GPCR ligand dendrimer (GLiDe) conjugates: Adenosine receptor interactions of a series of multivalent xanthine antagonists

Article abstract of DOI:10.1021/bc1005812

Previously, G protein-coupled receptor (GPCR) agonists were tethered from polyamidoamine (PAMAM) dendrimers to provide high receptor affinity and selectivity. Here, we prepared GPCR ligand-dendrimer (GLiDe) conjugates from a potent adenosine receptor (AR)

Full text of DOI:10.1021/bc1005812

ARTICLE
pubs.acs.org/bc
GPCR Ligand Dendrimer (GLiDe) Conjugates: Adenosine Receptor
Interactions of a Series of Multivalent Xanthine Antagonists
Angela Kecskꢀes,† Dilip K. Tosh,† Qiang Wei, Zhan-Guo Gao, 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, United States
S Supporting Information
b
ABSTRACT: Previously, G protein-coupled receptor (GPCR)
agonists were tethered from polyamidoamine (PAMAM) den-
drimers to provide high receptor affinity and selectivity. Here,
we prepared GPCR ligandꢀdendrimer (GLiDe) conjugates
from a potent adenosine receptor (AR) antagonist; such agents
are of interest for treating Parkinson’s disease, asthma, and other
conditions. Xanthine amine congener (XAC) was appended with
an alkyne group on an extended C8 substituent for coupling by
Cu(I)-catalyzed click chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. These conjugates
also contained triazole-linked PEG groups (8 or 22 moieties per 64 terminal positions) for increasing water-solubility and optionally
prosthetic groups for spectroscopic characterization and affinity labeling. Human AR binding affinity increased progressively with the
degree of xanthine substitution to reach Ki values in the nanomolar range. The order of affinity of each conjugate was hA2AAR > hA3AR >
hA1AR, while the corresponding monomer was ranked hA2AAR > hA1AR g hA3AR. The antagonist activity of the most potent
conjugate 14 (34 xanthines per dendrimer) was examined at the Gi-coupled A1AR. Conjugate 14 at 100 nM right-shifted the AR agonist
concentrationꢀresponsecurveinacyclicAMPfunctionalassayinaparallelmanner,butat10nM(lower thanitsKi value), it significantly
suppressed the maximal agonist effect in calcium mobilization. This is the first systematic probing of a potent AR antagonist tethered on a
dendrimer and its activity as a function of variable loading.
’ INTRODUCTION
A2A and A3 subtypes) or antagonize P2Y receptors (specifically
the P2Y1 subtype) to modulate intracellular signaling,1,2,9 with
both of these receptor families being GPCRs. These GLiDe
conjugates are stable and biologically active without requiring the
release of small molecules or cellular internalization, which are
often an integral part of other polymeric drug delivery schemes.10
Recently, we showed that a potent A3AR agonist conjugate
displayed cytoprotective effects in a cardiomyocyte culture
heterologously expressing the A3AR.11 The present study ex-
tends the GLiDe conjugate approach for the first time from AR
agonists to multivalent conjugates of AR antagonists. AR antago-
nists, the naturally occurring xanthines theophylline 1 and caffeine 2
being prototypical examples (Figure 1), are of therapeutic interest
in the treatment of Parkinson’s disease, diabetes, asthma, cancer,
and other conditions.4,12 The structureꢀactivity relationship (SAR)
of xanthine derivatives as AR antagonists has been exhaustively
explored.13,14 One of the earliest high affinityARligandprobeswas
the 8-phenyl derivative xanthine amine congener (3, XAC), which
is a moderately selective antagonist of the A1AR subtype in the
rat and is relatively nonselective in binding to human (h) ARs.15
XAC contains an amine-terminal chain placed at an insensitive site
on the pharmacophore and was designed as a functionalized
We have used poly(amidoamine) (PAMAM) dendrimers as
polymeric nanocarriers for drugs that bind to G protein-coupled
receptors (GPCRs) located on the cell surface.1ꢀ3 These recep-
tors are an important area of drug discovery. Agonists and
antagonists of rhodopsin-like GPCRs constitute a large fraction
(∼30%) of the 324 molecular targets of FDA-approved drugs.4
Multivalent GPCR ligandꢀdendrimer (GLiDe) conjugates, in
which a strategically derivatized agonist or antagonist is cova-
lently tethered from a high molecular weight PAMAM dendri-
mer, have displayed enhanced pharmacodynamic properties in
comparison to those of the monomeric ligands.2,3,5 One of the
objectives of this approach is to favorably alter the pharmacoki-
netics of the drug in vivo when bound to the nanocarrier. It is also
feasible to target the binding of a polymeric drug to aggregates of
GPCRs, which have been shown to exist and have been postu-
lated to be major participants in the varied biological effects of a
given receptor.6 GPCR dimers, including heterodimers of two
different receptor classes, have already been established to have
an altered pharmacology compared to that of homomeric GPCRs.7
We have shown with molecular modeling that GLiDe conjugates
of sufficient molecular size can adopt a suitable geometry to bridge
multiple binding sites in such receptor aggregates.8
We initially reported the use of PAMAM dendrimers as
scaffolds for the presentation of nucleosides and nucleotides that
selectively activate adenosine receptors (ARs, particularly the
Received: December 17, 2010
Revised:
May 2, 2011
Published: May 03, 2011
This article not subject to U.S. Copyright.
Published 2011 by the American Chemical Society
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ARTICLE
Figure 1. Structures of nonselective AR antagonists (1ꢀ3) and the design of a series of multivalent dendrimer conjugates of a potent xanthine,
represented by the triazole derivative of unspecified stoichiometry in the general formula (5). Compound 3 is a functionalized XAC congener designed
for attachment to carrier moieties with retention of the ability to bind potently to ARs. The conjugation of alkyne derivative 4 is performed using click
chemistry, which forms a triazole linker on an insensitive site on the xanthine with respect to receptor recognition.
congener,16 i.e., for attachment to larger carrier moieties and
reporter groups with retention of the ability to bind potently to ARs.
In this study, we have identified a means of linking XAC to
water-soluble PAMAM dendrimers by Cu(I)-catalyzed click
chemistry between an alkyne and an azide,17 which we found
to preserve the high receptor affinity in this series. The degree of
substitution by XAC and by polyethylene glycol (PEG) water-
solubilizing chains was systematically varied and the effects on
receptor affinity studied. This analysis suggests that each GLiDe
conjugate molecule may bind to multiple AR binding sites in the
vicinity on the cell surface. Furthermore, the ability to introduce
prosthetic groups for receptor localization or characterization on
the carrier without interfering with the high affinity in receptor
binding was shown.
optimized Q-TOF-2 (Micromass-Waters) using external cali-
bration with polyalanine and matrix-assisted laser desorption
ionization (MALDI) time-of-flight MS experiments on a
Waters LCT Premier mass spectrometer at the Mass Spectro-
metry Facility, National Institute of Diabetes and Digestive and
Kidney Diseases (NIDDK), NIH. Observed mass accuracies are
those expected on the basis of known performance of the
instrument as well as the trends in masses of standard com-
pounds observed at intervals during the series of measurements.
Reported masses are observed masses uncorrected for this time-
dependent drift in mass accuracy. IR spectra were recorded
using a PerkinElmer Spectrum One FT-IR spectrometer
(applied as a DMSO solution).
