4-Alkyloxyimino-cytosine nucleotides: Tethering approaches to molecular probes for the P2Y6 receptor

Article abstract of DOI:10.1039/c3md00132f

4-Alkyloxyimino derivatives of pyrimidine nucleotides display high potency as agonists of certain G protein-coupled P2Y receptors (P2YRs). In an effort to functionalize a P2Y₆R agonist for fluorescent labeling, we probed two positions (N⁴ and γ-phosphate of cytidine derivatives) with various functional groups, including alkynes for click chemistry. Functionalization of extended imino substituents at the 4 position of the pyrimidine nucleobase of CDP preserved P2Y₆R potency generally better than γ-phosphoester formation in CTP derivatives. Fluorescent Alexa Fluor 488 conjugate 16 activated the human P2Y₆R expressed in 1321N1 human astrocytoma cells with an EC₅₀ of 9 nM, and exhibited high selectivity for this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific labeling with 16 to P2Y ₆R-expressing but not to wild-type 1321N1 cells. Additionally, confocal microscopy indicated both internalized 16 (t_1/2 of 18 min) and surface-bound fluorescence. Known P2Y₆R ligands inhibited labeling. Theoretical docking of 16 to a homology model of the P2Y₆R predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we have identified the N⁴-benzyloxy group as a structurally permissive site for synthesis of functionalized congeners leading to high affinity molecular probes for studying the P2Y₆R.

Full text of DOI:10.1039/c3md00132f

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4-Alkyloxyimino-cytosine nucleotides: tethering
approaches to molecular probes for the P2Y6 receptor†
Cite this: Med. Chem. Commun., 2013,
4, 1156
P. Suresh Jayasekara,a Matthew O. Barrett,b Christopher B. Ball,b Kyle A. Brown,b
Eszter Kozma,a Stefano Costanzi,c Lucia Squarcialupi,a
Ramachandran Balasubramanian, Hiroshi Maruoka and Kenneth A. Jacobson
a
a
a
*
4-Alkyloxyimino derivatives of pyrimidine nucleotides display high potency as agonists of certain G protein-
coupled P2Y receptors (P2YRs). In an effort to functionalize a P2Y6R agonist for fluorescent labeling, we
probed two positions (N4 and g-phosphate of cytidine derivatives) with various functional groups,
including alkynes for click chemistry. Functionalization of extended imino substituents at the 4 position
of the pyrimidine nucleobase of CDP preserved P2Y6R potency generally better than g-phosphoester
formation in CTP derivatives. Fluorescent Alexa Fluor 488 conjugate 16 activated the human P2Y6R
expressed in 1321N1 human astrocytoma cells with an EC50 of 9 nM, and exhibited high selectivity for
this receptor over other uridine nucleotide-activated P2Y receptors. Flow cytometry detected specific
labeling with 16 to P2Y6R-expressing but not to wild-type 1321N1 cells. Additionally, confocal
microscopy indicated both internalized 16 (t1/2 of 18 min) and surface-bound fluorescence. Known
P2Y6R ligands inhibited labeling. Theoretical docking of 16 to a homology model of the P2Y6R
predicted electrostatic interactions between the fluorophore and extracellular portion of TM3. Thus, we
have identified the N4-benzyloxy group as a structurally permissive site for synthesis of functionalized
congeners leading to high affinity molecular probes for studying the P2Y6R.
Received 10th May 2013
Accepted 30th May 2013
DOI: 10.1039/c3md00132f
www.rsc.org/medchemcomm
Potent P2Y6R agonists (EC50 values, nM) include 3-phenacyl-
UDP 2 (70),11 derivatives of the 5 position 3 (15) and 4 (300),12,13
Introduction
The P2Y6 receptor (P2Y6R) belongs to a family of eight G
protein-coupled receptors (GPCRs) activated by extracellular
nucleotides.1 The P2Y6R, which is both cytoprotective and
proinammatory, promotes inositol lipid signaling through
Gq mediated activation of phospholipase C-b (PLC-b) isozymes
and is found in intestinal epithelial, endocrine, skeletal
muscle, bone, neuronal, vascular, immune and other cells.1–10
Pharmacological modulation of the P2Y6R has been proposed
to be useful in treatment of osteoporosis, neurodegeneration,
gout, ocular hypertension, glaucoma, inammation, intestinal
disorders, diabetes, and other diseases.2–10
a bicyclic analogue 5 (MRS2795, 42) of UDP that maintains a
South (S) conformation of the ribose-like ring,14 N4-benzyloxy-
CDP 6 (MRS2964, 26) and various dinucleoside triphos-
phates,14–16 including Up3U
7 8
(270), N4-methoxy-Cp3U
(MRS2957, 12), and INS48823 9 (130). Here, we considered
structural extensions of UDP that would both preserve P2Y6R
agonist potency and permit the coupling of reporter groups or
other large chemical carrier moieties.
A functionalized
congener approach to ligand derivatization has been explored
for a variety of GPCRs, leading to uorescent probes,18 affinity
labels, cross-linking ligands, and specialized radioactive
probes.39 SAR analyses suggested that the steric constraints of
the pharmacophoric binding site of the receptor likely could
be circumvented by chain elongation through two specic
sites in P2Y6R agonists. Thus, we introduced a 4-alkyloxyimino
group in the nucleobase, and the terminal phosphate group
was extended in g-ester derivatives of UTP. The potent
N4-benzyloxy derivative 6, which was 82- and 44-fold P2Y6R-
selective vs. P2Y2 and P2Y4Rs, respectively,14 served as the lead
compound for a series of 4-oxyimino analogues of cytidine 50-
diphosphate in the present study. Similarly, several O-alkyl
and aryl ester derivatives14 of the g-phosphate of UTP sug-
gested extension in that region as a possible tethering
approach for P2Y6R agonists.
