Biotin ergopeptide probes for dopamine receptors

Article abstract of DOI:10.1021/jm101566d

The incorporation of chemical modifications into the structure of bioactive compounds is often difficult because the biological properties of the new molecules must be retained with respect to the native ligand. Ergopeptides, with their high affinities at D₁ and D₂ dopamine receptors, are particularly complex examples. Here, we report the systematic derivatization of two ergopeptides with different peptide-based spacers and their evaluation by radioligand binding assays. Selected spacer-containing ergopeptides with minimal biological alteration and a proper anchoring point were further derivatized with a biotin reporter. Detailed characterization studies identified 13 as a biotin ergopeptide maintaining high affinity and agonist behavior at dopamine receptors, being a useful tool for the study of heteromers involving D₁R, D₂R, or D₃R.

Full text of DOI:10.1021/jm101566d

1080 J. Med. Chem. 2011, 54, 1080–1090
DOI: 10.1021/jm101566d
Biotin Ergopeptide Probes for Dopamine Receptors
‡
‡
‡
‡
Marc Vendrell,†,^ Anabel Molero,†,§ Sergio Gonzalez, Kamil Perez-Capote, Carme Lluis, Peter J. McCormick,
ꢀ
ꢀ
Rafael Franco, Antoni Cortes, Vicent Casado, Fernando Albericio, ,§ and Miriam Royo*,†
‡
‡
ꢀ
ꢀ ‡
†Combinatorial Chemistry Unit, Barcelona Science Park, University of Barcelona, 08028 Barcelona, Spain, ‡Centro de Investigacioꢀn Biomeꢀdica en
Red sobre Enfermedades Neurodegenerativas (CIBERNED), and Department of Biochemistry and Molecular Biology, Faculty of Biology,
University of Barcelona, 08028 Barcelona, Spain, §CIBER-BBN, Networking Centre on Bioengineering, Biomaterials, and Nanomedicine,
Barcelona Science Park, 08028 Barcelona, Spain, and Institute for Research in Biomedicine, 08028 Barcelona, Spain. ^ Current address:
Laboratory of Bioimaging Probe Development, Singapore Bioimaging Consortium, 138667 Singapore
Received December 8, 2010
The incorporation of chemical modifications into the structure of bioactive compounds is often difficult
because the biological properties of the new molecules must be retained with respect to the native ligand.
Ergopeptides, with their high affinities at D1 and D2 dopamine receptors, are particularly complex
examples. Here, we report the systematic derivatization of two ergopeptides with different peptide-
based spacers and their evaluation by radioligand binding assays. Selected spacer-containing ergopep-
tides with minimal biological alteration and a proper anchoring point were further derivatized with a
biotin reporter. Detailed characterization studies identified 13 as a biotin ergopeptide maintaining high
affinity and agonist behavior at dopamine receptors, being a useful tool for the study of heteromers
involving D1R, D2R, or D3R.
Introduction
reported.2-4 Dual ligands have been successfully applied
to the study of G-protein coupled receptors (GPCRs)
oligomerization.5,6 While ergopeptides may provide insights
into the D1 or D2 receptors containing heteromers, they require
a proper reporter tag prior to their use in protein localization
and profiling studies.7-10 The modification of hit compounds
is a critical step as the biological properties of the labeled
moleculescanbe significantlyalteredwithrespecttothenative
ligand.11 This step is particularly important when applied to
small molecule ligands, such as ergopeptides, because the
ligand affinity and efficacy of the pharmacophore is likely to
becompromised.12 In the present work, wereport a systematic
study to optimize the length and chemical nature of different
spacer moieties attached to two ergopeptides with high affi-
nity at D1R and D2R (1 and 2, Chart 1) and identify those
linkers that retain their binding profile. Trifunctional amino
acids, such as lysine and glutamic acid, are excellent scaffolds
for the synthesis of peptide-based spacers. In addition to their
low toxicity, they can be easily adapted to a solid-phase
synthesis approach to allow the incorporation of a range of
functional groups (amines, anilides, and carboxamides) with-
in the spacer structure. A number of peptide-based spacers
were incorporated to 1 and 2 to render a 40-member library of
new ergopeptides, and the evaluation of their affinities at D1R
and D2R identified two linker moieties with minimal biologi-
cal interference. The subsequent incorporation of a biotin
reporter led to identification of 13 as a biotin ergopeptide for
dopamine receptors with nanomolar binding affinities and
agonist behavior.
Ergopeptides, with their high affinity at D1 and D2 dopa-
mine receptors (D1Rs and D2Rsa), are valuable molecules to
study dopamine receptors.1 The therapeutic significance of
dopamine receptors containing heteromers has been extensively
*To whom correspondence should be addressed: Phone: 0034
934037122. Fax: 0034 934037126. E-mail: mroyo@pcb.ub.cat.
a Abbreviations: A2AR, adenosine A2A receptor; A1R, adenosine A1
receptor; Ac2O, acetic anhydride; ACN, acetonitrile; Alloc, allyloxy-
carbonyl; All, allyl ester; Ahx, aminohexanoic acid; Akt, protein kinase
B; Boc, t-butoxycarbonyl; CDI, 1,10-carbonyldiimidazole; CHO,
Chinese hamster ovary; DCM, dichloromethane; cDNA, cDNA; [3H]-
CP55940, tritium labeled 2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)-
cyclohexyl]-5-(2-methyloctan-2-yl)phenol; DIPCDI, N,N0-diisopropyl-
carbodiimide; DIEA, diisopropylethylamine; DMF, dimethylforma-
mide; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; D1R, dopamine
D1 receptor; D2R, dopamine D2 receptor; D3R, dopamine D3 receptor;
ERK, extracellular-signal-regulated kinases; Fmoc, fluorenylmethylox-
ycarbonyl; GPCR, G-protein coupled receptor; HEPES, 4-(2-hydro-
xyethyl)-1-piperazineethanesulfonic acid; HOBt, hydroxybenzotriazole;
HOAt, 1-hydroxy-7-azabenzotriazole; HRMS, high resolution mass
spectrometry; MAPK, mitogen-activated protein kinases; PEG, poly-
ethylene glycol; PEI, polyethylenimine; Rink-MBHA-PS, 4-(20,40dimethoxy-
phenyl-Fmoc-aminomethyl)-pheoxyacetamido p-methylbenhidryla-
mine resin; RP-HPLC, reversed phase-high performance liquid chro-
matography; RP-HPLC-MS, reversed phase-high performance liquid
chromatography-mass spectroscopy; SKF81297, (()-6-chloro-2,3,4,5-
tetrahydro-1-phenyl-1H-3-benzazepine hydrobromide; TFA, trifluor-
oacetic acid; TMUCl Cl, N-[chloro(dimethylamino)methylene]-N-
methylmethanaminium chloride; TBTU, O-(benzotriazol-1-yl)-N,N,
N0,N0-tetramethyluronium tetrafluoroborate; Tris-HCl, tris(hydroxy-
methyl)aminomethane hydrochloric acid; [3H]-RAMH, tritium labeled
R-methyl histamine; [3H]-R-PIA, tritium labeled R-phenylisopropyla-
denosine; [3H]-SCH23390, tritium labeled [R-(þ)-8-chloro-2,3,4,5-tet-
rahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol); [3H]-YM09151-2,
tritium labeled nemonapride (N-(1-benzyl-2-methylpyrrolidin-3-yl)-5-
chloro-2-methoxy-4-(methylamino)benzamide); [3H]-ZM241358, tri-
tium labeled 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-
5-ylamino]ethyl)phenol. Abbreviations used for amino acids follow the
IUPAC-IUB Commission of Biochemical Nomenclature in Jones, J. H.
