Molecular modeling of the human P2Y2 receptor and design of a selective agonist, 2′-amino-2′-deoxy-2-thiouridine 5′-triphosphate

Article abstract of DOI:10.1021/jm060903o

A rhodopsin-based homology model of the nucleotide-activated human P2Y ₂ receptor, including loops, termini, and phospholipids, was optimized with the Monte Carlo multiple minimum conformational search routine. Docked uridine 5′-triphosphate (UTP) formed a nucleobase π-π complex with conserved Phe3.32. Selectivity-enhancing 2′-amino-2′-deoxy substitution interacted through π-hydrogen-bonding with aromatic Phe6.51 and Tyr3.33. A "sequential ligand composition" approach for docking the flexible dinucleotide agonist Up₄U demonstrated a shift of conserved cationic Arg3.29 from the UTP γ position to the δ position of Up₄U and Up₄ ribose. Synthesized nucleotides were tested as agonists at human P2Y receptors expressed in 1321N1 astrocytoma cells. 2′-Amino and 2-thio modifications were synergized to enhance potency and selectivity; compound 8 (EC₅₀ = 8 nM) was 300-fold P2Y ₂-selective versus P2Y₄. 2′-Amine acetylation reduced potency, and trifluoroacetylation produced intermediate potency. 5-Amino nucleobase substitution did not enhance P2Y₂ potency through a predicted hydrophilic interaction possibly because of destabilization of the receptor-favored Northern conformation of ribose. This detailed view of P2Y ₂ receptor recognition suggests mutations for model validation.

Full text of DOI:10.1021/jm060903o

1166
J. Med. Chem. 2007, 50, 1166-1176
Molecular Modeling of the Human P2Y2 Receptor and Design of a Selective Agonist,
2′-Amino-2′-deoxy-2-thiouridine 5′-Triphosphate
Andrei A. Ivanov,†,‡ Hyojin Ko,†,‡ Liesbet Cosyn,§ Savitri Maddileti,# Pedro Besada,† Ingrid Fricks,# Stefano Costanzi,|
T. Kendall Harden,# Serge Van Calenbergh,§ and Kenneth A. Jacobson*,†
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and DigestiVe and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland 20892, Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences (FFW),
Ghent UniVersity, Harelbekestraat 72, B-9000 Ghent, Belgium, Department of Pharmacology, UniVersity of North Carolina School of Medicine,
Chapel Hill, North Carolina 27599, and Laboratory of Biological Modeling, National Institute of Diabetes and DigestiVe and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland 20892
ReceiVed July 28, 2006
A rhodopsin-based homology model of the nucleotide-activated human P2Y2 receptor, including loops, termini,
and phospholipids, was optimized with the Monte Carlo multiple minimum conformational search routine.
Docked uridine 5′-triphosphate (UTP) formed a nucleobase π-π complex with conserved Phe3.32. Selectivity-
enhancing 2′-amino-2′-deoxy substitution interacted through π-hydrogen-bonding with aromatic Phe6.51
and Tyr3.33. A “sequential ligand composition” approach for docking the flexible dinucleotide agonist Up4U
demonstrated a shift of conserved cationic Arg3.29 from the UTP γ position to the δ position of Up4U and
Up4 ribose. Synthesized nucleotides were tested as agonists at human P2Y receptors expressed in 1321N1
astrocytoma cells. 2′-Amino and 2-thio modifications were synergized to enhance potency and selectivity;
compound 8 (EC50 ) 8 nM) was 300-fold P2Y2-selective versus P2Y4. 2′-Amine acetylation reduced potency,
and trifluoroacetylation produced intermediate potency. 5-Amino nucleobase substitution did not enhance
P2Y2 potency through a predicted hydrophilic interaction possibly because of destabilization of the receptor-
favored Northern conformation of ribose. This detailed view of P2Y2 receptor recognition suggests mutations
for model validation.
Introduction
and INS37217 (Up4dC) 4b are potent and relatively stable
agonists of the P2Y2 receptor.11,14
P2 nucleotide receptors are activated by a range of naturally
occurring extracellular nucleotides and consist of two families:
the eight subtypes of P2Y receptors, which are G-protein-
coupled receptors (GPCRs) (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11,
P2Y12, P2Y13, and P2Y14), and the seven subtypes of P2X
ligand-gated cation channels.1-5 The P2Y2 receptor is activa-
ted by endogenous uridine 5′-triphosphate (UTP 1), adenosine
5′-triphosphate (ATP 2), and various dinucleotides (Chart 1).
It is preferentially Gq-coupled and stimulates phospholipase
Câ (PLCâ). The P2Y2 receptor, first cloned from mice,6 is
expressed in epithelial cells, smooth-muscle cells, endothelial
cells, leukocytes, osteoblasts, and cardiomyocytes. In the central
nervous system, activation of the receptor is associated with
neuronal differentiation.7 The P2Y2 receptor appears to interact
with integrins to stimulate chemotaxis and to enhance R-secre-
tase-dependent amyloid precursor protein processing.8,9
Activation of the P2Y2 receptor causes a pronociceptive effect.10
Agonists of the P2Y2 receptor are of interest in the develop-
ment of therapeutic agents for pulmonary diseases (e.g.,
cystic fibrosis), salivary gland dysfunction, and ophthalmic
diseases.11,12
The first site-directed mutagenesis and molecular modeling
of transmembrane helical domains 6 and 7 (TMs 6 and 7) of
the P2Y2 receptor were reported by Erb et al.15 Modeling
methods have subsequently undergone significant development.
Furthermore, a variety of newly synthesized potent analogues
of UTP are available for docking studies. Hence, in the present
study, we carried out new molecular modeling studies to predict
sites of interaction of the ligands with the P2Y2 receptor and to
provide hypotheses for the design of additional analogues. Our
recent publications1,13 provided an initial rhodopsin-based
homology model, which was refined in the present study. We
also have optimized agonist selectivity for the P2Y2 receptor
by synthesizing new UTP analogues, which bear modifications
at the uracil moiety and the ribose ring. Thus, this study
integrates the synthesis of a highly selective agonist with
modeling studies that use a molecular dynamics (MD) simula-
tion in a phospholipidic and aqueous environment to refine the
receptor and a Monte Carlo approach to ligand docking.
Results and Discussion
1. Structure of the P2Y2 Receptor. A molecular model of
the P2Y2 receptor was recently published.1 In contrast to the
earlier model constructed by Erb et al.,15 this model of the
P2Y2 receptor is a rhodopsin-based homology model con-
taining not only seven TMs but also all extracellular and
intracellular hydrophilic loops. Here, we present a further refined
model of the P2Y2 receptor, updated by insertion of a segment
of the carboxyl-terminal (CT) domain including the H8 helix
(Gly310-Met346) and the amino-terminal (NT) domain. The
formal geometry of the model was tested with the ProTable
command of Sybyl as well as the Procheck software (Supporting
Information).16
The SAR (structure-activity relationship) of nucleotides in
activating the human P2Y2 receptor has been probed.2,3,13 UTP-
γ-S 3 and the dinucleoside tetraphosphates INS365 (Up4U) 4a
* To whom correspondence should be addressed. Phone: 301-496-9024.
Fax: 301-480-8422. E-mail: kajacobs@helix.nih.gov.
† Laboratory of Bioorganic Chemistry, National Institute of Diabetes and
Digestive and Kidney Diseases.
‡ These authors contributed equally to this work.
§ Ghent University.
# University of North Carolina School of Medicine.
| Laboratory of Biological Modeling, National Institute of Diabetes and
Digestive and Kidney Diseases.