Uridine 50-diphosphate (UDP 1) is the endogenous P2Y6R
agonist (EC50 ¼ 0.30 mM), and various structure activity rela-
tionship (SAR) studies have identied sites for modication of
UDP that improve its potency and/or selectivity (Chart 1).11–17
aMolecular Recognition Section, Laboratory of Bioorganic Chemistry, National
Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, Bethesda, MD 20892-0810, USA. E-mail: kajacobs@helix.nih.gov; Fax: +1
301-480-8422; Tel: +1 301-496-9024
bDepartment of Pharmacology, University of North Carolina, School of Medicine,
Chapel Hill, North Carolina 27599-7365, USA
cDepartment of Chemistry, American University, Washington, DC 20016, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3md00132f
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Chart 1 Structures of prototypical agonist ligands for studying P2Y6Rs.
This work has a two-fold goal: (1) the exploration of SAR of mixture was treated with phosphoric acid to produce 50-
pyrimidine nucleotides at the P2YRs leading to a successful diphosphates. The desired N4-alkoxycytidine 50-triphosphates
strategy for tethering large reporter groups to the pharmaco- 19 and 21 were synthesized from corresponding N4-alkoxy-
phore, and (2) the characterization of a new uorescent probe as cytidines 32 and 33 aer treatment with phosphorous oxy-
a useful tool for studying the P2Y6R. We chose to study a uo- chloride followed by bis(tri-n-butylammonium) pyrophosphate.
rescent probe initially using ow cytometry (FCM) and confocal The g-phosphoester derivatives 20, 22, and 23 were prepared by
microscopy. Similar to previous studies of uorescent agonists the condensation of corresponding alkyl monophosphoric acid
of the A2A and A3 adenosine receptors,18a,34 there was signicant with the corresponding N4-alkoxycytidine 50-diphosphate in the
internalization of this uorescent GPCR agonist in P2Y6R- presence of N,N0-diisopropylcarbodiimide (DIC).
expressing cells.
The uorescent 50-diphosphates (N4-(3-(AlexaFluor-NH-
(CH2)6-triazole-(CH2)2-C^C)-benzyloxy))-CDP 16 and N4-(3-
(Cy5-NH-(CH2)6-triazole-(CH2)2-C^C)-benzyloxy)-CDP 17 and
model compound g-(CH3CONH-(CH2)2-triazole-(CH2)2)-N4-ben-
Results and discussion
Novel nucleotide derivatives for testing at P2YRs (Table 1) were zyloxy-CTP 24 were synthesized from alkynes 15 and 22 using
prepared by the synthetic routes shown in Schemes 1 and 2. The copper-catalyzed click cycloaddition reactions (Scheme 2).22 The
nucleotide products for biological testing were puried by 50-monophosphates 42 and 44 corresponding to the two uo-
HPLC to assure high purity (>95%). The types of nucleotide rescent analogues 16 and 17, respectively, were also isolated as
modications include: CTP and CDP analogues containing a N4- byproducts of the reaction.
alkoxy group (Schemes 1 and 2); Cp3 analogues containing a
Functional assays of the nucleotide analogues (Table 1)
substituted g-alkyl group at the terminal phosphate (Scheme 1). consisted of measuring potency for activation of phospholipase
Click chemistry, especially [2 + 3] cycloaddition of acetylene and C (PLC) in 1321N1 human astrocytoma cells stably expressing
azide groups,19 is oen used as a linking reaction for preparing the human (h) P2Y6R (1321N1-P2Y6R cells),23 and selectivity was
conjugates of biologically active small molecules, and therefore, assessed by quantication of PLC activation in 1321N1 cells
we chose to incorporate alkynyl groups in the nucleotide stably expressing the hP2Y2R or hP2Y4R. Previously reported
analogues.
data for 50-diphosphate analogue 6, dinucleotides 7 and 8, and
The synthesis of N4-alkoxycytidines 32–38 from cytidine was 50-triphosphate analogues 19 and 21 were included for
performed using corresponding alkoxyamines 25–31. The comparison (Table 1).14
resulting N4-alkoxycytidines were phosphorylated by standard
High potency at the P2Y6R was achieved with N4-benzyloxy-
methods14,16,20 to give the desired N4-alkoxycytidine 50-diphos- cytidine 50-diphosphates, including various benzyl-substituted
phates 10–15 (Scheme 1). N4-3-(1,5-Hexadiynyl)-benzyloxy- analogues. Halogenated analogues 10–13 were compared with
cytidine 39 was prepared from N4-3-iodobenzyloxycytidine 35 14, which has a strong electron withdrawing nitro group and is
using a palladium-catalyzed cross coupling Sonogashira reac- highly selective for the P2Y6R. 3-Bromo derivative 10 was 160-
tion.21 In each case, the unprotected nucleoside was rst treated and >260-fold selective toward the P2Y6R, in comparison to
with phosphorous oxychloride, and aer 2 h the reaction the P2Y2R and P2Y4R, respectively. N4-(3-iodobenzyloxy)-CDP
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Table 1 Potency of a series of pyrimidine nucleotide derivatives at three subtypes of hP2YRs
Potency, EC50, nM or % activation
Structure, R1 ¼
P2Y6
P2Y2
P2Y4
a
a
a
No.
