Caged agonist of P2Y1 and P2Y12 receptors for light-directed facilitation of platelet aggregation

Article abstract of DOI:10.1016/j.bcp.2007.10.037

We have prepared a caged form (MRS2703) of a potent dual agonist of the P2Y₁ and P2Y₁₂ nucleotide receptors, 2-MeSADP, by blocking the β-phosphate group with a 1-(3,4-dimethyloxyphenyl)eth-1-yl phosphoester. Although MRS2703 is itsel

Full text of DOI:10.1016/j.bcp.2007.10.037

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 4 1 – 1 3 4 7
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journal homepage: www.elsevier.com/locate/biochempharm
Caged agonist of P2Y1 and P2Y12 receptors for light-directed
facilitation of platelet aggregation
a
Zhan-Guo Gao , Beatrice Hechler b,c,d, Pedro Besada a, Christian Gachet b,c,d
,
´
Kenneth A. Jacobson a,
*
a Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Building 8A, Room B1A-19, Bethesda, MD 20892-0810, USA
b INSERM U.311, Strasbourg, France
c EFS-Alsace, Strasbourg, France
d Universite´ Louis Pasteur, Strasbourg, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We have prepared a caged form (MRS2703) of a potent dual agonist of the P2Y1 and P2Y12
nucleotide receptors, 2-MeSADP, by blocking the b-phosphate group with a 1-(3,4-dimethy-
loxyphenyl)eth-1-yl phosphoester. Although MRS2703 is itself inactive at human P2Y1 and
P2Y12 receptors expressed heterologously in 1321N1 astrocytoma cells or in washed human
platelets, this derivative readily regenerates the parent agonist upon mild irradiation with
long-wave UV light (360 nm). The functional effect of the regenerated agonist was demon-
strated by a rise in intracellular calcium mediated by either P2Y1 or P2Y12 receptors in
transfected cells. Washed human platelets exposed to a solution of MRS2703 were induced
to aggregate upon UV irradiation. At 1.0 mM MRS2703, full aggregation was achieved within
1 min of irradiation. Thus, this caged nucleotide promises to be a useful probe for potent P2Y
receptor activation with light-directed spatial and temporal control.
Received 31 August 2007
Accepted 27 October 2007
Keywords:
Aggregation
Nucleotides
G-protein-coupled receptors
Phospholipase C
Calcium
Published by Elsevier Inc.
1.
Introduction
term ‘caged’ was first coined by Kaplan et al. [19,20], and
caged-ATP was the first to be introduced to study organized
Caged molecules, in which light is able to rapidly and site-
specifically generate a reactive or biologically active chemical
species, have been designed based on the photocleavage of the
2-nitrobenzyl protecting group. The 2-nitrobenzyl group was
originally introduced as an orthogonal photoremovable
protecting group for use in peptide synthesis by Patchornik
et al. [1]. Since then various chemically improved modifica-
tions of the photolabile 2-nitrobenzyl group, such as the 1-(3,4-
dimethyloxyphenyl)eth-1-yl group, have been utilized [2]. The
biological systems. A caged-ATP derivative (P3-1-(2-nitrophe-
nylethyladenosine 50-triphosphate)) has been used to probe
the fast kinetics of muscle contraction, etc. [9]. This principle
of photocleavage has also been effectively applied to selective
deprotection for synthesis in a spatially addressable fashion
on a surface array [3], masking of intracellular and extra-
cellular signaling molecules for biological studies [4–6], and
the light-reversible rapid activation of proteins [7,8]. In the
area of platelet biology, an oligonucleotide aptamer consisting
* Corresponding author. Tel.: +1 301 496 9024; fax: +1 301 480 8422.
E-mail address: kajacobs@helix.nih.gov (K.A. Jacobson).