10.1021/jm060903o CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/16/2007

Modeling of Receptor and Design of Agonist
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1167
Chart 1. Structures of Native Nucleoside 5′-Triphosphate (UTP and ATP) Ligands and a Dinucleotide (UpnU) Ligand of the P2Y2
Receptora
a EC50 values reported at the human P2Y2 receptor are shown.13,23,24,42
Figure 1. (a) The rmsf of the P2Y2 receptor residues calculated from the 10 ns MD trajectory. (b) The rmsf of a part of the C-terminal region of
the P2Y2 receptor. A helical structure of H8 remained stable during the MD simulation.
Inclusion of the natural environment of the receptor was
previously shown to significantly improve the results of MD
simulation, providing a more accurate structure of the receptor,
especially in its loop and terminal regions.17 Therefore, the
model was subjected to 10 ns of MD simulation in a hydrated
phospholipid bilayer.
analysis of the variation of the rmsd and the total energy along
the trajectory suggested that during MD simulation the receptor
structure became stable after 7.5 ns. Thus, we prolonged the
simulation up to 10 ns and used the final typical structure for
our docking studies. Also, the rms fluctuations (rmsf) of the
P2Y2 receptor residues were calculated from the MD trajectory.
As shown in Figure 1, the lowest rmsf values were obtained
for amino acid residues located in TM domains and the highest
values corresponded to residues located in the hydrophilic loops
The MD trajectory was analyzed, and the root-mean-square
deviations (rmsd) of the P2Y2 receptor atoms, as well as its
total energy, were calculated (Supporting Information). The

1168 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
IVanoV et al.
E29(NT), and D185(EL2)-R265(6.55). Throughout this paper
we use the GPCR residue indexing system for TM regions,
which allows comparison among receptors, as explained in detail
elsewhere.17 In addition, E190 can interact with R194; both
residues are located at the beginning of EL2. Several other
bridges in the extracellular region of the P2Y2 receptor appeared
in the model. R24(NT) can interact with D97(EL1) and with
D30 (NT). Furthermore, E20(NT) interacted not only with R177-
(EL2) but also with R26(NT). Finally, C25, located in the NT
domain, and C278, located in EL3, formed a disulfide bridge
characteristic of all P2Y receptors.
1.2. Intracellular Regions. Three possible pairs of charged
residues were found in the intracellular region of the P2Y2
receptor: D319-R340, R334-D342, and R335-D345 (Figure
3B). All these residues are located in the CT domain. Our
molecular model suggests that ionic interactions between these
residues keep the long CT domain in a more compact and
configurationally restricted form, giving the entire receptor a
more densely packed structure.
The rmsf values obtained for the residues located in the H8
helix (Figure 1b) showed that this part of the receptor remained
stable during MD simulation, and H8 was found to be helical
in the typical structure of the P2Y2 receptor.
Figure 2. Superimposition of the initial structure of the human P2Y2
receptor (white) and its structure obtained after MD simulation (colored
by residue position: N-terminus in red, TM 1 in orange, TM 2 in ochre,
TM 3 in yellow, TM 4 in green, TM 5 in cyan, TM 6 in blue, TM 7
and C-terminus in purple). Lipid and water molecules are not shown.
Table 1. Predicted Electrostatic and Disulfide Bridges Observed in the
1.3. Putative Interhelical Disulfide Bridges. An analysis
of the model obtained after MD simulation allowed us to
hypothesize that two pairs of cysteine residues, C132(3.51)-
C217(5.57) and C44(1.43)-C300(7.47), can be involved in the
formation of disulfide bridges (Figure 3B). The distances
observed between Câ atoms of these cysteines (6.2 and 4.5 Å,
respectively) are in good agreement with the distances measured
experimentally for disulfide-bonded Cys-Cys pairs.19 However,
C1.43 and C3.51 are present in the P2Y2 receptor only; in other
subtypes of P2Y receptors these residues are F1.43 and Y3.51
(except F3.51 in the P2Y13). For this reason, these two proposed
disulfide bridges could only exist in the P2Y2 receptor. It has
been shown experimentally that in rhodopsin the formation of
a disulfide bond between residues located at the 3.51 and 5.57
positions is possible and that a cross-link between Y3.51C and
C5.57 inhibits rhodopsin activation.20
2. Putative Binding Modes of P2Y2 Receptor Agonists. 2.1.
Binding Mode of UTP and Its Analogues. To study the
putative binding modes of P2Y2 receptor agonists, we performed
molecular docking combined with the Monte Carlo multiple
minimum (MCMM) conformational search analysis on several
known nucleotide ligands of this receptor subtype. As described
in the Experimental Section, UTP 1 initially was manually
docked inside the putative binding site of the P2Y2 receptor.
The published data of site-directed mutagenesis combined with
computational studies of P2Y receptors were taken into ac-
count.1,13,18 Because the Northern (N) conformation of the ribose
ring of the ligand was proposed to be important for recognition,21
UTP and all other studied ligands were sketched and initially
docked in their (N)-conformation. In addition, the anti confor-
mation of the ligand base ring was used during the modeling
studies. Interestingly, in the original rhodopsin-based model of
the P2Y2 receptor,1 the side chain of one of the key cationic
residues, namely R3.29, was oriented in the opposite direction
from the putative binding pocket, but during the simulation it
shifted toward the binding cavity.
P2Y2 Receptor Model Obtained after MD Simulation
residue
residue
Electrostatic Bridges
negatively charged
positively charged
Glu20 NT
Glu20 NT
Glu29 NT
Asp30 NT
Asp97 EL1
Asp185 EL2
Glu190 EL2
Asp319 CT
Asp342 CT
Asp345 CT
Arg26 NT
Arg177 EL2
Arg180 EL2
Arg24 NT
Arg24 NT
Arg 265 6.55
Arg194 EL2
Arg340 CT
Arg334 CT
Arg335 CT
Disulfide Bridges
Cys25 NT
Cys106 3.25
Cys278 EL3
Cys183 EL2
Putative Disulfide Bridges
Cys217 5.57
Cys300 7.47
Cys132 3.51
Cys44 1.43
and terminal domains. The superimposition of the CR atoms of
the TM domains of the initial structure of the P2Y2 receptor
and its typical structure calculated from the final 100 ps of the
MD trajectory (Figure 2) demonstrated that the configuration
of the loops and terminal domains was most significantly
changed.
1.1. Extracellular Regions. Recent MD simulation of the
P2Y6 receptor revealed that EL2 moved toward TM 3 and
further up into the extracellular space, opening the putative
nucleotide binding cavity.18 However, in the case of the P2Y2
receptor, EL2 did not show a significant displacement during
the MD simulation. The analysis of the configuration of EL2
in the P2Y2 receptor structure obtained after MD simulation
allowed us to propose several key residues that could fix this
loop near the TM domain (Table 1). In addition to the disulfide
bridge conserved among all GPCRs of the rhodopsin family
and formed between two cysteine residues located in TM 3 and
EL2 (C106 and C183 in the P2Y2 receptor), we found that
several charged residues located in EL2 of the P2Y2 receptor
can interact with some oppositely charged residues located
around the loop (Figure 3A). In particular, three pairs of such
residues were observed: R177(EL2)-E20(NT), R180(EL2)-
After MCMM refinement of the initially obtained docking
complex, the R-phosphate group of the UTP triphosphate chain
was bonded to R7.39 whereas R6.55 could interact with both
R- and γ-phosphate groups and R3.29 interacted with the
γ-phosphate group of UTP (Figure 4A). In addition, the

Modeling of Receptor and Design of Agonist
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1169
Figure 3. Putative electrostatic and disulfide bridges found in the extracellular (A) and intracellular (B) regions of the model of the P2Y2 receptor.