Diphosphates
6b
]N–O–Bn
]N–O–(3-Br–Bn)
]N–O-(3-I–Bn)
]N–O–(4-Br–Bn)
]N–O–(4-I–Bn)
]N–O–(4-NO2–Bn)
]N–O–(3-(HC^C(CH2)2C^C)–Bn)
]N–O–(3-(AlexaFluor–NH–(CH2)6-
triazole–(CH2)2–C^C)–Bn)
]N–O–(3-(Cy5–NH–(CH2)3–
triazole–(CH2)2–C^C)–Bn)
26 ꢀ 2
2130 ꢀ 640
6200 ꢀ 2100
2500 ꢀ 800
5200 ꢀ 90
NE
3300 ꢀ 510
3900 ꢀ 1200
2500 ꢀ 660
1150 ꢀ 150
>10 000
352 ꢀ 62
7440 ꢀ 500
<50%c
2200 ꢀ 320
952 ꢀ 205
<50%c
10
11
12
13
14
15
16
39 ꢀ 2.2
19 ꢀ 5
32 ꢀ 4.1
148 ꢀ 37
41 ꢀ 2.0
100 ꢀ 11
9 ꢀ 1.9
17
ND
ND
ND
Dinucleoside triphosphates
7b,d
]O
]N–O–Me
]N–O–Bn
270 ꢀ 70
12 ꢀ 3
1310 ꢀ 210
170 ꢀ 40
870 ꢀ 110
790 ꢀ 120
636 ꢀ 42
8b
18
126 ꢀ 17
1700 ꢀ 500
Triphosphates and g-phosphoesters
R1
R2
H
P2Y6
P2Y2
P2Y4
a
a
a
Compd
19b
20
]N–O–Me
]N–O–Me
]N–O–Bn
]N–O–Bn
]N–O–Bn
]N–O–Bn
130 ꢀ 21
731 ꢀ 28
230 ꢀ 37
1750 ꢀ 280
>10 000
28 ꢀ 4
25 ꢀ 3
2-Naphthyl
H
HC^C(CH2)2
HC^C(CH2)3
CH3CONH–(CH2)2–
triazole(CH2)2
2900 ꢀ 200
620 ꢀ 75
<50%c
<50%c
21b
22
23
97 ꢀ 14
<50%c
NE
<50%c
24
879 ꢀ 32
3800 ꢀ 690
3300 ꢀ 1200
a
Functional assays were conducted with 1321N1 astrocytoma cells expressing recombinant hP2Y2, hP2Y4, or hP2Y6. Values are expressed as the
b
c
d
mean ꢀ SEM. ND, not determined. Data from Maruoka et al., 2010, 2011.14,20 Percent of maximal activation by full agonist at 10 mM. 7,
Up3U, 10, MRS4141; 16, MRS4129; 17, MRS4137; 42, MRS4158 (50-monophosphate derivative of 16). NE – no effect. ND – not determined.
(11, EC50 19 nM) was more potent than the corresponding derivatives, the enlargement of the N4-methyloxy in 8 to
4-iodo isomer 13. This nding led to the introduction at the N4 N4-benzyloxy in 18 reduced both potency and selectivity at the
position in 15 of a terminal alkyne for tethering a sterically P2Y6R.
bulky group by click chemistry. P2Y6R potency was largely
UDP and its 50-diphosphate derivatives are known to potently
retained in this 3-dialkyne, with an EC50 value of 100 nM and at activate the P2Y14R.40 However, CDP analogues containing a
least one order of magnitude subtype selectivity. In general, the N4-alkoxy group were shown to be only weakly active at this
50-diphosphate derivatives containing various N4-benzyloxy subtype.14 Consistently, 16 at concentrations up to 10 mM did
substitutions at the m- or p-position were potent and selective at not activate the hP2Y14R to inhibit forskolin-stimulated ade-
the P2Y6R, suggesting that this region of the ligand protrudes nylate cyclase (in C6 rat glioma cells stably expressing the
outside of the steric and electronic constraints of the principal receptor,40 Fig. S1†). Thus, the Alexa Fluor 488 conjugate 16
binding site. The P2Y6R selectivity of uorescent agonist 16 in promises to be a potent and relatively selective ligand probe of
comparison to the P2Y2R and P2Y4R was 280- and >1100-fold, the P2Y6R.
respectively. This was especially striking, and its potency sur-
Derivatization at the g-phosphate moiety of 50-triphosphates
passed that of the immediate precursor 15 lacking the uo- was less favorable than at the N4 position for retention of
rophore by 11-fold. However, among dinucleoside triphosphate potency and selectivity at the P2Y6R. A naphthyl ester of the
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Scheme 1 Synthesis of various pyrimidine ribonucleoside 50-tri and 50-diphosphates.
Scheme 2 Synthesis of fluorescent conjugates 16 and 17 click-linked through the N4 position and a model compound 24 click-linked through the b-phosphate.
