Influence of fluorophore and linker composition on the pharmacology of fluorescent adenosine A1 receptor ligands: Themed section: Imaging in pharmacology research paper

Article abstract of DOI:10.1111/j.1476-5381.2009.00488.x

Background and purpose: The introduction of fluorescence-based techniques, and in particular the development of fluorescent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level. This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties of fluorescent agonists and antagonists. Experimental approach: Chinese hamster ovary cells stably expressing the human adenosine A₁ receptor and a cyclic 3′,5′ adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with whole cell [³H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5′ (N-ethylcarboxamido) adenosine (NECA). Key results: Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant differences in their binding properties to the adenosine A₁ receptor. The BODIPY 630/650 derivatives had the highest affinity. Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives, respectively. Conclusions and implications: The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe. This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x 2010 The Authors. Journal compilation

Full text of DOI:10.1111/j.1476-5381.2009.00488.x

British Journal of Pharmacology (2010), 159, 772–786
© 2010 The Authors
Journal compilation © 2010 The British Pharmacological Society All rights reserved 0007-1188/10
www.brjpharmacol.org
THEMED SECTION: IMAGING IN PHARMACOLOGY
RESEARCH PAPER
Influence of fluorophore and linker composition on
the pharmacology of fluorescent adenosine A1
receptor ligandsbph_488 772..786
Jillian G Baker1, Richard Middleton2*, Luke Adams2*, Lauren T May1, Stephen J Briddon1,
Barrie Kellam2 and Stephen J Hill1
2
1Institute of Cell Signalling, School of Biomedical Sciences, Medical School and School of Pharmacy, Centre for Biomolecular
Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham, UK
Background and purpose: The introduction of fluorescence-based techniques, and in particular the development of fluores-
cent ligands, has allowed the study of G protein-coupled receptor pharmacology at the single cell and single molecule level.
This study evaluated how the physicochemical nature of the linker and the fluorophore affected the pharmacological properties
of fluorescent agonists and antagonists.
Experimental approach: Chinese hamster ovary cells stably expressing the human adenosine A1 receptor and a cyclic 3′,5′
adenosine monophosphate response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene, together with
whole cell [3H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX) radioligand binding, were used to evaluate the pharmacological
properties of a range of fluorescent ligands based on the antagonist xanthine amine congener (XAC) and the agonist 5′
(N-ethylcarboxamido) adenosine (NECA).
Key results: Derivatives of NECA and XAC with different fluorophores, but equivalent linker length, showed significant
differences in their binding properties to the adenosine A1 receptor. The BODIPY 630/650 derivatives had the highest affinity.
Linker length also affected the pharmacological properties, depending on the fluorophore used. Particularly in fluorescent
agonists, higher agonist potency could be achieved with large or small linkers for dansyl and BODIPY 630/650 derivatives,
respectively.
Conclusions and implications: The pharmacology of a fluorescent ligand was critically influenced by both the fluorophore
and the associated linker. Furthermore, our data strongly suggest that the physicochemical properties of the fluorophore/linker
pairing determine where in the environment of the target receptor the fluorophore is placed, and this, together with the
environmental sensitivity of the resulting fluorescence, may finally decide its utility as a fluorescent probe.
British Journal of Pharmacology (2010) 159, 772–786; doi:10.1111/j.1476-5381.2009.00488.x; published online 26
January 2010
This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section
visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x
Keywords: GPCRs; fluorescent ligands; molecular pharmacology; reporter genes; ligand binding; adenosine
Abbreviations: ABEA, N6-(4-aminobutyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; ADOEA, N6-(8-amino-3,6-dioxaoctyl)-5′-
ethylamino-5′-oxo-5′-deoxyadenosine; AEAO, 8-(2-aminoethylamino)-8-oxooctanoyl; AHH, 6-(6-aminohex-
anamido)hexanoyl; AO, 8-aminooctanoyl; AOEA, N6-(8-aminooctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine;
APEA, N6-(5-aminopentyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; APrEA, N6-(3-aminopropyl)-5′-ethy-
lamino-5′-oxo-5′-deoxyadenosine; AUEA, N6-(11-aminoundecyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine;
cAMP, cyclic AMP; CGS 15943, 5-amino-9-chloro-2-(2-furyl)1,2,4-trizolo[1,5-c]quinazoline; CHO, Chinese
hamster ovary; CRE, cyclic AMP response element; DMEM/F12, Dulbecco’s modified Eagle’s medium/nutrient
mix F12; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; FCS, fluorescence correlation spectroscopy; GPCR, G
protein coupled receptor; HBS, HEPES-buffered salt solution; HEPES, N-2-Hydroxyethylpiperazine-N′-2-
ethanesulfonic acid; NECA, 5′ (N-ethylcarboxamido)adenosine; pNPP, 4-nitrophenyl phosphate; PTX, Pertussis
toxin; SPAP, secreted placental alkaline phosphatase; X, 6-aminohexanoyl; XAC, xanthine amine congener

Fluorescent A1 receptor ligands
JG Baker et al
773
et al., 2004; Middleton and Kellam, 2005; Cordeaux et al.,
2008).
Introduction
Adenosine A1 receptors are
G protein-coupled receptors
We have designed and synthesized a number of fluorescent
agonist and antagonist probes of the human adenosine A1
receptor, which vary in both their fluorescent label and also
in the composition and length of the linker that attaches
the fluorophore to the parent molecule. Our aim was to
investigate the effect of these variations on the pharmacol-
ogy of these probes, and also their usefulness as A1 receptor
probes in live cell imaging. Here, we show that the nature of
both the linker and the fluorophore can exert major effects
on the pharmacological properties of both agonists and
antagonists.
(GPCRs) that signal via Gi/o proteins to inhibit adenylyl cyclase
activity and reduce the cellular levels of the second messenger,
cyclic 3′,5′ adenosine monophosphate (cyclic AMP; Libert
et al., 1992; Olah and Styles, 1995; Fredholm et al., 2001). Our
current understanding of the molecular pharmacology of the
adenosine A1-receptor has been deduced primarily from the
use of radioligand binding techniques for studying ligand-
receptor interactions and the measurement of intracellular
second messenger generation. These experiments require large
numbers of cells, and the pharmacological parameters that are
deduced from them therefore represent the average for the cell
population. However, it is now evident that adenosine A1
receptors are not uniformly distributed in the plasma mem-
brane of individual cells but instead are located within mem-
brane compartments and microdomains (Gines et al., 2001;
Zajchowshi and Robbins, 2002; Helmreich, 2003). The intro-
duction of fluorescence-based techniques, and in particular the
development of fluorescent ligands, has now advanced the
study of GPCR pharmacology to the single-cell and single
molecule level (Emmerson et al., 1997; Harikumar et al., 2002;
Baker et al., 2003; Briddon et al., 2004; Cordeaux et al., 2008;
Daly and McGrath, 2003). For example, the application of
techniques such as fluorescence correlation spectroscopy (FCS)
has provided the means by which the diffusion of receptor
species within defined membrane microdomains can by moni-
tored in real time (Briddon and Hill, 2007), and we have used
both fluorescent agonists and antagonists to study the adenos-
ine A1 receptor using this technique (Briddon et al., 2004;
Middleton et al., 2007).
The general approach to the design of a fluorescent ligand
has been to take a parent molecule with known pharmaco-
logical properties and to add a fluorophore to it. Given the
size of most fluorophores in relation to the active parent
molecule, it would not be surprising to observe that this
process may result in alterations to the pharmacology of the
parent molecule (Baker et al., 2003; Middleton and Kellam,
2005; Middleton et al., 2007). An ideal fluorescent molecule
would retain the properties of the parent ligand. For an
antagonist, this would mean retaining high affinity and selec-
tivity for the receptor under study, as well as the neutral
antagonist or inverse agonist properties of the parent ligand.
For an agonist, the fluorescent molecule must retain the effi-
cacy of the parent ligand, and enough potency still to be
pharmacologically useful. However, the addition of a fluoro-
phore to a known ligand clearly alters its structure, and the
pharmacology of the subsequent molecule needs to be care-
fully studied in order to determine its usefulness (Briddon
Methods
Cell culture
Chinese hamster ovary (CHO) cells stably expressing both the
human A1-adenosine receptor and a cyclic AMP response
element-secreted placental alkaline phosphatase (CRE-SPAP)
reporter gene were used throughout this study (CHO-A1;
Baker and Hill, 2007). Cells were grown in Dulbecco’s modi-
fied Eagle’s medium/nutrient mix F12 (DMEM/F12) contain-
ing 10% fetal calf serum and 2 mM L-glutamine in
humidified 5% CO2: 95% air atmosphere at 37°C.
a
Synthesis of the fluorescent ligands
The chemical structures of all the compounds assessed in
these experiments are shown in Figures 1 and 2. The synthesis
of compounds (1)–(4) has been previously described (Middle-
ton et al., 2007). Compound (5) was synthesized in an iden-
tical manner to that of (1)–(4), except the linker was installed
using
N1-benzyloxycarbonyl-1,8-diamino-3,6-dioxooctane.
Compounds (6), (7) and (8) were synthesized in an identical
manner to (2) via the key intermediate N6-(4-aminobutyl)-
5′-ethylamino-5′-oxo-5′-deoxyadenosine (Middleton et al.,
2007), except the fluorophores coupled to this inter-
mediate were 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-
indacene-3-propionic acid, succinimidyl ester (BODIPY FL SE,
Invitrogen, Carlsbad, CA, USA) for (6) and dansyl chloride for
(7), respectively. Compound (8) was synthesized in an iden-
tical manner to (2), except the modified fluorophore,
N-dansyl-8-aminooctanoic acid was coupled to N6-(4-
aminobutyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine using
1,1′-carbonyldiimidazole (CDI) in N,N-dimethylformamide
(DMF). Compound (9) was synthesized in an identical
manner to (2), except the terminal fluorophore was
coupled using 6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-
diaza-s-indacene-3-propionyl)amino)hexanoic acid, succin-
imidyl ester (BODIPY FL-X, SE). Compound (10) was
synthesized in an identical manner to (7), except the linker
was installed using N1-benzyloxycarbonyl-1,11-diaminound
ecane. Compounds (11), (12) and (13) were all synthesized
Correspondence: Professor SJ Hill, Institute of Cell Signalling, Queen’s Medical
Centre, Nottingham NG7 2UH, UK. E-mail: stephen.hill@nottingham.ac.uk or
Dr Barrie Kellam, School of Pharmacy, Centre for Biomolecular Sciences, Uni-
versity of Nottingham, University Park NG7 2RD, UK. E-mail: barrie.kellam@
nottingham.ac.uk
*Present address: CellAura Technologies Ltd, BioCity, Nottingham.
Re-use of this article is permitted in accordance with the Terms and Conditions
set out at http://www3.interscience.wiley.com/authorresources/onlineopen.
html
via
the
key
intermediate
N6-(4-aminobutyl)-5′-
(Middleton et al.,
ethylamino-5′-oxo-5′-deoxyadenosine
2007), except the fluorophores coupled to this intermediate
were Texas Red-X, succinimidyl ester (Invitrogen), Cy5
monosuccinimidyl ester (GE Healthcare, Little Chalfont,
Received 22 May 2009; revised 21 July 2009; accepted 31 July 2009
British Journal of Pharmacology (2010) 159 772–786