Figure 4. Binding modes of various agonists to the human P2Y2 receptor following MCMM calculations: (A) UTP 1; (B) 2′-amino-2′-deoxy
analogue 8 of UTP, where the 2′-amino group is shown in its protonated form, which can be involved in cation-π interactions with F6.51 and can
form an additional H-bond with Y3.33; (C) 3-methyl-UTP,13 where the 3-methyl group has an unfavorable position between hydroxyl groups of
Y1.39 and S7.43, and backbone oxygen atom of P7.40; (D) 5-methyl-UTP 19,13 where, in the model obtained, the 5-methyl group of 19 is unfavorably
located between hydroxyl groups of T182 (EL2) and Y1.39 and is close to the methyl groups of V181 (EL2); (E) ATP 2; (F) Up4U 4a.
γ-phosphate group of the ligand formed H-bonds with H184,
located in EL2 directly after the conserved cysteine residue,
with the backbone nitrogen atom of D185 and with the hydroxyl
group of Y6.59. Another tyrosine residue, Y3.33, was H-bonded
to the â-phosphate group.
the P2Y11 and P2Y14 receptors, this position is Y6.51). This
observation suggests the possibility of OH-π H-bonding
between the 2′-hydroxyl group and the aromatic ring of F6.51.
Several examples of similar interactions seen in protein crystal-
lographic structures were reviewed by Meyer et al.22 The
hypothesis of H-bonding of the 2′-hydroxyl group to the receptor
is consistent with the experimental SAR.13 For example, 2′-
In the model obtained after MCMM calculations, the 2′-
hydroxyl group of UTP appeared near the conserved F6.51 (in

1170 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
IVanoV et al.
Table 2. In Vitro Pharmacological Data for UTP 1 and Its Analogues in the Stimulation of PLC at Recombinant Human P2Y2, P2Y4, and P2Y6
Receptors Expressed in 1321N1 Astrocytoma Cells (Unless Otherwise Noted, R1 ) OH, X ) O, and R2 ) H)
EC50,a nM
compd
modification
structure
at hP2Y2 receptor
at hP2Y4 receptor
at hP2Y6 receptor
1b
2b
4a
5
()UTP)
()ATP)
()Up4U)
49 ( 12
85 ( 12
210 ( 30
1880 ( 30
62 ( 8
35 ( 4
8 ( 2
470 ( 60
6500 ( 1400
5600 ( 1200
1800 ( 400
480 ( 100
73 ( 20
antagonistd
130 ( 10
NDh
>10000e
NEg
20000e
NDh
()Up4[5]ribose)
6b
7b
8c
9
2′-deoxy-2′-amino
2-thio
2-thio-2′-deoxy-2′-amino
2-thio-2′-deoxy-2′-trifluoroacetylamino
2-thio-2′-deoxy-2′-acetylamino
5-amino
R1 ) NH2
1200 ( 300
350 ( 10
2400 ( 800
8300 ( 1200
NEg
>100000
∼1500f
>10000
NEg
X ) S
X ) S, R1 ) NH2
X ) S, R1 ) CF3CONH
X ) S, R1 ) CH3CONH
R2 ) NH2
10
17
18
19b
NEg
NDh
333 ( 88
467 ( 120
140 ( 30
5-azido
5-methyl
R2 ) N3
NDh
R2 ) CH3
3900 ( 1600
a Agonist potencies reflect stimulation of PLC determined as reported,13,21,30 unless otherwise noted, and were calculated using a four-parameter logistic
equation and the GraphPad software package (GraphPad, San Diego, CA). EC50 values (mean ( standard error) represent the concentration at which 50%
of the maximal effect is achieved. The potency of UDP at the P2Y6 receptor was 23 ( 4 nM. N ) 3. b Agonist potencies from ref 13. c MRS2698. d ATP
antagonized the human P2Y4 receptor with a KB of 708 nM. e Agonist potencies in mobilization of intracellular [Ca2+], from refs 23 and 24. f Agonist
potency from ref 26. g NE: no effect at 10 µM. h ND: not determined.
deoxy-UTP (EC50 ) 1.08 µM) is 22-fold less potent than UTP
(EC50 ) 0.049 µM), whereas the replacement of the 2′-hydroxyl
group by a 2′-methoxy group (EC50 ) 14.3 µM) reduced the
potency further (290-fold weaker than UTP). In the latter case,
our model showed that not only was the suggested OH-π
H-bond lost but the 2′-methyl group also interfered sterically
with the aromatic ring of F6.51.
derivatives: UTP ∼ 2′-NH2-2′-dUTP > 2′-dUTP > 2′-OCH3-
2′-dUTP . 3′-OCH3-3′-dUTP.
In our model the oxygen atom at position 4 of the UTP uracil
ring was H-bonded to the hydroxyl group of Y1.39, and S7.43
accepted a H-bond from the 3-NH group of the ligand. In
contrast, the oxygen atom at position 2 was not involved in
H-bonding with the receptor. In addition, F3.32, conserved
among all subtypes of P2Y receptors, was found to be involved
in π-π interaction with the uracil ring of UTP. Interestingly,
in the model obtained for the complex of the P2Y2 receptor
with CTP (EC50 ) 5.63 µM),13 the hydroxyl group of S7.43
was involved in H-bonding with the nitrogen atom at the 3
position of the nucleobase of CTP, but the NH2 group at the 4
position was unable to form a H-bond with Y1.39. Furthermore,
the nucleoside moiety of zebularine 5′-triphosphate (180-fold
weaker than UTP)13 docked to the P2Y2 receptor was signifi-
cantly shifted from the initial position of UTP outwardly and
toward Y1.39, although the ligand still maintained a H-bond
with S7.43. In addition, it was shown that a thiouracil ring can
form H-bonded pairs with other nucleobases, which are only
slightly less stable than the pairs formed by unmodified uracil.39
This means that 4-thio-UTP could be involved in the same
interactions with Y1.39 as UTP. These data argue in favor of
the hypothesis that in the case of the P2Y2 receptor, Y1.39 plays
a role in the recognition of the functional group at position 4
of the uracil moiety. Available experimental data indicate that
3-methyl-UTP (EC50 ) 1.20 µM) is 24-fold weaker than UTP.13
With the aim of explaining the observed differences in activity,
we used MCMM calculations to establish a favored binding
mode of 3-methyl-UTP, which was compared to the binding
mode of UTP. According to this model, the methyl group at
position 3 was located between the hydroxyl groups of Y1.39
and S7.43, and the side chain of the serine residue was shifted
Agonist activities of the ligands, taken together with the
binding mode obtained, strongly suggest that the hydroxyl group
at the 2′ position is important as a donor but not as an acceptor
of a H-bond. The binding mode of 2′-deoxy-2′-amino-UTP 6
(Table 2, EC50 ) 0.062 µM), which was found to maintain
potency in activation of the P2Y2 receptor,13 was studied with
MCMM calculations. The 2′-amino group (pKa ) 6.2 in a related
derivative)40,41 was examined in both unprotonated and proto-
nated positively charged forms. Similar to the 2′-hydroxyl group
of UTP, the 2′-amino group of 6 was found near and oriented
toward the aromatic ring of F6.51. Both protonated and
unprotonated forms of the ligands interacted with this residue.
However, the protonated form of this amino group was involved
in a stronger cation-π interaction with F6.51. In addition, the
+
protonated 2′-NH3 group acted as a H-bond donor to the
hydroxyl of Y3.33 in our model (Figure 4B).
In the receptor-ligand complexes obtained for all studied
agonists, the 3′-hydroxyl group of the ligand appeared to be
H-bonded to the R-phosphate group of the triphosphate chain.
This could be an important intramolecular interaction to stabilize
the ribose ring in its (N)-conformation, to facilitate the interac-
tion of the 2′-hydroxyl group with F6.51. This assumption is in
agreement with the ligand activities;13,21 both 3′-deoxy-3′-
methoxy-UTP and (S)-methanocarba ATP are essentially inac-
tive at the P2Y2 receptor.