Abbreviations: FBS, fetal bovine serum; MRS2500, (10R,20S,40S,50S)-4-(2-iodo-6-methylamino-purin-9-yl)-1-[(phosphato)-methyl]-2-
(phosphato)-bicyclo[3.1.0]hexane; MRS2703, P1-2-(methylthio)adenosyl-P2-(RS)-1-(4,5-dimethoxy-2-nitrophenyl)ethyl pyrophosphate,
triethylammonium salt; 2-MeSADP, 2-methylthioadenosine 50-diphosphate; DMEM, Dulbecco’s modified Eagle’s medium.
0006-2952/$ – see front matter. Published by Elsevier Inc.
doi:10.1016/j.bcp.2007.10.037

1342
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 4 1 – 1 3 4 7
of a ssDNA 15mer was caged, by preparation of a thymidine 4-
O-(2-nitrophenylethyl) derivative, to provide a UV-light acti-
vatable derivative [10]. This derivative when unmasked with
light bound to and inactivated R-thrombin, which is a key
player in the blood-clotting cascade. Recently, coumarin-
based photosensitive protecting groups have been applied to
cage biologically active molecules [11].
Alto, CA). System A: linear gradient solvent system: 5 mM
TBAP (tetrabutylammonium dihydrogenphosphate)–CH3CN
from 80:20 to 40:60 in 20 min; the flow rate was 1 ml/min.
Peaks were detected by UV absorption with a diode array
detector. High-resolution mass measurements were per-
formed on Micromass/Waters LCT Premier Electrospray Time
of Flight (TOF) mass spectrometer coupled with a Waters HPLC
system. Purification of the caged nucleotide was carried out by
HPLC using a Luna 5 mm RP-C18(2) semipreparative column
(250 mm ꢀ 4.6 mm; Phenomenex, Torrance, CA). System B:
10 mM TEAA (triethylammonium acetate)–CH3CN from 90:10
to 60:40 in 30 min; the flow rate was 2 ml/min.
The P2Y1 and P2Y12 receptors are G-protein-coupled
receptors of the rhodopsin family present on the surface of
platelets that respond to extracellular ADP to induce aggrega-
tion [12–14]. This activation is an essential step in the process
of platelet aggregation in vivo. In in vitro studies of platelet
function, the activation of both receptors by ADP is required
for complete aggregation. Thus, application of a selective
antagonist of either the P2Y1 or the P2Y12 receptor inhibits
aggregation. A potent and selective P2Y1 receptor nucleotide
antagonist MRS2500, in which the ribose moiety is conforma-
tionally constrained to ‘‘fit’’ the preferred geometry of the
binding site, inhibited both aggregation and the initial shape
change in platelets, which are characteristic of the effects of
ADP. MRS2500 was also shown to act as a P2Y1 receptor
antagonist in vivo to impede systemic thromboembolism and
localized arterial thrombosis [14,15].
1-(4,5-Dimethoxy-2-nitrophenyl)-diazoethane Generation
Kit was purchased from Invitrogen Molecular Probes (Eugene,
OR). 2-MeSADP (1) and other reagents and solvents were
purchased from Sigma–Aldrich (St. Louis, MO).
2.2.
Chemical synthesis
2.2.1. P1-2-(methylthio)adenosyl-P2-(RS)-1-(4,5-dimethoxy-2-
nitrophenyl)ethyl pyrophosphate, triethylammonium salt (4)
To prepare the reactive diazoethane (3) a solution of 4,5-
dimethoxy-2-nitroacetophenone hydrazone (2, 25 mg,
0.10 mmol) in DMF (1 ml) was treated with MnO2 (100 mg,
1.15 mmol). After stirring for 30 min at room temperature, the
reaction mixture was filtered through Celite1 diatomaceous
earth (100 mg) contained in a Pasteur pipette to obtain a red
solution that contains the 1-(4,5-dimethoxy-2-nitrophenyl)-
diazoethane (3). This solution was used in the next step
without further purification. To a solution of 2-MeSADP (1,
5 mg, 0.008 mmol) in H2O (1 ml) was added the red solution
that contains the 1-(4,5-dimethoxy-2-nitrophenyl)-dia-
zoethane (3). To avoid light the reaction flasks are covered
with aluminum foil. The reaction mixture was stirred at room
temperature for 24 h. The aqueous phase was treated with
CH2Cl2. After removal of the solvent, the residue was purified
by semipreparative HPLC as described above to obtain the
mixture of diastereoisomers 4ab (4.8 mg, 66%) as a white solid.