J. Pept. Sci. 2003, 9, 1-8.
Results
Design of the Library. The incorporation of a spacer
moiety into a bioactive molecule requires the selection of a
suitable attachment point, which must be well separated
r
pubs.acs.org/jmc
Published on Web 01/31/2011
2011 American Chemical Society

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1081
Chart 1. Ergopeptides with High Affinity at D1 and D2 Dopamine Receptors
from the pharmacophore in order to prevent any interference
on the binding between biomolecule and receptor. The
binding mode analysis of 1 and 2 revealed the interaction
of the ergolene scaffold at the transmembrane binding sites
while the peptide moieties interacted with adjacent amino
acids.1 Bearing this in mind, the attachment of distinct spacer
moieties into 1 and 2 was designed by means of the C-term-
inal carboxamide group modification, which would presum-
ably produce a minor change to the biological properties of
the final compounds.
Several spacer moieties were designed on the basis of two
trifunctional amino acids, lysine and glutamic acid, which
are key scaffolds for the derivatization of biomolecules when
a suitable protecting group scheme is used.13 The introduc-
tion of a number of building blocks onto the lysine and
glutamic acid side chains afforded 19 spacer moieties with
various length and chemical functionalities (Table 1), includ-
ing aromatic and saturated rings, primary amines, carbox-
amides, anilides, and poliethylenglycol (PEG) units. Moreover,
a common NR-group at the C-terminus was acetylated to
mimic the further incorporation of a reporter molecule.
Synthesis of the Spacer-Containing Ergopeptides. The synthe-
sis of the peptide-based spacers was entirely performed on solid-
phase, using Rink-MBHA-PS as a polymeric support.
Coupling Fmoc-Lys(Alloc)-OH or Fmoc-Glu(OAll)-OH onto
the resin, Fmoc elimination, acetylation of the NR-group, and
removal of the side chain protecting groups led to resins 3 and 4,
which were used for the construction of the 19 peptide-based
spacers (Scheme S1 in Supporting Information (SI)).
The introduction of distinct units (amino acids, diamines,
anhydrides, and dicarboxylic acids) onto resins 3 and 4 was
carried out using standard solid-phase peptide synthesis
(SPPS) protocols and 1,10-carbonyldiimidazole (CDI) for
the activation of supported carboxylic acids. The poor nucleo-
philicity of some anilines required the use of a previously
described procedure, based on N-[chloro(dimethylamino)-
methylene]-N-methylmethanaminium chloride (TMUCl Cl)
as the activating reagent, for the solid-phase synthesis of
anilides14 (compounds 1p, 2p, 1r, and 2r). The incorporation
of the 19 spacer moieties into the ergopeptides structure was
carried out in two steps: (1) NR-Boc-tripeptides 5 and 6 were
attached to the supported spacers in a convergent protocol
instead of a stepwise procedure to favor the parallelization
synthetic process; (2) the crude mixtures released after
cleavage (TFA-H2O, 95:5) were further coupled to the D-
lysergic acid in solution using DIPCDI and HOAt (Scheme 1).
Although this method involved an extra step compared to
the direct coupling of ergopeptides onto the spacer moieties,
the amount of the costly D-lysergic acid could be minimized.
The purification of the whole library by semipreparative RP-
HPLC afforded the 38 final products with excellent purities
(1a-s and 2a-s, Table S1 in SI).
Biological Assays: Binding Properties of Spacer-Contain-
ing Ergopeptides. The binding properties of 1a-s and 2a-s
were assayed by displacement experiments of D1R or D2R
radiolabeled ligands (concentration indicated in the legends
of Figures 1 and 2) by 25 μM 1a-s or 2a-s. The binding
screening at D1R and D2R (Figures 1 and 2) indicated a
significant decrease of binding affinity when aromatic rings
were included within the spacer moieties (1p (mainly at D2R),
2i, 2j, 2p, and 2r). In contrast, the incorporation of linear
aliphatic spacers resulted in compounds showing a more
favorable binding, with slightly stronger interactions in the
presence of medium-length spacers (1c, 1f, 1n at D1R; 1l, 1n,
2f, and 2k at D2R) and some shorter ones (1a, 1m at D1R; 1a,
1m, 2a at D2R). Regarding the inclusion of primary amines
within the spacer structure, the results were both ergopeptide
and receptor-dependent; 1d did not show a significantly differ-
ent binding at D1R, but 1d and 1e binding affinities were
remarkably improved at D2R. Whereas determining the
exact nature of this enhancement would require further studies,
the incorporation of medium-sized aliphatic spacers (c and f,
Table 1) at the C-terminus of ergopeptides 1 and 2 proved
to be successful in preserving their binding affinities at both
D1R and D2R.
We further investigated the behavior of the modified
ergopeptides (1c, 1f, 2c, and 2f) at A1 and A2A adenosine
receptors because heteromers containing adenosine-dopa-
mine receptors have been very well described.15-17 The binding
affinities of 1c, 1f, 2c, and 2f were assayed by displacement
experiments of A1R or A2AR radiolabeled ligands (con-
centration indicated in the Figure S1 and S2 legends in SI)
by 25 μM ergopeptides. The binding of 1c, 1f, 2c, and 2f at
adenosine receptors (Figures S1 and S2 in SI) proved to be
much lower than at D1R and D2R and confirmed their
specificity for dopamine receptors.
Synthesis and Pharmacological Characterization of Biotin
Ergopeptides (9-16). Biotin has been extensively used for
protein profiling studies because its tight interaction with
avidin can facilitate the isolation of protein complexes.18-20
Moreover, the availability of numerous biotin/streptavidin-
labeled antibodies and reagents makes biotin one of the most
versatile reporters for protein characterization. To construct
the corresponding biotin ergopeptide probes, a biotin molecule

1082 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Vendrell et al.
Table 1. Chemical Structures of Ergopeptides after the Incorporation of Peptide-Based Spacers
was incorporated to the C-terminus of 1c, 2c, 1f, and 2f.
Biotin ergopeptides were synthesized using a slightly mod-
ified procedure from the previously described (Scheme 2),
and the biotin reporter was coupled to the NR-group of the
C-terminus of ergopeptides using TBTU and HOBt.
The larger size of the biotin ergopeptides facilitated the
isolation of the two ergolene diastereomers derived from the
epimerization of D-lysergic acid under the coupling con-
ditions,21 and eight biotin ergopeptides (9-16, Chart 2) were
subjected to primary radioligand binding assays at D1R and

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1083
Scheme 1. Synthesis of Representative Compounds 1c and 2s
a Conditions: (a) Fmoc-Ahx-OH, DIPCDI/HOBt; (b) piperidine-DMF (2:8); (c) 5, DIPCDI/HOBt; (d) TFA-H2O (95:5); (e) D-lysergic acid,
DIPCDI/HOAt, DIEA; (f) (Fmoc-4-amino) piperidine HCl, DIEA, CDI; (g) piperidine-DMF (2:8); (h) 6, DIPCDI/HOBt.