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Fig. 1 (A) FCM histogram following 3 h incubation of 1321N1 astrocytoma cells expressing the P2Y6R in 400 nM 16 (MRS4129), showing the total binding (red),
autofluorescence (blue) and nonspecific binding (green). The histogram represents data of one of the three independent experiments. (B–E) FCM histograms following
60 min incubation of: (B) control 1321N1 astrocytoma cells with various concentrations of P2Y6R agonist 16; (C) control 1321N1 astrocytoma cells with various
concentrations of inactive 50-monophosphate derivative 42; (D) 1321N1 astrocytoma cells expressing the P2Y6R with various concentrations of P2Y6R agonist 16; (E)
1321N1 astrocytoma cells expressing the P2Y6R with various concentrations of inactive 50-monophosphate derivative 42.
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g-phosphate moiety 20 failed to enhance potency at the P2Y6R
in comparison to 19. Several alkynyl g-phosphoester model
compounds exhibited substantially reduced P2Y6R potency in
comparison to the corresponding 50-triphosphate 21. A terminal
alkynyl ester 22 was signicantly more potent than the higher
homologue 23, which was nearly inactive at the P2Y6R. A model
click product, triazole 24 with an amide-containing terminal
chain, had improved potency compared to precursor 23 but still
had an EC50 value of only 879 nM.
Thus, the pyrimidine N4 position of CDP was thought to
provide a better prospect than the g-phosphate moiety of CTP
derivatives, for chain extension with retention of potency at the
P2Y6R. N4-linked uorescent conjugates, such as the Alexa Fluor
488 conjugate 16, were considered for use as uorescent probes
in assays using 1321N1-P2Y6R cells. Compound 16 proved to be
a useful uorescent probe for FCM, a technique that has proven
effective for studying other GPCRs in intact cells.18 The uo-
rescent dye Alexa Fluor 488 is a suitable uorophore for FCM
binding assays and live cell imaging.18,34
The visible uorescence spectrum of aqueous 16 displayed
absorption and emission maxima at 494 and 518 nm,
respectively (ESI, Fig. S2†). This uorescent probe was
initially tested in FCM aer a long incubation period to allow
internalization. A FCM histogram of the total binding and
nonspecic binding to 1321N1-P2Y6R cells using 400 nM 16
aer 3 h incubation is shown in Fig. 1A. The peak for total
binding was signicantly higher than the autouorescence
and was located within the optimal window for analysis, but
it was not completely separated from the peak for auto-
uorescence. However, the corresponding uorescent 50-
monophosphate 42 at the same concentrations did not label
the cells during a 60 min incubation (Fig. 1B–E and S3†). This
is consistent with the inactivity of 50-monophosphate deriv-
atives in general at the P2Y6R. There was no specic labeling
by 16 or 42 of control 1321N1 cells. Also, we observed no
specic labeling by 16 of 1321N1 cells expressing the hP2Y1R
(data not shown).
Increasing the concentration of 16 from 10 nM to 10 mM
increased uorescent labeling of 1321N1-P2Y6R cells during a
60 min incubation, and a comparison by FCM of specic and
nonspecic binding shows a clear separation over a wide
concentration range (10 nM to 1.5 mM, 3 h incubation at 37 ꢁC,
Fig. 2A). The uorescence values were converted to molecules of
equivalent soluble uorochrome (MESF).18 Cells expressing the
P2Y6R in the absence of 16 were used to measure cellular
autouorescence. Nonspecic binding of 16 was measured in
the presence of 100 mM UDP, and the ratio of specic to
nonspecic binding was high (ꢂ4 : 1 at 500 nM 16). Fluorescent
labeling of the cells with 16 approached saturation. The
measured cell uorescence is a composite of the surface-bound
and the internalized uorescence due to the agonist nature of
16. Therefore, it was not possible to calculate an apparent
equilibrium binding constant (Kd) from this curve, which
represents a multistep process. Also, the metabolic stability of
these nucleotide derivatives was not studied; it is possible that
hydrolysis of the diphosphate during the incubations affects the
uorescent labeling of whole cells.
Fig. 2 (A) Saturation binding experiment with Alexa Fluor 488 conjugate 16 at
the P2Y6R expressed on 1321N1 astrocytoma cells using FCM (3 h incubation).
Nonspecific binding was measured in the presence of 100 mM UDP. (B) Internal-
ization kinetics of 400 nM P2Y6R-bound 16 at 37 ꢁC. Internalized amount (%int
)
was calculated as the acid insensitive fluorescence at x time point (MESFx) is
compared to total MESF (MESFtotal) corrected with the nonspecific binding
(MESFnonspec): %int¼(MESFx ꢃ MESFnonspec)/(MESFtotal ꢃ MESFnonspec). Fluores-
cence of internalized 16 was measured by FCM after removing cell-surface bound
ligand by 3 ꢄ 5 min acid wash (pH 3.5). The t1/2 of the internalization was 18 min.
(C) Comparison of the nonspecifically bound, surface bound, internalized and
total bound 16 (sum of surface bound and internalized) after 60 min incubation.
In this plot, nonspecific has been subtracted from surface bound, internalized and
total bound values. 1321N1-P2Y6R was incubated with 400 nM fluorescent
ligand 16 at 37 ꢁC. Nonspecific binding was measured in the presence of 100 mM
UDP, the surface bound fraction was measured with incubation of 0.4 M sucrose-
containing media, and the internalized ligand was measured as the acid-insen-
sitive fraction following acid wash. After a 60 min incubation period, the 17% of
the total binding was found on the cell surface, 77% was internalized and 23%
was nonspecifically bound.