Azido-Derivatized G4 PAMAM dendrimer (6b). The G4
PAMAM dendrimer containing an ethylene diamine core was
obtained from Sigma-Aldrich and terminal amines reacted with
imidazole-1-sulfonyl azide hydrochloride.3 K2CO3 (3.48 g,
’ EXPERIMENTAL PROCEDURES
Chemical Synthesis. Materials and Methods. All reactions
were carried out under a nitrogen atmosphere. We purchased
N-ethyl-N0-dimethylaminopropylcarbodiimide hydrochloride
(EDC.HCl) and the G4 PAMAM dendrimer (5 wt % solution
in methanol) with an ethylenediamine core 6a from Sigma-
Aldrich (St. Louis, MO). All other reagents and solvents, except
those indicated, came from Sigma-Aldrich. We purchased dialysis
membranes (Spectra/Pore Membrane, MWCO 3500, flat width
18 mm) from Spectrum Laboratories (Rancho Dominguez, CA).
Imidazole-1-sulfonyl azide hydrochloride was prepared.18 Synth-
esis of XAC 3 is described elsewhere.14 Active esters IR-
Dye800CW 23 and IRDye700DX 24 were obtained from LI-
COR Biosciences (Lincoln, NE). Amicon centrifugal ultrafilters
(molecular weight cutoff 3000) were used.
Proton nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker DRX-400 spectrometer with d6-DMSO
as a solvent. The chemical shifts are reported in parts per million
(ppm) relative to tetramethylsilane (δ 0.0 ppm) or in D2O
relative to HOD (4.80 ppm). ESIꢀhigh resolution mass spectro-
scopic (HRMS) measurements were performed on a proteomics
30.15 mmol), CuSO4 5H2O (34.6 mg, 0.163 mmol), and
3
imidazole-1-sulfonyl azide hydrochloride (3.0 g, 1.04 mol) were
added to a solution of G4 PAMAM dendrimer 6a, (20 g, 10 w% in
methanol, [NH2] = 16.3 mmol) in anhydrous methanol (11 mL),
and the reaction mixture was stirred at room temperature over-
night. The mixture was then dialyzed in deionized water for 7 days
(changing water 4 times per day). Lyophilization after dialysis
provided the azide-derivatized G4 dendrimer 6b (3.2 g, 58%) as a
solid. IR νmax 2110 cmꢀ1. ESI-MS: calcd, 15,611; found, 15,545.
G4 PAMAM, Conjugated with PEG [8] (8). The azide-deriva-
tized G4 PAMAM dendrimer 6b (111 mg, 7.1 μmol) and a
poly(ethylene glycol)methyl ether derivative of butynoic acid 7
(MW 2000, Aldrich Chemical Co., 114 mg, 57 μmol) were added
to a mixture of DMSO (4 mL) and water (4 mL). The solution
was treated with freshly prepared aqueous sodium ascorbate
(1 M solution, 228 μL), followed by the addition of 7.5% aqueous
cupric sulfate (380 μL, 113 μmol). The reaction mixture was
stirred at room temperature (rt) overnight, and the product was
purified by extensive dialysis in water (changing water 4 times per
day for 2 days). The mixture was lyophilized to give compound 8
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Bioconjugate Chemistry
ARTICLE
(153 mg, 67%) containing ∼8 equivalents of PEG per dendrimer
molecule as a colorless foamy solid. 1H NMR (DMSO-d6,
400 MHz) δ 8.10 (br s), 4.35 (s), 4.13 (s), 3.5 (s), 3.41ꢀ3.44
(m), 3.33 (br s), 3.24 (s), 2.67ꢀ2.87 (m), 2.26 (br s). ESI-MS:
calcd, 31,545; found, 32,077.
(br m), 2.05ꢀ2.33 (m), 1.39ꢀ1.81 (br m), 0.86ꢀ0.92 (br m).
ESI-MS: calcd, 70,050; found, 70,074.
G4 PAMAM, conjugated with PEG [22] and XAC [23]
derivative (13). Compound 13 (78%) was synthesized from
PEG conjugated dendrimer 9 following the same procedure as
that for compound 10 using 23 equivalents of the XAC alkyne
derivative 4. 1H NMR (DMSO-d6, 400 MHz) δ 8.13 (br s), 8.09
(d, J = 8.8 Hz), 7.91ꢀ7.86 (m), 7.11 (d, J = 9.2 Hz), 4.54 (s),
4.32ꢀ4.34 (m), 4.12 (br s), 4.04 (t, J = 7.6 Hz), 3.86 (t, J =
7.6 Hz), 3.58ꢀ3.68 (m), 3.44 (br s), 3.14ꢀ3.44 (br m), 2.67ꢀ
2.87 (br m), 2.03ꢀ2.33 (m), 1.49ꢀ1.75 (br m), 0.86ꢀ0.92
(br m). ESI-MS: calcd, 72,197; found, 72,320.
G4 PAMAM, Conjugated with PEG [22] (9). Compound 9
(74%) containing ∼22 equivalents of PEG per dendrimer
molecule was synthesized from dendrimer 6b following the
1
same method as that for compound 8. H NMR (DMSO-d6,
400 MHz) δ 8.03 (br s), 4.35 (s), 4.25 (s), 3.59 (s), 3.39ꢀ3.44
(m), 3.32 (br s), 3.24 (s), 2.87 (br s), 2.66 (br s), 2.33 (s), 2.24
(br s). ESI-MS: calcd, 59,545; found, 59,855.
N-(2-(2-(4-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-
purin-8-yl)phenoxy)-acetamido)ethyl)hept-6-ynamide (4).
6-Heptynoic acid (33 μL, 0.27 mmol) and EDC (68.8 mg,
0.36 mmol) were added to a solution of compound 3 (77 mg,
0.18 mmol) in anhydrous DMF (3 mL) and stirred overnight at
rt. Solvent was evaporated, and the residue was purified using
flash silica gel column chromatography to give compound 4
(69 mg, 72%) as a colorless solid. 1H NMR (CD3OD, 400 MHz)
δ 8.05 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 8.8 Hz, 2H), 4.61ꢀ4.63
(m, 2H), 4.14 (t, J = 7.2 Hz, 2H), 4.01 (t, J = 7.6 Hz, 2H), 3.41ꢀ
3.65 (m, 6H), 2.19ꢀ2.13 (m, 5H), 1.86ꢀ1.70 (m, 1H), 1.69ꢀ
1.64 (m, 4H), 1.52ꢀ1.46 (m, 1H), 1.15 (d, J = 6.4 Hz, 1H),
1.17ꢀ0.95 (m, 6H). HRMS calculated for C28H37N6O5 (M þ
H) þ: 537.2825; found, 537.2823.
G4 PAMAM, Conjugated with PEG [22] and the XAC [6]
Derivative (10). The azide-derivatized G4 PAMAM dendrimer
6b (10.5 mg, 0.17 μmol) and a XAC alkyne derivative 4 (0.54 mg,
1.02 μmol) were added to a mixture of DMSO (0.4 mL) and
water (0.4 mL). The solution was treated with freshly prepared
aqueous sodium ascorbate (1 M solution, 3 μL), followed by the
addition of 7.5% aqueous cupric sulfate (2.3 μL, 0.7 μmol). The
reaction mixture was stirred at rt overnight, and the product was
purified by extensive dialysis in water (changing water 4 times per
day for 2 days). The mixture was lyophilized to give compound
10 (6.9 mg, 66%) containing ∼22 equivalents of PEG and ∼6
xanthine equivalents per dendrimer molecule as a foamy solid.