Fluorescent A1 receptor ligands
774
JG Baker et al
Fluorescent NECA derivatives
R1
H
HN
N
N
HN
N
n
N
HN
N
O
R1
H
HN
N
N
N
N
R2
N
N
O
N
N
N
O
O
O
O
O
O
N
N
ABEA-BYFL (6)
H
H
N
ADOEA-X-BY630 (5)
OH
HO
OH
HO
H
OH
HO
n = 3 (APrEA-X-BY630) (1)
n = 4 (ABEA-X-BY630) (2)
n = 5 (APEA-X-BY630) (3)
n = 8 (AOEA-X-BY630) (4)
HN
H
HN
H
R3
N
N
N
N
N
R3
N
N
N
N
7
H
O
O
O
N
N
O
O
N
H
N
ABEA-dansyl (7)
H
OH
OH
HO
O
ABEA-AO-dansyl (8)
HN
H
HO
N
R2
N
R3
N
N
5
HN
N
N
H
H
11
O
O
N
N
O
O
N
N
N
HN
N
N
ABEA-X-BYFL (9)
H
HN
O
O
N
OH
HO
N
N
H
O
N
O
HN
N
OH
HO
N
N
N
HN
O S O
N
H
AUEA-dansyl (10)
OH
HO
O
HN
O
HN
N
ABEA-X-Texas Red (11)
H
N
N
SO3
OH
O3S
HO
O
HN
N
N
N
O
O
N
N
N
O
N
H
OH
HO
N
ABEA-Cy5 (12)
O
ABEA-EVOBlue 30 (13)
N
O3S
N
O3S
O
O
S
N
N
R3 =
N
F
B
R2 =
N
R1 =
B
F
F
F
S
O
NMe2
O
O
N
H
O
Figure 1 Chemical structures of fluorescent NECA derivatives. ABEA, N6-(4-aminobutyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; ADOEA,
N6-(8-amino-3,6-dioxaoctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; AO, 8-aminooctanoyl; AOEA, N6-(8-aminooctyl)-5′-ethylamino-5′-oxo-
5′-deoxyadenosine; APEA, N6-(5-aminopentyl)- 5′-ethylamino-5′-oxo-5′-deoxyadenosine; APrEA, N6-(3-aminopropyl)-5′-ethylamino-5′-oxo-5′-
deoxyadenosine; AUEA, N6-(11-aminoundecyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; X, 6-aminohexanoyl.
British Journal of Pharmacology (2010) 159 772–786

Fluorescent A1 receptor ligands
JG Baker et al
775
Buckinghamshire, UK) and EVOBlue 30 succinimidyl ester
(Novabiochem, Nottingham, UK), respectively. Compound
(14) has been previously described (Briddon et al., 2004).
Compounds (15), (16), (18) and (19) were synthesized in an
identical manner to (14), except the fluorophores coupled to
xanthine amine congener (XAC) were Texas Red-X succinim-
idyl ester (Invitrogen), Cy5 monosuccinimidyl ester (GE
Healthcare), EVOBlue 30 succinimidyl ester (Novabiochem)
and dansyl chloride, respectively. Compound (17) was pre-
pared by reacting XAC and suberic acid monomethyl ester in
DMF in the presence of CDI. The crude 7-carboxyheptanoyl-
XAC methyl ester product was saponified and reacted with
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-
propionyl ethylenediamine, hydrochloride (BODIPY FL EDA,
Invitrogen) using CDI as the coupling reagent in DMF.
Compound (20) was synthesized by coupling XAC with N-
dansyl-8-aminooctanoic acid using 1,1′-carbonyldiimidazole
in DMF. Compound (21) was synthesized by reacting
N-tbutyloxycarbonylaminocaproic acid with XAC using dicy-
clohexylcarbodiimide (DCC) as the coupling reagent in DMF.
Following acidolytic removal of the tbutyloxycarbonyl
protecting group, aminocaproyl-XAC was coupled with
N-dansyl-6-aminohexanoic acid using diisopropylcarbodiim-
ide as the coupling reagent in DMF. All compounds were
purified using preparative thin-layer chromatography (pTLC),
as previously described (Briddon et al., 2004; Middleton et al.,
2007). Single peak purity of all compounds was confirmed by
analytical reversed-phase high-performance liquid chroma-
tography (recorded on a Waters Millennium 995 LC system,
Milford, MA, USA) and the structures confirmed using high-
resolution mass spectrometry (recorded on a Waters 2795
Separation Module/Micromass LCT platform).
replaced with 100 mL serum-free media (DMEM/F12 contain-
ing 2 mM L-glutamine), and the cells incubated for a further
24 h. Where used, Pertussis toxin (PTX, 100 ng·mL–1) was
added for this 24 h period. On the day of experimentation,
the serum-free media was removed and replaced with 100 mL
serum-free media Ϯ antagonist at the final required concen-
tration, and the cells incubated for 1 h at 37°C (5% CO2).
Agonists (10 mL, diluted in serum free media) were then
added to each well, and the plate incubated at 37°C for
10 min. Forskolin (10 mL) was then added to all but the basal
wells, and the cells incubated for a further 5 h at 37°C (5%
CO2). After 5 h, the media and drugs were removed, 40 mL
serum-free media was added to each well, and the cells incu-
bated for a further 1 h at 37°C. The plates were then incu-
bated at 65°C for 30 min to destroy any endogenous
phosphatases before the plates were then cooled to 37°C.
4-nitrophenyl phosphate (pNPP; 100 mL of 5 mM) in dietha-
nolamine buffer was added to each well, and the plates incu-
bated at 37°C until the yellow colour developed. The plates
were then read on a Dynex MRX plate reader (Chelmsford,
MA, USA) at 405 nm.
Confocal microscopy
Cells were grown to 100% confluency in Labtek 8-well
chambered-coverglasses (Nalgene Nunc International, Scien-
tific Laboratory Supplies, Nottingham, UK) in 400 mL DMEM-
F12 media containing 10% FCS and 2 mM glutamine. The
media was replaced with 400 mL N-2-Hydroxyethylpiperazine-
N′-2-ethanesulfonic acid (HEPES)-buffered saline (HBS,
Briddon et al., 2004) at room temperature. Confocal micros-
copy was performed using a Zeiss LSM510 laser-scanning
microscope using either a Zeiss 40 ¥ 1.3NA oil-immersion lens
(Zeiss, Jena, Germany) or (for experiments using 364 nm exci-
tation) a C-Apochromat 60 ¥ 1.2NA water-immersion lens.
Images of ligands were captured using the following optical
configurations: BODIPY 630/650, Cy5 and EVOBlue 30 con-
jugates, 633 nm excitation, with emission captured through a
650LP filter; Texas Red conjugates, 543 nm excitation and
560LP emission filter; BODIPY-FL conjugates, 488 nm excita-
tion and LP505 emission and dansyl conjugates 364 nm exci-
tation and 475-525BP emission. In each case, a pinhole
diameter of 1 Airy unit was used.
Initial images were captured in the absence of ligand to
determine the level of auto-fluorescence under each excita-
tion wavelength, and settings adjusted to minimize this.
Single equatorial images were subsequently captured follow-
ing a 5 min incubation of the indicated concentration of
fluorescent ligands (in HBS). If membrane fluorescence was
seen, the media and fluorescent drug were removed from the
well and the cells washed by the addition and removal of
400 mL HBS. A further 400 mL HBS was added, and a further
image captured. This was to ensure that any apparent
membrane-localized fluorescence was indeed bound to the
membrane and not just due to fluorescent ligand present in
the media between cells. For any given experimental condi-
tion, a minimum of three different cell preparations on three
different days with different drug dilutions were used. In the
text, ‘n’ refers to the number of different complete experi-
ments performed.
[3H]-8-cyclopentyl-1,3-dipropylxanthine (DPCPX)
whole cell binding
CHO-A1 cells were grown to confluence over 24 h in white-
sided 96 well view plates. The following day, the media was
removed and replaced with 100 mL serum-free media (i.e.
DMEM/F12 containing 2 mM L-glutamine only) containing
the competing ligand. This was immediately followed by the
addition of 100 mL serum-free media containing 3H-DPCPX
(to give a final concentration of 3H-DPCPX of 0.99–1.77 nM).
Total and non-specific binding (as defined by 10 mM XAC)
were measured in each experiment. Cells were then incu-
bated for 2 h at 37°C in a humidified 5% CO2: 95% room air
atmosphere. After 2 h, the media and drugs were removed,
and the cells washed twice by the addition and removal of
phosphate-buffered saline at 4°C (2 ¥ 200 mL per well). A
white base was then added to the plate, followed by 100 mL
Microscint 20 (Perkin Elmer, Shelton, CT, USA) per well and
a sealant film placed over the wells. The plates were then
counted the following day on a Topcount (Perkin Elmer) for
2 min per well.
CRE-SPAP gene transcription
Cells were grown to confluence in 96-well plates in 100 mL
DMEM/F12 containing 10% fetal calf serum and 2 mM
L-glutamine. Once confluent, the media was removed and
British Journal of Pharmacology (2010) 159 772–786