1H NMR (D2O) d 8.05 (s, 1H), 7.97 (s, 1H), 7.44 (s, 1H), 7.39 (s, 1H),
7.32 (s, 1H), 7.19 (s, 1H), 6.04 (m, 2H), 5.90 (d, J = 4.8 Hz, 1H), 5.82
(d, J = 4.8 Hz, 1H), 4.50 (m, 1H), 4.41 (m, 1H), 4.33 (m, 1H), 4.30
(m, 1H), 4.21 (m, 1H), 4.13 (m, 3H), 3.99 (m, 2H), 3.94 (s, 1H), 3.91
(s, 1H), 3.71 (s, 1H), 3.68 (s, 1H), 2.56 (s, 1H), 2.54 (s, 1H), 1.50 (d,
J = 6.3 Hz, 6H); 31P NMR (D2O) d ꢁ11.17 (d, J = 20.8 Hz), ꢁ11.33 (d,
J = 18.3 Hz), ꢁ11.82 (d, J = 20.8 Hz), ꢁ12.18 (d, J = 18.9 Hz); HRMS
m/z found 681.0742 (M ꢁ H+)ꢁ. C21H27N6O14P2S requires
681.0781; HPLC (system A) 15.2 min.
We previously applied the 2-nitrobenzyl group in the form
of a nucleoside 50-ether to prepare a caged agonist of the A1
adenosine receptor [16]. In the present study we have
synthesized and characterized pharmacologically a 2-nitro-
benzyl-type derivative of the potent proaggregatory receptor
probe, 2-methylthio-ADP (2-MeSADP), which was shown to
activate both P2Y1 and P2Y12 receptors on the surface of
platelets. 2-MeSADP is approximately three orders of magni-
tude more potent than ADP as an agonist of these two P2Y
receptor subtypes and displays nanomolar-binding affinity at
each. Structure–activity studies have been extensively
reviewed [12]. This caged nucleotide allows spatial and
temporal control over the proaggregatory signal, as demon-
strated in in vitro models.
2.
Materials and methods
2.1.
Materials
The 1321N1 astrocytoma cells stably transfected with human
P2Y1 and P2Y12 receptors were generously provided by Prof.
T.K. Harden (University of North Carolina, Chapel Hill, NC).
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine
serum (FBS) were purchased from Life Technologies (Rockville,
MD). Plastic collagen-coated cellware was purchased from
Becton Dickinson (Bedford, MA). Calcium Mobilization Assay
Kit was purchased from Molecular Devices (Sunnyvale, CA).
1H NMR spectra were obtained with a Varian Gemini 300
spectrometer using D2O as a solvent. The chemical shifts are
expressed as relative ppm from HOD (4.80 ppm). 31P NMR
spectra were recorded at room temperature by the use of
Varian XL 300 spectrometer (121.42 MHz); orthophosphoric
acid (85%) was used as an external standard. Purity of
compounds was checked using a Hewlett-Packard 1100 HPLC
equipped with a Zorbax Eclipse 5-mm XDB-C18 analytical
column (250 mm ꢀ 4.6 mm; Agilent Technologies Inc., Palo
HPLC analysis using System A was carried out on a solution
of MRS2703 (50 mM in PBS) before and after irradiation (as
described in Section 2.4) for 2 min. Following this irradiation
all of the MRS2703, having a retention time (r.t.) of 15.2 min,
had disappeared and the major peak observed was a new peak
with the identical r.t. as the parent 2-MeSADP (12.9 min).
2.3.