3
D2R. The binding properties of 9-16 were initially evaluated
using displacement experiments of a fixed concentration of
D1R, D2R, A1R or A2AR radiolabeled ligands (indicated
in the legends of Figures S3-S6 in SI) by 25 μM 9-16
(Figures S3-S6 in SI). The incorporation of the biotin
molecule retained the binding properties of the parent com-
pounds 1 and 2 and confirmed that both selected spacers
(c and f) separate well the pharmacophore from the reporter
tag. We selected compounds 9, 12, 13, and 14 for their
pharmacological characterization, and their affinity con-
stants at D1R and D2R were calculated by competition
experiments: brain membranes (0.5 mg/mL) were incubated
with a fixed concentration of D1R or D2R radiolabeled
antagonists in the absence or in the presence of increasing
concentrations of compounds 9, 12, 13, or 14, as described in
the Experimental Section. From the resulting competition
curves (Figure 3 for compound 13, and Figures S7-S9 in SI
for compounds 9, 12, and 14), their corresponding KD values
were determined (Table 2). The biotin ergopeptide 13 proved
to be the compound that best maintained the nanomolar
range affinities at both dopamine receptor subtypes.
D1R or D2R in a dose-dependent manner and to a similar
extent than a D1R full agonist (e.g., SKF 81297)22 or a D2R
agonist (e.g., quinpirole).23 The 13-mediated effect (at 0.1 or
1 μM) was also reverted when cells were preincubated with 5
or 50 μM of D1R (e.g., SCH 23390) or D2R antagonists (e.g.,
raclopride), further demonstrating the agonist behavior of
13 at both dopamine receptor subtypes. Regarding the Akt
(PKB) pathway, both SKF 81297 and quinpirole (D1R and
D2R agonists, respectively) induced a decrease in the Akt
Ser473 phosphorylation, a signaling that has been also observed
in mouse brain.24,25 Similarly, 13 decreased the Akt phosphor-
ylation in a dose-dependent manner in cells expressing D1R
or D2R, and its effect was also reverted upon preincubation
with D1R (e.g., SCH 23390) or D2R antagonists (e.g.,
raclopride), confirming the agonist behavior of 13 at both
D1R and D2R (Figure 4c,d).
Since it has been described that D1Rs can also heteromer-
ize with dopamine D3Rs,26 we analyzed the binding affinity
of compound 13 at D3R. Because of the low expression level
of D3R in the striatum when compared to D2R, we transi-
ently transfected CHO cells with D3R to study the binding
affinity of compound 13 to this receptor. Membranes
(0.5 mg/mL) from transfected cells were incubated with
4 nM [3H]-raclopride, a D3R antagonist, in the absence or
in the presence of increasing concentrations of compound 13.
Thecompetitioncurvewasusedtodeterminethecorresponding
To test whether the compound 13 behaved as an agonist,
we examined two different signal transduction pathways
(e.g., MAPK and Akt (PKB)) in cells that were separately
transfected with D1R and D2R. As shown in Figure 4a,b, 13
increased the ERK1/2 phosphorylation in cells expressing

1084 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Vendrell et al.
Figure 2. Displacement experiments at D2R. Specific binding of
0.7 nM D2R antagonist [3H]-YM 09151-2 in the absence or in the
presence of competing ligands was measured as indicated in the
Experimental Section. C, control of radioligand binding without
competing ligand; 1a-2s, radioligand specific binding in the pre-
sence of 25 μM ergopeptides; 1-2, radioligand specific binding in
the presence of 25 μM ergopeptides 1 and 2, respectively. Values are
represented means ( SD (n = 3). Student’s t-test for unpaired
samples showed significant differences (*p < 0.05; **p < 0.01)
compared to the ergopeptides 1 or 2.
Figure 1. Displacement experiments at D1R. Specific binding of 0.9
nM D1R antagonist [3H]-SCH23390 in the absence or in the
presence of competing ligands was measured as indicated in the
Experimental Section. C, control of radioligand binding without
competing ligand; 1a-2s, radioligand specific binding in the pre-
sence of 25 μM ergopeptides; 1-2, radioligand specific binding in
the presence of 25 μM ergopeptides 1 and 2, respectively. Values are
represented as means ( SD (n = 3). Student’s t-test for unpaired
samples showed significant differences (**p < 0.01) compared to
the ergopeptides 1 or 2.
with agonist behavior and significantly higher binding affi-
nities at D1R, D2R, and D3R when compared to adenosine
(A1/A2A), histamine H3, metabotropic glutamate 1/5, soma-
tostatin SST, and cannabinoid CB1 receptors. These results
attest the potential application of 13 to study heteromer com-
plexes involving dopamine receptors. Experiments with biotin
ergopeptides to study dopamine receptors heteromers are
currently ongoing and will be reported in due course.
KD values (KDB1 = 23 ( 9 nM and KDB2 = 93 ( 36 nM),
proving that 13 can be a useful tool for the study of hetero-
mers involving D1, D2, or D3 dopamine receptors (Figure 5).
The binding properties of 13 were also examined at other
GPCRs that can form heteromers with D1Rs or D2Rs (e.g.,
histamine H3, metabotropic glutamate 5, somatostatin SST5,
and cannabinoids CB1 receptors).27-31 Displacement experi-
ments (Figure S10 in SI) showed that high concentrations of
13 did not significantly decrease the radioligand binding at
the studied receptors and proved that the binding of 13 at
other GPCRs was very low when compared to dopamine
receptors.
Experimental Section
Materials and Equipment. All Fmoc-amino acids were pur-
chased from Neosystem (Strasbourg, France), and Fmoc-Rink-
PS and 2-chlorotrityl resins were supplied by Calbiochem-
Novabiochem AG. DIPCDI was obtained from Fluka Chemika
(Buchs, Switzerland) and HOBt from Albatross Chem, Inc.
(Montreal, Canada). Solvents for peptide synthesis and RP-
HPLC equipment were obtained from Scharlau (Barcelona,
Spain). Trifluoroacetic acid was supplied by KaliChemie (Bad
Wimpfen, Germany). Other chemicals of the highest commer-
cially available purity were purchased from Aldrich (Milwaukee,
WI). All commercial reagents and solvents were used as received.
Adenosine deaminase (EC 3.5.4.4) was purchased from
Roche (Basel, Switzerland), and [3H]-R-PIA was supplied by
Amersham Biosciences (Buckinghamshire, UK). Raclopride,
polyethylenimine (PEI), MgCl2, DPCPX, mouse antiphospho-
ERK1/2 antibody, rabbit anti-ERK1/2 antibody, IRDye 800
antimouse antibody, and IRDye 680 antirabbit antibodies were
purchased from Sigma (St Louis, MO). Rabbit anti-P-Ser473Akt
antibody was purchased from SAB Signalway (Pearland, USA).