Fluorescent A1 receptor ligands
776
JG Baker et al
Excitation and emission spectra for fluorescent ligands
Ligand solutions were prepared in methanol or HBS (1 mM for
XAC-X-BY630 and XAC-X-TR; 50 mM for XAC-dansyl). Exci-
tation and emission spectra (2 nm intervals) were collected
using a Flexstation 96-well fluorescence plate reader (Molecu-
lar Devices Corporation, Sunnyvale, CA, USA) using 100 mL of
ligand dispensed in to UV-transparent flat-bottomed 96-well
plates (Costar, Fisher Scientific, Loughborough, UK). Data
analysis was performed in Softmax Pro 4.2 (Molecular Devices
Corporation) and GraphPad Prism 4.0 (GraphPad, San Diego,
CA, USA).
was required for most ligands. This was performed using the
following equation:
A
[ ]
[ ]
A + IC50
⎡
⎤
(
)
Response = Basal + FK − Basal 1−
⎢
⎣
⎥
⎦
(
)
A
[ ]
A + EC50
⎡
⎤
+ SMAX
⎢
⎣
⎥
⎦
(
)
[ ]
where basal is the response in the absence of agonist, FK is the
response to a fixed concentration of forskolin, [A] is the con-
centration of A1-receptor agonist, IC50 is the concentration of
agonist that inhibits 50% of the response to forskolin, SMAX is
the maximum stimulation of the Gs-component of the
response to the agonist and EC50 is the concentration of
agonist that stimulates a half maximal Gs-response.
A maximal forskolin concentration (3 mM) was included in
each CRE-gene transcription plate for each separate experi-
ment. All data are presented as mean Ϯ SEM of triplicate
determinations, and n in the text refers to the number of
separate experiments.
Data analysis
3H-DPCPX whole cell binding. Curves for inhibition of specific
binding of 3H-DPCPX by a range of concentrations of 5′
(N-ethylcarboxamido)adenosine (NECA), XAC and the fluo-
rescent adenosine A1 receptor ligands were fitted to the
following equation:
100 × A
[ ]
[ ]
A + IC50
% of specific binding = 100 −
Materials
where [A] is the concentration of competing ligand, and IC50
is the concentration that inhibits specific binding by 50%.
Antagonist dissociation constants (KB) were then determined
from the following expression:
CGS 15943, DPCPX and NECA were from Tocris Cookson
(Avonmouth, Bristol, UK). 3H-DPCPX was obtained from
Amersham International (Buckinghamshire, UK). Fetal calf
serum was from PAA laboratories (Teddington, Middlesex,
UK). XAC was from Sigma Chemicals (Poole, Dorset, UK),
who also supplied all other reagents. Pertussis toxin was from
Calbiochem, Nottingham, UK. The receptor nomenclature
used in this manuscript follows Alexander et al. (2008).
IC50 × KD
KB =
KD + L
[ ]
where [L] and KD are the concentration and dissociation con-
stant of 3H-DPCPX, respectively. The KD value of 3H-DPCPX
was previously determined in this cell line to be 3.61 nM
(Baker and Hill, 2007).
Results
In order to systematically test the effect of fluorophore, linker
length and composition on ligand pharmacology, we synthe-
sized a number of fluorescent derivatives of the archetypal
adenosine A1-receptor agonist, NECA and the corresponding
antagonist, XAC (Figures 1 and 2, Table 1; compounds 1–21).
We have previously demonstrated the use of the red-excited
fluorophore BODIPY 630/650 to generate functional fluores-
cent A1-receptor agonists and antagonists (Briddon et al.,
2004; Middleton et al., 2007). We have extended the range of
red-emitting fluorophores tested to include the cyanine dye,
Cy5 and also EVOBlue30. In addition, we have used dyes
spread over the full UV-visible excitation wavelength range
using dansyl (excitation maximum = 320 nm), BODIPY FL
(BYFL, 488 nm) and Texas Red (TXR, 590 nm). Furthermore,
we have varied the length of the linker between the active
pharmacophore and fluorophore. Initially, these compounds
were assessed for A1-receptor affinity using whole-cell radioli-
gand binding (3H-DPCPX) and also for functional potency
and efficacy using a CRE-based reporter gene assay.
Functional experiments
One-site concentration responses curves. Sigmoidal agonist
concentration-response curves were fitted to the data using
the following equation through computer-assisted non-linear
regression using the programme GraphPad Prism 2:
EMAX
EC50
×
+
[
[
A
]
Response =
A
]
where EMAX is the maximal response, [A] is the agonist con-
centration and EC50 (or IC50) is the concentration of agonist
that produces 50% of the maximal response.
Antagonist KD values were then calculated from the shift of
the agonist concentration responses in the presence of a fixed
concentration of antagonist using the following equation:
B
[ ]
DR = 1+
KD
where DR (dose ratio) is the ratio of the agonist concentration
required to stimulate an identical response in the presence
and absence of a fixed concentration of antagonist [B].
Fluorescent XAC derivatives
Whole-cell binding in CHO-A1 cells using 3H-DPCPX con-
firmed that the affinity of XAC itself was similar to that
previously shown (Table 2; Figure 3). In line with our previous
Two-site agonist curves. As many concentration response
curves clearly contained two components, two-site analysis
British Journal of Pharmacology (2010) 159 772–786