Cell culture
Human 1321N1 astrocytoma cells stably transfected with the
human P2Y1 or P2Y12 receptors were grown at 37 8C in a
humidified incubator with 5% CO2/95% air in DMEM/F-12
medium (1:1) supplemented with 10% FBS, 100 units/ml

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 4 1 – 1 3 4 7
1343
penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine.
The cells were passaged using trypsinization every 4–5 days.
and finally resuspended at a density of 3 ꢀ 105 platelets/ml in
the same buffer in the presence of 0.02 U/ml of the ADP
scavenger apyrase (adenosine 50-triphosphate diphosphohy-
drolase, EC 3.6.1.5), a concentration sufficient to prevent
desensitization of platelet ADP receptors during storage.
Platelets were kept at 37 8C throughout all experiments.
2.4.
Calcium mobilization assay
Cells were grown overnight in 100 ml of medium in 96-well flat-
bottom plates at 37 8C at 5% CO2 or until they reached ꢂ60–80%
confluency. The calcium assay kit (Molecular Devices, Sunny-
vale, CA, USA) was used as directed with no washing of cells
and with probenecid added to the loading dye at a final
concentration of 2.5 mM to increase dye retention. Cells were
loaded with 50 ml dye with probenecid in each well and
incubated for 45 min at room temperature. The compound
plate was prepared with dilutions of various compounds in
Hank’s buffer at pH 7.2. For irradiation of the caged agonist, a
solution of MRS2703 in PBS was exposed to UV light under
various conditions of wavelengths and for a range of times. A
UV light (Spectroline model, UV-400; Spectronics Co., West-
2.6.
Platelet aggregation studies
Aggregation was measured at 37 8C by a turbidimetric method
in a dual-channel Payton aggregometer (Payton Associates,
Scarborough, Ontario, Canada). A solution of MRS2703 in PBS
was exposed to UV light (12 W light source, with a peak UV
wavelength of 254 nm) for various time periods and immedi-
ately added to a 450 ml aliquot of platelet suspension stirred at
1100 rpm, in the presence of human fibrinogen (0.8 mg/ml), in
a final volume of 500 ml.
bury, USA) was outfitted with
a
360 nm filter (Edmund
2.7.
Statistical analysis
Industrial Optics, Barrington, NJ, USA), and a PBS solution of
MRS2703 (20 mM) in a glass cuvette was irradiated at a distance
of 1.0 cm. Room lighting was dimmed during the experiments
using MRS2703 under non-irradiated conditions. Samples
were run in duplicate with a Flexstation I (Molecular Devices)
at room temperature. Cell fluorescence (excitation = 485 nm;
emission = 525 nm) was monitored following exposure to a
compound. Increases in intracellular calcium are reported as
the maximum fluorescence value after exposure minus the
basal fluorescence value before exposure.
Pharmacological parameters were analyzed with the Graph-
PAD Prism software (version 4.0, GraphPAD Prism, San Diego,
CA). Data were expressed as mean ꢃ standard error. Statistical
significance was calculated using the Student’s t-test. P values
less than 0.05 (P < 0.05) were considered to be statistically
significant.
3.
Results
2.5.
Preparation of human washed platelets
3.1.
Design of reagent and chemical synthesis
Human washed platelets were prepared as previously
described [22]. Briefly, fresh blood obtained from healthy
donors was centrifuged at 175 ꢀ g for 15 min at 37 8C, platelet
rich plasma was removed, and centrifuged at 1570 ꢀ g for
15 min at 37 8C. The platelet pellet was washed twice in
Tyrode’s buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO3,
0.3 mM NaH2PO4, 1 mM MgCl2, 2 mM CaCl2, 5.5 mM glucose,
5 mM Hepes, pH 7.3) containing 0.35% human serum albumin,
The caged nucleotide MRS2703 (4) was prepared from 2-
MeSADP (1) and the in situ generated phenyl diazoethane
derivative (3) as shown in Fig. 1 [2]. The presence of the veratryl
group is intended to allow unmasking by light of longer
wavelength than is required for simple 2-nitrobenzyl deriva-
tives. The presence of the ethyl moiety reduces side reactions
as a result of the generation of photoproducts, which in this
case would be an acetylaryl compound that is less reactive
Fig. 1 – Structure and preparation of caged nucleotide MRS2703 4. The caged nucleotide could be reconverted to the starting
material, agonist 2-MeSADP 1 upon irradiation. Reagents and conditions: (i) MnO2, DMF, r.t. (ii) H2O, r.t.

