Human P2Y6 receptor: Molecular modeling leads to the rational design of a novel agonist based on a unique conformational preference

Article abstract of DOI:10.1021/jm050911p

Combining molecular dynamics (MD) in a hydrated phospholipid (DOPC) bilayer, a Monte Carlo search, and synthesis of locked nucleotide analogues, we discovered that the Southern conformation of the ribose is preferred for ligand recognition by the P2Y

Full text of DOI:10.1021/jm050911p

8108
J. Med. Chem. 2005, 48, 8108-8111
Chart 1
Human P2Y6 Receptor: Molecular
Modeling Leads to the Rational Design of
a Novel Agonist Based on a Unique
Conformational Preference
Stefano Costanzi,*,† Bhalchandra V. Joshi,‡
Savitri Maddileti,§ Liaman Mamedova,‡
also referred to as θ).4,5 Using conformationally locked
nucleotide analogues, we deduced a preference for the
Northern (N) hemisphere of the pseudorotational cycle
(2′-exo, 3′-endo) for the P2Y1, P2Y2, P2Y4, and P2Y11
receptors.4,6,7,8 However, the UDP analogue 2 locked in
the (N) conformation by a (N)-methanocarba system was
found completely inactive (Table 1, Figure 1).7 Hence,
prior to this work, the P2Y6 receptor has been an elusive
subtype of the P2Y1-like receptors and the conformation
of the ribose moiety of UDP required for the recognition
of the ligand was not established.
Maria J. Gonzalez-Moa,| Victor E. Marquez,|
T. Kendall Harden,§ and Kenneth A. Jacobson*,‡
Computational Chemistry Core Laboratory and Molecular
Recognition Section, NIDDK, National Institutes of Health,
DHHS, Bethesda, Maryland 20892, Department of
Pharmacology, University of North Carolina, School of
Medicine, Chapel Hill, North Carolina 27599, and
Laboratory of Medicinal Chemistry, NCI, National
Institutes of Health, DHHS, Frederick, Maryland 21702
Received September 14, 2005
Here we report the discovery of the biologically active
conformation of the sugar moiety of UDP, which we
accomplished by means of an interdisciplinary approach
combining advanced molecular modeling techniques
with the synthesis of conformationally locked nucleotide
analogues. This finding represents a breakthrough
toward the understanding of the biology of the P2Y6
receptor, as it may be the key to the design of the long
needed pharmacological tools.
Abstract: Combining molecular dynamics (MD) in a hydrated
phospholipid (DOPC) bilayer, a Monte Carlo search, and
synthesis of locked nucleotide analogues, we discovered that
the Southern conformation of the ribose is preferred for ligand
recognition by the P2Y6 receptor. 2′-Deoxy-(S)-methanocar-
baUDP was found to be a full agonist of the receptor and
displayed a 10-fold higher potency than that for the corre-
sponding flexible 2′-deoxyUDP. MD results also suggested a
conformational change of the second extracellular loop conse-
quent to agonist binding.
To obtain a refined model of the P2Y6 receptor that
could be exploited to identify the conformation of the
ribose moiety required for the molecular recognition of
UDP, we submitted our rhodopsin-based homology
model of the receptor1 to 10 ns of molecular dynamics
(MD) in a fully hydrated phospholipid (dioleoylphos-
phatidylcholine, DOPC) bilayer, which was constructed
around the receptor according to a modification of the
stepwise procedure developed by Woolf and Roux.9 The
simulation was conducted with the program CHARMM10
(details given in Supporting Information).
To date, bovine rhodopsin is the only GPCR for which
an experimental structure has been elucidated and
therefore is the only suitable template for the construc-
tion of homology models of the other members of the
superfamily. Homology modeling has proven to be a very
powerful technique, even when the template and the
protein to be modeled are only distantly related.11
However, although a common general structure is likely
to be shared by all GPCRs, specific differences do exist
between rhodopsin and the other receptors. In particu-
lar, many GPCRs, including the P2Y receptors, show a
different distribution of Pro residues within the seven
transmembrane (TM) domains when compared to rhodop-
sin, suggesting the likelihood of conformational differ-
ences.1 Moreover, the intracellular and extracellular
regions of the GPCRs show a high variability in the
amino acid sequence, which suggests a very limited
structural similarity to rhodopsin. An MD simulation
of a membrane protein in a hydrated lipid bilayer
permits an unconstrained relaxation of the protein
model within the context of its natural environment,
allowing the backbone and the side chains to search for
a stable conformation.
P2Y receptors are a family of class A G protein-
coupled receptors (GPCRs), variously activated by ex-
tracellular purine and pyrimidine nucleotides.1,2 The
family is composed of two distinct subgroups, which
differ in overall sequence similarity, mechanism of
ligand recognition, and coupling to second messengers.