ZM241385, SCH23390, RAMH, quisqualic acid, somatostatin,
and CP55940 were supplied by Tocris Biosciences (Avonmouth,
UK). [3H]-SCH23390, [3H]-YM09151-2, [3H]-quisqualic acid,
Conclusions
The derivatization of two ergopeptides showing high affi-
nity at dopamine receptors has been optimized using a com-
binatorial chemistry approach to develop of a novel biotin
ergopeptide that maintained both nanomolar binding affi-
nities and an agonist behavior at dopamine receptors. The
systematic modification of the two parent ergopeptides using
a solid-phase synthesis approach afforded a 40-member li-
brary including different peptide-based spacers at the C-ter-
minus of the ergopeptides. The binding analysis of the library
identified two modified ergopeptides incorporating medium-
length aliphatic spacers as the compounds that best retained
the affinity profile at D1R and D2R. Subsequent derivatiza-
tion of the spacer-containing ergopeptides with a biotin mole-
cule rendered a set of biotin ergopeptides that bound at D1R
and D2R with KD values in the nanomolar range. Further
characterization studies identified 13 as a biotin ergopeptide

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1085
Scheme 2. Synthesis of Representative Biotin Ergopeptides 11 and 13. a
a Conditions: (a) Pd(PPh3)4-PhSiH3 in DCManh; (b) biotin, TBTU/DIEA, HOBt; (c) piperidine-DMF (2:8); (d) sequential peptide synthesis:
couplings in DIPCDI/HOBt, Fmoc removal in piperidine-DMF (2:8); (e) TFA-H2O (95:5); (f) D-lysergic acid, DIPCDI/HOAt.
[3H]-CP55940, [125I]-Tyr11-somatostatin 14, and [3H]-ZM241385
were supplied by Perkin-Elmer (Boston, MA). [3H]-RAMH was
purchased from GE Healthcare (Buckinghamshire, U.K). Ecos-
cint H scintillation cocktail was purchased from National Diagnos-
tics(Atlanta,GA).Bradfordassaykitwaspurchasedfrom Bio-Rad
(Munich, Germany). All other supplements were purchased from
Invitrogen (Paisley, UK).
Analytical RP-HPLC-MS was performed using 2795 Waters
(Milford, MA) Alliance with a Micromass ZQ mass spectro-
meter and a 996 PDA detector. Semipreparative RP-HPLC was
performed on a 2767 Waters chromatography system with a
Micromass ZQ mass spectrometer. Multiple sample evapora-
tion was carried out in a Discovery SpeedVac ThermoSavant
(Waltham, MA). Radioligand binding experiments were per-
formed using a Brandel (Gaithersburg, MD) cell harvester and a
Packard 1600 TRI-CARB scintillation counter. Fitting data
binding program GRAFIT was obtained from Erithacus Soft-
ware (Surrey, UK). For ERK1/2 or P-Ser473Akt phosphoryla-
tion determination, the Odyssey infrared scanner (LI-COR
Biosciences, Lincoln, Nebraska, USA) was used. Band densities
were quantified using the scanner software and exported to
Excel (Microsoft, Redmond, WA, USA).
out with DMF (5 ꢀ 1 min) and DCM (5 ꢀ 1 min) using 10 mL of
solvent/g of resin each time.
Coupling using DIPCDI and HOBt-HOAt. Fmoc-AA-OH,
carboxylic acids or Boc-peptides (3 equiv) were coupled using
DIPCDI (3 equiv) as coupling reagent and HOBt (3 equiv) or
HOAt (3 equiv) as additives in DCM-DMF (1:1) for 2-4 h at rt.
After each coupling, the resin was washed with DMF (5 ꢀ 1 min)
and DCM (5 ꢀ 1 min). Reaction completion was checked by
means of the Kaiser or chloranil tests.32
Anhydrides Coupling. Anhydrides (10 equiv) were coupled
using DIEA (10 equiv) in DCM for 2-4 h at rt. After each
coupling, the resin was washed with DMF (5 ꢀ 1 min) and DCM
(5 ꢀ 1 min). Reaction completion was checked by means of the
Kaiser or chloranil tests.
Coupling using CDI.33 Solid-supported carboxylic acids were
washed with DCM (5 ꢀ 1 min) and DMF (5 ꢀ 1 min) and treated
with CDI (25 equiv) in DMF (30 min). After filtering the resin
and washing with DMF, amines (5 equiv) were added in DCM-
DMF (1:1) and kept under orbital agitation for 2-4 h at rt. After
each coupling, the resin was washed with DMF (5 ꢀ 1 min) and
DCM (5 ꢀ 1 min). Reaction completion was checked by means
of the malachite green test.34
Coupling using TMUCl Cl.14 Solid-supported carboxylic
acids were washed with DCM (5 ꢀ 1 min) and treated with
TMUCl Cl (10 equiv) and DIEA (10 equiv) in DCM (10 min).
After filtering the resin and washing with DCM and DMF,
anilines (5 equiv) were added in DCM-DMF (1:1) and left
Synthesis. Solid-Phase General Procedure. Peptide syntheses
were performed manually in a polypropylene syringes, each
fitted with a polyethylene porous disk. Solvents and soluble
reagents were removed by filtration. Washings between depro-
tection, coupling, and subsequent deprotection steps were carried

1086 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Chart 2. Structures of Biotin Ergopeptides 9-16
Vendrell et al.
under orbital agitation for 2 h at rt. After each coupling, the
resin was washed with DMF (5 ꢀ 1 min) and DCM (5 ꢀ
1 min).
Synthesis of Boc-pNO2Phe-pFPhe-Nip-OH (6). Starting from
2-chlorotrityl resin (6.0 g, loading: 1.3 mmol/g), the peptide was
synthesized as described above. Cleavage and lyophilization
in H2O-ACN (2:1) yielded a white solid powder; 3.58 g (yield:
68%); Mexp 587.0 (Mcalc 586.2), 95%.
Coupling of D-Lysergic Acid. Evaporated crude cleavages for
the different peptide moieties (23.7-79.0 mg, 0.03-0.09 mmol)
were dissolved in individual vials using 0.5 mL of DMF, treated
with DIEA (1 equiv, 5-15 μL, 0.03-0.09 mmol), and stirred for
5 min at rt. Second, D-lysergic acid (1.2 equiv, 11-29 mg,
0.04-0.11 mmol) and HOAt (1.5 equiv, 6-18 mg, 0.05-0.14
mmol) were dissolved in 1 mL of DMF (each reaction) and
added to every vial. Finally, DIPCDI was added (2 equiv, 9-
27 μL, 0.06-0.18 mmol) and the set of reactions was stirred in a
parallel synthesizer for 16 h. Multiple sample evaporation
rendered the crude mixtures further purified by semipreparative
RP-HPLC.
Library Characterization. The purified compounds 1a-1s
and 2a-2s were characterized by analytical RP-HPLC-MS,
using a reverse-phase Symmetry C18 (5 μm, 3.9 mm ꢀ 150 mm)
column and 1 mL/min flow. Elution system A: H2O-TFA,
99.9:0.1; B: ACN-TFA, 99.9:0.1; gradient 0% B to 100% B in
25 min. The purities of all library compounds (average g95%)
were determined by UV absorption at 220 nm (HPLC-MS data,
quantities, and purities of all library compounds are included in
Table S1 in SI).
