Loratadine and analogues: Discovery and preliminary structure-activity relationship of inhibitors of the amino acid transporter B0AT2

Article abstract of DOI:10.1021/jm501086v

B⁰AT2, encoded by the SLC6A15 gene, is a transporter for neutral amino acids that has recently been implicated in mood and metabolic disorders. It is predominantly expressed in the brain, but little is otherwise known about its function. To identify inhibitors for this transporter, we screened a library of 3133 different bioactive compounds. Loratadine, a clinically used histamine H₁ receptor antagonist, was identified as a selective inhibitor of B⁰AT2 with an IC₅₀ of 4 μM while being less active or inactive against several other members of the SLC6 family. Reversible inhibition of B⁰AT2 was confirmed by electrophysiology. A series of loratadine analogues were synthesized to gain insight into the structure-activity relationships. Our studies provide the first chemical tool for B⁰AT2.

Full text of DOI:10.1021/jm501086v

Article
pubs.acs.org/jmc
Loratadine and Analogues: Discovery and Preliminary Structure−
Activity Relationship of Inhibitors of the Amino Acid Transporter
B0AT2
Serena Cuboni,†,# Christian Devigny,†,∥,# Bastiaan Hoogeland,†,⊥ Andrea Strasser,‡ Sebastian Pomplun,†
Barbara Hauger,† Georg Hofner,§ Klaus T. Wanner,§ Matthias Eder,† Armin Buschauer,‡
̈
Florian Holsboer,† and Felix Hausch*,†
†Max Planck Institute of Psychiatry, 80804 Munich, Germany
‡Institute of Pharmacy, University of Regensburg, 93053 Regensburg, Germany
§Department of Pharmacy, Ludwig-Maximilians-University Munchen, 80539 Munich, Germany
̈
S
* Supporting Information
ABSTRACT: B0AT2, encoded by the SLC6A15 gene, is a
transporter for neutral amino acids that has recently been
implicated in mood and metabolic disorders. It is predom-
inantly expressed in the brain, but little is otherwise known
about its function. To identify inhibitors for this transporter,
we screened a library of 3133 different bioactive compounds.
Loratadine, a clinically used histamine H1 receptor antagonist, was identified as a selective inhibitor of B0AT2 with an IC50 of 4
μM while being less active or inactive against several other members of the SLC6 family. Reversible inhibition of B0AT2 was
confirmed by electrophysiology. A series of loratadine analogues were synthesized to gain insight into the structure−activity
relationships. Our studies provide the first chemical tool for B0AT2.
approved second-generation antihistaminic drug, has substan-
tial activity at B0AT2.
INTRODUCTION
■
Transporters of the solute carrier family 6 (SLC6) play an
essential role in removing neurotransmitters from the synaptic
cleft as well as in amino acid homeostasis.1−3 In this family, the
transporter B0AT2 (also known as v7-3, NTT73, or SBAT1)
has a preference for neutral amino acids such as proline,
leucine, and methionine.4,5 It is highly expressed in regions of
the brain that are important for mood, behavior, appetite
regulation, and energy homeostasis.6−9 Recently, a single-
nucleotide polymorphism linked to SLC6A15, the gene
encoding B0AT2, has been associated with depression.10 The
same polymorphism was also associated with changes in gray
matter volume in the median cingulate gyrus.11 Patients
carrying the risk genotype in SLC6A15 also had an increased
activation of the hypothalamus−pituitary−adrenal axis (HPA)
upon stress.12 Moreover, another recent genetic study identified
two rare nonsynonymous variants of SLC6A15, which were
associated with an increase in the proline uptake compared to
that of the wild-type protein.13 In addition to its role in stress-
related disorders, B0AT2 has also been implicated in the
regulation of food intake and weight gain after dietary leucine
supplementation.7,14
RESULTS
■
To explore the amenability of B0AT2 for drug discovery, we
first developed an assay suitable for medium throughput.
Toward this end, an HEK293 cell line stably expressing eGFP-
