(2.61) in the P2Y4 receptor) and a residue in EL2 (T182 in the
P2Y2 receptor corresponding to L184 in P2Y4 receptor). Both of
these non-conserved residues are located in proximity to the
uracil moiety of UTP, and their presence results in an
apparently smaller binding pocket of the P2Y4 receptor for
the cognate agonist.
The P2Y2 receptor is broadly distributed in mammalian
tissues, and its expression in epithelial cells of the lung, eye,
and other tissues make it a potentially important therapeutic
target in cystic fibrosis, eye disease, and other pathophysiol-
ogies [9,10]. Although the P2Y4 receptor is less prominent than
the P2Y2 receptor in mammals, the presence of this signaling
protein on neuronal, vascular, and epithelial tissues also
makes the P2Y4 receptor a potentially important drug target.
Simultaneous expression of P2Y2 and P2Y4 receptors occurs in
a number of cell types and tissues. These receptors theore-
tically can be delineated in human tissues since whereas both
UTP and ATP activate the human P2Y2 receptor, only UTP
activates the human P2Y4 receptor. However, both of these
receptors are potently activated by ATP and UTP in rodents
[11–13], and the complexities introduced by metabolism and
interconversion of nucleotides also makes pharmacological
delineation of the P2Y2 and P2Y4 receptors difficult in human
tissues using the natural triphosphate agonists [15].
The docking models suggested other ligand recognition
elements. In agreement with observations for the P2Y1
receptor [31,32], the triphosphate moiety of UTP is putatively
coordinated by three conserved cationic residues (Fig. 5).
These residues, all Arg in P2Y2 and P2Y4 receptors, are located
in TM3 (3.29), TM6 (6.55), and TM7 (7.39). A fourth cationic
residue located at the 7.36 position is in proximity to the ligand
but, consistent with mutagenesis data [34], apparently does
not directly interact. The NH at the 3-position of the uracil ring
donates an H-bond to a Ser in TM7 (7.43). Alternatively, in
other docking poses Ser(7.43) donated an H-bond to the
oxygen at the 2-position of the uracil ring. This Ser residue is
highly conserved within the P2Y family. Mutagenesis data
revealed its fundamental role in the recognition of agonists
and antagonists at the P2Y1 receptor, where it is probably
involved in the coordination of the adenine base [31].
Few studies have addressed selectivity of synthetic
nucleotide analogues at the P2Y2 and P2Y4 receptors, and no
systematic comparison of structure activity relationships has
been made for the P2Y2 receptor versus P2Y4 receptor using a
unified assay system. Certain UTP analogues with substitu-
tions in the 4-position of the base have been reported, and
several of these, e.g. 4-SH or 4-S-hexyl analogues of UTP,
retained potencies similar to UTP at the P2Y2 receptor [35]. To
our knowledge the activities of these molecules at the P2Y4
receptor have not been reported. 5-Br-UTP is an agonist at both
P2Y2 and P2Y4 receptors, but relative potencies have not been
compared simultaneously in the same test system. We
4.
Discussion
Results from this study provide the first systematic structure
activity analysis designed to distinguish the P2Y2 versus P2Y4
receptor selectivity of base- and ribose-modified analogues of
UTP. The UTP receptor selectivity of molecules with phosphate

b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 5 4 0 – 5 4 9
547
Fig. 5 – Models of the complexes formed by UTP (1) with the P2Y2 (a) and P2Y4 (b) receptors. The nucleotide binding pockets
are highly conserved in the two receptors [23]. The only two sites of divergence can be found in two residues located in TM2
and EL2 (represented with balls and sticks) in proximity to the uracil-binding pocket. To the left of each detailed binding site
is a schematic representation of the entire receptor structure complexed with UTP. In the tube representations the receptor
is colored according to residue positions, with a spectrum of colors that ranges from red (N-terminus) to purple (C-
terminus): TM1 is in orange, TM2 in ochre, TM3 in yellow, TM4 in green, TM4 in cyan, TM5 in blue, TM7 in purple.
previously directly compared at the human P2Y2 and P2Y4
receptors analogues of both UTP and ATP in which the ribose
moiety was replaced by a fixed ring (methanocarba) system,
i.e. a cyclopentane fused in the Northern confirmation with a
cyclopropane ring [33]. (N)-Methanocarba UTP was equipotent
to UTP at both the P2Y2 and P2Y4 receptors, and this analogue
was approximately five-fold more potent at the P2Y2 receptor
than the P2Y4 receptor. In contrast, whereas (N)-methano-
carba-ATP is a very potent agonist at the human P2Y2 receptor,
(N)-methanocarba-ATP like ATP itself is inactive at the human

548
b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 5 4 0 – 5 4 9
P2Y4 receptor. UTPgS is a reasonably potent agonist at both
P2Y2 and P2Y4 receptors [36,37]. Receptor activity also is
retained in UTP analogues with imido- and methylene-
modifications of the phosphate side chain [18]. Finally, both
diadenosine and diuridine polyphosphate molecules are
agonists at the P2Y2 receptor with the most potent of these
reported to be dinucleotides with four phosphates [29]. Up4U
also is a potent P2Y4 receptor agonist.