Synthesis of Biotin Ergopeptides (9-16). Rink-MBHA PS
(1 g, loading: 0.56 mmol/g) was swollen with DCM (1 ꢀ
1 min, 2 ꢀ 10 min) and DMF (5 ꢀ 1 min, 1 ꢀ 15 min) before
Fmoc Group Removal involved the following sequence: (i)
DMF (5 ꢀ 1 min); (ii) piperidine-DMF (2:8) (1 ꢀ 1 min þ 2 ꢀ
15 min); (iii) DMF (5 ꢀ 1 min).
Alloc/All Group Removal involved the following sequence: (i)
DCM (5 ꢀ 1 min); (ii) Pd(PPh3)4 (0.1 equiv) and PhSiH3
(10 equiv) in anhydrous DCM (3 ꢀ 15 min); (iii) anhydrous
DCM (5 ꢀ 1 min); (iv) DCM (5 ꢀ 1 min); (v) DMF (5 ꢀ 1 min);
(vi) 0.02 M solution of sodium diethyldithiocarbamate in DMF
(3 ꢀ 15 min), DMF (5 ꢀ 1 min), DCM (5 ꢀ 1 min), and DMF
(5 ꢀ 1 min).
Nr-Terminus Acetylation. The resins were treated with Ac2O
(10 equiv) and DIEA (10 equiv) in DCM (2 ꢀ 15 min). Reaction
completion was checked by the Kaiser test.
Cleavage Conditions. A. 2-Chlorotrityl-Based Resins. The resins
were treated with a solution of TFA-DCM (5:95) (5 ꢀ 1 min).
Filtrates were collected, washed with DCM (3 ꢀ 1 min), and
evaporated under vacuum.
B. Rink-Based Resins. The resins were treated with a solution
of TFA-H2O (95:5) and orbitally shaken for 2 h at rt. Filtrates
were collected, washed with TFA (2 ꢀ 1 min) and DCM (3 ꢀ
1 min), and evaporated under vacuum.
Synthesis of Boc-Val-pNO2Phe-Pro-OH (5). Starting from
2-chlorotrityl resin (6.0 g, loading: 1.3 mmol/g), the peptide was
synthesized as described above. Cleavage and lyophilization
in H2O-ACN (2:1) rendered a white solid powder; 1.75 g
(yield: 39%); Mexp 507.1 (Mcalc 506.3), 96%.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1087
Figure 3. Competition curves of dopamine receptors antagonists binding versus increasing concentrations of compound 13. Competition
experiments of 0.9 nM D1R antagonist [3H]-SCH23390 (a) or 0.7 nM D2R antagonist [3H]-YM 09151-2 (b) versus increasing concentrations of
compound 13 were performed with brain striatal membranes (0.5 mg prot/mL) as indicated in the Experimental Section. Data are represented
as means ( SD from a representative experiment (n = 3) performed in triplicate.
Identity of compound 13 was confirmed by 1H NMR
Table 2. KD Values of Selected Biotin Ergopeptides at D1R and D2R
(400 MHz, DMSO-d6): 10.7 (s, 1H), 8.44 (s, 1H), 8.37 (s, 1H),
D1R
KDB1 (nM)
D2R
KDB1 (nM)
8.29 (s, 2H), 8.17 (d, 2H), 7.90 (s,1H), 7.54 (d, 2H), 7.3-6.9 (m,
7H), 7,30 (s,1H), 7.09 (s, 1H), 7.02 (s, 1H), 6.42 and 6.36 (s, 2H),
6.26 (s, 1H), 4.98 (m, 1H), 4.64 (m, 1H), 4.4-3.8 (m, 7H), 3.00
(dd, 1H), 2.78 (m, 1H), 2.59 (m, 2H), 2.51 (s, 3H), 2.3-1.24
(m, 41H).
KDB2 (μM)
KDB2 (μM)
9
107 ( 9
2100 ( 300
56 ( 6
3.2 ( 0.3
>50
260 ( 60
2000 ( 400
17 ( 5
7 ( 2
23 ( 8
9 ( 3
12
13
14
1.5 ( 0.2
>50
490 ( 80
160 ( 50
10 ( 4
Biological Assays. Radioligand Binding Experiments. Gener-
al Procedure. Membrane suspensions from lamb brain striatum
or D3R transiently transfected CHO cells were obtained by
following the method previously described.35 Radioligand bind-
ing assays using membrane suspensions (0.5 mg prot/mL deter-
mined with bicinchoninic acid kits) were carried out at 22 ꢀC in
50 mM Tris-HCl buffer, pH 7.4 (see conditions below used for
each receptor). After radioligand incubation, free and mem-
brane-bound ligand were separated by rapid filtration of 500 μL
aliquots in a cell harvester through Whatman GF/C filters
embedded in 0.3% polyethylenimine (PEI) that were subse-
quently washed for 5 s with 5 mL of ice-cold Tris-HCl buffer.
The filters were incubated with 10 mL of Ecoscint H scintillation
cocktail overnight at room temperature, and radioactivity
counts were determined using a scintillation counter with an
efficiency of 62% for tritium labeled compounds and 99% for
the 125I-labeled compound.
a KDB1 and KDB2 are respectively the equilibrium dissociation con-
stants of the high and low binding affinities of ergopeptides to the
dimeric D1R and D2R.
use. After washing, Alloc-L-Lys(Fmoc)-OH (3 equiv) was
coupled to the resin, using DIPCI (3 equiv) and HOBt (3 equiv)
as a coupling system in DMF. The resin was washed and the
Fmoc group removed, yielding Alloc-L-Lys(NH2)-AM-MBHA.
At this point, the resin was split into two equal aliquots. Ac-
Lys(Fmoc)-OH (3 equiv) and the Fmoc-Ahx-OH (3 equiv) were
respectively coupled on each one as previously described to
render the resins 7 and 8. After that, Alloc group was eliminated
in both resins and biotin (3 equiv) was introduced using TBTU
(3 equiv), DIEA (6 equiv), and HOBt (3 equiv) in DMF for 1 h at
rt. After the Fmoc group removal, the resins were again divided
in two parts, obtaining at that point four different resins. From
this point onward, the attachment of the three different amino
acids (corresponding to the tripeptides of 1 and 2) was per-
formed according to the procedure described previously. The
biotin peptide moieties were cleaved following the B cleavage
conditions, and the coupling of D-lysergic acid was carried out as
described above. Evaporated crude cleavages for the different
biotin peptide moieties (95-127 mg, 0.10-0.13 mmol) were
dissolved in individual vials using 0.5 mL of DMF, treated with
DIEA (1 equiv), and stirred for 5 min at rt. Second, D-lysergic
acid (1.2 equiv) and HOAt (1.5 equiv) were dissolved in 1 mL of
DMF (each reaction) and added to every vial. Finally, DIPCDI
was added (2 equiv) and the set of reactions were stirred for 16 h
at rt. Evaporation rendered the crude mixtures further purified
by semipreparative RP-HPLC-MS under basic conditions
(A: 20 mM NH4COOCH3, pH 9; B: ACN) using a reverse-
phase X-Bridge C18 column (5 μm, 19 mm ꢀ 100 mm2) to yield
the biotin ergopeptides 9-16.
Screening of the Library. Binding experiments of the whole
library were performed at a concentration of 25 μM for compounds
1, 2, 1a-1s, 2a-2s and 9-16.