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Fig. 3 Fluorescent micrographs using a Zeiss LSM 700 confocal microscope of P2Y6R-expressing 1321N1 astrocytoma cells exposed to the fluorescent agonist 16
(2 mM, 60 min incubation at 37 ꢁC). (A) Control cells in the absence of 16; (B) incubation with 1 mM 16 at 37 ꢁC for 60 min in medium without sucrose; (C) incubation
with 1 mM 16 at 37 ꢁC for 60 min in medium containing 0.4 M sucrose.
Another uorophore (Cy5) with a longer emission wave- providing further evidence of P2Y6R internalization upon
length than Alexa Fluor 488 was introduced in 17 at the same agonist binding (Fig. 3). Also, the addition of hyperosmolaric
position as Alexa Fluor 488 in 16. Unfortunately, the labeling of sucrose to the medium clearly le the uorescent labeling on
1321N1 astrocytoma cells with 17 (100 nM to 1 mM) was inde- the cell surface and prevented its internalization. The appear-
pendent of the expression of the P2Y6R, and it could not be ance of the labeling on the cell surface in Fig. 3C is punctate,
blocked by 100 mM UDP (Fig. S4†). Therefore, this conjugate was suggesting that the receptor is organized into concentrated
not suitable for further studies of P2Y6R labeling.
regions on the cell surface rather than being evenly distributed.
Kinetics of cell labeling by 16 was also determined using a Micrographs in Fig. 4 illustrate the ability of known P2Y6R
FCM assay (Fig. 2B). Binding of 400 nM 16 to 1321N1-P2Y6R ligands, agonist 8 and diisothiocyanate antagonist MRS2578, to
cells approached a maximum slowly, as observed previously inhibit uorescent labeling in 1321N1-P2Y6R cells by
with a uorescent antagonist of the hA3 adenosine receptor.18 compound 16. Several known P2Y6R ligands were compared for
An association rate constant (k1) was determined to be 0.019 the ability to inhibit the uorescent binding of 16 to 1321N1-
minꢃ1 (Fig. S5†). Nonspecic P2Y6R binding was measured in P2Y6R cells were compared aer a 30 min pre-incubation fol-
the presence of 100 mM UDP, the surface bound fraction was lowed by a brief treatment with the uorescent ligand 16
measured with incubation of 0.4 M sucrose-containing media, (2 min). Agonists UDP (10 mM) and MRS2957 (0.5 mM) and
and the internalized ligand was measured as the acid-insensi- antagonist MRS2578 (1 mM) caused a signicant reduction of
tive fraction following acid wash. The internalization of the labeling determined using FCM (Fig. S6†).
P2Y6R (determined by the comparison of the acid-insensitive
Other uorescent agonist ligands for GPCRs have been
fraction and the total binding) occurred with a t1/2 of 18 min. characterized, such as adenosine receptor agonists,24,34 which
The fraction of uorescence associated with the internalized internalized in a similar fashion.34 It has been shown that a
label was much greater (77% of the total binding) than the uorescently labeled A2A adenosine receptor agonist Alexa488-
fraction on the cell surface (17% of the total binding) at the end APEC internalized upon agonist stimulation via clathrin-coated
of the 60 min incubation period (Fig. 2C).
pit endocytosis, promoting a localization of the receptor in
In confocal microscopy, aer 60 min incubation at 37 ꢁC, the Rab5-positive early endosomes in both CHO and HEK293
cell-associated uorescence of compound 16 (2 mM) in 1321N1- cells.34 It was also demonstrated that desensitization of the
P2Y6R cells was receptor-dependent and mainly intracellular, P2Y6R is delayed compared to the P2Y4R,35 and that prolonged
Fig. 4 Inhibition of fluorescent labeling by known P2Y6R ligands, using a Keyence BZ-9000 fluorescent microscope equipped with filters for green fluorescence with
excitation at 495 nm and emission at 519 nm. Fluorescent micrographs of P2Y6R-expressing 1321N1 astrocytoma cells exposed to (A) the fluorescent agonist 16
(500 nM, 60 min incubation at 37 ꢁC) in the absence of inhibitor or sucrose; (B) same conditions, except a 30 min preincubation with P2Y6R agonist MRS2957
(8, 500 nM) was performed at 37 ꢁC; (C) same conditions, except a 30 min preincubation with P2Y6R antagonist MRS2578 (500 nM) was performed at 37 ꢁC P2Y6R-
expressing 1321N1 astrocytoma cells in the absence of 16 showed a lack of fluorescence, similar to Fig. 3A. Control experiments using 1321N1 astrocytoma cells not
expressing a P2YR showed no increase in fluorescence when incubated with 16.
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be a suitable template for the modeling of the P2YRs, since it is
relatively close in sequence and shares common structural
features with P2YRs. It was rst necessary to align the
sequences of the two receptors (Fig. S7†). Aer the construction
of the model, we subjected the uorescent ligand 16 to a Monte
Carlo Multiple Minima (MCMM) conformational search within
the binding cavity of the P2Y6R, granting full exibility to this
ligand as well as all of the surrounding amino acid residues.
The conformational search suggested that while the nucleotide
portion of the uorescent ligand bound rigidly to the receptor,
the large N4 substituent retains some exibility (Fig. 5A).