Taken together, these findings are in good agreement with
the SAR data and can explain the experimentally observed
differences in activities of UTP and its 3′- and 2′-substituted

Modeling of Receptor and Design of Agonist
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1171
Scheme 1. Preparation of the Synthetic Intermediate 15, a
2′-Amino-2′-deoxy-2-thio Nucleosidea
otide tetraphosphates, such as Up4U 4a or Up4dC 4b (Chart 1),
have the optimal phosphate chain length for activation of the
P2Y2 receptor and tend to be more resistant to nucleotidase
degradation than the nucleoside triphosphates. In contrast, Up2U,
containing only two phosphate groups, is nearly inactive as an
agonist, and Up6U is 98-fold weaker than Up4U at the P2Y2
receptor.
To study the binding mode of dinucleotides within the P2Y2
receptor, we docked Up4U to the putative binding site. Docking
of molecules that are as complex as Up4U is challenging because
of their large size and great flexibility. In particular, this process
is especially difficult in the absence of X-ray structures or even
molecular models of the receptor complexed with similar
ligands. Several methods for docking of large ligands were
described previously.25 In particular, fragmental-guided docking
methods decompose a ligand into small fragments and then use
favorable binding modes of these fragments to determine the
most favorable orientation of the whole ligand.
Here, we introduce an alternative “sequential ligand composi-
tion” technique, which we applied to the molecular docking of
Up4U. With this technique, the structure of UTP docked in the
binding site of the P2Y2 receptor and subjected to MCMM
calculations was sequentially converted to Up4, Up4-ribose, and
Up4U. The MCMM conformational search analysis of the ligand
and all receptor residues located within 5 Å of the ligand was
performed at each stage of the computational conversion of UTP
to Up4U to provide an energetically favorable orientation and
configuration of the ligand. In contrast to the docking of the
entire structure of the ligand (e.g., Up4U) at once, this method
can avoid undesirable changes in the configuration of the
receptor binding site.
Thus, “sequential ligand composition” provided us with a
putative binding mode of Up4U at the P2Y2 receptor (Figure
4F and Supporting Information). In our model, the common
uridine triphosphate subset of the Up4U structure had almost
the same binding mode as UTP. However, three residues that
interacted with the γ-phosphate group of UTP, i.e., R3.29, D185
(EL2), and Y6.59, were found to be bonded to the δ-phosphate
group of Up4U. The uracil ring of UTP (uracilI) was involved
in a π-π interaction with F3.32, and the uracil ring of the
second uridine moiety (uracilII) of Up4U formed π-π interac-
tions with Y3.33, which was also bonded to the â-phosphate
group of the ligand. The oxygen atom at the 2 position of the
uracilII ring could form H-bonds with Q4.57 and Y3.37. The
oxygen atom of the hydroxyl group of Y3.37 also appeared near
the 3-NH groupII of the ligand and could be involved in
H-bonding with this group. As was observed with UTP, neither
of the ribose ring oxygen atoms was H-bonded to the receptor.
However, the 3′-hydroxyl groups of both uridineI and uridineII
of Up4U were H-bonded to the R and δ phosphate groups,
respectively.
a Reagents and conditions: (a) NaN3, DMF, 150 °C, 15 h; (b) MsCl,
pyridine, 0 °C, 61%; (c) NaHCO3/EtOH, reflux, 36 h, 57%; (d) H2S,
pyridine, 50 °C, 250 psi, 24 h, 77%.
by 1.5 Å away from the ligand. Moreover, the backbone oxygen
atom of P7.40 also appeared near the 3-methyl group (Figure
4C).
The binding modes of 5-bromo- (EC50 ) 0.75 µM), 5-iodo-
(EC50 ) 0.83 µM), and 5-methyl-UTP (EC50 ) 0.48 µM) were
determined (Figure 4D) indicating that a hydrophobic group at
position 5 of UTP was located unfavorably between the
hydrophilic hydroxyl groups of T182 (EL2) and Y1.39. The
methyl groups of V181 (EL2) were also located approximately
4 Å from the substituent at position 5. Therefore, our model
suggested that the introduction of a small hydrophilic group,
such as an amino or hydroxyl group, at position 5 could provide
additional H-bonds with T182 and Y1.39 and thus favor potency.
Also, we predicted that an azido group would interact favorably
within the available space between Y40, R180, V181, T182,
and P293.
2.2. Binding Mode of ATP. The question of whether UTP
and ATP bind to the same site in the P2Y2 receptor has already
been considered.3 Because ATP 2 (EC50 ) 0.085 µM) and UTP
1 (EC50 ) 0.049 µM) are nearly equipotent at the P2Y2 receptor,
ATP was initially docked (Figure 4E) at the P2Y2 receptor with
the ribose in the (N)-conformation and the adenine ring in the
anti conformation and subsequently subjected to an MCMM
conformational search. Superimposition of the ATP-P2Y2 and
the UTP-P2Y2 complexes revealed that the phosphate chains
and the ribose rings of these two ligands have identical positions
and configurations inside the receptor. The residues interacting
with UTP were also found to interact with ATP. In agreement
with the binding mode obtained for UTP, F3.32 was involved
in a π-π interaction with the adenine ring of ATP. Also, Y1.39
was H-bonded with the N6-amino group, whereas S7.43 was
H-bonded with the nitrogen atom at position 1 of the adenine
ring of ATP. In addition, Y2.53, conserved among P2Y1,2,4,6
receptors, formed a H-bond with the nitrogen atom at position
1. On this basis, we propose that Y2.53 is likely more important
for interactions of the P2Y2 receptor with ATP and its
derivatives than with UTP analogues.
The model of receptor-docked Up4U is consistent with a
tetraphosphate chain having the optimal length for dinucleotide
binding. The phosphate side chain of diuridine diphosphates
apparently is too short to allow uridineII to reach its binding
pocket. In contrast, ligands with longer phosphate chains, such
as Up6U, are too large to be fully accommodated inside the
receptor. Also, as discussed by Brunschweiger and Mu¨ller,2
among various linear dinucleotides only dinucleoside tetraphos-
phates have the same number of negatively charged oxygen
atoms as UTP.
2.3. Binding Mode of Dinucleotide Agonists. The P2Y2
receptor can be activated not only by uracil or adenine
triphosphates but also by dinucleotide molecules.23,24 Dinucle-
Although H-bonds between the P2Y2 receptor and the ribose
ring oxygen of uridineII of Up4U are absent, both 2′- and 3′-
hydroxyl groups were found to be H-bonded to the receptor.

1172 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
IVanoV et al.
Scheme 2. Synthesis of 2′-Amino-2′-deoxy-2-thio-UTP 8 and Its N-Trifluoroacetyl Derivative 9a
a Reagents and conditions: (a) DIEA, ethyl trifluoroacetate, DMF, room temperature, 13 h, 88%; (b) (i) POCl3, PO(OCH3)3, Proton Sponge, 0 °C, 2 h;
(ii) (Bu3NH+)2P2O7H2, Bu3N, DMF, 10 min; (iii) 0.2 M triethylammonium bicarbonate solution, room temperature, 1 h, 12% (8), 7% (9).
Scheme 3. Synthesis of 2′-Acetylamino-2′-deoxy-2-thio-UTP
The 2′-hydroxyl group was found to be H-bonded with the
hydroxyl group of Y5.38, and the 3′-hydroxyl group interacted
with the backbone NH group of V4.60. Also, the hydroxyl group
of Y6.59 appeared near the 3′-hydroxyl group of the ligand.
These observations suggest that Up4-ribose could be active at
the P2Y2 receptor, as UDP-glucose is active at the P2Y14
receptor. In the complex obtained for Up4U, both the hydroxyl
group of Tyr and the backbone NH group of Val were found
5.5 Å from the ligand.
10a
2.4. Selective Recognition for Nucleoside Tri- and Diphos-
phates at P2Y2 and P2Y6 Receptors. Although UDP is almost
inactive at the P2Y2 receptor,2 it is the cognate agonist of the
P2Y6 receptor. Although UTP analogues may display consider-
able potency at the P2Y6 receptor,26 UTP itself is nearly inactive.