Dopamine D1, D2, and D3, Histamine H3, and Metabotropic
Glutamate 1/5 Receptors. Membranes were incubated with
0.9 nM [3H]-SCH23390 (85 Ci/mmol), 0.7 nM [3H]-YM09151-
2 (85.5 Ci/mmol), 4 nM [3H]-raclopride (82.8 Ci/mmol), 2.5 nM
[3H]-RAMH (34 Ci/mmol), or 27 nM [3H]-quisqualic acid
(30.9 Ci/mmol) in 50 mM Tris-HCl buffer (pH 7.4) containing
10 mM MgCl2 for 2 h in the absence or in the presence of tested
compounds. Nonspecific binding was measured in the presence
of 10 μM SCH23390, raclopride, RAMH, or quisqualic acid,
respectively.
Somatostatin Receptors. Membranes were incubated with
0.1 nM [125I]-Tyr11-somatostatin 14 (2,200 Ci/mmol) in 50 mM
Tris-HCl buffer (pH 7.4) containing 10 mM MgCl2 for 3 h in the
absence or in the presence of tested compound. Nonspecific
binding was measured in the presence of 100 nM somatostatin.
Adenosine A1 and A2A Receptors. Membranes were incubated
with 1.0 nM [3H]-R-PIA (30.5 Ci/mmol) or 1.6 nM [3H]-
ZM241385 (27.4 Ci/mmol) in 50 mM Tris-HCl buffer (pH 7.4)
containing 10 mM MgCl2 and 0.2 U/mL ADA for 2 h in the
absence or in the presence of tested compounds. Nonspecific
binding was measured in the presence of 10 μM DPCPX or ZM
241385, respectively.
Biotin Ergopeptides Characterization (9-16). Biotin ergopep-
tides 9-16 were characterized by analytical RP-HPLC-MS and
RP-HPLC using a reverse-phase XBridge C18 column (3.5 μm,
4.6 mm ꢀ 50 mm2) at 2 mL/min (two separate elution solvent
systems: (a) A, H2O-HCOOH (99.9:0.1); B, ACN-HCOOH
(99.93:0.07). (b) A, H2O-TFA (99.9:0.1); B, ACN-TFA
(99.9:0.1). The purities of 9-16 were determined by RP-HPLC
at 220 nm UV absorption using as elution system b and a
gradient 5% B to 50% B in 4.5 min. All purities were confirmed
to be g95%. HRMS spectra for 9-16 were recorded confirming
the identity of each biotin ergopeptides (Table 3).
Cannabinoid CB1 Receptor. Membranes were incubated in
siliconated tubes with 0.5 nM [3H]-CP 55940 (144 Ci/mmol) in

1088 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Vendrell et al.
Figure 4. Functional characterization of the biotin ergopeptide 13. CHO cells expressing D1R (a and c) or D2R (b and d) were cultured
in serum-free medium for 16 h prior to the addition of any ligand. Cells were treated (or not) with 50 μM of the D1R antagonist SCH23390
(a and c) or the D2R antagonist raclopride (b and d). After 5 min, increasing concentrations of compound 13, D1R agonist SKF 81297, or the
D2R agonist quinpirole were added for further 5 min of incubation, and ERK1/2 phosphorylation (a and b) or P-Ser473Akt (c and d) were
determined as indicated in the Experimental Section. Results are expressed as means ( SEM of four independent experiments. Student’s t-test
for unpaired samples showed significant increases (a and b) or decreases (c and d) over basal (not treated cells, *p < 0.05, **p < 0.01) or
significant decreases (a and b) or increases (c and d) respect to cells stimulated with the same concentration of compound 13 in the absence of
antagonist (#p < 0.05, ##p < 0.01).
13, or 14 were performed by incubating membranes under the
same conditions as described above for D1R, D2R, or D3R
binding. Nonspecific binding was determined as previously
outlined. Radioligand displacement curves were analyzed by
nonlinear regression using the commercial program GRAFIT
(Erithacus Software, Surrey, UK) by fitting the specific binding
data to the mechanistic two-state dimer receptor model.35,36 To
calculate the macroscopic equilibrium dissociation constants,
the equations used for a competition binding experiment were
ꢀ 35
deduced by Casado. Goodness-of-fit was tested following the
reduced χ2 value given by the nonlinear regression program
GRAFIT. A modified F test was used to analyze whether the fit
to cooperativity model significantly improved upon the fit to
noncooperative model, and p < 0.05 was taken as a criterion of
Figure 5. Competition curve of a D3R antagonist binding versus
increasing concentrations of compound 13. Competition experi-
ments of 4 nM D3R antagonist [3H]-raclopride versus increasing
concentrations of compound 13 were performed with membranes
(0.5 mg prot/mL) from CHO cells transiently transfected with 2 μg
of the cDNA corresponding to D3R, as indicated in the Experi-
mental Section. Data are represented as means ( SD from a
representative experiment (n = 3) performed in triplicate.
significance; when no significant improvement over the non-
cooperative model was detected, the p values were >0.30.
CellCulture, TransientTransfection, andProtein Determination.
Chinese hamster ovary (CHO) cells were cultured in MEM
R medium without nucleosides supplemented with 100 U/mL
penicillin/streptomycin and 10% (v/v) heat inactivated fetal
bovine serum (FBS). CHO cells were maintained at 37 ꢀC in a
humidified atmosphere of 5% CO2 and were passaged when
they were 80-90% confluent, i.e., approximately twice a week.
CHO cells were transiently transfected with 2 μg cDNA corre-
sponding to human D1R, D2R, or D3R by the ramified PEI
method. Cells were incubated for 4 h with the corresponding
cDNA together with ramified PEI (5 mL/mg cDNA of 10 mM
PEI) and 150 mM NaCl in a serum-starved medium. After 4 h,
the medium was changed to a fresh complete culture medium.
Forty-eight h after transfection, cells were washed twice in quick
succession in HBSS (Hanks’ balanced salt solution: 137 mM
50 mM Tris-HCl buffer (pH 7.4) containing 10 mM MgCl2 and 1
mg/mL fatty acid-free bovine serum albumin (BSA) for 2 h in
the absence or in the presence of tested compounds. In this case,
filters were presoaked with buffer containing BSA, and BSA was
maintained in the washing medium. Nonspecific binding was
measured in the presence of 10 μM CP55940.
K
D Determination. Competition experiments of 0.9 nM [3H]-
SCH23390, 0.7 nM [3H]-YM09151-2, or 4 nM [3H]-raclopride
binding versus increasing concentrations of compounds 9, 12,

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4 1089
Table 3
compd
t
R (min)
molecular formula
Mcalc
Mexp (HRMS)
purity (220 nm) (%)
mg
9
3.33
3.68
3.20
3.58
3.88
3.85
3.81
3.77
C57H78N12O10
C57H78N12O10
S
S
S
S
1122.5685
1122.5685
1179.5899
1179.5899
1202.5747
1202.5747
1259.5961
1259.5961
1123.5747
1123.5747
1180.5961
1180.5963
1203.5815
1203.5804
1260.6015
1260.6016
95
99
99
98
99
94
95
96
12.6
14.3
14.5
13.6
14.7
15.5
12.9
25.1
10
11
12
13
14
15
16
C59H81N13O11
C59H81N13O11
C
62H79FN12O10
C62H79FN12O10
64H82FN13O11
C64H82FN13O11
S
S
S
S
C
NaCl, 5 mM KCl, 0.34 mM Na2HPO4 12H2O, 0.44 mM
KH2PO4, 1.26 mM CaCl2 2H2O, 0.4 mM MgSO4 7H2O,
characterization data for the ergopeptide library, radioligand
binding assays of 1c, 1f, 2c, and 2f (A1R and A2AR) and 9-16
(D1R, D2R, A1R, and A2AR), competition curves of 9, 12, and
14 at D1R and D2R and binding experiments of 13 at different
GPCRs. This material is available free of charge via the Internet
at http://pubs.acs.org.