The P2Y1-like subgroup encompasses the P2Y1, P2Y2,
P2Y4, P2Y6, and P2Y11 receptors, which couple mainly
to the stimulation of phospholipase C (PLC) via Gq. On
the other hand, the P2Y12-like subgroup encompasses
the P2Y12, P2Y13, and P2Y14 receptors, which couple
mainly to the inhibition of adenylyl cyclase (AC) via Gi.
The P2Y6 receptor is the only P2Y receptor whose
natural agonist is uridine-5′-diphosphate (UDP, 1)
(Chart 1). It has been one of the least studied subtype
of the P2Y1-like subgroup, due to the lack of potent
ligands. It has recently been shown that the activation
of the P2Y6 receptor induces Ca2+ and Cl- secretion in
mouse trachea. Hence, P2Y6 agonists may be useful to
stimulate electrolyte transport in the airways of cystic
fibrosis patients.3
It is known that the five-membered ring of the ribose
moiety of nucleosides and nucleotides can adopt a range
of conformations, which can be exhaustively described
by two parameters indicative of puckering: P (phase
angle of pseudorotation, schematically represented as
a pseudorotational cycle) and ν (puckering amplitude,
* Corresponding authors. (S.C.) Email: stefanoc@mail.nih.gov.
Phone: 301-451-7353. Fax: 301-443-8000. (K.J.) Email: kajacobs@
helix.nih.gov. Phone: 301-496-9024. Fax: 301-480-8422.
† Computational Chemistry Section, NIDDK.
‡ Molecular Recognition Section, NIDDK.
§ University of North Carolina.
| Laboratory of Medicinal Chemistry, NCI.
10.1021/jm050911p CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/23/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8109
conserved throughout the MD simulation, a root-mean-
square deviation (RMSD) of 2.70 Å was found between
the backbone of the 7 TMs of the initial and the final
structures. In particular, the P2Y6 receptor contains four
Pro residues, not conserved in rhodopsin, at positions
1.36, 1.37, 2.58, and 7.40. These Pro residues caused
the formation of kinks in the upper segments of TM1,
TM2, and TM7 during the MD simulation. The amphi-
pathic helix 8 (H8), located at the cytoplasmic end of
TM7, at the interface between the lipid bilayer and the
cytosolic water, remained stable throughout the trajec-
tory (Figure 2a). Among the extracellular regions, the
second extracellular loop (EL2) has been recognized as
particularly important for the ligand recognition and for
the activation of various GPCRs, including the P2Y
receptors.12-15 In rhodopsin, and hence in our initial
homology model of the P2Y6 receptor, EL2 forms a
â-hairpin parallel to the plane of the membrane, which
occludes the entry to the cavity within the helical
bundle. In our MD simulation of the P2Y6 receptor, EL2
moved toward TM3 and further up into the extracellular
space, making the putative nucleotide binding site
accessible to an approaching ligand.
Figure 1. Activation of the human P2Y6 receptors by uracil
nucleotides. PLC activity was measured as described in the
Supporting Materials in 1321N1 human astrocytoma cells
stably expressing the receptor. The data are the means of
triplicate determinations and are representative of results
obtained in three separate experiments.
The typical structure of the system occurring during
the time frame from 9.9 to 10 ns of the MD was used
for the ligand docking experiments. UDP was positioned
into the putative binding pocket in line with our
previously published binding mode of nucleotides at the
P2Y1-like receptors.1,4 According to this experimentally
supported binding mode, the sugar moiety is located
between TM3 and TM7, the base points toward TM1
and TM2, while the diphosphate moiety points in the
direction of TM6.
A conformational analysis of the ligand inside the
binding pocket, with particular attention to the sugar
puckering, was conducted by means of the Monte Carlo
Multiple Minimum (MCMM) conformational sampling
as implemented in MacroModel (Schro¨dinger, LLC). In
order to keep the bias on the ribose puckering as low
as possible, the search was seeded with one UDP
molecule in the (N) and one in the Southern ((S), 2′-
endo, 3′-exo) conformation, minimized within the bind-
ing pocket. All the torsional angles of the ligand and
the side chains of the residues within 5 Å from the
ligand were subjected to the Monte Carlo search. At the
same time, the ligand was subjected to Monte Carlo
rotations and translations.