Fluorescent A1 receptor ligands
JG Baker et al
777
Fluorescent XAC derivatives
O
O
NH
NH
O
N
O
HN
O
HN
H
N
H
N
NH
O2S
NH
N
N
O
O
O
O
N
N
O
O
N
SO3
O
O
XAC-X-Texas Red (15)
N
B
N
F
XAC-X-BY630 (14)
N
F
S
N
NH
O
H
N
HN
O
O
O
N
O
6
NH
NH
NH
N
N
O
N
O
N
HN
O
O
SO3
N
N
O
XAC-Cy5 (16)
N
O
H
O
N
N
B
F
N
F
XAC-AEAO-BYFL (17)
SO3
O
N
N
N
O
O
O
S
N
NH
N
H
H
O
HN
H
N
N
O
N
O
O
O
NH
N
O
N
N
O
O
XAC-
dansyl (19)
O3S
N
N
XAC-EVOBlue 30 (18)
O
N
HN
O
H
N
HN
H
NH
O
N
NH
N
N
O
O
O
O
N
O
N
O
O
N
Me2N
HN
O
HN
S
Me2N
S NH
O
O
O
O
XAC-AHH-dansyl (21)
XAC-AO-dansyl (20)
Figure 2 Chemical structures of fluorescent XAC derivatives. AEAO, 8-(2-aminoethylamino)-8-oxooctanoyl; AHH, 6-(6-
aminohexanamido)hexanoyl; AO, 8-aminooctanoyl; X, 6-aminohexanoyl; XAC, xanthine amine congener.
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JG Baker et al
Table 1 Fluorescent analogues of NECA and XAC
Compound no.
Compound name
Parent ligand
Mass spectroscopy
1
2
3
4
5
6
7
8
APrEA-X-BY630
ABEA-X-BY630
APEA-X-BY630
AOEA-X-BY630
ADOEA-X-BY630
ABEA-BYFL
ABEA-dansyl
ABEA-AO-dansyl
ABEA-X-BYFL
AUEA-dansyl
ABEA-X-Texas Red
ABEA-Cy5
ABEA-EVOBlue 30
XAC-X-BY630
XAC-X-Texas Red
XAC-Cy5
XAC-AEAO-BYFL
XAC-EVOBlue 30
XAC-dansyl
NECA
NECA
NECA
NECA
NECA
NECA
NECA
NECA
NECA
NECA
NECA
NECA
NECA
XAC
XAC
XAC
XAC
XAC
XAC
XAC
XAC
C44H49BF2N10O7S (MH)+ requires 911.3647, found 911.3718
C45H51BF2N10O7S (MH)+ requires 925.3803, found 925.3842
C46H53BF2N10O7S (MH)+ requires 939.3960, found 939.4054
C49H60BF2N10O7S (MH)+ requires 981.4428, found 981.4470
C47H56BF2N10O9S (MH)+ requires 985.4014, found 985.4111
C30H39BF2N9O5 (MH)+ requires 654.3135, found 654.3098
C28H37N8O6S (MH)+ requires 613.2557, found 613.2532
C36H52N9O7S (MH)+ requires 754.3710, found 754.3744
C36H50BF2N10O6 (MH)+ requires 767.3976, found 767.3962
C35H51N8O6S (MH)+ requires 711.3652, found 711.3609
C53H65N10O11S2 (MH)+ requires 1081.4270, found 1081.4360
C49H63N9O11S2 (MH)+ requires 1018.4161, found 1018.4229
C37H49N10O9S (MH)+ requires 809.3399, found 809.3427
C50H55BF2N9O7S (MH)+ requires 974.4006, found 974.4041
C58H68N9O11S2 (MH)+ requires 1130.4480, found 1130.4530
C54H67N8O11S2 (M2H)+ requires 1067.4365, found 1067.4495
9
10
11
12
13
14
15
16
17
18
19
20
21
C
45H60BF2N10O7 (MH)+ requires 901.4708, found 901.4691
C42H52N9O9S (MH)+ requires 858.3609, found 858.3646
C33H40N7O6S (MH)+ requires 662.2761, found 662.2740
C41H55N8O7S (MH)+ requires 803.3914, found 803.3971
C45H65N9O8S (MH)+ requires 888.4442, found 888.4612
XAC-AO-dansyl
XAC-AHH-dansyl
The confirmed molecular weight from mass spectroscopy is also compared with the predicted molecular weight for the final product.
ABEA, N6-(4-aminobutyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; ADOEA, N6-(8-amino-3,6-dioxaoctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; AEAO,
8-(2-aminoethylamino)-8-oxooctanoyl; AHH, 6-(6-aminohexanamido)hexanoyl (see Figures 1 and 2 for complete structures); AO, 8-aminooctanoyl; AOEA,
N6-(8-aminooctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; APEA, N6-(5-aminopentyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; APrEA, N6-(3-aminopropyl)-5′-
ethylamino-5′-oxo-5′-deoxyadenosine; AUEA, N6-(11-aminoundecyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; NECA, 5′(N-ethylcarboxamido)adenosine; X,
6-aminohexanoyl; XAC, xanthine amine congener.
Table 2 Inhibition of specific binding of 3H-DPCPX to the adenosine A1-receptor by fluorescent analogues of NECA and XAC
1
Log KD
Log IC50
% Inhibition2
n
NECA
-5.97 Ϯ 0.03
8
APrEA-X-BY630
ABEA-X-BY630
APEA-X-BY630
AOEA-X-BY630
ADOEA-X-BY630
ABEA-dansyl
ABEA-AO-dansyl
AUEA-dansyl
ABEA-X-Texas Red
ABEA-EVOBlue 30
ABEA-Cy5
-6.62 Ϯ 0.09 (apparent)
-5.93 Ϯ 0.10 (apparent)
-6.01 Ϯ 0.25 (apparent)
-6.15 Ϯ 0.21 (apparent)
-6.19 Ϯ 0.24 (apparent)
-6.51 Ϯ 0.10
-5.81 Ϯ 0.10
-5.89 Ϯ 0.25
-6.04 Ϯ 0.21
-6.23 Ϯ 0.23
>-4
55.4 Ϯ 2.5%
46.2 Ϯ 4.9%
45.3 Ϯ 4.2%
19.7 Ϯ 3.6%
24.3 Ϯ 4.8%
10
10
9
7
7
5
3
6
6
NI
-4.97 Ϯ 0.15 (apparent)
-4.83 Ϯ 0.15
44.8 Ϯ 2.3%
>-4
>-5
2
4
4
NI
NI
ABEA-BYFL
ABEA-X-BYFL
XAC
>-4
5
-7.38 Ϯ 0.05
11
10
7
5
6
6
7
6
6
XAC-X-BY630
XAC-X-Texas Red
XAC-AEAO-BYFL
XAC-EVOBlue 30
XAC-Cy5
XAC-dansyl
XAC-AO-dansyl
XAC-AHH-dansyl
-7.42 Ϯ 0.05 (apparent)
-5.72 Ϯ 0.07
-7.28 Ϯ 0.05
82.3 Ϯ 1.4%
NI
-5.27 Ϯ 0.11
-5.59 Ϯ 0.02
-6.71 Ϯ 0.05
-6.91 Ϯ 0.09 (apparent)
-6.87 Ϯ 0.05 (apparent)
-6.77 Ϯ 0.09
-6.73 Ϯ 0.04
91.0 Ϯ 0.5%
81.0 Ϯ 0.9%
All values are mean Ϯ SEM.
1Unless otherwise stated, KD values were calculated as described under Methods. In those situations where a maximal inhibition of specific binding was obtained
that was less than 100% (defined with 10 mM XAC), an apparent KD was calculated from the IC50 of the displaceable component of binding.
2Where indicated, maximum inhibition of specific binding was obtained that was less than that defined with 10 mM XAC.
ABEA, N6-(4-aminobutyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; ADOEA, N6-(8-amino-3,6-dioxaoctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; AEAO,
8-(2-aminoethylamino)-8-oxooctanoyl; AHH, 6-(6-aminohexanamido)hexanoyl (See Figures 1 and 2 for complete structures); AO, 8-aminooctanoyl; AOEA,
N6-(8-aminooctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; APEA, N6-(5-aminopentyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; APrEA, N6-(3-aminopropyl)-5′-
ethylamino-5′-oxo-5′-deoxyadenosine; AUEA, N6-(11-aminoundecyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; NECA, 5′(N-ethylcarboxamido)adenosine; NI, no
inhibition of specific binding at concentrations up to 0.1 mM; X, 6-aminohexanoyl; XAC, xanthine amine congener.
British Journal of Pharmacology (2010) 159 772–786