3
3
3
0.5 mM MgCl2, 10 mM HEPES, pH 7.4) supplemented with
0.1% glucose (w/v), detached by gently pipetting and resus-
pended in the same buffer. To control the cell number, sample
protein concentration was determined using a Bradford assay
kit using BSA dilutions as standards.
ERK and Akt Phosphorylation Assay. Transfected CHO cells
were cultured in serum-free medium for 16 h before the addition
of any agent. Cells were treated or not with 5 or 50 μM of the
D1R antagonist SCH 23390 or the D2R antagonist raclopride.
After 5 min, increasing concentrations of compound 13, 1 μM
D1R agonist SKF 81297, or 1 μM D2R agonist quinpirole were
added for further 5 min incubation. Cells were rinsed with ice-
cold phosphate-buffered saline and lysed by the addition of
500 μL of ice-cold lysis buffer (50 mM Tris-HCl pH 7.4, 50 mM
NaF, 150 mM NaCl, 45 mM β-glycerophosphate, 1% Triton
X-100, 20 μM phenyl-arsine oxide, 0.4 mM NaVO4, and pro-
tease inhibitor cocktail). The cellular debris was removed by
centrifugation at 13000g for 5 min at 4 ꢀC, and the protein was
quantified by the bicinchoninic acid method using bovine serum
albumin dilutions as standard. To determine the level of ERK1/
2 or Akt-phosphorylation, equivalent amounts of protein (10 μg)
were separated by electrophoresis on a denaturing 7.5% SDS-
polyacrylamide gel and transferred onto PVDF-FL membranes.
Odyssey blocking buffer was then added, and the membrane was
rocked for 90 min. The membranes were then probed with a
mixture of antiphospho-ERK1/2 antibody (1:2500) or anti-P-
Ser473Akt antibody (1:2500) and anti-ERK1/2 antibody that
recognizes both phosphorylated and nonphosphorylated ERK1/2
(1:40000) for 2-3 h. Bands were visualized by the addition of a
mixture of IRDye 800 (antimouse) antibody (1:10000) and
IRDye 680 (antirabbit) antibody (1:10000) for 1 h and scanned
by the Odyssey infrared scanner. Bands densities were quanti-
fied using the scanner software, exported to Excel. The level of
phosphorylated ERK1/2 isoforms or P-Ser473Akt was normal-
ized for differences in loading using the total ERK protein band
intensities.
References
ꢀ
(1) Vendrell, M.; Angulo, E.; Casado, V.; Lluis, C.; Franco, R.;
Albericio, F.; Royo, M. Novel ergopeptides as dual ligands for
adenosine and dopamine receptors. J. Med. Chem. 2007, 50, 3062–
3069.
(2) Maggio, R.; Aloisi, G.; Silvano, E.; Rossi, M.; Millan, M. J.
Heterodimerization of dopamine receptors: new insights into
functional and therapeutic significance. Parkinsonism Relat. Dis-
ord. 2009, 15 (S4), S2–S7.
(3) So, C. H.; Verma, V.; O’Dowd, B. F.; George, S. R. Desensitization
of the dopamine D1 and D2 receptor hetero-oligomer mediated
calcium signal by agonist occupancy of either receptor. Mol.
Pharmacol. 2007, 72, 450–462.
(4) So, C. H.; Varghese, G.; Curley, K. J.; Kong, M. M.; Alijaniaram,
M.; Ji, X.; Nguyen, T.; O’Dowd, B. F.; George, S. R. D1 and D2
dopamine receptors form heterooligomers and cointernalize after
selective activation of either receptor. Mol. Pharmacol. 2005, 68,
568–578.
(5) Bhushan, R. G.; Sharma, S. K.; Xie, Z.; Daniels, D. J.; Portoghese,
P. S. A bivalent ligand (KDN-21) reveals spinal delta and kappa
opioid receptors are organized as heterodimers that give rise to
delta(1) and kappa(2) phenotypes. Selective targeting of del-
ta-kappa heterodimers. J. Med. Chem. 2004, 47, 2969–2972.
(6) Waldhoer, M.; Fong, J.; Jones, R. M.; Lunzer, M. M.; Sharma,
S. K.; Kostenis, E.; Portoghese, P. S.; Whistler, J. L. A hetero-
dimer-selective agonist shows in vivo relevance of G protein-
coupled receptor dimers. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
9050–9055.
(7) Adam, G. C.; Sorensen, E. J.; Cravatt, B. F. Proteomic profiling of
mechanistically distinct enzyme classes using a common chemotype.
Nature Biotechnol. 2002, 20, 805–809.
(8) Speers, A. E.; Adam, G. C.; Cravatt, B. F. Activity-based protein
profiling in vivo using a copper(I)-catalyzed azide-alkyne [3 þ 2]
cycloaddition. J. Am. Chem. Soc. 2003, 125, 4686–4687.
(9) Walsh, D. P.; Chang, Y. T. Chemical genetics. Chem. Rev. 2006,
106, 2476–2530.
(10) Middleton, R. J.; Briddon, S. J.; Cordeaux, Y.; Yates, A. S.; Dale,
C. L.; George, M. W.; Baker, J. G.; Hill, S. J.; Kellam, B. New
fluorescent adenosine A1-receptor agonists that allow quantifica-
tion of ligand-receptor interactions in microdomains of single
living cells. J. Med. Chem. 2007, 50, 782–793.
(11) Lokey, R. S. Forward chemical genetics: progress and obstacles on
the path to a new pharmacopoeia. Curr. Opin. Chem. Biol. 2003, 7,
91–96.
Acknowledgment. We acknowledge the technical help ob-
ꢀ
tained from Jasmina Jimenez (Molecular Neurobiology La-
boratory, University of Barcelona), from Serveis Cientıfic-
Tecnics of University of Barcelona for their support in the
HRMS, and Marıa Macıas (Institute for Research in
´
ꢁ
´
´
Biomedicine) for their support in NMR analysis. This work
was partially supported by grants from Spanish Ministerio de
(12) Middleton, R. J.; Kellam, B. Fluorophore-tagged GPCR ligands.
Curr. Opin. Chem. Biol. 2005, 9, 517–525.
ꢀ
Ciencia e Innovacion MICINN (SAF2008-00146, SAF2008-
(13) Gellerman, G.; Elgavi, A.; Salitra, A.; Kramer, M. Novel Gly
building units for backbone cyclization: synthesis and incorpora-
tion into model peptides. J. Pept. Res. 2001, 57, 277–291.
(14) Vendrell, M.; Ventura, R.; Ewenson, A.; Royo, M.; Albericio, F.
N-[Chloro(dimethylamino)methylene]-N-methylmethanaminium
chloride (TMUCl Cl), the reagent of choice for the solid-phase
synthesis of anilides. Tetrahedron Lett. 2005, 46, 5383–5386.