1H NMR (DMSO-d6, 400 MHz) δ 8.15 (br s), 8.09 (d, J =
8.8 Hz), 7.71ꢀ7.97 (m), 7.16 (d, J = 9.2 Hz), 4.54 (s), 4.24ꢀ4.36
(m), 4.12 (br s), 4.04 (t, J = 7.6 Hz), 3.81 (t, J = 7.6 Hz),
3.54ꢀ3.67 (m), 3.49 (br s), 3.14ꢀ3.47 (br m), 2.63ꢀ2.71 (br
m), 2.07ꢀ2.33 (m), 1.42ꢀ1.81 (br m), 0.84ꢀ0.92 (br m). ESI-
MS: calcd, 63,074; found, 63,069.
G4 PAMAM, Conjugated with PEG [22] and the XAC [8]
Derivative (11). Compound 11 (70%) was synthesized from
PEG conjugated dendrimer 9 following the same procedure as
that for compound 10 using 8 equivalents of the XAC alkyne
derivative 4. 1H NMR (DMSO-d6, 400 MHz) δ 8.13 (br s), 8.10
(d, J = 8.8 Hz), 7.87ꢀ7.93 (m), 7.11 (d, J = 9.2 Hz), 4.55 (s),
4.32ꢀ4.36 (m), 4.12 (br s), 4.04 (t, J = 7.6 Hz), 3.85 (t, J =
7.6 Hz), 3.59ꢀ3.67 (m), 3.46 (br s), 3.14ꢀ3.42 (br m),
2.67ꢀ2.86 (br m), 2.05ꢀ2.33 (m), 1.51ꢀ1.75 (br m), 0.86ꢀ
0.91 (br m). ESI-MS: calcd, 64,147; found, 64,251.
G4 PAMAM, Conjugated with PEG [22] and the XAC [19]
Derivative (12). Compound 12 (75%) was synthesized from
PEG conjugated dendrimer 9 following the same procedure as
that for compound 10 using 19 equivalents of the XAC alkyne
derivative 4. 1H NMR (DMSO-d6, 400 MHz) δ 8.17 (br s), 8.07
(d, J = 8.8 Hz), 7.69ꢀ7.93 (m), 7.11 (d, J = 9.2 Hz), 4.54 (s),
4.30ꢀ4.35 (m), 4.13 (br s), 4.02 (t, J = 7.6 Hz), 3.84 (t, J =
7.6 Hz), 3.58ꢀ3.68 (m), 3.51 (br s), 3.13ꢀ3.48 (br m), 2.62ꢀ2.7
G4 PAMAM, Conjugated with PEG [22] and the XAC [34]
Derivative (14). Compound 14 (84%) was synthesized from
PEG conjugated dendrimer 9 following the same method as that
for compound 10 using 34 equivalents of the XAC alkyne
1
derivative 4. H NMR (DMSO-d6, 400 MHz) δ 8.11 (br s),
8.09 (d, J = 9.2 Hz), 7.90ꢀ7.85 (m), 7.11 (d, J = 9.2 Hz), 4.54 (s),
4.02 (t, J = 6.0 Hz), 3.87 (t, J = 7.6 Hz), 3.12ꢀ3.71 (br m),
2.67ꢀ2.74 (m), 2.30ꢀ2.38 (m), 2.10ꢀ2.14 (m), 2.05 (t, J =
7.2 Hz), 1.72ꢀ1.79 (m), 1.53ꢀ1.61 (m), 1.37ꢀ1.41 (m), 0.86ꢀ
0.92 (br m). ESI-MS: calcd, 78,100; found, 77,999.
G4 PAMAM, Conjugated with PEG [8] and the XAC [4]
Derivative (15). Compound 15 (67%) was synthesized from
PEG conjugated dendrimer 8 following the same method as that
for compound 10 using 4 equivalents of the XAC alkyne
1
derivative 4. H NMR (DMSO-d6, 400 MHz) δ 8.14 (br s),
8.08 (d, J = 8.8 Hz), 7.72ꢀ7.90 (m), 7.10 (d, J = 8.8 Hz), 4.54 (s),
4.34 (br s), 4.13 (br s), 4.02 (t, J = 7.6 Hz), 3.86 (t, J = 7.5 Hz),
3.60ꢀ3.67 (m), 3.49 (br s), 3.08ꢀ3.44 (br m), 2.67ꢀ2.87 (br
m), 2.22ꢀ2.33 (br m), 1.48ꢀ1.68 (br m), 0.86ꢀ0.92 (br m).
ESI-MS: calcd, 34,223; found, 34,377.
G4 PAMAM, Conjugated with PEG [8] and the XAC [16]
Derivative (16). Compound 16 (72%) was synthesized from
PEG conjugated dendrimer 8 following the same method as
that for compound 10 using 17 equivalents of the XAC alkyne
1
derivative 4. H NMR (DMSO-d6, 400 MHz) δ 8.15 (br s),
8.09 (d, J = 8.8 Hz), 7.81ꢀ7.86 (m), 7.11 (d, J = 8.8 Hz), 4.54
(s), 4.34 (s), 4.19 (t, J = 7.5 Hz), 4.04 (t, J = 7.6 Hz), 3.52 (br s),
3.14ꢀ3.44 (br m), 2.62ꢀ2.74 (m), 2.03ꢀ2.33 (m), 1.39ꢀ1.75
(br m), 0.89ꢀ0.92 (br m). ESI-MS: calcd, 40,662; found,
40,461.
G4 PAMAM, Conjugated with PEG [8] and the XAC [25]
Derivative (17). Compound 17 (80%) was synthesized from
PEG conjugated dendrimer 8 following the same method as that
for compound 10 using 26 equivalents of the XAC alkyne
1
derivative 4. H NMR (DMSO-d6, 400 MHz) δ 8.16 (br s),
8.09 (d, J = 8.8 Hz), 7.86ꢀ7.88 (m), 7.11 (d, J = 8.8 Hz), 4.54 (s),
4.02 (t, J = 6.8 Hz), 3.89 (t, J = 6.7 Hz), 3.51 (br s), 3.14ꢀ3.32
(m), 2.10ꢀ2.14 (m), 2.04 (t, J = 7.2 Hz), 1.72ꢀ1.77 (m),
1.51ꢀ1.61 (m), 1.35ꢀ1.43 (m), 0.81ꢀ0.92 (br m). ESI-MS:
calcd, 45,492; found, 45,223.
G4 PAMAM, Conjugated with PEG [8] and the XAC [37]
Derivative (18). Compound 18 (84%) was synthesized from
PEG conjugated dendrimer 8 following the same method as that
for compound 10 using 37 equivalents of the XAC alkyne
1
derivative 4. H NMR (DMSO-d6, 400 MHz) δ 8.18 (br s),
8.08 (d, J = 9.2 Hz), 7.86ꢀ7.88 (m), 7.11 (d, J = 8.8 Hz), 4.54 (s),
4.02 (t, J = 6.0 Hz), 3.87 (t, J = 6.8 Hz), 3.12ꢀ3.62 (br m),
2.67ꢀ2.74 (m), 2.33 (br s), 2.10ꢀ2.13 (m), 2.05 (t, J = 6.8 Hz),
1.72ꢀ1.77 (m), 1.53ꢀ1.61 (m), 1.37ꢀ1.43 (m), 0.88ꢀ0.92 (br
m). ESI-MS: calcd, 51,931; found, 52,035.