To explain these differences, we compared residues of the P2Y2
receptor directly involved in ligand interactions with the
corresponding residues of the P2Y6 receptor (details are given
in Supporting Information). The results obtained allow us to
propose that residues R/K6.55, Y/L6.59, and H184/Y178 of the
P2Y2/P2Y6 receptors play a critical role in UTP versus UDP
recognition. This hypothesis will be tested in future experiments
with site-directed mutagenesis.
a Reagents and conditions: (a) acetic anhydride, H2O, room temperature,
6 h, 57%.
3. Exploration of SAR at the P2Y2 Receptor with the Aid
of Modeling. To further examine determinants of P2Y2 receptor
selectivity, we synthesized several nucleotide derivatives and
tested them for activation of the human P2Y2 and P2Y4 receptors
expressed in 1321N1 astrocytoma cells (Table 2). The 2′-amino
modification of 6, which maintained potency at the P2Y2
receptor,13 was combined with another P2Y2-favorable modi-
fication of UTP, i.e., 2-thio, leading to 8. The requirement of a
free amine at this position was explored through N-acylation
reactions, leading to 9 and 10. Although a 5-methyl modification
of the uracil ring of UTP was found to reduce P2Y2 receptor
potency,13 we also examined whether introduction of a polar,
H-bonding group such as amino at this position would be
beneficial. Thus, the 5-amino 17 and 5-azido 18 analogues were
evaluated at the receptors.
3.1. Chemical Synthesis. 2′-Amino-2′-deoxy-2-thiouridine
5′-triphosphate 8 was prepared by standard phosphorylation of
the corresponding nucleoside 15. The synthesis of 2′-amino-
2′-deoxy-2-thiouridine 15 is depicted in Scheme 1. 2′-Azido-
2′-deoxyuridine 12 was obtained via opening of 2,2′-anhydrou-
ridine with NaN3 in DMF.27 After selective mesylation of the
5′-hydroxyl group of 12, the resulting 5′-methanesulfonate ester
13 was converted to 2′-azido-2′-deoxy-2-O-ethyluridine (14)
according to the method of Manoharan and co-workers.28 In
this reaction, 2,5′-anhydro-2′-azido-2′-deoxyuridine was gener-
ated in situ and subsequently opened with ethoxide. Treatment
of 14 with H2S in anhydrous pyridine allowed simultaneous
2-thionation and reduction of the 2′-azido group, resulting in
2′-amino-2′-deoxy-2-thiouridine (15). The 2-thionation was
confirmed by the 13C NMR resonance signal of C-2, which shifts
Scheme 4. Synthesis of Up4-5′-ribose 5a
a Reagents and conditions: (a) (i) DCC, DMF, room temperature, 1 h;
(ii) D-ribose 5-phosphate, DMF, room temperature, 48 h, 18%.
to a low magnetic field (177.66 ppm) compared to uridine
(150.66 ppm).
Attempts to directly phosphorylate compound 15 by a
standard method failed to yield the desired 5′-triphosphate 8.
Therefore, the 2′-amino group was protected by trifluoroacety-
lation. The phosphorylation of the protected nucleoside 16 was
performed as shown in Scheme 2.29 During purification by ion
exchange chromatography, partial hydrolysis yielded a mixture
of 8 and 9, which was readily separable by HPLC. The
acetylation of the 2′-amino group of 8 to obtain 10 was
performed as shown in Scheme 3. Up4ribose 5 was prepared as
shown in Scheme 4.
3.2. Pharmacological Activity. Activation of PLC by a range
of concentrations of each nucleotide derivatives was studied in
1321N1 astrocytoma cells stably expressing the human P2Y2,
P2Y4, and P2Y6 receptors (Table 2), by reported meth-
ods.13,21,30,31 Tritiated inositol phosphates produced from a
radiolabeled myo-inositol precursor were measured using a
standard ion exchange method.

Modeling of Receptor and Design of Agonist
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1173
P2Y2 receptor action. Modeling provided an explanation for the
general stabilizing effect of the 2′-amino modification of UTP
in P2Y2 receptor recognition. A “sequential ligand composition”
approach was adopted for docking flexible dinucleotide agonists
such as Up4U. This modeling approach, in which ligands are
docked to the receptor (including loops and terminal regions),
embedded in a phospholipid bilayer, and optimized with
MCMM, promises to be helpful in the design of additional
potent and selective P2Y2 receptor agonists.
Experimental Section
Chemical Synthesis. All reagents were from standard com-
mercial sources and of analytic grade. Compounds 17 and 18 were
purchased from ALT, Inc. (Lexington, KY). 2,2′-Anhydrouridine
(11) was obtained from Wako Chemicals GmbH (Neuss, Germany).
Precoated Merck silica gel F254 plates were used for TLC, and
spots were examined under ultraviolet light at 254 nm and further
visualized by sulfuric acid-anisaldehyde spray. Column chroma-
tography was performed on ICN silica gel (63-200 µm, 60 Å, ICN
Biochemicals, Eschwege, Germany). For compounds 11-15, NMR
spectra were obtained with a Varian Mercury 300 MHz spectrom-
eter. Chemical shifts are given in ppm (δ) relative to the residual
solvent signal; in the case of DMSO-d6, the shift is 2.54 ppm for
1H and 40.5 ppm for 13C. Structural assignment was confirmed with
correlation spectroscopy (COSY) and distortionless enhancement
by polarization transfer (DEPT) experiments. All signals assigned
to hydroxyl groups were exchangeable with D2O. Exact mass
measurements were performed on a quadruple orthogonal-accelera-
tion time-of-flight (Q/oaTOF) tandem mass spectrometer (qToF 2,
Micromass, Manchester, U.K.) equipped with a standard electro-
spray ionization (ESI) interface. Samples were infused in a
2-propanol/water (1:1) mixture at 3 µL/min. For compounds 16
Figure 5. Activation by compound 8 of PLC in 1321N1 astrocytoma
cells expressing the human P2Y2 receptor or P2Y4 receptor.
An intermediate structure related to the modeling by “se-
quential ligand composition”, Up4-[5′]-ribose 5, activated the
P2Y2 receptor with an EC50 of 1.88 µM, i.e., 9-fold less potent
than Up4U.
The 5′-triphosphate 8, which combined potency-enhancing
features derived from our previous studies, was 300-fold
selective in activation of the P2Y2 receptor in comparison with
the P2Y4 receptor (Figure 5). Compound 8 and other 2′-deoxy-
2′-amino analogues, 6, 9, and 10, were nearly inactive at the
P2Y6 receptor. The binding mode of this potent and selective
analogue was studied with MCMM calculations, and the results
were similar to those for 6, in which the 2′-amino group in its
protonated form was involved in a cation-π interaction with
F6.51 and as a H-bond donor to Y3.33 (Figure 4B). The potency
of 8 was greatly reduced upon acetylation (10) and reduced to
a lesser degree upon trifluoroacetylation (9). These observations
are consistent with the binding modes obtained for compound
10. In the model of the P2Y2 receptor complex with compound
10 (Supporting Information), the H-bond between F6.51 and
the NH group of the acetamide moiety was not observed.
Moreover, the methyl group of this moiety undesirably appeared
near the OH group of Y3.33 and the NH2 group of R6.55. The
oxygen atom of the acetyl group was not involved in interactions
with the receptor. We speculate that in the case of compound
9, the CF3 group, which is more electronegative than CH3,
provides some favorable interactions with positively charged
R6.55, improving the potency of compound 10.
The 5 position was predicted to accommodate an azido group
as well as a small, polar, H-bonding group, such as an amino
group. Building on these observations, we measured the
activities of 5-amino 17 and 5-azido 18 UTP derivatives at the
human P2Y2 receptor (Table 2). However, the results indicated
that both structures are less active than 5-methyl-UTP 19 at
this receptor.13 Similar to certain other uracil-substituted uridine
5′-triphosphates,26 compounds 17-19 also potently activated
the human P2Y6 receptor.