1344
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 4 1 – 1 3 4 7
Fig. 2 – The ability of 2-MeSADP, and the caged 2-MeSADP
(MRS2703) to activate the human P2Y1 receptor stably
expressed in 1321N1 astrocytoma cells. Cells were loaded
with 50 ml dye with 2.5 mM probenecid in each well and
incubated for 45 min at room temperature. The agonists
were tested without exposure to UV light. 2-MeSADP
induced a concentration-dependent rise in [Ca2+]i levels,
with an EC50 value of 2.03 W 0.35 nM (n = 3), similar to
previously reported values [18].
Fig. 4 – The ability of MRS2703 and the un-caged MRS2703
(upon 120 s irradiation) to activate the human P2Y12
receptor stably expressed in 1321N1 astrocytoma cells.
Cells were loaded with 50 ml dye with 2.5 mM probenecid
in each well and incubated for 45 min at room
temperature. Activation of the P2Y12 receptor by either 2-
MeSADP or the irradiated MRS2703 induced a
concentration-dependent increase in [Ca2+]i that was not
observed in control 1321N1 astrocytoma cells. The
methods used were described in Section 2. Results are
from three separate experiments performed in duplicate.
than the corresponding benzaldehyde. HPLC analysis indi-
cated a nearly complete conversion of a dilute solution of
MRS2703 to the parent 2-MeSADP during a 2 min irradiation.
control experiment these cells were exposed to 2-MeSADP,
which induced
a
concentration-dependent rise in [Ca2+
]
i
3.2.
Characterization in astrocytoma cells
levels, with an EC50 value of 2.03 ꢃ 0.35 nM (n = 3), similar to
previously reported values [18]. MRS2703, without exposure to
UV light, was weak in the stimulation of calcium mobilization,
with an EC50 value estimated to be ꢄ10,000 nM (Fig. 2).
For irradiation of the caged agonist, a dilute solution of
MRS2703 (20 mM) in PBS in a quartz cuvette was exposed to
360 nm UV light (from a portable light source equipped with a
filter) for a range of times. The production of 2-MeSADP upon
irradiation was determined using a bioassay consisting of
stimulation of calcium mobilization in astrocytoma cells
stably expressing the human P2Y1 receptor. At 10 mM
MRS2703, a partial (ꢂ50% of maximal) stimulation of calcium
mobilization was noted. The potency of the solution gradually
increased over a period of 5 min, such that the final activity
reached the same potency and maximal effect as 2-MeSADP
(Fig. 3). This indicated that most of the MRS2703 was
effectively un-caged upon irradiation to provide the biologi-
cally active P2Y1 receptor agonist, 2-MeSADP. As a control, the
activity of 2-MeSADP before and after 5 min irradiation under
the same condition was not changed. The EC50 values of the
MRS2703 after exposure to UV light for 0, 5, 10, 30, 60, 120 and
300 s were demonstrated to be ꢄ10,000, 3420 ꢃ 586, 633 ꢃ 128,
122 ꢃ 36, 22 ꢃ 7, 3.9 ꢃ 1.4 and 2.8 ꢃ 0.7 nM (n = 3), respectively.
As a control, the EC50 value of 2-MeSADP after 300 s exposure
to UV light was shown to be 2.6 ꢃ 0.8 nM, which is similar to
that of the non-irradiated. The results suggest that the UV-
light exposure only induces the uncaging not the breakdown
of 2-MeSADP.