Specically, it suggested that the nucleotide portion of the
uorescent ligand bound to the receptor with a mode consistent
with that seen in our previous models of the P2YRs
(Fig. 5B).12,20,27–29 In particular, the 50-diphosphate moiety of the
ligand interacts, in our model, with three cationic residues
located in TM3, TM6 and TM7: Arg1033.29, Lys2596.55 and
Arg2877.39 (using a standard numbering convention30). These
residues are conserved as cationic residues in all subtypes of the
P2Y1-like subfamily of the P2YRs and, according to mutagenesis
data gathered for the P2Y1 and P2Y2Rs, are fundamental for
ligand recognition.31–33 A fourth cationic residue located in TM7
and conserved as a Lys in all the subtypes of the P2Y1-like
receptors that bind uracil nucleotides, namely Lys2847.36 in the
P2Y6R, interacted in the model with the phosphate as well as
with the pyrimidine base of the ligand.
The conformational search also suggested that the large N4
substituent of 16 protruded from the interhelical binding cavity
of the receptor toward the extracellular space to wrap around
the second extracellular loop and to end in proximity of the
extracellular portion of TM3, interacting with Asp963.22 and
Arg1003.26 (Fig. 5C). Notably, a visualization of the molecular
surface of the residues that surround the extended N4 substit-
uent, colored according to their electrostatic potential, sug-
gested a substantial steric and electrostatic complementarity
between the distal portions of the ligand and the receptor. A
sulfonate group of the Alexa Fluor 488 forms an electrostatic
interaction with R100, and an amino group is in proximity to
Asp96 (both in TM3 near EL2). By analogy, the enhanced affinity
of a uorescent probe for the A3 adenosine receptor also
correlated with predicted charged interactions of a bulky Alexa
Fluor 488 moiety with EL2.18
Fig. 5 Molecular model of the lowest energy conformation of the hP2Y6R
complexed with the Alexa Fluor 488-labeled agonist 16, as obtained after a fully
flexible MCMM conformational search, (A) viewed from the plane of the phos-
pholipid bilayer (with other conformations of 16 obtained), or (B) and (C) from the
extracellular side. (C) shows the molecular surface of 16 (mesh) and the residues
that surround its N4 substituent (solid), colored by electrostatic potential (red:
negative; blue: positive). The schematic representation of the backbone of the
receptor is colored by residue number (TM1: orange; TM2: dark yellow; TM3:
yellow; TM4 light green; TM5: green; TM6: blue; TM7: purple/blue). The ligand is
shown as a ball and stick structure, colored by element (carbon atoms colored in
gray). The residues located in proximity to the ligand are labeled and shown as
sticks, colored by residue number. The fluorophore is located near the outer
segment of TM3.
incubation with UDP caused a loss of surface P2Y6R, and rapid
recovery of surface P2Y6R did not occur following removal of the
agonist.36 The desensitization and internalization of two other
Conclusions
Gq-coupled receptors, P2Y2R and P2Y4R, have also been In an effort to functionalize a P2Y6R agonist for uorescent
described.36–38 Nevertheless, in this study the uorescent labeling, we probed two positions of pyrimidine nucleotides
agonist 16 was clearly internalized in P2Y6R-expressing cells.
Molecular modeling was used to probe the structural basis
for the signicantly enhanced potency and selectivity of the
Alexa Fluor conjugate 16. To generate a hypothesis of the
interactions of the uorescent ligand 16 with the receptor, we
constructed a new P2Y6R model based on our recently pub-
by introducing various functional groups, including alkynes
for click chemistry. Functionalization of extended imino
substituents at the N4 position of the pyrimidine nucleobase
of CDP retained P2Y6R potency to a higher degree than
g-phosphoester formation in UTP derivatives. Thus, we have
identied a pyrimidine N4-alkoxy group as a site for tethering
lished homology model of the P2Y4R, constructed using the P2Y6R agonists to a uorescent label. The bifunctional
CXCR4 chemokine receptor structure as a template.20,25 As we conjugate 16 containing a uorophore (Alexa Fluor 488) and
explained recently,20,26 among the GPCRs structures that have spacer chain constructed through click chemistry, was potent
been solved crystallographically, the CXCR4 receptor appears to
and selective agonist at the P2Y6R. It was suitable for the study
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of binding and internalization kinetics on whole cells using
FCM and microscopy. The binding of 16 to puried
membranes of cells overexpressed the P2Y6R remains to be
characterized. Thus, we introduce the rst high affinity uo-
rescent ligand specic for a P2YR, although additional phar-
macological characterization to dene the complex cell
labeling is needed.
UDP
UTP
Uridine 50-diphosphate;
Uridine 50-triphosphate.
Acknowledgements
This research was supported in part by the Intramural Research
Program of the NIH, National Institute of Diabetes and Diges-
tive and Kidney Diseases and a grant (GM38213) from the
National Institute of General Medical Sciences. We thank Noel
Whittaker (NIDDK) for mass spectral determinations. EK
thanks the Hungarian-American Enterprise Scholarship Foun-
dation (HAESF) for nancial support. LS thanks the University
of Florence, Italy for nancial support.