Introduction of an amino or azido group at position 5 of UTP
apparently influences the conformation of the ribose ring. It was
shown previously,32 through NMR analysis combined with
semiempirical calculations, that electron-donating groups at
position 5 of uracil nucleotides can destabilize the P2Y2
receptor-preferred (N)-conformation of the ribose ring.
1
and 8-10, H NMR spectra were obtained with a Varian Gemini
300 spectrometer (Varian, Inc., Palo Alto, CA). 31P NMR spectra
were recorded at room temperature with a Varian XL 300
spectrometer (121.42 MHz); orthophosphoric acid (85%) was used
as an external standard. Purity of compounds was checked with a
Hewlett-Packard 1100 HPLC equipped with a Zorbax Eclipse 5
µm XDB-C18 analytical column (250 mm × 4.6 mm; Agilent
Technologies Inc., Palo Alto, CA). Purity was measured with two
different solvent systems. (System A: 5 mM TBAP (tetrabutylam-
monium dihydrogen phosphate)/CH3CN from 80:20 to 40:60 in 20
min; flow rate of 1 mL/min. System B: 10 mM TEAA (triethy-
lammonium acetate)/CH3CN from 80:20 to 60:40 in 20 min; flow
rate of 1 mL/min. System C: 10 mM TEAA/CH3CN from 100:0
to 80:20 in 20 min; flow rate of 1 mL/min.) Purifications by HPLC
were performed under the following conditions: Luna 5 µm RP-
C18 (2) semipreparative column, 250 mm × 10.0 mm, Phenom-
enex, Torrance, CA; flow rate of 2 mL/min. System D: 10 mM
TEAA/CH3CN from 100:0 to 90:10 in 20 min. System E: 10 mM
TEAA/CH3CN from 100:0 to 95:5 in 30 min. High-resolution mass
measurements were performed on a Micromass/Waters LCT
Premier electrospray time-of-flight mass spectrometer coupled with
a Waters HPLC system (Waters Corp., Milford, MA).
2′-Azido-2′-deoxy-5′-O-methanesulfonyluridine (13). Meth-
anesulfonyl chloride (210 µL, 2.7 mmol) was added to a solution
of 12 (610 mg, 2.3 mmol) in 10 mL of pyridine at -78 °C. The
mixture was stirred for 1 h at 0 °C, quenched with aqueous saturated
NaHCO3, extracted with CH2Cl2, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by column
chromatography (CH2Cl2/MeOH, 96:4) to obtain compound 13 as
1
a white solid (472 mg, 61%). H NMR (DMSO-d6): δ 3.23 (s,
3H, CH3), 4.06 (m, 1H, H-4′), 4.25-4.48 (m, 4H, H-2′, H-3′, H-5′A,
and H-5′B), 5.68 (dd, J ) 7.9, 2.1 Hz, 1H, H-6), 5.82 (d, J ) 5.3
Hz, 1H, 3′-OH), 6.2 (d, J ) 5.6 Hz, 1H, H-1′), 7.62 (d, J ) 8.2
Hz, 1H, H-5), 11.47 (s, 1H, NH). HRMS (ESI-MS) for
C10H14N5O7S: [M + H]+ found, 348.0618; calcd, 348.0613.
2′-Azido-2′-deoxy-2-O-ethyluridine (14). Compound 13 (465
mg, 1.3 mmol) was refluxed in absolute ethanol (60 mL) in the
presence of anhydrous sodium bicarbonate (281 mg, 3.3 mmol)
Conclusions
This detailed view of molecular recognition at the P2Y2
receptor suggests mutations for further model validation. In this
study we report a highly potent and selective agonist, the 2′-
amino-2-thio derivative of UTP, compound 8, which should
prove to be very useful as a pharmacological probe for studying

1174 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
IVanoV et al.
under a nitrogen atmosphere for 36 h. After cooling to room
temperature, the mixture was diluted with ethyl acetate and the
precipitated sodium salt was removed by filtration. The filtrate was
concentrated in vacuo and purified on a silica gel column (CH2-
Cl2/MeOH, 95:5) to obtain compound 14 (222 mg, 57%) as a white
solid. 1H NMR (DMSO-d6): δ 1.33 (t, J ) 6.9 Hz, 3H, CH3), 3.56-
3.63 (m, 1H, H-5′A), 3.67-3.74 (m, 1H, H-5′B), 3.92 (m, 1H, H-4′),
4.12 (app t, J ) 4.8 Hz, 1H, H-3′), 4.27-4.39 (m, 3H, CH2 and
H-2′), 5.23 (t, J ) 4.8 Hz, 1H, 5′-OH), 5.85 (m, 2H, H-6 and 3′-
OH), 6.01 (d, J ) 5.4 Hz, 1H, H-1′), 7.98 (d, J ) 7.8 Hz, 1H,
H-5). HRMS (ESI-MS) for C11H15N5O5Na: [M + Na]+ found,
320.0974; calcd, 320.0971.
4.80 (hidden by the water peak, 1H, H-3′), 6.26 (d, J ) 8.1 Hz,
1H, H-6), 6.92 (d, J ) 5.4 Hz, 1H, H-1′), 8.13 (d, J ) 8.4 Hz, 1H,
H-5). 31P NMR (D2O): δ -22.28 (t, J ) 19.6 Hz), -11.04 (d, J )
19.6 Hz), -8.54 (m). HRMS m/z: found 593.9364 (M - H+)-.
C11H14N3O14P3F3S requires 593.9361. Purity, >98% by HPLC
(system A, 18.3 min; system B, 7.7 min).
2′-Acetylamino-2′-deoxy-2-thiouridine 5′-Triphosphate Tri-
ethylammonium Salt (10). Acetic anhydride (0.07 mL, 0.74 mmol)
was added to a solution of 8 (0.5 mg, 0.55 µmol) in H2O (0.5 mL)
at room temperature. After the reaction mixture was stirred for 6
h, the solvent was removed in vacuo. The residue was purified by
HPLC (system D) to obtain 10 (0.3 mg, 57%) as the triethylam-
monium salts. 1H NMR (D2O): δ 1.28 (t, J ) 7.5 Hz, 36H,
N(CH2CH3)3), 3.20 (q, J ) 7.5 Hz, 24H, N(CH2CH3)3), 4.29 (m,
2H, H-5′A and H-5′B), 4.36 (m, 1H, H-4′), 4.53 (m, 2H, H-2′ and
H-3′), 6.27 (d, J ) 8.1 Hz, 1H, H-6), 6.98 (d, J ) 5.6 Hz, 1H,
H-1′), 8.08 (d, J ) 8.1 Hz, 1H, H-5). 31P NMR (D2O): δ -21.31
(t, J ) 20.5 Hz), -10.84 (d, J ) 20.8 Hz), -7.62 (m). HRMS m/z:
found 539.9660 (M - H+)-. C11H17N3O14P3S requires 539.9644.
Purity, >98% by HPLC (system A, 17.2 min; system B, 6.7 min).
Diuridine 5′,5′-Tetraphosphate Ammonium Salt (4a). Com-
pound 4a was synthesized by following the procedures of ref 23.
HRMS m/z: found 788.9842 (M - H+)-. C18H25N4O23P4 requires
788.9860. Purity, >98% by HPLC (system A, 20.5 min; system
C, 7.7 min).