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G4 PAMAM, Conjugated with PEG [8], XAC [37], and Alexa
Fluor 488 (20). A solution of Alexa Fluor 488 alkyne 19 (0.5 mg,
0.64 μmol) in water (0.5 mL) and freshly prepared sodium
ascorbate (1 M, 5 μL) were added to a solution of the XAC-
dendrimer conjugate 18 (8.2 mg, 0.16 μmol) in DMSO
(0.5 mL). Then, 7.5% aqueous cupric sulfate (6 μL, 113 μmol)
was added to the reaction mixture, which was stirred overnight at
rt. The dendrimer derivative was purified by extensive dialysis in
water, and lyophilization of the resulting solution gave com-
pound 19 (5.9 mg, 66%) as an orange colored solid. ESI-MS:
calcd, 57,065; found, 57,079.
PerkinElmer (Waltham, MA). DMEM/F12 medium, 1 M Tris-
HCl (pH 7.5), and G418 Sulfate were purchased from Media-
tech, Inc. (Herndon, VA). The calcium assay kit was from
Molecular Devices (Sunnyvale, CA). All other reagents were
from standard sources and are of analytical grade.
We prepared test compounds as 0.1 mM stock solutions
in DMSO and stored them at 4 °C. Adenosine deaminase
(25.3 U/mg) was purchased from Worthington Biochemical
Corporation (Lakewood, NJ.)
Cell Culture and Membrane Preparation. We cultured Chi-
nese hamster ovary (CHO) cells stably expressing the recombi-
nant hA1AR, hA2BAR, and hA3AR and human embryonic kidney
(HEK) 293 cells stably expressing the hA2AAR in DMEM and
F12 (1:1) supplemented with 10% fetal bovine serum (FBS),
50 U/mL penicillin, and 50 μg/mL streptomycin.19 In addition,
we added 800 μg/mL Geneticin to the hA2AAR media and
500 μg/mL Hygromycin B to the hA1AR, hA2BAR, and hA3AR
media. After harvesting the cells, we centrifuged them at 250g for
5 min at 4 °C. The pellet was resuspended in 50 mM Tris-HCL
buffer (pH 7.5), containing 10 mM MgCl2. The suspension was
homogenized with an electric homogenizer for 10 s and was then
recentrifuged at 20,000g for 30 min at 4 °C. The resultant pellet
was homogenized again, resuspended in the buffer mentioned
above in the presence of 3 U/mL adenosine deaminase, finally
pipetted into 1 mL vials, and then stored at ꢀ80 °C until the
binding experiments were conducted. We measured the protein
concentration with BCA Protein Assay Kit from Pierce Biotech-
nology (Rockford, IL).20
Binding Assays. Binding assays using standard AR ligands were
performed using the general methods reported.21ꢀ23 Each tube
in the binding assay contained 50 μL of increasing concentra-
tions of the test ligand in Tris-HCl buffer (50 mM, pH 7.5)
containing 10 mM MgCl2, 50 μL of the appropriate agonist
radioligand, and 100 μL of membrane suspension, added
sequentially. For the hA1AR (20 μg protein/tube), we used the
radioligand [3H]R-PIA (0.2 nM, precise final concentration is
calculated for each experiment). For the hA2AAR (20 μg protein/
tube), we used the radioligand [3H]CGS21680 (10 nM). For the
hA3AR (20 μg protein/tube), we used the radioligand [125I]I-
AB-MECA (0.89 nM). We determined nonspecific binding with
a final concentration of 10 μM unlabeled NECA diluted with the
buffer. We incubated the mixtures at 25 °C for 60 min in a
shaking water bath. We terminated binding reactions by filtration
through Brandel GF/B filters under reduced pressure with an
M-24 cell harvester (Brandel, Gaithersburg, MD). We washed
filters three times with 3 mL of 50 mM ice-cold Tris-HCl buffer
(pH 7.5). We then placed filters for hA1AR and hA2AAR binding
in scintillation vials containing 5 mL of Hydrofluor scintillation
buffer and counted with a PerkinElmer Liquid Scintillation
Analyzer (Tri-Carb 2810TR). We counted filters for hA3AR
binding with a Packard Cobra II γ-counter (PerkinElmer).
Cyclic AMP Accumulation Assay. CHO cells expressing the
A1AR or A2BAR were seeded in 24-well plates and incubated at
37 °C overnight. The following day, the medium was removed
and replaced with DMEM containing 50 mM HEPES, 10 μM
rolipram, 3 U/mL adenosine deaminase, and increasing concen-
trations of agonists. For measurements at the A1AR, forskolin
(10 μM) was added 30 min after the addition of agonists and
incubated for another 15 min. Antagonists were added 20 min
before the addition of agonists. The medium was removed, and
the cells were lysed with 200 μL of 0.1 M HCl. One hundred
microliters of the HCl solution was used in the Sigma Direct
4-(2-Hept-6-ynamidoethyl)benzene-1-sulfonyl Fluoride (21).
6-Heptynoic acid (11 μL, 0.09 mmol), HATU (31 mg,
0.08 mmol), and diisopropyl ethylamine (14 μL, 0.08 mmol)
were added to a solution of 4-(2-aminoethyl)benzene-1-sulfo-
nyl fluoride (Aldrich, 15 mg, 0.06 mmol) in anhydrous DMF
(1 mL). The reaction mixture was stirred overnight at rt. The
solvent was evaporated, and the residue was purified using flash
silica gel column chromatography (hexane/ethyl acetate = 2:1)
1
to give compound 21 (15.2 mg, 78%) as a colorless solid. H
NMR (CD3OD, 400 MHz) δ 8.00 (d, J = 8.4 Hz, 2H), 7.60 (d,
J = 8.0 Hz, 2H), 3.52ꢀ3.48 (m, 2H), 2.98 (t, J = 6.8 Hz, 2H),
2.32 (t, J = 7.2 Hz, 2H), 2.23ꢀ2.20 (m, 2H), 1.75ꢀ1.44 (m,
þ
5H). HRMS calculated for C15H19NO3SF (M þ H)
:
312.1070; found, 312.1073.
G4 PAMAM, Conjugated with PEG [8], XAC [37], Alexa Fluor
488, and the Benzene-sulfonyl Fluoride Derivative (22). Ben-
zene-sulfonyl fluoride alkyne derivative 21 (0.5 mg, 1.6 μmol)
and freshly prepared sodium ascorbate (1 M, 3 μL) were added
to a solution of XAC-dendrimer conjugate 20 (5.6 mg, 0.1 μmol)
in DMSO (0.4 mL). Then, 7.5% aqueous cupric sulfate (4 μL,
0.9 μmol) was added to the reaction mixture, which was stirred
overnight at rt. The dendrimer derivative was purified by extensive
dialysis in water, and lyophilization of the resulting solution gave
compound 22 (4.2 mg, 73%) as an orange colored solid. ESI-MS:
calcd, 58,945; found, 59,155.
G4 PAMAM, Conjugated with PEG [22], XAC [34], and the IR
Dye 800 Derivative (25). A solution of IR dye 800CW NHS ester
23 (0.5 mg, 0.42 μmol) in DMF (0.3 mL) was added to a solution
of compound 14 (2.66 mg, 0.05 μmol) in DMF (1 mL) followed
by triethylamine (10 μL, 0.42 μmol) and stirred overnight at rt.
The dendrimer derivative was purified by extensive dialysis in
water, and lyophilization of the resulting solution gave com-
pound 25 (2.13 mg, 76%) as a green colored foamy solid. ESI-
MS: calcd, 81,943; found, 82,258.