The search produced 10 receptor-ligand complexes
compatible with our previously published1,4 binding
mode. The 10 complexes fell into two distinct clusters,
which identified two overlapping binding modes of UDP
within the exofacial side of the cavity delineated by
TM1, TM2, TM3, TM6, and TM7 (Figure 2b). The more
external pocket (Figure 2c) could be a meta-binding site
to which the ligand binds in its path to the principal
binding site. Similar meta-binding sites have been
previously hypothesized for the P2Y1 receptor.15 The
meta-binding site cluster was composed of three recep-
tor-ligand complexes, two with UDP in the (S) confor-
mation and one with UDP in the (N) conformation. The
more deeply buried binding site cluster consisted of
seven receptor-ligand complexes, all with UDP in the
(S) conformation (Figure 2d). Thus, our conformational
search indicated a clear preference of P2Y6 for the (S)
Figure 2. (a) The P2Y6 receptor before (thin tube) and after
(thick tube) MD. (b) Tube representation of P2Y6 with ten UDP
molecules docked at the meta and the principal binding sites.
(c and d) Details of two complexes representative of the meta
(c) and the principal (d) binding sites. (e) Typical structure of
the P2Y6-UDP complex after 5 ns of MD. (f) An electronic
interaction between R103(3.29) and D179(EL2) is present in
the unoccupied P2Y6 receptor (thin tube, cyan carbon atoms)
and is lost upon MD of the receptor-ligand complex (thick
tube, brown carbons). 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.
In Figure 2a we report a superimposition of the P2Y6
homology model and the typical structure occurring in
the time frame from 9.9 to 10 ns of the MD simulation.
To facilitate the comparison among receptors, through-
out this paper we use the GPCR residue indexing
system, as explained in the Supporting Information.
Although the overall structure of the helical bundle was

8110 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Letters
conformation of the ribose moiety of UDP. Although
UDP could bind to the meta-binding site in the (N)
conformation, the (S) conformation seemed to be exclu-
sively required for binding to the principal binding site.
In all the docking poses (Figure 2b-d), the phosphate
moiety of UDP bound to the same positively charged
subpocket formed by three cationic amino acids from
TM3, TM6, and TM7, namely R103(3.29), K259(6.55),
and R287(7.39). These residues, which are conserved as
positive charges in all the subtypes of the P2Y1-like
subgroup of the P2Y receptors, were found essential for
the activation of the P2Y1 receptor by nucleotides in site-
directed mutagenesis experiments.16 Our previous mo-
lecular modeling studies suggested their involvement
in the coordination of the phosphate moieties in all the
P2Y1-like receptors.1 The fact that in the meta-binding
site the diphosphate moiety of UDP was already bound
to the aforementioned positively charged pocket sug-
gests that the electrostatic attraction between this
pocket and the negatively charged diphosphate could
be the driving force of the ligand binding process.
In the principal binding site the conserved S291(7.43)
was involved in the coordination of the 2-O of the
pyrimidine moiety of UDP (Figure 2). S7.43 was also
found essential for the activation of P2Y1 by nucle-
otides16 and was suggested to be involved in the
coordination of the nucleobase on the basis of molecular
modeling results.1 Conversely, in the meta-binding site
S291(7.43) was hydrogen bonded to Y33(1.39). Molecular
modeling results had previously suggested the impor-
tance of the H-bond between the conserved Y1.39 and
S7.43 in the stabilization of the ground state of the P2Y
receptors.1 The loss of this H-bond consequent to shift-
ing of UDP from the meta to the principal binding site,
could play a role in the activation of the receptor.
Mutagenesis studies aimed at further investigating this
working hypothesis are currently ongoing in our labo-
ratories.
To refine the receptor-ligand interactions and to
study the stability of the (S) conformation of UDP within
the receptor binding pocket, a P2Y6-UDP complex
representative of the principal binding site was submit-
ted to 5 ns of MD in the DOPC bilayer environment.
The typical structure occurring in the time frame from
14.9 to 15 ns of the MD simulation is represented in
Figure 2e. The interactions among the diphosphate
moiety of UDP and the three cationic residues from
TM3, TM6, and TM7 remained stable throughout the 5
ns of MD. Y283(7.35) also engaged a stable H-bond with
the diphosphate. Consistently, we previously found that
Ala and Phe mutations of the corresponding residue in
the P2Y1 receptor led to 10-fold reduced affinity for the
agonist 2-MeSADP.1 The role of Y7.35 in the ligand
binding could not be established in docking experiments
conducted prior to MD. The 3′-OH group of UDP
established stable H-bonds, mediated by a water mol-
ecule, with K259(6.55) and, intramolecularly, with the
5′-O. The interaction between S291(7.43) and the 2-O
of UDP remained stable throughout the simulation, and
Y33(1.39) established a stable H-bond with the 3-NH
of UDP. The uracil ring was stabilized by hydrophobic
interactions with the conserved residues F106(3.32),
L29(1.35), and I83(2.61).