Uridine 5′-Tetraphosphate 5′-Ribose Triethylammonium Salt
(5). Uridine 5′-triphosphate trisodium salt (25 mg, 0.05 mmol) and
D-ribose 5-monophosphate disodium salt (59 mg, 0.19 mmol) were
converted to the tributylammonium salts by treatment with the ion-
exchange resin (DOWEX 50WX2-200 (H)) and tributylamine. After
removal of the solvent, the resulting residue was dried under high
vacuum overnight. To a solution of uridine 5′-triphosphate tribu-
tylammonium salt (0.05 mmol) in DMF (2 mL) was added N,N′-
dicyclohexylcarbodiimide (22 mg, 0.11 mmol), and the mixture was
stirred for 1 h at room temperature. A solution of d-ribose
5-monophosphate tributylammonium salt (0.19 mmol) in DMF (2
mL) was added to the reaction mixture, with stirring continuing at
room temperature for 48 h. After removal of the solvent, the residue
was purified by ion-exchange column chromatography with Sepha-
dex DEAE A-25 resin using a linear gradient (0.01-0.5 M) of 0.5
M ammonium bicarbonate as the mobile phase. Compound 5 (7.0
mg, 18%) was additionally purified by HPLC (system E). 1H NMR
(D2O): δ 1.28 (t, J ) 7.5 Hz, 36H, N(CH2CH3)3), 3.20 (q, J ) 7.5
Hz, 24H, N(CH2CH3)3), 4.11 (m, 3H, H-ribose), 4.28 (m, 4H, H-4′,
H-5′, 1H-ribose), 4.42 (m, 3H, H-2′, H-3′, 1H-ribose), 5.22 (d, J
) 2.4 Hz, 3/5H, H-1′ribose), 5.41 (d, J ) 3.6 Hz, 2/5H, H-1′ribose),
5.99 (d, J ) 8.3 Hz, 1H, H-5), 6.02 (d, J ) 5.4 Hz, 1H, H-1′), 7.99
(d, J ) 8.3 Hz, 1H, H-6). 31P NMR (D2O): δ -24.55 (m), -12.83
(m), -12.43 (m). HRMS m/z: found 694.9720 (M - H+)-.
C14H23N2O22P4 requires 694.9693. Purity, >99% by HPLC (system
A, 18.6 min; system C, 7.1 min).
2′-Amino-2′-deoxy-2-thiouridine (15). In a Parr apparatus
compound 14 (210 mg, 0.71 mmol) was dissolved in pyridine (40
mL), cooled to -50 °C, and saturated with H2S. The mixture was
heated at 50 °C for 24 h, resulting in a pressure of 250 psi. After
the mixture was cooled to room temperature, the remaining H2S
was released, the solvent was evaporated, and the residue was
purified on a silica gel column (CH2Cl2/MeOH, 90:10), yielding
compound 15 (141 mg, 77%) after subsequent crystallization from
1
MeOH. H NMR (DMSO-d6): δ 3.32 (dd, J ) 6.1, 5.0 Hz, 1H,
H-2′), 3.56-3.67 (m, 2H, H-5′A and H-5′B), 3.94 (m, 2H, H-3′
and H-4′), 5.19 (t, J ) 4.7 Hz, 5′-OH), 5.41 (br s, 1H, 3′-OH),
6.01 (d, J ) 8.2 Hz, 1H, H-6), 6.53 (d, J ) 6.0 Hz, 1H, H-1′), 8.09
(d, J ) 8.2 Hz, 1H, H-5). 13C NMR (DMSO-d6): δ 59.74 (C-2′),
61.43 (C-5′), 70.91 (C-3′), 86.50 (C-4′), 93.49 (C-1′), 107.32 (C-
5), 141.66 (C-6), 160.24 (C-4), 177.66 (C-2). HRMS (ESI-MS)
for C9H14N3O4S1: [M + H]+ found, 260.0692; calcd, 260.0704.
2′-Trifluoroacetylamino-2′-deoxy-2-thiouridine (16). N,N-Di-
isopropylethylamine (0.006 mL, 0.035 mmol) and ethyl trifluoro-
acetate (0.004 mL, 0.035 mmol) were added sequentially to a
solution of 15 (6 mg, 0.023 mmol) in DMF (1 mL). The reaction
mixture was stirred for 13 h at room temperature. The solvent was
removed in vacuo, and the residue was purified by preparative thin-
layer chromatography (CH2Cl2/MeOH, 90:10) to afford 16 (7.2 mg,
88%) as a white solid. 1H NMR (CD3OD): δ 3.84 (m, 2H, H-5′A
and H-5′B), 4.14 (dd, J ) 5.4, 2.7 Hz, 1H, H-4′), 4.34 (dd, J )
5.7, 3.0 Hz, 1H, H-3′), 4.58 (dd, J ) 6.6, 6.3 Hz, 1H, H-2′), 6.01
(d, J ) 8.1 Hz, 1H, H-6), 7.12 (d, J ) 6.9 Hz, 1H, H-1′), 8.25 (d,
J ) 8.1 Hz, 1H, H-5). HRMS m/z: found 356.0520 (M + H+)-.
C11H13N3O5F3S requires 356.0528.
2′-Amino-2′-deoxy-2-thiouridine 5′-Triphosphate Triethy-
lammonium Salt (8) and 2′-Trifluoroacetylamino-2′-deoxy-2-
thiouridine 5′-Triphosphate Triethylammonium Salt (9). Phos-
phorus oxychloride (0.004 mL, 0.04 mmol) was added to a solution
of 16 (7.2 mg, 0.020 mmol) and Proton Sponge (4 mg, 0.033 mmol)
in trimethyl phosphate (1 mL) at 0 °C. The reaction mixture was
stirred for 2 h at 0 °C. Then a mixture of tributylamine (0.02 mL,
0.08 mmol) and tributylammonium pyrophosphate (1.6 mol of
C12H27N per mol of H4PO7, 62 mg, 0.131 mmol) in DMF (0.3 mL)
was added at once. After 10 min, 0.2 M triethylammonium
bicarbonate solution (2 mL) was added, and the clear solution was
stirred at room temperature for 1 h. The latter was lyophilized
overnight, and the resulting residue was purified by ion-exchange
column chromatography with a Sephadex DEAE A-25 resin with
a linear gradient (0.01-0.5 M) of 0.5 M ammonium bicarbonate
as the mobile phase to get a mixture of 8 and 9 as the ammonium
salts. The mixture was purified by HPLC (system D) to obtain 8
(2.2 mg, 12%) and 9 (1.4 mg, 7%) as the triethylammonium salts.
Molecular Modeling. The published molecular model of the
human P2Y2 receptor1 was updated by insertion of a part of the
C-terminal (CT) domain including the H8 helix and the N-terminal
(NT) domain. The P2Y2 CT domain (residues Gly310-Met346)
was modeled by homology to bovine rhodopsin. We used the
published sequence alignment1 to superimpose the backbone atoms
of the TM domain of the P2Y2 receptor on the corresponding atoms
of bovine rhodopsin (PDB code 1U19)33 with Sybyl 7.1.34 The
rhodopsin TM domains and its hydrophilic loops (Ala26-Met309)
as well as Gly310 of the P2Y2 receptor (last residue in the published
model) subsequently were removed from the receptor structures.
The remaining rhodopsin NT (Met1-Glu25) and CT (residues
starting from Asn310) domains were connected to Gly22 and
Ala309 of the P2Y2 receptor, respectively. The residues of the CT
domain were replaced with the corresponding residues of the P2Y2
receptor (residues Gly310-Met346) with Sybyl 7.1.
1
Compound 8. H NMR (D2O): δ 1.28 (t, J ) 7.5 Hz, 36H,
N(CH2CH3)3), 3.20 (q, J ) 7.5 Hz, 24H, N(CH2CH3)3), 4.15 (dd,
J ) 6.9, 5.7 Hz, 1H, H-2′), 4.26 (m, 1H, H-5′A), 4.37 (m, 1H,
H-5′B), 4.48 (m, 1H, H-4′), 4.86 (m, 1H, H-3′), 6.31 (d, J ) 8.1
Hz, 1H, H-6), 7.22 (d, J ) 7.2 Hz, 1H, H-1′), 8.12 (d, J ) 8.1 Hz,
1H, H-5). 31P NMR (D2O): δ -22.64 (t, J ) 20.2 Hz), -11.22 (d,
J ) 20.2 Hz), -9.63 (d, J ) 19.6 Hz). HRMS m/z: found 497.9529
(M - H+)-. C9H15N3O13P3S requires 497.9538. Purity, >98% by
HPLC (system A, 15.2 min; system B, 6.6 min).