G4 PAMAM, Conjugated with PEG [22], XAC [34], the IR Dye
700 Derivative (26). A solution of IR dye 700DX NHS ester 24
(0.5 mg, 0.25 μmol) in DMF (0.3 mL) was added to a solution of
compound 14 (2.32 mg, 0.03 μmol) in DMF (1 mL) followed by
sodium tetraborate buffer (20 μL, 0.25 μmol), and the reaction
mixture was stirred overnight at rt. The dendrimer derivative was
purified by extensive dialysis in water, and lyophilization of the
resulting solution gave compound 26 (2.19 mg, 83%) as a green
colored foamy solid. ESI-MS: calcd, 88,511; found, 89,063.
Biological Methods. Materials. PenicillinꢀStreptomycinꢀ
Glutamine and Hygromycin B were purchased from Invitrogen
(Carlsbad, CA). [3H]R-N6-(2-Phenylisopropyl)adenosine ([3H]R-
PIA, 42.6 Ci/mmol) was obtained from Moravek Biochemicals
(Brea, CA). [125I]4-Amino-3-iodobenzyl-50-N-methylcarboxami-
doadenosine ([125I]I-AB-MECA, 2200 Ci/mmol) and [3H]-2-
[p-(2-carboxyethyl)phenylethylamino]-50-N-ethylcarboxamidoade-
nosine ([3H]CGS21680, 40.5 Ci/mmol) were purchased from
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ARTICLE
Scheme 1. Use of Click Chemistry in Sequential Steps to Synthesize PAMAM Dendrimer Derivatives 9ꢀ18, Containing a
Functionalized AR Antagonist (Average Structures Shown)a
a (A) PEG moieties were added as water-solubilizing groups, followed by conjugation of the xanthine alkyne derivative 4 (B). Compound 7 was
calculated to contain approximately 43 PEG groups on the basis of its molecular weight (2000 D).
cAMP Enzyme Immunoassay following the instructions pro-
vided with the kit. The results were interpreted using a BioTek
ELx808 Ultra Microplate reader (BioTek, Winooski, VT) at
405 nm.
of the xanthine moieties for graphic presentation. We repeated all
experiments at least three times.
’ RESULTS
Intracellular Calcium Mobilization. CHO cells expressing
the A1AR cells were grown overnight in 100 μL of media in
96-well flat bottom plates at 37 °C at 5% CO2 or until 80ꢀ
90% confluency. The calcium assay kit (Molecular Devices,
Sunnyvale, CA) was used as directed without washing 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 μL of dye with probenecid to each well and incubated for
60 min at rt. The compound plate was prepared using dilutions of
various compounds in Hanks Buffer (pH 7.4). Samples were run
on a FLIPRTETRA (Molecular Devices) in duplicate at rt. Anta-
gonists were added 20 min before the addition of agonists. Cell
fluorescence was measured (excitation at 485 nm; emission at
525 nm) following exposure to agonists. Increases in intracellular
calcium are reported as the maximum fluorescence value after
exposure minus the basal fluorescence value before exposure.
Data Analysis. We initially measured the concentrations of the
dendrimerꢀligand complexes by the concentration of the den-
drimer, not the attached ligand. We determined EC50 and
apparent Ki values with Prism software (GraphPad, San Diego,
CA); they are presented as the mean ( SE. The number of
xanthine moieties on a given dendrimer that are accessible to
receptors is uncertain; the data are therefore shown as apparent
Ki values. Later, the same data were converted to the concentration
Chemical Synthesis. We prepared generation 4 (G4) PA-
MAM dendrimer conjugates of the potent, nonselective hAR
antagonist XAC 3 (Scheme 1 and Table 1, 9ꢀ18) to act as multiva-
lent ligands of the receptors. The peripheral groups of the
precursor dendrimer 6a were modified to contain predominantly
azido groups in place of the 64 terminal primary amines by
reacting with imidazole-1-sulfonyl azide hydrochloride in metha-
nol to give compound 6b.3,18 The same approach of preparing
the PAMAM dendrimer for click chemistry by the substitution of
amino groups with azides was taken in the synthesis of conjugates
of AR agonists and P2Y1 receptor antagonists.3,9 Mass spectral
data of 6b indicated that 8 amino groups out of the 64 remained
unreacted, and the presence of azido groups was confirmed by a
strong IR peak at 2110 cmꢀ1. The presence of these residual
amines was later used for synthetic advantage in the coupling of
additional prosthetic groups. Prior to coupling of the pharma-
cophoric species, the dendrimer was appended with PEG moi-
eties by a Cu(I)-catalyzed click cyclization using an alkyne-
derivatized PEG 7 (end-capped by a methyl ether and a butynoyl
ester) to yield the water-soluble and mainly uncharged PAMAM
derivatives 8 and 9, having 8 or 22 PEG moieties per dendrimer,
respectively. Thus, two degrees of substitution with PEG of the
64-terminal groups of the G4 dendrimer were compared.
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Table 1. Binding Affinity of a Series of XAC Dendrimer Conjugates (10ꢀ22), Dendrimer Precursors (9, (PEG)22 series; 8,
(PEG)8 series), and Small Moleculal Monomers (3 and 4) at Three Subtypes of Human ARsa
compound
# of XAC moieties/dendrimer
Ki,app or % inhibition, A1AR
Ki,app or % inhibition, A2AAR
Ki,app or % inhibition, A3AR
3b
4b
9c
(monomer)
8.3 ( 1.3 nM
107 ( 13 nM
21.8 ( 17.8%
34.3 ( 14.2%
59.2 ( 3.5%
91.0 ( 15.6 nM
47.1 ( 3.4 nM
15.0 ( 4 nM
10.3 ( 5.2%
193 ( 117 nM
95.4 ( 35.3 nM
54.3 ( 17.1 nM
29.0 ( 7.1 nM
20.2 ( 6.5 nM
29.3 ( 8.2 nM
ND
5.8 ( 2.3 nM
23.2 ( 11.2 nM
29.7 ( 4.1%
45.9 ( 2.3%
61.5 ( 23.0 nM
11.4 ( 5.0 nM
4.2 ( 1.7 nM
2.6 ( 0.6 nM
4.4 ( 5.8%
13.8 ( 4.4 nM
175 ( 15 nM
70.2 ( 3.7%
(monomer)
0
10
11
12
13
14b,c
8c
6
71.7 ( 6.7%
8
219 ( 67 nM
30.6 ( 3.8 nM
18.1 ( 5.2 nM
8.9 ( 3.3 nM
51.9 ( 3.6%
19
23
34
0
15
16
17
18b,c
20
22
25b
26b
4
32.7 ( 6.0 nM
15.1 ( 1.7 nM
4.6 ( 0.4 nM
4.2 ( 0.3 nM
4.1 ( 0.2 nM
5.4 ( 1.8 nM
ND
99.7 ( 25.0 nM
48.7 ( 20.2 nM
25.2 ( 3.8 nM
14.5 ( 4.4 nM
6.2 ( 0.9 nM
7.8 ( 1.2 nM
6.8 ( 2.1 nM
20.3 ( 16.4 nM
15
25
37
37
37
34
34
ND
3.7
a All experiments were done on CHO (A1 and A3AR) or HEK293 (A2AAR) cells stably expressing one of four subtypes of human ARs. The binding
affinity for A1, A2A, and A3ARs was expressed as Ki values (n = 3ꢀ5) and was determined by using agonist radioligands ([3H]R-PIA, [3H]CGS21680, or
[
125I]I-AB-MECA, respectively), unless noted. The percentage inhibition refers to the inhibition of radioligand binding at the concentration of 1 μM.