Fluorescent A1 receptor ligands
JG Baker et al
779
easier to see, forskolin was added to the experiments in order
to raise the basal adenylyl cyclase activity, and thus cAMP and
CRE-gene transcription levels. In the presence of forskolin,
the agonist response to NECA is comprised of both an inhibi-
tory Gi-mediated inhibition of gene transcription and, at
higher agonist concentrations, a Gs-mediated enhancement
of the response to forskolin (Figure 4). We have previously
shown that both the Gi and Gs responses to NECA in this cell
line are mediated via the adenosine A1 receptor (Baker and
Hill, 2007). Inverse agonists, however, result in an increase in
adenylyl cyclase activity and an increase in CRE-mediated
gene transcription (Figure 4; Baker and Hill, 2007). Log KD
values obtained from antagonism of the Gs and Gi phases of
the NECA response for each fluorescent antagonist were very
similar (Table 3). The Gs phase of the NECA response could be
isolated by pretreatment with PTX, and the KD values for
fluorescent antagonists were again similar (Table 3).
All of the fluorescent derivatives of XAC that were able to
antagonize NECA-stimulated Gi-mediated responses, also
acted as inverse agonists (Table 4; Figure 4). Table 4 shows the
mean log EC50 values for an increase in CRE-mediated gene
transcription that are in good agreement with the antagonist
KD values obtained from antagonism of NECA-stimulated
responses (Table 3). Interestingly, similar KD values were
obtained for XAC-X-BY630 (14), XAC-X-TXR (15) and Dansyl
derivatives of XAC (19, 20, 21) from inhibition of DPCPX
binding (Table 2).
A
100
50
0
XAC
XAC-dansyl
XAC-AO-dansyl
XAC-X-BY630
XAC-AEAO-BYFL
–12 –11 –10 –9
–8
–7
–6
–5
–4
log [XAC or derivative] M
B
100
50
0
NECA
AprEA-X-BY630
APEA-X-BY630
AOEA-X-BY630
–9
–8
–7
–6
–5
–4
log [NECA or derivative] M
3
Figure 3 Inhibition of the specific binding of H-DPCPX by fluores-
cent (A) antagonists and (B) agonists in CHO-A1 cells expressing the
human adenosine A1 receptor. Non-specific binding was defined with
10 mM XAC. 3H-DPCPX was used at (A) 1.30 nM or (B) 1.11 nM.
Values represent mean Ϯ SEM from triplicate determinations in a
single experiment. These separate experiments are representative
of (A) five and (B) seven separate experiments. AEAO, 8-(2-
aminoethylamino)-8-oxooctanoyl; AO, 8-aminooctanoyl; AOEA,
N6-(8-aminooctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; APEA, N6-
Fluorescent NECA derivatives
In whole cell radioligand binding assays, the parent
A1-receptor agonist, NECA, fully inhibited specific binding
with a log KD value of -5.97 (see Table 2; Figure 3). The pre-
viously characterized A1 receptor agonist ABEA-X-BY630 (2)
had a similar apparent log KD value to NECA (Table 2:
Figure 3), but displaced only 50% of the specific binding as
previously reported (Middleton et al., 2007). Maintaining a
similar linker length, but altering the fluorophore to Texas
Red (ABEA-X-TR, 11), Cy5 (ABEA-Cy5, 12), EVOBlue30
(ABEA-EVOBlue30, 13) or BODIPY FL (ABEA-X-BYFL, 9) effec-
tively abolished any measurable affinity of the agonists for the
A1 receptor. The dansyl equivalent of ABEA-X-BY630, AUEA-
dansyl (10), did show measurable affinity for the A1 receptor,
but was significantly less potent (Table 2).
For the BODIPY 630/650-labelled NECA derivatives, varying
the linker length from three to eight carbon atoms (APrEA-,
ABEA-, APEA- and AOEA-X-BY630, 1,2,3 and 4, respectively)
did not significantly change their affinity for the adenosine A1
receptor or their inability to displace all of the specific
binding. The exception to this was for the longest chain
length, AOEA-X-BY630 (4), which displaced only 20% of the
specific binding. This lack of displacement was unlikely to be
a solubility issue, as a compound with a diethylene glycol
linker of equivalent length (ADOEA-X-BY630, 5) showed
similar properties. In the case of the dansyl derivatives
varying the carbon chain from 4 carbon atoms in ABEA-
dansyl (7) to 7 carbon atoms in ABEA-AO-dansyl (8), or 11
carbon atoms in AUEA-dansyl (10), indicated that the longer
carbon chain length was required for measurable binding
affinity (Table 2).
(5-aminopentyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine;
APrEA,
N6-(3-aminopropyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; X, 6-
aminohexanoyl;
CHO,
Chinese
hamster
ovary;
DPCPX,
8-cyclopentyl-1,3-dipropylxanthine; NECA, 5′ (N-ethylcarboxamido)
adenosine; X, 6-aminohexanoyl; XAC, xanthine amine congener.
studies, XAC-X-BY630 (14) showed a comparable affinity for
the adenosine A1 receptor (Table 2), and inhibited over 80% of
specific binding (Figure 3). Derivatives in which the fluoro-
phore was changed, but which had an equivalent linker
length, showed significant differences in their binding prop-
erties (Table 2). XAC-AO-dansyl (20) showed
a reduced
but reasonable affinity. In contrast, XAC-Cy5 (16), XAC-
EVOBlue30 (18) and XAC-X-TXR (15) were at least 10-fold
less potent than the BODIPY630/650 derivative. The
BODIPY-FL derivative of XAC (XAC-AEAO-BYFL; 17) showed
no displacement of specific binding at concentrations up to
10 mM (Figure 3).
The effect of linker length was investigated using three
dansyl derivatives (XAC-dansyl (19), XAC-AO-dansyl (20)
and XAC-AHH-dansyl (21)), with relative chain lengths of 0,
9 and 14 atoms, respectively. Interestingly, increasing the
chain length had no significant effect on the binding affinity
of the ligand, but did seem to decrease slightly the maximum
3
displacement of H-DPCPX (Table 2).
The adenosine A1 receptor is primarily a Gi-coupled recep-
tor, and, as such, agonist responses result in a decrease in
cAMP and CRE-mediated gene transcription responses (Baker
and Hill, 2007). In order to make these inhibitory responses
British Journal of Pharmacology (2010) 159 772–786

Fluorescent A1 receptor ligands
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JG Baker et al
Basal
Forskolin 3μM
XAC-X-BY630 1μM
NECA
D
A
Basal
Forskolin 3μM
XAC-X-BY630
1.5
1.0
NECA + XAC-X-BY630 1μM
0.8
0.6
0.4
0.2
0.0
1.0
0.5
0.0
–11 –10 –9
–8
–7
–6
–5
–11 –10 –9
–8
–7
–6
–5
log[NECA](M)
log[XAC-X-BY630](M)
Basal
E
B
Basal
Forskolin 3 μM
XAC-X-TXR 1μM
NECA
1.0
0.8
0.6
0.4
0.2
0.0
1.00
0.75
0.50
0.25
0.00
Forskolin 3 μM
XAC-X-TXR
NECA + XAC-X-TXR 1μM
–11 –10
–9
–8
–7
–6
–5
–11 –10
Basal
–9
–8
–7
–6
–5
log[NECA](M)
log[XAC-X-TXR](M)
Forskolin 3 μM
XAC-AEAO-BYFL 1μM
NECA
Basal
C
F
Forskolin 3μM
XAC-AEAO-BYFL
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.00
0.75
0.50
0.25
0.00
NECA + XAC-AEAO-BYFL 1μM
–11 –10
–9
–8
–7
–6
–5
–11 –10
–9
–8
–7
–6
–5
log[NECA](M)
log[XAC-AEAO-BYFL](M)
Figure 4 CRE-SPAP production in CHO-A1 cells in response to (A) NECA in the presence and absence of 1 mM XAC-X-BY630 (14); (B) NECA
in the presence and absence of 1 mM XAC-X-Texas Red (XAC-X-TXR; 15); (C) NECA in the presence and absence of 1 mM XAC-AEAO-BYFL (17);
(D) XAC-X-BY630 (14); (E) XAC-X-Texas Red (XAC-X-TXR) and (F) XAC-AEAO-BYFL (17). Bars show basal CRE-SPAP production, that in
response to 3 mM forskolin, and for A–C, that in response to 1 mM of the XAC-derivative in the presence of 3 mM forskolin. Data points are mean
Ϯ SEM from triplicate values from a single experiment. These separate experiments are representative of (A) 9, (B) 6, (C) 10, (D) 9, (E) 6 and
(F) 6 separate experiments. AEAO, 8-(2-aminoethylamino)-8-oxooctanoyl; CHO, Chinese hamster ovary; CRE-SPAP, cyclic 3′,5′ adenosine
monophosphate response element-secreted placental alkaline phosphatase; NECA, 5′ (N-ethylcarboxamido)adenosine; X, 6-aminohexanoyl;
XAC, xanthine amine congener.
Several of the fluorescent NECA derivatives were also able to
stimulate agonist responses via the adenosine A1 receptor
(Table 5). In each case, the Gi-coupled agonist response was
abolished following pretreatment of the cells with PTX
(Table 5), and could be competitively antagonized by the
non-fluorescent A1 receptor antagonists DPCPX, XAC and
CGS 15943 (Table 6). BODIPY 630/650 derivatives of NECA
were able to stimulate both Gi- and Gs- mediated responses
(Figure 5). However, the increased length of the linker
resulted in lower potency and reduced Gs responses (Figure 5).
The dansyl derivatives of NECA elicited Gi-mediated
responses, but no significant activation of Gs-mediated
responses was observed over the concentration range studied
(Table 5; Figure 6). This was probably a consequence of their
lower potency and the inability to reach concentrations at
which a Gs response would be visible.
It was notable that while BODIPY 630/650 derivatives of
NECA became less potent as the carbon chain length
increased, the opposite was true for the dansyl derivatives,
with the most potent compound being AUEA-dansyl (10),
with an 11 carbon linker (Table 5; Figures 5 and 6). This was
in keeping with AUEA-dansyl providing the only measurable
binding affinity in whole cell ligand-binding studies. It was
notable that IC50 values for the Gi-mediated function gene
transcription responses were generally much lower than the
KD values obtained in binding experiments for all agonists.
This reflects the fact that this is a well-coupled system and
explains why weak agonist responses were obtained for
British Journal of Pharmacology (2010) 159 772–786