An attempt to apply the same technique for modeling of the NT
domain failed because of overlap of the rhodopsin NT domain and
the extracellular loops (ELs) of the P2Y2 receptor. For this reason,
the configuration of the P2Y2 receptor NT domain (Met1-Gly22)
1
Compound 9. H NMR (D2O): δ 1.28 (t, J ) 7.5 Hz, 36H,
N(CH2CH3)3), 3.20 (q, J ) 7.5 Hz, 24H, N(CH2CH3)3), 4.32 (m,
2H, H-5′A and H-5′B), 4.58 (m, 1H, H-4′), 4.68 (m, 1H, H-2′),

Modeling of Receptor and Design of Agonist
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1175
was predicted with the Loopy program from the Jackal package35
and 1000 initial conformations were generated. The option of energy
minimization of the generated candidates was used.
residues located within 5 Å of the ligand, with a shell of constrained
atoms with a radius of 2 Å. The following parameters were used:
MMFFs force field, water as an implicit solvent, maximum of 1000
iterations of the Polak-Ribier conjugate gradient (PRCG) mini-
The P2Y2 receptor model was minimized with Sybyl 7.1 in the
Amber7 FF99 force field. The energy minimization of side chains
of the terminal domains initially was performed with constrained
positions of all other atoms of the P2Y2 receptor until an energy
gradient lower than 0.05 kcal‚mol-1‚Å-1 was reached. The mini-
mization was continued without any constraints in the terminal
domains but with constraint of the atoms of the TM helices and
hydrophilic loops. The structure was then minimized, fixing only
the backbone atoms of the TM R-helices until an energy gradient
lower than 0.05 kcal‚mol-1‚Å-1 was reached. Finally, an uncon-
strained energy minimization of the whole P2Y2 receptor model
was performed until the energy gradient was lower than 0.05
kcal‚mol-1‚Å-1. The formal geometry of the model was tested with
the ProTable command of Sybyl 7.1 as well as the Procheck
software.18
Molecular Dynamics Simulation of the P2Y2 Receptor. The
molecular dynamics (MD) simulation of the P2Y2 receptor was
performed on the NIH Biowulf Cluster (Bethesda, MD) with
CHARMM 32a2 software.36 The protocol used for the system
construction and MD simulation was described previously.18,37 The
constructed system included the P2Y2 receptor surrounded by 100
DOPC lipids, 5964 TIP3 water molecules, and 107 Cl- and K+
ions, for a total of 37 242 atoms. The MD simulation was performed
for 10 ns under conditions of CPT (constant pressure and temper-
ature), as previously described.18 A hexagonal unit cell (69.3 Å ×
69.3 Å × 85 Å) and hexagonal periodic boundary conditions in all
directions were used.
For the first 4.5 ns of MD simulation, nuclear Overhauser effect
(NOE) restraints were applied within TM7 to the distances between
each backbone carbonyl oxygen atom of residue n and the backbone
NH group of the residue n + 4. The restraints were applied to all
residues of TM7 with the exception of prolines and residues before
and after prolines. MD simulation performed without such restraints
led to a disordered secondary structure of TM7. Similar findings
were described previously for the P2Y6 receptor.18 After 4.5 ns,
the MD simulation was continued without any restraints and the
helical structure of the TM7 remained stable until the end of the
simulation. The typical structure of the P2Y2 receptor calculated
from the last 100 ps of the MD trajectory was used as the final
model.
Manual Molecular Docking. The structure of UTP containing
all hydrogen atoms initially was sketched with Sybyl 7.1. The ligand
geometry was optimized in the Tripos force field with Gasteiger-
Hu¨ckel atomic charges.34 Available site-directed mutagenesis data
and previously published results of molecular modeling1,13,18 were
used (with the DOCK command of the Sybyl 7.1 package34) to
manually preposition UTP (with an anti conformation of the uracil
ring and an (N)-conformation of the ribose ring) inside the putative
binding site of the P2Y2 receptor. The triphosphate chain of the
ligand was placed between the R3.29, R6.55, and R7.39. The
nucleobase ring was located near Y1.39, Y2.53, and S7.43.
The manual molecular docking procedure was performed in
several stages. During the first stage of the molecular docking
process, only the bonds of the ligand were flexible. When the most
energetically favorable location and conformation of the ligand were
found, the molecular docking procedure was repeated with flexible
bonds of the receptor. During each iteration of the docking process,
the minimization of the binding site with the ligand inside was
performed until an rms of 0.01 kcal‚mol-1‚Å-1 was reached. Finally,
the energy of the entire obtained protein-ligand complex was
minimized until an energy gradient lower than 0.01 kcal‚mol-1‚Å-1
was reached.
mization method with a convergence threshold of 0.05 kJ‚mol-1‚Å-1
,
number of conformational search steps ) 100, energy window for
saving structures ) 1000 kJ‚mol-1. The ligand-receptor complex
obtained after MCMM calculations was subjected to an additional
100 steps of the mixed torsional/low-mode conformational search.
The structure of UTP docked in the receptor subsequently was
transformed to other studied UTP analogues by substitution of
functionalities, and MCMM calculations were performed for each
analogue, setting the number of steps to 100 and the energy window
for saving structures to 100 kJ‚mol-1
.
Molecular Docking of Up4U. The “subsequent ligand composi-
tion” technique was introduced to dock the dinucleotide Up4U to
the P2Y2 receptor. The UTP binding mode obtained after MCMM
calculations was used as a starting point. Initially, the δ-phosphate
group was added to UTP located inside the receptor. The resulting
uridine 5′-tetraphosphate and all residues located within 5 Å of
the ligand were subjected to MCMM calculations with a shell of
constrained atoms within a radius of 2 Å. The following parameters
were used: MMFFs force field, water as an implicit solvent,
maximum of 1000 iterations of the (PRCG) minimization method
with a convergence threshold of 0.05 kJ‚mol-1‚Å-1, number of
conformational search steps ) 100, energy window for saving
structures ) 100 kJ‚mol-1. A ribose moiety in its (N)-conformation
was then attached to the δ-phosphate group of the ligand. The
obtained molecule and all residues located within 5 Å were also
subjected to MCMM calculations with the same parameters. The
uracil ring in its anti conformation then was attached to the ligand.
The resulting Up4U and all receptor residues located within 5 Å
from the ligand were subjected to MCMM calculations with the
specified parameters. Finally, Up4U and all receptor residues located
within 5 Å of the ligand were subjected to an additional 100 steps
of the mixed torsional/low-mode conformational search.
Acknowledgment. Mass spectral measurements were carried
out by John Lloyd, and NMR measurements were carried out
by Wesley White (NIDDK). This research was supported in
part by the Intramural Research Program of the NIH, National
Institute of Diabetes and Digestive and Kidney Diseases. This
work was supported by National Institutes of Health Grants
GM38213 and HL34322 to T. K. Harden. We thank C.
Hoffmann (University of Wu¨rzburg, Germany) and C. Mu¨ller
and A. El-Tayeb (University of Bonn, Germany) for helpful
discussions.
Supporting Information Available: Details on the P2Y2
receptor model geometry; details on selective recognition; additional
references; figures illustrating the change in energy and rmsd of
the P2Y2 receptor during the MD simulation, UDP and Up4U
docked inside the P2Y2 receptor, and UTP docked in a P2Y2/P2Y6
chimeric receptor; and purity data of target compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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