Based on the bulk tolerance of some of these nucleotide
derivatives, the functionalized chain appears to be accessing
the extracellular regions of the receptor. Although only one
uorescent conjugate 16 showed high potency at the P2Y6R,
this site on the nucleotide promises to be a general site for
derivatization with bulky substituents, including other uo-
rophores and specialized reporter groups for receptor detec-
tion and characterization.39 A molecular modeling study was
carried out to explore the recognition of the agonist 16 in
binding to the P2Y6R to highlight the key putative interactions
between charged groups of a large uorophore moiety and the
outer regions of TM3 in the P2Y6R, which evidently enhance
the potency of 16. In conclusion, we have identied the
N4-benzyloxy group as a structurally permissive region for
synthesis of a family of functionalized congeners as uores-
cent conjugates and other pharmacological probes for
studying the P2Y6R. This will enable further drug discovery
related to this receptor.
Notes and references
1 M. P. Abbracchio, G. Burnstock, J. M. Boeynaems,
E. A. Barnard, J. L. Boyer, C. Kennedy, G. E. Knight,
M. Fumagalli, C. Gachet, K. A. Jacobson and
G. A. Weisman, Pharmacol. Rev., 2006, 58, 281–341.
2 R. P. Vieira, T. Muller, M. Grimm, V. von Gernler, B. Vetter,
T. Durk, S. Cicko, C. K. Ayata, S. Sorichter, B. Robaye,
R. Zeiser, D. Ferrari, A. Kirschbaum, G. Zissel,
J. C. Virchow, J. M. Boeynaems and M. Idzko, Am. J. Respir.
Crit. Care Med., 2011, 184, 215.
¨
¨
3 L. K. Mamedova, R. Wang, P. Besada, B. T. Liang and
K. A. Jacobson, Pharmacol. Res., 2008, 58, 232.
4 R. Balasubramanian, I. Ruiz de Azua, J. Wess and
K. A. Jacobson, Biochem. Pharmacol., 2010, 79, 1317.
5 I. R. Orriss, N. Wang, G. Burnstock, T. R. Arnett, A. Gartland,
B. Robaye and J.-M. Boeynaems, Endocrinology, 2011, 152,
3706.
6 S. Koizumi, Y. Shigemoto-Mogam, K. Nasu-Tada,
Y. Shinozaki, K. Ohsawa, M. Tsuda, B. V. Joshi,
K. A. Jacobson, S. Kohsaka and K. Inoue, Nature, 2007, 446,
1091.
Abbreviations
1321N1-P2Y6R 1321N1 Human astrocytoma cells expressing
cells
CDP
CTP
DCC
DIC
the human P2Y6 receptor;
Cytidine 50-diphosphate;
Cytidine 50-triphosphate;
N,N0-Dicyclohexylcarbodiimide;
N,N0-Diisopropylcarbodiimide;
N,N-Dimethylformamide;
DMF
EDC
N-(3-Dimethylaminopropyl)-N0-
ethylcarbodiimide;
´
7 D. M. Grbic, E. Degagne, J. F. Larrive, M. S. Bilodeau,
FCM
Flow cytometry;
V. Vinette, G. Arguin, V. Stankova and F. P. Gendron,
Inammatory Bowel Dis., 2012, 18, 1456.
GPCR
HBSS
HEPES
G protein-coupled receptor;
Hank's balanced salt solution;
N-2-Hydroxyethylpiperazine-N0-2-
ethanesulfonic acid;
´
8 A. Markovskaya, A. Crooke, A. I. Guzman-Aranguez, A. Peral,
A. U. Ziganshin and J. Pintor, Eur. J. Pharmacol., 2008, 579,
93.
9 I. Bar, P. J. Guns, J. Metallo, F. Wilkin, D. Cammarata,
J. M. Boeynaems, H. Bult and B. Robaye, Mol. Pharmacol.,
2008, 74, 777.
10 H. Uratsuji, Y. Tada, T. Kawashima, M. Kamata, C. S. Hau,
Y. Asano, M. Sugaya, T. Kadono, A. Asahina, S. Sato and
K. Tamaki, J. Immunol., 2012, 188, 436.
HPLC
MCMM
MESF
High performance liquid chromatography;
Monte Carlo Multiple Minima;
Molecules of equivalent soluble uorochrome;
P1-(Uridine 50-)-P4-(N4-methoxycytidine 50-)
triphosphate;
MRS2957
MRS2578
N,N00-1,4-butanediylbis[N0-(3-
isothiocyanatophenyl)thiourea;
P2Y receptor;
¨
11 A. El-Tayeb, A. Qi and C. E. Muller, J. Med. Chem., 2006, 49,
P2YR
PLC
Phospholipase C;
7076.
SAR
Structure activity relationship;
Tetrabutylammonium dihydrogenphosphate;
Triethylammonium acetate;
Tetrahydrofuran;
Thin layer chromatography;
Transmembrane helical domain;
´
12 P. Besada, D. H. Shin, S. Costanzi, H. J. Ko, C. Mathe,
TBAP
TEAA
THF
TLC
TM
J. Gagneron, G. Gosselin, S. Maddileti, T. K. Harden and
K. A. Jacobson, J. Med. Chem., 2006, 49, 5532–5543.
13 T. Ginsburg-Shmuel, M. Haas, M. Schumann, G. Reiser,
O. Kalid, N. Stern and B. Fischer, J. Med. Chem., 2010, 53,
1673.
1164 | Med. Chem. Commun., 2013, 4, 1156–1165
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Concise Article
MedChemComm
14 H. Maruoka, M. O. Barrett, H. Ko, D. K. Tosh, A. Melman,
L. E. Burianek, R. Balasubramanian, B. Berk, S. Costanzi,
P. Kuhn, T. M. Handel, V. Cherezov and R. C. Stevens,
Science, 2010, 330, 1066.