Fluorescent A1 receptor ligands
JG Baker et al
781
Table 3 Log KD values from antagonism of NECA-mediated inhibition (Gi) and enhancement (Gs) of forskolin-stimulated CRE reporter
gene responses
Antagonist
Log KD
n
Log KD
n
+PTX
n
Gi
Gs
Gs
XAC
-7.86 Ϯ 0.05
-6.92 Ϯ 0.09
-6.00 Ϯ 0.15
-7.37 Ϯ 0.08
-7.43 Ϯ 0.07
-7.14 Ϯ 0.15
14
9
6
8
6
-7.92 Ϯ 0.04
-6.94 Ϯ 0.06
-6.09 Ϯ 0.08
-7.31 Ϯ 0.03
-7.42 Ϯ 0.09
-7.30 Ϯ 0.10
14
12
7
7
6
-7.99 Ϯ 0.05
-7.14 Ϯ 0.05
-6.31 Ϯ 0.10
-7.22 Ϯ 0.17
-7.02 Ϯ 0.10
-6.99 Ϯ 0.09
6
6
4
7
6
6
XAC-X-BY630
XAC-X-Texas Red
XAC-dansyl
XAC-AO-dansyl
XAC-AHH-dansyl
5
5
Values represent mean Ϯ SEM of n separate experiments. KD values were determined from the parallel shifts obtained in the inhibitory (Gi) and stimulatory (Gs)
phases of the NECA concentration response curves. In some experiments (+PTX), cells were incubated overnight with PTX (100 ng·mL–1), and under these
conditions, only a stimulatory response (Gs) was obtained. No antagonism was seen with XAC-AEAO-BYFL, XAC-EVOBlue 30 or XAC-Cy5 at concentrations up to
1 mM.
AEAO, 8-(2-aminoethylamino)-8-oxooctanoyl; AO, 8-aminooctanoyl; CRE, cyclic AMP response element; NECA, 5′(N-ethylcarboxamido)adenosine; PTX, Pertussis
toxin; X, 6-aminohexanoyl; XAC, xanthine amine congener.
Table 4 Inverse agonist effects of fluorescent XAC derivatives
pared with methanol was 0.89 for XAC-X-TR (15), 0.26 for
XAC-dansyl (19) and 0.07 for XAC-X-BY630 (14). It was also
notable that clear membrane binding could be detected with
XAC-X-BY630 when confocal imaging was undertaken in
the presence of Brilliant Black BN (50 mM), which heavily
quenches the extracellular aqueous fluorescence (Figure 9).
Antagonist
(log EC50
)
n
Fold increase
XAC
-7.15 Ϯ 0.14
-6.93 Ϯ 0.12
-5.82 Ϯ 0.22
-6.84 Ϯ 0.16
-7.52 Ϯ 0.17
-6.80 Ϯ 0.14
9
9
6
9
7
5
1.41 Ϯ 0.07
1.32 Ϯ 0.04
1.26 Ϯ 0.06
1.47 Ϯ 0.08
1.20 Ϯ 0.03
1.40 Ϯ 0.10
XAC-X-BY630
XAC-X-Texas Red
XAC-dansyl
XAC-AO-dansyl
XAC-AHH-dansyl
Discussion
Values represent mean Ϯ SEM of n separate experiments.
AHH, 6-(6-aminohexanamido)hexanoyl (see Figures 1 and 2 for complete
structures); AO, 8-aminooctanoyl; X, 6-aminohexanoyl; XAC, xanthine amine
congener.
XAC and its antagonist fluorescent derivatives
XAC is a well-known antagonist of the human adenosine A1
receptor. It bound with high affinity to the human adenosine
A1 receptor in this transfected cell system (log KD -7.38). In
keeping with previous findings, the inverse agonist nature of
this ligand was also evident (Baker and Hill, 2007). The addi-
tion of EVOBlue30, Cy5 and BODIPY-FL fluorophores to XAC
resulted in a large loss in affinity of the resultant molecules,
with only very weak binding being detected for XAC-Cy5 (16)
and XAC-EVOBlue30 (18). None of these XAC derivatives
were able to inhibit any of the NECA agonist responses (at a
concentration up to 1 mM for the fluorescent XAC ligand).
The addition of the EVOBlue, Cy5 and BODIPY-FL fluoro-
phores to XAC therefore resulted in a loss of pharmacological
activity of the XAC molecule that was due to a large reduction
in affinity for the adenosine A1 receptor.
When the XAC-X-Texas Red (15) molecule was examined,
the affinity as measured by 3H-DPCPX binding was about
30-fold lower than that seen with the parent XAC. XAC-X-
Texas Red (15) was also able to inhibit the NECA agonist
responses, and this included both those occurring via the
Gi-and Gs-coupled states of the receptor (Table 3). However,
when XAC-X-Texas Red (15) was monitored at the single-cell
level, no specific binding was seen at concentrations up to
30 nM. At 30 nM, bright fluorescence was seen between cells,
suggesting that enough fluorescent molecules were present to
be detected by confocal microscopy, but this fluorescence was
lost immediately on washout of the label. This suggests that
the affinity of XAC-X-Texas Red (15) for the adenosine A1
receptor is too low for successful imaging studies.
several compounds when no displacement of 3H-DPCPX
binding was detectable. This was particularly true of the
BODIPY FL derivatives ABEA-BYFL (6) and ABEA-X-BYFL (9).
Visualizing ligand binding to live cells
All of the fluorescent ligands were examined under the con-
focal microscope for their ability to bind to CHO-A1 cells
(Figure 7). When XAC-X-TXR (15) was monitored at the
single-cell level, no specific binding was seen at concentra-
tions up to 30 nM. At 30 nM, bright fluorescence was seen
between cells (Figure 7E), suggesting that enough of the
fluorescent molecules were present to be detected by confocal
microscopy, but this fluorescence was lost immediately on
washout of the label (Figure 7F). In the case of the dansyl
derivatives (both agonist and antagonist), no increase in fluo-
rescence was seen above the level of autofluoresence from the
cells (Figure 7G,H). Furthermore, unlike the situation with
XAC-X-TXR (15), no dansyl fluorescence was detected in solu-
tion. When XAC-X-BY630 (14) and the BODIPY 630/650
derivatives of NECA (1–4) were visualized, clear membrane
binding was observed even in the continued presence of fluo-
rescent ligand (Figure 7A–D). This latter result suggests that the
BODIPY 630/650 fluorophore is heavily quenched in aqueous
solution. This suggestion was reinforced when excitation and
emission spectra for XAC-dansyl (19), XAC-X-TR (15) and
XAC-X-BY630 (14) were determined in HBS and methanol
(Figure 8). The ratio of peak emission intensities in HBS com-
The addition of the smaller dansyl fluorophore to XAC
resulted in a molecule that maintained high affinity at the A1
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Table 5 Agonist responses to fluorescent NECA analogues at the adenosine A1 receptor
Agonist
Gi (log IC50
)
n
Gs (log EC50
)
n
+PTX
Gs (log EC50
)
Gs EMAX (fold increase)
n
Log KD - Log IC50
NECA
-8.43 Ϯ 0.04
-8.21 Ϯ 0.11
-7.92 Ϯ 0.06
-7.97 Ϯ 0.07
-7.32 Ϯ 0.09
-7.20 Ϯ 0.17
-5.95 Ϯ 0.09
-6.64 Ϯ 0.08
-7.08 Ϯ 0.08
-6.05 Ϯ 0.17
-5.45 Ϯ 0.11
-5.27 Ϯ 0.35
-6.03 Ϯ 0.09
-6.57 Ϯ 0.08
15
8
8
8
6
6
7
10
8
3
-6.13 Ϯ 0.03
15
8
6
-6.37 Ϯ 0.04
1.81 Ϯ 0.07
1.67 Ϯ 0.06
1.44 Ϯ 0.03
1.41 Ϯ 0.06
13
7
8
1.92
2.40
2.03
1.93
1.28
0.97
APrEA-X-BY630
ABEA-X-BY630
APEA-X-BY630
AOEA-X-BY630
ADOEA-X-BY630
ABEA-dansyl
ABEA-AO-dansyl
AUEA-dansyl
ABEA-X-Texas Red
ABEA-EVOBlue 30
ABEA-Cy5
-6.18 Ϯ 0.13
-6.62 Ϯ 0.13
-6.38 Ϯ 0.17
-6.40 Ϯ 0.06
-6.23 Ϯ 0.13
5
-6.58 Ϯ 0.06
11
NS
>-5
-5.79 Ϯ 0.11
4
-5.97 Ϯ 0.07
1.31 Ϯ 0.06
4
7
7
7
3
3
3
7
7
NS
NS
NS
NS
NS
NS
NS
NS
>-5
>-6
NS
NS
NS
NS
NS
>-5
2.25
3
3
3
7
7
3
3
7
7
ABEA-BYFL
ABEA-X-BYFL
Values represent mean Ϯ SEM of n separate experiments. Values are log (IC50) and log (EC50) determinations from the agonist-mediated inhibition (Gi) or
enhancement (Gs), respectively, of forskolin-stimulated CRE-reporter gene activity. In some experiments (+PTX), cells were incubated overnight with PTX
(100 ng·mL-1), and under these conditions, only a stimulatory response (Gs) was obtained. An indication of agonist efficacy is provided by the difference between
log IC50 values and the log KD values reported for each agonist in Table 2.
ABEA, N6-(4-aminobutyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; ADOEA, N6-(8-amino-3,6-dioxaoctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; AEAO,
8-(2-aminoethylamino)-8-oxooctanoyl; AO, 8-aminooctanoyl; AOEA, N6-(8-aminooctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; APEA, N6-(5-aminopentyl)-
5′-ethylamino-5′-oxo-5′-deoxyadenosine; APrEA, N6-(3-aminopropyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; AUEA, N6-(11-aminoundecyl)-5′-ethylamino-5′-
oxo-5′-deoxyadenosine; PTX, Pertussis toxin; NECA, adenosine-5′-N-ethyluronamide; NS, no stimulation; X, 6-aminohexanoyl.
Table 6 Antagonism of fluorescent agonist Gi-mediated responses by DPCPX, XAC and CGS15943
Agonist
log KD DPCPX
n
tog KD XAC
n
log KD CGS
n
NECA
-9.13 Ϯ 0.10
-8.70 Ϯ 0.09
-8.69 Ϯ 0.06
-8.61 Ϯ 0.06
-8.97 Ϯ 0.07
-8.67 Ϯ 0.09
-8.76 Ϯ 0.06
-8.78 Ϯ 0.15
-8.80 Ϯ 0.12
-8.73 Ϯ 0.16
7
8
8
8
3
6
6
5
7
5
-7.86 Ϯ 0.05
-7.62 Ϯ 0.07
-7.55 Ϯ 0.08
-7.56 Ϯ 0.08
-7.87 Ϯ 0.09
-7.71 Ϯ 0.07
-7.90 Ϯ 0.06
-7.74 Ϯ 0.12
-7.97 Ϯ 0.10
-7.83 Ϯ 0.13
14
7
8
8
3
6
6
5
6
-8.80 Ϯ 0.10
-8.58 Ϯ 0.12
-8.54 Ϯ 0.09
-8.57 Ϯ 0.10
-8.87 Ϯ 0.15
-8.67 Ϯ 0.08
-8.78 Ϯ 0.07
-8.90 Ϯ 0.19
-8.96 Ϯ 0.11
-8.69 Ϯ 0.15
3
7
8
8
3
6
6
6
7
6
APrEA-X-BY630
ABEA-X-BY630
APEA-X-BY630
AOEA-X-BY630
ADOEA-X-BY630
ABEA-dansyl
ABEA-AO-dansyl
AUEA-dansyl
ABEA-X-BYFL
5
Values represent mean Ϯ SEM from n separate experiments.
ABEA, N6-(4-aminobutyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; ADOEA, N6-(8-amino-3,6-dioxaoctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; AO,
8-aminooctanoyl; AOEA, N6-(8-aminooctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; APEA, N6-(5-aminopentyl)- 5′-ethylamino-5′-oxo-5′-deoxyadenosine; APrEA,
N6-(3-aminopropyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; AUEA, N6-(11-aminoundecyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; CGS 15943, 5-amino-9-
chloro-2-(2-furyl)1,2,4-trizolo[1,5-c]quinazoline; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; NECA, 5′(N-ethylcarboxamido)adenosine; X, 6-aminohexanoyl;
XAC, xanthine amine congener.
receptor, comparable with that of the parent XAC molecule.
This was true whether the affinity was deduced from inhibi-
tion of specific H-DPCPX binding or from inhibition of the
ments along with a blue shift in emission wavelength
(Hayashida et al., 2007). The increase in fluorescence intensity
in environments of lower polarity (methanol) was also dem-
onstrated directly in the present study (Figure 8), although
the emission wavelength increased slightly. These data
suggest that when the dansyl derivatives are bound to the
adenosine A1 receptor, the fluorophore is likely to be located
within an aqueous and/or highly quenched environment.
The addition of BODIPY 630/650 fluorophore to the XAC
molecule made no difference to the affinity of the molecules
as determined from 3H-DPCPX whole cell binding. The
inverse agonist activity of the molecule was maintained, as
was the affinity when measured at both the Gi- and
Gs-coupled states of the receptor. Furthermore, when XAC-X-
BY630 (14) was visualized using the confocal microscope,
clear membrane binding was observed, which remained even
3
NECA-stimulated reporter gene responses (Tables 2 and 3).
The addition of the dansyl fluorophore therefore had little
effect on the pharmacological properties of XAC. However,
when the ligands were visualized, no binding was seen at all.
In fact, no increase in fluorescence was seen above the level of
cellular autofluoresence (Figure 7). Furthermore, unlike the
situation with XAC-X-Texas Red (15), no dansyl fluorescence
was detected in solution. This is consistent with the relatively
weak fluorescence intensity normally observed with dansyl
fluorophores in aqueous solution (Hayashida et al., 2007).
This fluorophore has been observed previously to demon-
strate sensitivity to the microenvironment polarity such that
its fluorescence intensity increases in lower polarity environ-
British Journal of Pharmacology (2010) 159 772–786