The concentrations of the dendrimerꢀligand complexes were measured by the concentration of the dendrimer, not the ligand. Therefore, all binding Ki
values of dendrimers are expressed as Kiapp values. ND: not determined. b 3, XAC; 4, MRS5398; 14, MRS5359; 18, MRS5385; 25, MRS5421; 26,
c
MRS5422. PEGylated dendrimer 9 is the precursor of 10ꢀ14; PEGylated dendrimer 8 is the precursor of 15ꢀ18. Xanthine-derivatized 18 is the
dendrimer precursor of 20 and 22. Xanthine-derivatized 14 is the dendrimer precursor of 25 and 26.
The synthetic method for cross-linking the receptor ligand to
the nanocarriers 8 and 9 by click chemistry3 required the
introduction of an alkyne at an insensitive site on the xanthine
moiety, and for this purpose, the hexynoyl amide derivative of
XAC 4 was prepared. We have explored in detail the SAR at ARs
at the distal region of the chain extended from the para position
of the 8-phenylxanthines.15,16 This is a site for unlimited chain
derivatization, and thus, the alkyne-functionalized chain was used
to provide click products with the azide-functionalized dendrimers.3
We systematically varied the degree of substition of the dendrimer
carrier with the GPCR ligand, i.e., the nonselective AR antagonist,
ranging from substitution of an average of 4 to 37 of the terminal
groups with the xanthine moiety. The efficiency of incorporation
of the xanthine was nearly quantitative depending on the equiva-
lents of 4 added to the click reaction, as indicated by mass
spectroscopy. A fully XAC-substituted dendrimer was not pre-
pared due tothelimitation of aqueous solubility, whichpresented a
concern in the absence of tethered PEG groups.
XAC conjugate 18, which contained a random distribution of
an average of 37 xanthine moieties and 8 PEG groups, was further
derivatized with functional prosthetic groups that were coupled
by click chemistry (Scheme 2). A fluorescent prosthetic group for
spectroscopic characterization (Alexa Fluor 488) was included in
conjugates 20 and 22.24 Alexa Fluor 488, which has emission and
excitation maxima at 495 and 519 nm, respectively, has already
been used as a fluorophore in small ligand probes of the A2AAR.25
Conjugate 22 also contained an aryl sulfonyl fluoride (an average
of 5 moieties incorporated per PAMAM dendrimer) for affinity
labeling of the receptor.26 Aryl sulfonyl fluorides readily react
irreversibly with proximal nucleophilic amino acids on a receptor
yet are sufficiently stable to be applied in aqueous media. Two
near-infrared (NIR) dyes27 with absorption in the range of
800 nm (25) and 700 nm (26) were included in conjugates
containing an average of 34 xanthine moieties per dendrimer.
The NIR dyes were amide-linked to dendrimer 14 using the
active ester precursors 23 and 24, leading to an average degree of
incorporation of 4 or 6 dye moieties incorporated per PAMAM
dendrimer in 25 and 26, respectively, as shown in Scheme 3.
Radioligand Binding. We tested the xanthineꢀdendrimer
conjugates in binding assays at three hAR subtypes; we used
standard radioligands21ꢀ23 and membrane preparations from
CHO cells (hA1AR and hA3AR) or HEK293 cells (A2AAR) stably
expressing a hAR subtype.28,29 Binding affinity at the A2BAR was not
determined because of the lack of a facile radioligand binding assay,
but one representative conjugate was examined in a functional assay
at this subtype.19 Binding results shown in Table 1 indicated that
receptor affinity was maintained in many of the conjugates. We used
a previously reported monomeric antagonist of the ARs, XAC 3,16
and its hexynoyl derivative 4, the synthetic precursor of the
conjugates, for comparison in the binding assays. We included the
parent dendrimers 8 and 9, which contained a mixture of PEG,
azido, and amino terminal groups, as controls; they were inactive or
only weakly active in inhibiting the binding of hA1AR and hA2AAR
radioligands. The parent dendrimers 8 and 9 at 1 μM inhibited
binding at the hA3AR by 72% and 52%, respectively. However, we
were not able to plot a full sigmoidal curve in the competition
binding experiments to determine a Ki value. The relatively high
displacement of radioligand at the hA3AR by the parent dendrimers
in high concentration may be related to the presence of aromatic
triazolo rings on the surface of the dendrimer.
Inhibition of radioligand binding by dendrimer conjugates 14
and 18 in membranes of CHO cells expressing the hA1 and A3
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Scheme 2. Synthesis of PAMAM Dendrimer Derivatives That Combined an Antagonist of the ARs and Functional Prosthetic
Groups That Were Coupled by Click Chemistry (Average Structures Shown)a
a These consisted of conjugates for fluorescent detection (20) and for irreversible binding to the receptors (22). The dendrimer precursor 18 contained a
random distribution of 37 xanthine moieties.
ARs and HEK-293 cells expressing the hA2AAR is shown in
Figure 2B,C. Inhibition of radioligand binding by the xanthine
monomer 4 is shown for comparison in Figure 2A. The results were
also plotted graphically to illustrate the dependence of the affinity on
the degree of substitution (Figure 3). Two methods of calculating
the data were used: initially according to the concentration of the
dendrimer, which is a single multivalent molecule as was done
in previous studies,1,2,5,9 or according to the concentration of the
pendant xanthine moieties. The latter method is a mathematical
construct, not an authentic equilibrium constant, because the
xanthine moieties could not all be available to compete for receptor
binding sites, by virtue of spatial limitations.
There was a smooth dependence of the affinity on the degree
of substitution of the XAC-derivatized click-linked dendrimers in
both series containing 8 or 22 PEG groups out of a total of 64
terminal sites. The more highly substituted dendrimer derivatives
were considerably more potent than lightly substituted conju-
gates in binding assays at the hA1AR, hA2AAR, and hA3AR. Thus,
there was a regular progession toward increased affinity at each of
these AR subtypes for 10ꢀ14 (from 6 to 34 xanthine moieties) in
the more heavily PEGylated series and for 15ꢀ18 (from 4 to 37
xanthine moieties) in the more lightly PEGylated series. The
dendrimer conjugates having 8 PEG groups displayed Ki values at
the hA2AAR that varied from 33 nM to 4 nM (n = 3), for 15 and
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Scheme 3. Synthesis of PAMAM Dendrimer Conjugates That Combined an Antagonist of the ARs and Prosthetic Groups for NIR
Detection That Were Coupled through Amide Linkage (Average Structures Shown)a
a The dendrimer precursor 14 contained a random distribution of 34 xanthine moieties.
18, respectively. The gain of affinity for the more highly PEGylated
dendrimer series (Figure 3C and D) was steeper than the gain in the
less highly PEGylated dendrimer series (Figure 3A and B). Thus,
the affinity of the most highly substituted conjugate having 22 PEG
groups (14) in binding to the ARs was particularly high, with Ki
values of 15, 2.6, and 8.9 nM at hA1AR, hA2AAR, and hA3AR,
respectively. Therefore, the binding affinity at hAR subtypes
(inversely related to apparent Ki) of these multivalent conjugates
depended mainly on the degree of loading with the pharmacophore
and secondarily on the degree of PEG substitution.