In summary, these results indicate that agonist potency
and P2Y2 versus P2Y4 receptor selectivity can be differentially
modified in ribose- and base-modified analogues. Our work
identifies molecules that may prove useful in pharmacologi-
cally distinguishing these receptors in complex tissues and
that provide leads for rational new drug synthesis. Further
evolution of molecular models for these two uracil nucleotide-
activated receptors should proceed hand in hand with this
novel molecule development.
The current structure activity studies provide encouraging
new insight on agonist action at the P2Y2 versus P2Y4
receptors. Certain substitutions, e.g. thio-substitution in the
4-position of the uracil base, resulted in increases in potency
at both the P2Y2 and P2Y4 receptors. These increases in
potency are explainable on the basis of the favorable
interactions that the sulfur atom establishes with hydro-
phobic residues from TM1, TM7, and EL2 in the binding
pockets of both receptors (Fig. 5). These results strongly
suggest that relative affinity can be retained or enhanced in
future analogues modified in other positions to alter their P2Y
receptor subtype-selectivity. The stereoselectivity in recogni-
tion of a-thiophosphate analogues of UTP showed a small but
consistent preference at both P2Y2 and P2Y4 receptors for the
Rp isomer, which is the opposite of the diastereoselectivity
found at the P2Y1 receptor [39].
Acknowledgements
We thank Dr. Victor Marquez (NCI, Frederick, MD) for the gift
of zebularine 50-triphosphate and for helpful discussion. This
research was supported in part by the Intramural Research
Program of the NIH, National Institute of Diabetes and
Digestive and Kidney Diseases and by NIH grants GM38213
and HL34322. Susanna Tchilibon and Pedro Besada thank the
Cystic Fibrosis Foundation (Bethesda, MD) for financial
support. Mass spectral measurements were carried out by
Dr. John Lloyd and NMR by Wesley White (NIDDK).
r e f e r e n c e s
A large number of modifications of the base or ribose
moiety resulted in differential changes in the potency of
analogues at the two receptors. Several base modifications
differentially affected P2Y2 receptor versus P2Y4 receptor
selectivity, but in all such cases P2Y2 receptor potency was
better retained than activity at the P2Y4 receptor. As presented
in Section 3, molecular modeling indicates a larger size for the
base-interacting portion of the P2Y2 binding pocket compared
to that of the P2Y4 receptor, and the observed enhancement of
selectivity, for example, in the 5-substituted analogs is
consistent with this predicted difference between the steric
bulk tolerance of the P2Y2 receptor versus the P2Y4 receptor.
The observation that introduction of different functional
groups in the 20 position resulted in analogues that favored the
P2Y2 receptor in some cases, e.g. 20-amino, or the P2Y4 receptor
in another substitution, e.g. 20-azido, suggests both subtle
differences in the binding pocket of these two receptors and
avenues for developing analogues that favor binding to one of
these UTP-activated receptors. The 30-OH is intramolecularly
H-bonded to the b-phosphate (Fig. 5) and, according to our
molecular dynamics (MD) studies on the P2Y6–UDP complex
[38], could be involved in an interaction with the cationic
residues, possibly mediated by a water molecule. Therefore,
substitutions at this position are likely to be detrimental for
activity. Conversely, the 20-OH apparently does not interact
directly with the receptor. Hence, substitutions at this position
are likely to be tolerated and can be exploited to modulate the
P2Y2/P2Y4 receptor selectivity. The residues that interact
directly with the ribose moiety are conserved in the P2Y2
and the P2Y4 receptors (Fig. 5). Thus, the selectivities observed
with various 20-substituted compounds for the P2Y2 or P2Y4
receptors must arise from non-conserved residues located
further away from the ligand that indirectly affect the shape of
the binding pocket. Prolonged MD simulations of the receptor–
ligand complexes in a phospholipid bilayer should reveal
these residues.
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