Fluorescent A1 receptor ligands
JG Baker et al
783
Basal
Basal
Forskolin 3μM
DPCPX 10nM
APrEA-X-BY630
Forskolin 3μM
DPCPX 30nM
APrEA-X-BY630
D
2.5
A
1.2
APrEA-X-BY630 + DPCPX 10nM
APrEA-X-BY630 + DPCPX 30nM
1.0
0.8
0.6
0.4
0.2
0.0
2.0
1.5
1.0
0.5
0.0
+ PTX
–11 –10 –9
–8
–7
–6
–5
–11 –10 –9
–8
–7
–6
–5
log[APrEA-X-BY630](M)
log[APrEA-X-BY630](M)
Basal
Basal
Forskolin 3μM
DPCPX 10nM
APEA-X-BY630
Forskolin 3 μM
DPCPX 30nM
APEA-X-BY630
APEA-X-BY630 + DPCPX 30nM
E
2.0
B
1.2
APEA-X-BY630 + DPCPX 10nM
1.0
0.8
0.6
0.4
0.2
0.0
1.5
1.0
0.5
0.0
+ PTX
–11 –10 –9
–8
–7
–6
–5
–11 –10
–9
–8
–7
–6
–5
log[APEA-X-BY630](M)
log[APEA-X-BY630](M)
Basal
Basal
Forskolin 3μM
DPCPX 10nM
ADOEA-X-BY630
Forskolin 3μM
DPCPX 30nM
ADOEA-X-BY630
C
1.2
F
2.0
ADOEA-X-BY630 + DPCPX 10nM
ADOEA-X-BY630 + DPCPX 30nM
1.0
0.8
0.6
0.4
0.2
0.0
1.5
1.0
0.5
0.0
+ PTX
–11 –10
–9
–8
–7
–6
–5
–11 –10 –9
–8
–7
–6
–5
log[ADOEA-X-BY630](M)
log[ADOEA-X-BY630](M)
Figure 5 CRE-SPAP production in CHO-A1 cells in response to APrEA-X-BY630 (A and D), APEA-X-BY630 (B and E), ADOEA-X-BY630 (C and
F) in the presence and absence of 30 nM DPCPX (A, B and C) and 10 nM DPCPX (D, E and F). The cells in D, E and F were incubated with
PTX (100 ng·mL-1) for 24 h before experimentation. Bars show basal CRE-SPAP production, that in response to 3 mM forskolin and that in
response to either 30 nM or 10 nM DPCPX in the presence of 3 mM forskolin. Data points are mean Ϯ SEM from triplicate values from a single
experiment. These separate experiments are representative of (A) 8, (B) 8, (C) 6, (D) 6, (E) 6 and (F) 6 separate experiments. ADOEA,
N6-(8-amino-3,6-dioxaoctyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; APEA, N6-(5-aminopentyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine;
APrEA, N6-(3-aminopropyl)-5′-ethylamino-5′-oxo-5′-deoxyadenosine; CHO, Chinese hamster ovary; CRE-SPAP, cyclic AMP response element-
secreted placental alkaline phosphatase; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; NECA, 5′ (N-ethylcarboxamido)adenosine; PTX, Pertussis
toxin; X, 6-aminohexanoyl.
when the extracellular fluorescent ligands were washed away.
Thus, the addition of BODIPY 630/650 to XAC had very little
effect on the pharmacology of the molecule, with all the
pharmacological properties being preserved in addition to
NECA and its fluorescent derivatives
NECA had a binding affinity as measured by 3H-DPCPX
whole-cell binding of 1 mM. The IC50 for activation of the
adenosine A1 receptor was, however, 38 nM. This is because
NECA is a highly efficacious agonist and needs to occupy only
a few receptors to stimulate a full agonist response. This
highly efficacious nature of NECA makes it an ideal choice for
the starting molecule from which to make fluorescent deriva-
tives. Adenosine A1 receptors have also been shown to couple
to Gs-coupled receptors in CHO cells (Cordeaux et al., 2000;
2004; Baker and Hill, 2007), and the EC50 for stimulation by
NECA of the Gs-coupled state of the receptor (794 nM) shows
that this coupling is much less efficient.
good fluorescent properties.
A striking property of the
BODIPY 630/650 ligands was that membrane binding to cells
expressing the adenosine A1 receptor could be determined in
the continued presence of the fluorescent ligand. These data
suggest that the fluorescence intensity of the BODIPY 630/650
fluorophore increases on binding to the receptor. Thus, unlike
the dansyl congeners, the BODIPY 630/650 fluorophore is
likely to be located within a non-polar environment when
bound to the A1 receptor.
British Journal of Pharmacology (2010) 159 772–786