T. K. Harden and K. A. Jacobson, J. Med. Chem., 2010, 53, 26 F. Deorian and K. A. Jacobson, J. Comput.-Aided Mol. Des.,
4488. 2011, 25, 329.
15 S. R. Shaver, J. L. Rideout, W. Pendergast, J. G. Douglass, 27 S. Costanzi, L. Mamedova, Z. G. Gao and K. A. Jacobson,
E. G. Brown, J. L. Boyer, R. I. Patal, C. C. Redick, J. Med. Chem., 2004, 47, 5393.
A. C. Jones, M. Picher and B. R. Yerxa, Purinergic Signalling, 28 S. Costanzi, B. Joshi, S. Maddileti, L. Mamedova,
2005, 1, 183.
M. Gonzalez-Moa, V. Marquez, T. K. Harden and
16 H. Ko, R. L. Carter, L. Cosyn, R. Petrelli, S. de Castro,
K. A. Jacobson, J. Med. Chem., 2005, 48, 8108.
P. Besada, Y. Zhou, L. Cappellacci, P. Franchetti, 29 K. A. Jacobson, S. Costanzi, A. Ivanov, S. Tchilibon,
M. Grifantini, S. Van Calenbergh, T. K. Harden and
K. A. Jacobson, Bioorg. Med. Chem., 2008, 16, 6319.
P. Besada, Z. Gao, S. Maddileti and T. K. Harden, Biochem.
Pharmacol., 2006, 71, 540.
¨
17 A. El-Tayeb, A. Qi, R. A. Nicholas and C. E. Muller, J. Med. 30 J. A. Ballesteros and H. Weinstein, Methods Neurosci., 1995,
Chem., 2011, 54, 2878. 25, 366.
18 (a) E. Kozma, T. S. Kumar, S. Federico, K. Phan, 31 P. Hillmann, G. Y. Ko, A. Spinrath, A. Raulf, I. von Kugelgen,
¨
¨
S. C. Wolff, R. A. Nicholas, E. Kostenis, H. D. Holtje and
R. Balasubramanian, Z. G. Gao, S. Paoletta, S. Moro,
¨
C. E. Muller, J. Med. Chem., 2009, 52, 2762.
G. Spalluto and K. A. Jacobson, Biochem. Pharmacol., 2012,
83, 1552; (b) E. Kozma, E. T. Gizewski, D. K. Tosh, 32 S. Moro, D. Guo, E. Camaioni, J. L. Boyer, T. K. Harden and
L. Squarcialupi, J. A. Auchampach and K. A. Jacobson,
Biochem. Pharmacol., 2013, 85, 1171.
19 J. E. Moses and A. D. Moorhouse, Chem. Soc. Rev., 2007, 36,
1249.
K. A. Jacobson, J. Med. Chem., 1998, 41, 1456.
33 Q. Jiang, D. Guo, B. X. Lee, A. M. Van Rhee, Y. C. Kim,
R. A. Nicholas, J. B. Schachter, T. K. Harden and
K. A. Jacobson, Mol. Pharmacol., 1997, 52, 499.
20 H. Maruoka, M. P. S. Jayasekara, M. O. Barrett, 34 F. Brand, A. Klutz, K. A. Jacobson, B. B. Fredholm and
D. A. Franklin, S. de Castro, N. Kim, S. Costanzi, G. Schulte, Eur. J. Pharmacol., 2008, 590, 36.
T. K. Harden and K. A. Jacobson, J. Med. Chem., 2011, 54, 35 B. Robaye, J. M. Boeynaems and D. Communi, Eur.
4018. J. Pharmacol., 1997, 329, 231.
21 R. Chinchilla and C. Najera, Chem. Rev., 2007, 107, 36 A. E. Brinson and T. K. Harden, J. Biol. Chem., 2001, 276,
874. 11939.
22 D. K. Tosh, L. S. Yoo, M. Chinn, K. Hong, S. M. Kilbey, 37 R. V. Flores, M. G. Hernandez-Perez, E. Aquino, R. C. Garrad,
´
´
´
M. O. Barrett, I. P. Fricks, T. K. Harden, Z. G. Gao and
K. A. Jacobson, Bioconjugate Chem., 2010, 21, 372.
G. A. Weisman and F. A. Gonzalez, Mol. Cell. Biochem., 2005,
280, 35.
23 D. M. Bourdon, M. R. Wing, E. B. Edwards, J. Sondek and 38 S. M. Sromek and T. K. Harden, Mol. Pharmacol., 1998, 54,
T. K. Harden, Methods Enzymol., 2006, 406, 489. 485.
24 C. L. Dale, S. J. Hill and B. Kellam, Med. Chem. Commun., 39 K. A. Jacobson, Bioconjugate Chem., 2009, 20, 1816.
2012, 3, 333.
40 R. L. Carter, I. P. Fricks, M. O. Barrett, L. E. Burianek,
Y. Zhou, H. Ko, A. Das, K. A. Jacobson, E. R. Lazarowski
and T. K. Harden, Mol. Pharmacol., 2009, 76, 1341.
25 B. Wu, E. Y. Chien, C. D. Mol, G. Fenalti, W. Liu, V. Katritch,
R. Abagyan, A. Brooun, P. Wells, F. C. Bi, D. J. Hamel,
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 1156–1165 | 1165