The order of affinity at AR subtypes for each conjugate was
generally hA2AAR > hA3AR > hA1AR. Therefore, a slight selectivity
of these xanthineꢀdendrimer conjugates for the hA2AAR in com-
parison to that of the other ARs was demonstrated (3- to 6-fold for
14 and 4- to 7-fold for 18). However, the corresponding monomers
3 and 4 displayed affinity in the order of hA2AAR > hA1AR g
hA3AR. Therefore, conjugation to the carrier altered the pharma-
cological profile of the xanthine by relatively increasing the A3AR
affinity. It is unknown whether this difference is due to the multi-
valency or to the chemical nature of the linker and its effect on
interaction with the extracellular regions of the receptor.
The association of binding activity of the conjugate with a
macromolecular species was confirmed by filtering a solution of
compound 14 through a 3000 MW cutoff centrifugal filter. Both
the UV absorption of the retained fraction and its binding activity
were maintained following ultrafiltration. Thus, the observed
activity was due to the intact conjugate, rather than a diffusable
small molecular weight impurity.
High affinity in AR binding was maintained in the conjugates
containing prosthetic groups for receptor characterization (20,
22, 25, and 26). In fact, the Ki values of these conjugates were
nearly the same as those of the precursors, i.e., XAC-conjugates
14 and 18. This was a striking observation considering the
structural diversity of the attached prosthetic groups. Compound
22 might have a component of nonequilibrium binding because
of the chemically reactive sulfonyl fluoride group, but this was not
evident from these binding results. The affinity of the 800 nm
NIR dye in 25 was identical to precursor 14, but the more
sterically bulky NIR dye in 26 caused a slight (3-fold) loss of
affinity at the A3AR.
We further examined the A1AR antagonist activity of com-
pound 14, one of the most potent conjugates for the AR subtypes
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A representative conjugate 14 was examined as an antagonist
in a functional adenylate cyclase assay at the Gs-coupled hA2BAR
expressed in CHO cells.19 The multivalent xanthine at a con-
centration of 10 nM produced an 18-fold right-shift of the NECA
response curve (Figure 5).
’ DISCUSSION
In this study, we used the Cu(I)-catalyzed alkyneꢀazide click
reaction as a chemically and biologically effective means of
linking a functionalized congener of a potent AR antagonist to
PAMAM dendrimers. Click chemistry was previously used to
link small molecular AR agonists to dendrimeric structures,30 but
this is the first systematic probing of PAMAM dendrimer conjugates
of small-molecular AR antagonists. As with the agonist conju-
gates, high affinity of the GLiDe conjugates was achieved. The
efficiency of the click reaction readily allowed a high degree of
substitution in the dendrimer derivatives that could be varied by
the proportion of PEG-alkyne or XAC-alkyne to dendrimer
molecules that was added to the reaction.
The effectiveness of PEGylation of polymeric drug conjugtes
to enhance polymer architecture, self-assembly, and bioavailabil-
ity has been demonstrated.31 In the present conjugates of a
hydrophobic xanthine pharmacophore, water-solubility was in-
creased, and the characteristic aggregation of PAMAM dendri-
mers was reduced by the presence of PEG groups. We compared
different degrees of loading of the GPCR ligand and of PEG
groups to the dendrimeric carrier. The ability to bind the ARs was
not prevented by attached PEG groups (each approximately 43
oxoethylene units in length), similar to previous findings with AR
agonist GLiDe conjugates.32
More highly XAC-substituted dendrimer derivatives prepared
by click chemistry were particularly potent in binding to the ARs,
with some selectivity for the hA2AAR subtype in comparison to
that for hA1 and hA3ARs. There was also indication of high
potency antagonist activity at the hA2B for a representative
derivative 14. Nearly nanomolar affinities in binding to three
AR subtypes were attained in the most highly XAC-substituted
conjugates, with affinity enhancement for the conjugates versus
monomer 4 being most pronounced for the A3AR. The phenom-
enon of progressively increased affinity upon greater xanthine
substitution was evident in both series of high and low PEG
content. However, the function of the gain in AR affinity with
increasing xanthine content did vary somewhat with different
degrees of PEG substitution. The conjugates with an average of
22 PEG units, corresponding to a total of 44,000 D added to the
molecular weight of the PAMAM, were initially weaker in
binding to ARs than the 8 PEG series but gained more rapidly
with increasing xanthine substitution. The molecular weights
of the highly substituted conjugates 14 and 18 were ∼78 and
∼53 kD, respectively. Thus, the PEG content of these two
conjugates amounted to approximately 56% and 30%, respec-
tively, of the total mass.
Substitution with additional prosthetic groups of varied struc-
ture attached either by click or amide-forming reactions did not
appreciably alter the binding affinity. For example, two sterically
bulky NIR dyes with absorption in the range of 800 and 700 nm
that are used in imaging in vivo33 were incorporated in conjugates
with retention of high receptor binding affinity. For example,
conjugate 25 is identical in A3AR affinity to its precursor and
contains an average of 4 equivalents of IR dye 800CW, which is
excited at 785 nm and fluorescence can be measured at 830 nm,
Figure 2. Inhibition of radioligand binding by xanthine monomer 4
(A) and dendrimer conjugates 14 (B) and 18 (C) (according to the
concentration of the dendrimer) in membranes of CHO cells expressing
the hA1 and A3 ARs and HEK-293 cells expressing the hA2AAR.
in two types of functional assays in stably transfected CHO cells.
We did not assay the functional effects at all the ARs; the Gi-coupled
A1AR is only representative of the full biological spectrum of the
xanthines, and the behavior of the conjugate could be different at
each of the AR subtypes. It was found that 14 at 100 nM right-
shifted the concentrationꢀresponse curve to the potent A1AR
agonist N6-cyclopentyladenosine (CPA) in the cyclic AMP func-
tional assay (inhibitory) in a parallel manner (Figure 4A). The
EC50 values of CPA in the absence and presence of compound 14 at
100 nM were 1.5 ( 0.3 and 45 ( 17.2 nM, respectively. The
activation of the A1AR is also reported to stimulate mobilization of
intracellular Ca2þ in various cells, which is likely dependent on Gβγ
subunits.30 CPA stimulated Ca2þ mobilization with an EC50 of 57.5
( 16.8nM(Figure 4B), and compound 14 at a concentration (10
nM) lower than its Ki value both right-shifted the CPA curve and
suppressed the maximal effect, suggesting qualitative pharmaco-
logical differences between the conjugates and monomers. In
contrast, the monomer XAC 3 at 10 nM or 100 nM (Figure 4C)
inhibited CPA-induced calcium mobilization in A1AR-expressing
CHO cells in a competitive fashion. In the presence of 100 nM
XAC, CPA displayed an EC50 of 346 ( 58 nM, corresponding to
a 6-fold reduced potency.
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Figure 3. Inhibition of radioligand binding by dendrimer nucleoside conjugates in membranes of CHO cells expressing the hA1 and A3 ARs and HEK-
293 cells expressing the hA2AAR showing dependence on the degree of substitution with the AR antagonist pharmacophore (XAC). Two methods of
calculating the data were used on the basis of the definition of antagonist concentration: according to the concentration of the dendrimer (A,C) or
according to the concentration of the pendant xanthine moieties (B,D). The conjugates were generally slightly selective for the hA2AAR in comparison to
the hA1 and hA3ARs.
which is suitable for whole body imaging in small animals.
Such GLiDe conjugates might eventually be useful for in vivo
diagnostic imaging of tissue overexpressing ARs, such as occurs
in disease states.34 Thus, the ability to substitute the dendrimer
with chemically diverse prosthetic groups in this series of AR
conjugates allows for considerable structural breadth for probing
and possibly targeting of the conjugates to the ARs or to cells
having specific markers on the surface.