The photoregeneration of the potent P2Y receptor agonist 2-
MeSADP was first tested using the recombinant human P2Y1
receptor expressed in 1321N1 astrocytoma cells (Fig. 2). In a
Fig. 3 – Concentration–response curves for the increase in
[Ca2+]i induced by the agonist mixtures, upon UV-light
irradiation, in hP2Y1-1321N1 cells. Cells were loaded with
50 ml dye with 2.5 mM probenecid in each well and
incubated for 45 min at room temperature. The compound
plate was prepared with dilutions of various compounds
in Hank’s buffer at pH 7.2. Samples were run with a
Flexstation I (Molecular Devices), and cell fluorescence
(excitation = 485 nm; emission = 525 nm) was monitored
following exposure to the agonist. Increases in
intracellular calcium are reported as the maximum
fluorescence value after exposure minus the basal
fluorescence value before exposure. The data shown are
from an experiment performed in duplicate representative
of two to four separate experiments of similar results
performed in duplicate.
2-MeSADP is an agonist of P2Y1 and P2Y12 receptors. Thus,
we next tested if the caged 2-MeSADP, MRS2703, was able to
activate P2Y12 receptors, either before or following irradiation.
The activity at the P2Y12 receptor was tested using a bioassay
consisting of stimulation of calcium mobilization in stably

b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 4 1 – 1 3 4 7
1345
transfected 1321N1 astrocytoma cells. Although in a previous
3.3.
Characterization in human platelets
study the 1321N1 cells were cotransfected with a chimeric G
protein, Gaq/i, to confer a capacity of the Gi-linked P2Y12
receptor to directly activate phospholipase C [26], in the
current study only the endogenous G proteins were present.
The coupling of the P2Y12 receptor to a calcium response in the
1321N1 cells was likely as reported previously [17] through G-
protein b,g subunits. Control 1321N1 cells did not display a rise
in [Ca2+]i levels in response to 2-MeSADP, but in those cells
expressing the P2Y12 receptor 2-MeSADP induced a concen-
tration-dependent rise with an EC50 value of 23 ꢃ 6 nM. Fig. 4
shows that MRS2703, at concentrations up to 10 mM, did not
induce a rise in [Ca2+]i levels. However, upon 120 s irradiation,
the un-caged nucleotide preparation showed an EC50 of
41.4 ꢃ 12.8 nM (n = 3).
The photoregeneration of 2-MeSADP was then studied in
washed human platelets (Fig. 5).
MRS2703 (1 mM), without exposure to UV light, did not
induce aggregation or shape change of human washed
platelets (Fig. 5A). In contrast, exposure of the compound (1
or 0.05 mM) to UV light for 5 s or longer resulted in platelet
aggregation (Fig. 5A and B). At the highest concentration of
MRS2703 tested (1 mM), full aggregation was achieved within
30 s of irradiation. The extent of aggregation was compar-
able to aggregation induced by 2-MeSADP at a concentration
of 1 mM. The ability to induce platelet aggregation was
slower at
(Fig. 5B).
a lower concentration of MRS2703 (0.05 mM)
Fig. 5 – Aggregation of human washed platelets induced by MRS2703 following UV irradiation. (A) Representative washed
human platelet aggregation measurements. (B) Effect of time exposure of MRS2703 (0.05 or 1 mM) to UV light on the extent of
platelet aggregation. The methods used were described in Section 2.