(15) Baraldi, P. G.; Tabrizi, M. A.; Gessi, S.; Borea, P. A. Adenosine
receptor antagonists: translating medicinal chemistry and pharma-
cology into clinical utility. Chem. Rev. 2008, 108, 238–263.
03229-E, SAF2009-07276, BQU2006-03794, CTQ2005-00315/
BQU, CTQ2008-00177, SAF2005-00170, and SAF2006-05481),
ꢀ
ꢀ
grant 060110 from Fundacio LaMarato deTV3, Generalitat de
Catalunya, CIBER-BBN, Networking Centre on Bioengi-
neering, Biomaterials, and Nanomedicine Institute for Re-
search in Biomedicine, and the Barcelona Science Park. P.J.
M. is a Ramon y Cajal Fellow.
ꢀ
(16) Agnati, L. F.; Ferre, S.; Lluis, C.; Franco, R.; Fuxe, K. Molecular
Supporting Information Available: Additional synthetic
schemes, tables with chemical structures, and complete
mechanisms and therapeutical implications of intramembrane
receptor/receptor interactions among heptahelical receptors with

1090 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 4
Vendrell et al.
examples from the striatopallidal GABA neurons. Pharmacol. Rev.
2003, 55, 509–550.
(17) Franco, R.; Casado, V.; Mallol, J.; Ferre, S.; Fuxe, K.; Cortes, A.;
Ciruela, F.; Lluis, C.; Canela, E. I. Dimer-based model for heptas-
panning membrane receptors. Trends Biochem. Sci. 2005, 300, 360–
366.
(18) Bayer, E. A.; Wilchek, M. Application of avidin-biotin techno-
logy to affinity-based separations. J. Chromatogr. 1990, 510, 3–11.
(19) Elia, G. Biotinylation reagents for the study of cell surface proteins.
Proteomics 2008, 8, 4012–4024.
(20) Bratthauer, G. L. The avidin-biotin complex (ABC) and other
avidin-biotin binding methods. Methods Mol. Biol. 1999, 115, 203–
214.
dopamine D1 and histamine H3 receptors. Br. J. Pharmacol. 2009,
157, 64–75.
(28) Ferrada, C.; Ferre, S.; Casado, V.; Cortes, A.; Justinova, Z.;
Barnes, C.; Canela, E. I.; Goldberg, S. R.; Leurs, R.; Lluis, C.;
Franco, R. Interactions between histamine H3 and dopamine D2
receptors and the implications for striatal function. Neuropharma-
cology 2008, 55, 190–197.
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(29) Cabello, N.; Gandıa, J.; Bertarelli, D. C.; Watanabe, M.; Lluıs, C.;
´ ´
ꢀ
ꢀ
Franco, R.; Ferre, S.; Lujan, R.; Ciruela, F. Metabotropic gluta-
mate type 5, dopamine D2 and adenosine A2A receptors form
higher-order oligomers in living cells. J. Neurochem. 2009, 109,
1497–1507.
(30) Kearn, C. S.; Blake-Palmer, K.; Daniel, E.; Mackie, K.; Glass, M.
Concurrent stimulation of cannabinoid CB1 and dopamine D2
receptors enhances heterodimer formation: a mechanism for receptor
cross-talk? Mol. Pharmacol. 2005, 67, 1697–1704.
(21) Smith, D. J.; Shappell, N. W. Technical note: epimerization of
ergopeptine alkaloids in organic and aqueous solvents. J. Anim.
Sci. 2002, 80, 1616–1622.
(22) Chausmer, A. L.; Katz, J. L. Comparison of interactions of D1-like
agonists, SKF 81297, SKF 82958, and A-77636, with cocaine:
locomotor activity and drug discrimination studies in rodents.
Psychopharmacology 2002, 159, 145–153.
(23) Levant, B.; Grigoriadis, D. E.; De Souza, E. B. Characterization of
[3H]-quinpirole binding to D2-like dopamine receptors in rat brain.
J. Pharmacol. Exp. Ther. 1992, 262, 929–935.
(24) Beaulieu, J.; Gainetdinov, R. R.; Caron, M. G. The Akt/GSK-3
signaling cascade in the actions of dopamine. Trends Pharmacol.
Sci. 2007, 28, 166–172.
(25) Beaulieu, J.; Gainetdinov, R. R.; Caron, M. G. Akt/GSK3 signal-
ing in the action of psychotropic drugs. Annu. Rev. Pharmacol.
Toxicol. 2009, 49, 327–347.
(31) Rocheville, M.; Lange, D. C.; Kumar, U.; Patel, S. C.; Patel, R. C.;
Patel, Y. C. Receptors for dopamine and somatostatin: formation
of hetero-oligomers with enhanced functional activity. Science
2000, 288, 154–157.
(32) Vazquez, J.; Qushair, G.; Albericio, F. Qualitative colorimetric
tests for solid phase synthesis. Methods Enzymol. 2003, 369, 21–35.
(33) Strohmeier, G. A.; Haas, W.; Kappe, C. O. Synthesis of functionalized
1,3-thiazine libraries combining solid-phase synthesis and post-clea-
vage modification methods. Chem.;Eur. J. 2004, 10, 2919–2926.
(34) Attardi, M. E.; Porcu, G.; Taddei, M. Malachite green, a valuable
reagent to monitor the presence of free COOH on the solid-phase.
Tetrahedron. Lett. 2000, 41, 7391–7394.
ꢀ
(35) Casado, V.; Ferrada, C.; Bonaventura, J.; Gracia, E.; Mallol, J.;
ꢀ
ꢀ
ꢀ
ꢀ
Canela, E. I.; Lluıs, C.; Cortes, A.; Franco, R. Useful pharmaco-
´
(26) Marcellino, D.; Ferre, S.; Casado, V.; Cortes, A.; Le Foll, B.;
Mazzola, C.; Drago, F.; Saur, O.; Stark, H.; Soriano, A.; Barnes,
C.; Goldberg, S. R.; Lluis, C.; Fuxe, K; Franco, R. Identification of
dopamine D1-D3 receptor heteromers. Indications for a role of
synergistic D1-D3 receptor interactions in the striatum. J. Biol.
Chem. 2008, 283, 26016–26025.
logical parameters for G-protein-coupled receptor homodimers
obtained from competition experiments. Agonist-antagonist
binding modulation. Biochem. Pharmacol. 2009, 78, 1456–1463.
ꢀ
ꢀ
ꢀ
(36) Casado, V.; Cortes, A.; Ciruela, F.; Mallol, J.; Ferre, S.; Lluıs, C.;
´
Canela, E. I.; Franco, R. Old and new ways to calculate the affinity
of agonists and antagonists interacting with G-protein-coupled
monomeric and dimeric receptors: the receptor-dimer cooperativity
index. Pharmacol. Ther. 2007, 116, 343–354.
ꢀ
ꢀ
(27) Ferrada, C.; Moreno, E.; Casado, V.; Bongers, G.; Cortes, A.;
ꢀ
Mallol, J.; Canela, E. I.; Leurs, R.; Ferre, S.; Lluı
Marked changes in signal transduction upon heteromerization of
´
s, C.; Franco, R.