Fluorescent A1 receptor ligands
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JG Baker et al
1.00
0.75
0.50
0.25
0.00
A
B
Basal
Forskolin 3μM
AUEA-dansyl
ABEA-AO-dansyl
ABEA-dansyl
–12 –11 –10 –9
–8
–7
–6
–5
log[NECA-dansyl-derivative](M)
C
D
Figure 6 CRE-SPAP production in CHO-A1 cells in response to
NECA-dansyl derivatives (AUEA-dansyl, ABEA-AO-dansyl and ABEA-
dansyl). Bars show basal CRE-SPAP production, that in response to
3 mM forskolin. Data points are mean Ϯ SEM from triplicate values
from a single experiment and this is representative of seven separate
experiments. ABEA, N6-(4-aminobutyl)-5′-ethylamino-5′-oxo-5′-
deoxyadenosine; AUEA, N6-(11-aminoundecyl)-5′-ethylamino-5′-
oxo-5′-deoxyadenosine; CHO, Chinese hamster ovary; CRE-SPAP,
cyclic AMP response element-secreted placental alkaline phos-
phatase; NECA, 5′ (N-ethylcarboxamido)adenosine.
E
F
The NECA agonist response was inhibited by non-
fluorescent adenosine A1 receptor antagonists to give values
similar to those previously published (Baker and Hill, 2007).
The affinity of non-fluorescent antagonists was the same for
both the NECA–Gi-induced state of the receptor, and the
NECA-induced Gs state of the receptor. The Gs-coupled state
could be isolated by pre-incubating the cells with PTX. This
resulted in a complete abolition of the inhibitory Gi-response
(i.e. the reduction in CRE-gene transcription), leaving only
the Gs-stimulatory response intact. The affinity of the non-
fluorescent antagonists measured under these conditions was
also identical.
H
G
The addition of the EVOBlue30, Texas Red and Cy5 fluoro-
phores to the parent NECA molecule completely abolished all
measurable pharmacological activity. The addition of the
BODIPY-FL fluorophore resulted in molecules with much
reduced potency, which was probably due to a reduction in
affinity, as no binding was detectable. The agonist response to
these molecules was mediated by the adenosine A1 receptor, as
it could be inhibited by non-fluorescent A1 receptor antago-
nists to give the same affinity (log KD values) as for the parent
NECA. Furthermore, the response was abolished following
PTX pretreatment confirming that it was occurring via the
Gi-coupled state of the receptor. However, when these NECA
derivatives were examined using confocal microscopy, no spe-
cific binding was seen with any of the ligands. Thus, although
ABEA-X-BYFL (9) had marginal functional activity, none of
the Texas Red, EVOblue30, Cy5 or BODIPY-FL derivatives
have useful functional or fluorescent use.
The addition of the dansyl derivatives to the parent NECA
molecule did result in functionally active molecules. These
molecules had reduced affinity for the adenosine A1 receptor
(Table 2); however agonist Gi-coupled inhibitory functional
responses were still seen. Although none of the dansyl deriva-
tives of NECA were sufficiently efficacious to induce the
Gs-coupled state of the A1 receptor, it is interesting to note
that lengthening the carbon chain in the linker between the
NECA moiety and fluorophore from C4 to C11 (AUEA-dansyl,
10) resulted in an increase in agonist potency of 10-fold, and
Figure 7 Confocal images of CHO-A1 cells incubated (5 min) with:
(A) APrEA-X-BY630 (10 nM), (B) ADOEA-X-BY630 (10 nM), (C, D)
XAC-X-BY630 (30 nM), (E, F) XAC-X-Texas Red (30 nM) and (G)
XAC-dansyl (10 nM). In D and F, images were collected immediately
after the fluorescent ligand had been removed from the cells. H
shows a representative image of autofluorescence collected under
identical conditions to image (G), but without the addition of
XAC-dansyl. ADOEA, N6-(8-amino-3,6-dioxaoctyl)-5′-ethylamino-5′-
oxo-5′-deoxyadenosine; APrEA, N6-(3-aminopropyl)-5′-ethylamino-
5′-oxo-5′-deoxyadenosine; CHO, Chinese hamster ovary; X,
6-aminohexanoyl; XAC, xanthine amine congener.
that this was most likely due to an increase in affinity
(Table 2). However, the combination of lower affinity for the
adenosine A1 receptor, together with the problems high-
lighted above for dansyl XAC derivatives regarding the low
fluorescence intensity of the dansyl fluorophore in polar
aqueous environments, is the most likely explanation of the
lack of success with these ligands in confocal microscopy
studies.
The addition of a BODIPY 630/650 fluorophore resulted in
a series of functionally active molecules. It was only possible
to determine the apparent affinity (log KD values) of the
fluorescent NECA BODIPY 630/650 derivatives from the
British Journal of Pharmacology (2010) 159 772–786

Fluorescent A1 receptor ligands
JG Baker et al
785
Brilliant Black (50mM)
Control
A
XAC-dansyl
B
A
HBS
MeOH
1.0
0.8
0.6
0.4
0.2
0.0
XAC-X-BY630
(10nM, 2min)
D
C
300
400
500
600
l (nm)
Phase
B
XAC-X-TR
HBS
MeOH
1.0
0.8
0.6
0.4
0.2
0.0
Figure 9 The effect of Brilliant Black on the fluorescence of XAC-X-
BY630 bound to CHO-A1 cells. CHO-A1 cells were incubated with
XAC-X-BY630 (10 nM, 37°C) for 2 min, and a single confocal (A) and
simultaneous phase image (C) were acquired. Immediately following
the addition of the fluorescence quencher, Brilliant Black (50 mM),
similar confocal (B) and phase contrast (D) images were taken, with
the darkening of the phase image indicating a successful addition of
the Brilliant Black. CHO, Chinese hamster ovary; X, 6-aminohexanoyl;
XAC, xanthine amine congener.
500
550
600
650
700
750
this may be partly due to differential penetration of the fluo-
rescent ligand and H-DPCPX into the intracellular environ-
l (nm)
3
ment of intact cells. Thus, permeabilization of cells did
increase the percentage of specific binding inhibited by this
fluorescent agonist by allowing access to intracellular
receptors (Middleton et al., 2007). An alternative interpreta-
tion is that these fluorescent analogues are acting allosteri-
cally to inhibit binding of 3H-DPCPX (Christopolous and
Kenakin, 2002; Kenakin, 2004; May et al., 2007).
C
XAC-X-BY630
HBS
1.0
0.8
0.6
0.4
0.2
0.0
MeOH
It is interesting, however, to note that the log IC50 values of
the BODIPY 630/650 derivatives in the functional Gi protein-
coupled CRE response increased as the linker lengthened. This
did not appear to be due to a decrease in binding affinity
(Table 2), but rather reflected a decrease in agonist efficacy. If
one takes the difference between log IC50 and apparent log KD
values as a measure of efficacy, then there was a clear decrease
in efficacy as the chain length increased (Table 5). All but
AOEA-X-BY630 (4) were able to stimulate an increase in CRE-
gene transcription by inducing a Gs-coupled state of the
adenosine A1 receptor at higher concentrations. Interestingly,
the ADOEA-X-BY630 (5) analogue was equally potent with
AOEA-X-BY630 (which has an identical linker length) at
inhibiting CRE-gene transcription, but retained some
Gs-stimulating activity (Table 5). These data suggest that the
latter compound has a reduced affinity and/or efficacy for the
Gs-coupled conformation of the adenosine A1 receptor com-
pared with the other fluorescent agonists.
500
550
600
650
700
750
l (nm)
Figure 8 Normalized excitation and emission spectra obtained in
HBS or methanol for (A) XAC-dansyl (50 mM), (B) XAC-X-Texas Red
(1 mM) and (C) XAC-X-BY630 (1 mM). Data have been normalized to
the maximum excitation or emission obtained with each ligand in
methanol. Excitation maxima were: (A) 320, 315 nm; (B) 590,
585 nm and (C) 628, 634 nm in HBS and methanol respectively.
Corresponding emission maxima were: (A) 500, 525 nm; (B) 605,
600 nm and (C) 645, 640 nm. The relative maximal emission inten-
sities (HBS; methanol) were: (A) 0.26; (B) 0.89 and (C) 0.07. HBS,
N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid-buffered sal-
ine; X, 6-aminohexanoyl; XAC, xanthine amine congener.
In the case of the dansyl NECA derivatives, there is a hint
that affinity and/or efficacy increases as the chain length
increases. This is opposite to the finding with the BODIPY
630/650 derivatives, and this observation may provide a
clue to the orientation of the fluorophore within the
3H-DPCPX whole cell-binding studies (Table 2) because the
fluorescent analogues did not inhibit all of the specific
binding. We have observed this previously for the ABEA-X-
BY630 (2) derivative (Middleton et al., 2007), and shown that
British Journal of Pharmacology (2010) 159 772–786

Fluorescent A1 receptor ligands
786
JG Baker et al
of BODIPY-TMR-CGP: a long acting fluorescent b2-adrenoceptor
agonist. Br J Pharmacol 139: 232–242.
receptor-binding pocket. A comparison of the affinity of XAC-
X-BY630 with all three XAC dansyl derivatives suggests that
binding affinity is retained with both fluorophores. However,
the marked differences in fluorescence intensity of ligand
binding at the single-cell level with these fluorescent ligands
suggest that the dansyl fluorophore is located within an
aqueous environment, whereas the BODIPY 630/650 fluoro-
phore is within a less polar membrane environment. This
suggestion is supported by the data obtained with XAC-dansyl
(19) and XAC-X-BY630 (14) in aqueous buffer or methanol
(Figure 7), which shows a marked quenching of both agents in
aqueous environments. The clear imaging of membrane bind-
ing of XAC-X-BY630 (Figure 9) in CHO-A1 cells in the presence
of Brilliant Black is also consistent with this conclusion.
The opposite influence of chain length on agonist activity
of the dansyl and BODIPY 630/650 NECA derivatives is
entirely consistent with this interpretation. That is, the
BODIPY 630/650 fluorophore could potentially be placed
within the transmembrane lipid environment of the A1 recep-
tor binding site without interfering with agonist action even
with short linker lengths, whereas the dansyl fluorophore
may alternatively need to exit the binding pocket into the
extracellular aqueous medium on a long linker to avoid
unfavourable interactions with either the surrounding lipid
environment or the receptor itself.
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In summary, the present study adds further weight to the
argument that design of a fluorescent ligand needs to take
into account the influence of both the fluorophore and the
associated linker on the pharmacology of the final product.
Furthermore, our data strongly suggest that the physico-
chemical properties of the fluorophore/linker pairing ulti-
mately determine where in the environment of the target
receptor the fluorophore is located, and this, together with
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Acknowledgments
JGB is a Wellcome Trust Clinician Scientist Fellow. LTM holds
an NHMRC Fellowship. We thank Tim Self, Richard Proud-
man and Marleen Groenen for technical assistance. We thank
Wellcome Trust, BBSRC and MRC for financial support.
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Conflicts of interest
SJH and BK are founding directors of the University of Not-
tingham spin-out company CellAura Technologies Ltd. RM
and LA are currently employed by CellAura Technologies Ltd.
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