The advantages of nanocarriers for small molecular weight
drugs include enhanced pharmacokinetic and pharmacodynamic
properties. Our approach derivatizes a small molecular GPCR
ligand for coupling to a carrier but allows it to remain active while
covalently attached.1,15 Internalization is not needed or desired
because the binding sites of the receptors are accessible only from
the extracellular medium. Molecular modeling indicated that
flexible PAMAM dendrimers are able to spread over the cell surface,
and purine conjugates of G3 dendrimers and higher generations
can readily adopt a conformation that can bridge multiple GPCR
binding sites.8 Inthe theoreticalmodel of a dendrimer conjugate of
an A2AAR agonist bridging adjacent binding sites of an A2AAR
homodimer, the distance between the two sites was ∼35 Å, which
was easily accommodated by two intermediate-spaced arms of the
G3 dendrimer in a nonextended conformation. This roughly
spherical G3 dendrimer conjugate had a diameter of ∼67 Å, and
the xanthine moieties of the present G4 conjugates in a non-
extended conformation might be expected to achieve an estimated
maximum separation of ∼80 Å. This distance would be increased
by stretching of the dendrimer over a surface.35
exist as complexes with other receptors and associated proteins.
The receptors to which a given GPCR pairs or forms a higher-
order aggregate can dramatically affect its pharmacology, and the
binding of an agonist to both protomers does not have equivalent
pharmacological effects.36 Thus, a multivalent GPCR ligand
might display unanticipated, complex effects.5 There is a lack
of GPCR ligands that are designed specifically to interact with
dimeric and oligomeric receptors. We do not have effective
ligand tools for separating the effects of one receptor dimeric
combination from another. The approach of mixing ligands for
two different receptors3,37,41 promises to be a means of achieving
selectivity for receptors in a given tissue, on the basis of
association or aggregation of GPCRs, in comparison to another
tissue in which the same receptor may be alternatively paired. At
this early stage, we do not know the topological requirements for
selectively targeting these combinations of receptors because we
are just beginning to detect their existence. Nevertheless, provid-
ing tools for studying these receptor aggregates is one of the
objectives of this study. The multivalent binding to other types of
cell surface receptors has been explored through sytematic
structural modification of the carrier polymer.42
There is evidence here for multivalent binding of the same
conjugate to more than one AR site. The fact that the AR affinity
rose substantially with increased xanthine content, even when
calculating IC50 values according to the concentration of the
pharmacophore rather than the dendrimer as shown in
Figure 3D, suggests that the same GLiDe conjugate molecule
may bind to multiple AR binding sites. The interaction of the
multivalent GLiDe conjugates can now be studied in cells
expressing varying levels of a given AR or with other receptors
coexpressed.
GPCRs tend not to be evenly or randomly distributed over the
surface of a given cell; we demonstrated this for the hA3AR
expressed heterologously in CHO cells.2 In general, GPCRs may
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Figure 5. Antagonist activity of compound 14 in the stimulation of
cyclic AMP accumulation induced by agonist NECA in CHO cells stably
expressing the hA2BAR. Each curve is representative of three determina-
tions. The EC50 values for NECA in the absence and presence of 10 nM
14 are 194 nM and 3.46 μM, respectively.
In conclusion, we have synthesized and characterized phar-
macologically a series of multivalent dendrimeric derivatives of a
potent strategically functionalized AR antagonist. GLiDe con-
jugates provide different quantitative and qualitative pharmaco-
logical properties than do monomers. Prosthetic groups designed
for therapeutic targeting or reporter groups for diagnostic
purposes might be introduced. Various therapeutic applications12
may be proposed for multivalent conjugates of AR antago-
nists. In vivo studies will be needed to fully characterize these
potent ligands biologically and determine their advantage over
monomeric drugs.
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR and mass spectra of
S
b
representative compounds 14 and 18. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Bldg. 8A, Rm. B1A-19, NIH, National Institute of Diabetes
and Digestive and Kidney Diseases, Bethesda, MD 20892-
0810. Tel. 301-496-9024, Fax 301-480-8422. E-mail: kajacobs@
helix.nih.gov.
Figure 4. Antagonist activity of compound 14 in the inhibition of
forskolin-stimulated cyclic AMP accumulation induced by agonist CPA
(A) and the inhibition by 14 of CPA-induced calcium mobilization (B)
in CHO cells stably expressing the hA1AR. Part C shows the comparable
inhibition by XAC 3 of CPA-induced calcium mobilization in hA1AR-
expressing CHO cells. Each curve is representative of three determina-
tions. The EC50 values are listed in the text.
Author Contributions
†Authors contributed equally to this study.
’ ACKNOWLEDGMENT
We thank Dr. John Lloyd and Dr. Noel Whittaker (NIDDK)
for the mass spectral determinations, Dr. Martin Garraffo
(NIDDK) for FT-IR measurements, and Khai Phan (NIDDK)
for pharmacological measurements. We thank Dr. Francesca
Deflorian (NIDDK), Dr. Michael Kilbey, III, and Dr. Kunlun
Hong (Oak Ridge National Laboratory, Knoxville TN) for
helpful discussions. This research was supported by the Intra-
mural Research Program of the NIH, NIDDK.
In previous studies, we have noted that the pharmacological
profile of GLiDe conjugates can differ from that of the mono-
meric ligands both quantitatively and qualitatively.5,11 For ex-
ample, the dendrimer conjugates of AR agonists have displayed a
shift in the AR subtype selectivity or in the correspondence
between the binding and functional potencies.2,11 Here, we have
detected a qualitative difference in the A1AR antagonistic re-
sponse between a potent, multivalent xanthine conjugate and its
corresponding monomer. Conjugate 14 acted as a competitive
antagonist of the GiR-dependent effect of inhibition of adenylate
cyclase, but at comparable concentrations acted as a noncompe-
titive inhibitor of calcium mobilization, which is likely indepen-
dent of GiR. The functional effects at other AR subtypes were not
determined here and will be the subject of further studies.
’ ABBREVIATIONS
AR, adenosine receptor; cAMP, adenosine 30,50-cyclic phos-
phate; CGS21680, 2-[p-(2-carboxyethyl)phenylethylamino]-50-
N-ethylcarboxamidoadenosine; CHO, Chinese hamster ovary;
CPA, N6-cyclopentyladenosine; DIPEA, diisopropylethylamine;
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Bioconjugate Chemistry
ARTICLE
DMEM, Dulbecco0s modified Eagle0s medium; DMF, N,N-di-
methylformamide; DMSO, dimethylsulfoxide; EDC, N-ethyl-N0-
dimethylaminopropylcarbodiimide; EDTA, ethylenediamine-
tetraacetic acid; GLiDe, GPCR ligandꢀdendrimer; GPCR, G
protein-coupled receptor; HATU, 2-(1H-7-azabenzotriazol-1-yl)-
1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium;
HEK, human embryonic kidney;HEPES, 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid; I-AB-MECA, 2-[p-(2-carbox-
yethyl)phenyl-ethylamino]-50-N-ethylcarboxamidoadenosine;
IB-MECA, N6-(3-iodobenzyl)-50-N-methylcarboxamidoadenosine;
MS, mass spectrometry; NECA, 50-N-ethylcarboxamidoadeno-
sine; NMR, nuclear magnetic resonance; NIR, near-infrared;
PAMAM, polyamidoamine; PEG, polyethyleneglycol; R-PIA,
R-N6-(2-phenylisopropyl)adenosine.
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