1346
4.
b i o c h e m i c a l p h a r m a c o l o g y 7 5 ( 2 0 0 8 ) 1 3 4 1 – 1 3 4 7
Discussion
to MRS2703 for the selective light-directed activation of P2Y1
receptors. Furthermore, caged P2Y receptor ligands, such as
MRS2703 and caged MRS2500, may have potential for
hemodynamic control in the clinic, in a manner that is more
spatially selective than with use of anti-thrombotic drugs. The
effects of the MRS2703 must now be evaluated in in vivo
models. Perhaps this probe will be found useful in electro-
physiological studies of the brain in which selected regions
can be irradiated, by analogy to caged excitatory amino acid
derivatives [24]. Methods for the rapid and noninvasive
liberation of transmitters using irradiation have been
shown to allow the manipulation of biochemical signals in
the brain with submicron spatial resolution [24]. The effects of
P2Y1 receptors, P2Y12 receptors, and other P2 receptors in
cerebral ischemia and in the central nervous system in general
are being studied [25]. Using a more sophisticated laser
irradiation device, we would expect the in situ photocleavage
of MRS2703 to occur on a much faster time scale than noted in
this study.
In the present study, we have synthesized and pharmacolo-
gically characterized the nucleotide derivative MRS2703. The
photosensitive 1-(3,4-dimethyloxyphenyl)eth-1-yl protecting
group was placed at the b-phosphate of 2-MeSADP. This 2-
nitrobenzyl-type group selected for ‘‘caging’’ the nucleotide
agonist derivative is additionally substituted with 4,5-
dimethoxy groups and an a-methyl moiety for light absorption
at longer wavelengths and less reactive photo-side products,
respectively [2,6]. Prior to irradiation, MRS2703 is inactive at
both P2Y1 and P2Y12 receptors. However, upon UV-light
exposure, it potently activated both P2Y1 and P2Y12 receptors
stably expressed in 1321N1 astrocytoma cells, and induced
platelet aggregation in vitro. Thus, it is suggested that the caged
agonist for P2Y1 and P2Y12 receptors should be useful in
controlling cell signaling. The inactivity of non-irradiated
MRS2703 at P2Y1 and P2Y12 receptors is consistent with the
hypothesis that blocking the b-phosphate as a terminal ester
precludes high-affinity binding to these two receptor sub-
types. We have not evaluated the light-directed activation of
the P2Y13 receptor by MRS2703. However, such activation is
expected because the parent nucleotide and photoproduct, 2-
MeSADP, is a potent agonist of the P2Y13 receptor [1,26].
Although caged-ADP and caged-ATP have been available
previously, they are not selective agonists for the P2Y1 and
P2Y12 receptors. Thus, potentially non-specific effects could
result using these nonselective agonists. Compared with the
non-caged 2-MeSADP, MRS2703 could be metabolically more
stable, because it is predicted that caged 2-MeSADP as a b-
phosphate ester would be resistant to certain phosphatases by
analogy to dinucleotides [21]. Thus, caged 2-MeSADP may be
used as a relatively stable source of 2-MeSADP to be used in
mechanistic studies.
In summary, the newly synthesized nucleotide derivative
MRS2703 acts as a caged agonist for both P2Y1 and P2Y12
receptors, which can be rapidly liberated in biologically active
form upon brief irradiation with UV light. Thus, MRS2703 and
similar derivatives of other P2Y receptor ligands should be
useful for precise control of signaling temporally and spatially
in future mechanistic studies.
Acknowledgments
This work was supported in part by the Intramural Research
Program of NIDDK, National Institutes of Health, Bethesda,
MD, USA. We thank Dr. Hyojin Ko (NIH) for HPLC analysis.
The caged molecules, such as MRS2703, have the potential
to achieve precise temporal control of signaling in mechanistic
studies. Investigation of the mechanisms of action of the
biological systems, such as cells, requires special techniques
to introduce effector molecules rapidly with known concen-
tration and location. Time-resolved measurements on such
systems may be impossible if the diffusion of target molecules
to the cells is slow or if complications resulted from
degradation of the molecules or desensitization of the
biological response. One approach to this problem is to
introduce the molecule rapidly and uniformly by pulse
photolysis of precursor compounds that are biologically
inactive. Thus, MRS2703 should be useful for the mechanistic
studies of P2Y1 and P2Y12 receptor-related biological func-
tions, such as platelet aggregation.
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