ns of the MD of the unoccupied P2Y6 and was lost in
the MD of the P2Y6-UDP complex. In fact, R103(3.29)
engaged in electrostatic interaction with the diphos-
phate moiety of UDP, while D179(EL2) was repelled
toward the extracellular space by the negative charge
of the ligand (Figure 2f). The R103(3.29)-D179(EL2)
interaction, which seems fundamental in the stabiliza-
tion of the unoccupied P2Y6 receptor, could not be
detected prior to the MD simulation. The presence of
the ligand in the P2Y6 binding pocket caused the
disruption of this interaction and a consequent sub-
stantial conformational change of EL2, whose backbone
showed an RMSD of 3.81 Å between the typical struc-
tures of the unoccupied and occupied receptors. A
movement of EL2 is associated with the activation of
other GPCRs.13,14 Previous studies suggested that the
corresponding R128(3.29) and D204(EL2) of the P2Y1
receptor are involved in the process of ligand recognition
and/or receptor activation, since in both cases a muta-
tion to Ala highly impaired the ability of the agonist
2-MeSATP to stimulate the receptor.12,16 Our MD
simulation suggests a similar role for the corresponding
residues of the P2Y6 receptor; however, for P2Y6 as well
as other subtypes of the P2Y receptors, this hypothesis
remains to be tested using mutagenesis experiments.
Besides EL2, the backbone of the P2Y6 receptor did not
show significant changes after the 5 ns of MD in
complex with the ligand. The average RMSD between
the backbone of the typical structures of the unoccupied
and the occupied receptors was 1.14 Å for the TMs and
2.29 Å for the loops, excluding EL2.
The ribose moiety of UDP maintained stable torsional
angles throughout the simulation, showing, in the final
typical structure, a P of 180° and a ν of 36°, indicative
a pure (S) conformation. We have reported elsewhere
the formulas to derive P and ν from the five torsional
angles of a five-membered ring.4,5 During the whole time
of the simulation, the (S) conformation of the ribose ring
ensured the optimal fitting of the sugar moiety of UDP
in the P2Y6 binding pocket. Furthermore, it conferred
to the phosphate and the nucleobase moieties the most
favorable orientation for the establishment of interac-
tions with the residues from TM1, TM3, TM6, and TM7
(Figure 2e).
On the basis of the molecular modeling results, we
devised the synthesis of a carbocyclic analogue of UDP
locked in the (S) conformation by a (S)-methanocarba
modification of the ribose ring. The methanocarba
system, introduced by Marquez and co-workers, consists
of a cyclopentane ring fused with a cyclopropane ring
and can be used to constrain a pseudosugar ring in the
(N) or the (S) conformations, depending on the position
of the fusion.17 The starting nucleoside necessary for the
synthesis of the enantiomerically pure (S)-2′-deoxy-
methanocarbaUDP was readily available in our labo-
ratories, since it was earlier synthesized and tested for
its antiviral activity by Marquez and co-workers.18
Furthermore, we inferred from the model that the 2′-
OH group, although important in the stabilization of
the ligand through an intramolecular H-bond with the
2-O, was not involved in crucial interactions with the
receptor. Therefore, we evaluated the potency and the
efficacy of 2′-deoxyUDP (3) in activation of PLC in
1321N1 astrocytoma cells stably transfected with the
human P2Y6 receptor, and discovered that, although
An electrostatic interaction between R103(3.29) and
the conserved D179(EL2) was stable throughout the 10

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8111
Table 1. EC50 Values for Stimulation of PLC in 1321N1
Astrocytoma Cells Stably transfected with the Human P2Y6
Receptor
modeling. This research was supported in part by the
Intramural Research Program of the NIH, NIDDK.
compd
PLC, EC50 (µM)
Supporting Information Available: Experimental pro-
cedures including spectral and analytical data of the new
compounds. Figure representing the P2Y6-UDP complex
embedded in the hydrated DOPC bilayer. Enlarged version of
Figure 2. This material is available free of charge via the
Internet at http://pubs.acs.org.
1, UDP
0.086 ( 0.007
inactive7
1.72 ( 0.76
0.23 ( 0.05
2
3, 2′deoxy-UDP
10
Scheme 1a
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less potent than UDP (1), it maintained full agonistic
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Supporting Information) began with the (S)-2′-deoxy-
methanocarba uridine (4), which was prepared accord-
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al.18
In agreement with the prediction by molecular model-
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P2Y6 receptor, but it displayed an EC50 of 0.23 µM and
proved to be 10-fold more potent than the corresponding
2-deoxyUDP 3 (Table 1, Figure 1).
In conclusion, compound 10 represents a successful
case of structure-based drug design and is the first
example of a nucleotide locked in the (S) conformation
found to activate any P2Y subtype. The discovery of the
activity of this novel locked nucleotide provides the first
lead for the development of potent and selective P2Y6
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receptor. The synthesis of the (S)-methanocarbaUDP,
which is expected to be more potent than its 2′-deoxy
analogue is currently in progress.
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Acknowledgment. The authors thank Dr. Anny-
Odile Colson for helpful the discussions on molecular
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