tagged B0AT2 was generated, and the [3H]proline uptake assay
was adapted to a 384-well format.
To assess the druggability of B0AT2, we next engaged a pilot
screening of seven small libraries containing commercially
available bioactive compounds, two of which were focused on
neuroactive substances. Surprisingly, in three sublibraries,
loratadine, a histamine H1 receptor (H1R) antagonist clinically
used to treat allergies,15,16 was identified as the predominant hit
(Figure 1A). In concentration-dependent assays, loratadine
inhibited [3H]proline uptake by B0AT2 with an IC50 value of 4
μM (Figure 1B). Interestingly, loratadine showed a clear
preference for B0AT2 within the SLC6 family. It was at least 3−
10 times weaker at transporters for GABA (human GAT1,
mouse GAT2−4). Loratadine was inactive at the transporters
for the monoamine neurotransmitters serotonin (SERT) and
dopamine (DAT) and at its closest homologue NTT4/
SLC6A17 (Figure 1C).
So far, the only ligands described for B0AT2 are amino acid
analogues with low affinity and unclear selectivity.4,5 As a first
step to allow pharmacological studies of the function of this
protein, we performed a pilot screening of a small library of
known bioactive compounds. This revealed that loratadine, an
Received: July 18, 2014
© XXXX American Chemical Society
A
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Figure 1. Loratadine is a specific inhibitor of B0AT2. (A) Chemical structure of loratadine. (B) Inhibition of [3H]proline uptake in B0AT2-
overexpressing HEK293 cells by loratadine. Individual data points represent the average of duplicates. (C) Selectivity profile of loratadine against
other transporters of the SLC6A family.
Figure 2. Loratadine reversibly inhibits electrogenic B0AT2 transport. (A, B) Proline (10 mM), but not loratadine (100 μM), induces an inward
current in B0AT2-overexpressing HEK293 cells. (C) Co-application of loratadine to the same cell inhibits the proline-induced inward current. (D)
After washout of loratadine, B0AT2 regains the ability for proline transport.
Table 1. Inhibition of B0AT2 ([3H]Proline Uptake) by Desloratadine Analogues
a
compd
R1
R2
R3
proline uptake (IC50, μM)
loratadine
H
H
4.0 0.4
>80
4-cyanoloratadine
H
H
H
H
H
CN
4-(hydroxymethyl)loratadine
3-hydroxyloratadine
H
CH2OH
18.3 3.6
17.6 1.9
4.5 1.1
>80
OH
OMe
H
H
H
H
H
H
3-methoxyloratadine
2-cyanoloratadine
CN
2-(hydroxymethyl)loratadine
2-(methoxycarbonyl)loratadine
isoloratadine
CH2OH
COOMe
H
>80
H
12.4 1.8
14.8 1.6
a
B0AT2 activity was measured by uptake of 80 nM [3H]proline by eGFP-B0AT2-expressing HEK293 cells within 10 min in the presence of 0.01−80
μM loratadine analogues.
The inhibition of B0AT2 by loratadine was independently
confirmed in patch-clamp experiments (n = 5, Figure 2).
Loratadine inhibited proline-induced sodium currents in
B0AT2-expressing HEK293 cells, but did not induce a current
in the absence of proline, indicating that it was not a B0AT2
substrate itself. Moreover, B0AT2 inhibition by loratadine was
reversible since the capacity of B0AT2-expressing HEK293 cells
for proline transport was fully restored after washout of
loratadine.
To explore the structure−activity relationships (SARs) for
B0AT2 inhibition, we first tested commercially available
analogues of loratadine (Table 1). Substituents in the 2- and
4-positions of the pyridine ring (A ring) of loratadine generally
reduced activity, although a hydroxymethyl group in the 4-
position and a methyl carboxylate in the 2-position were
tolerated. The 3-position of the pyridine ring was more tolerant
to substitutions, and the 3-methoxy derivative was equipotent
compared to loratadine. The activity of isoloratadine suggested
that the exocyclic double bond might be less important.
We next investigated various substituents at the piperidine
nitrogen (D ring, Table 2). These compounds were readily
accessible by derivatizing desloratadine (Figure 3). The
carbamate in loratadine was not essential, since the
corresponding amide 2 and thiocarbamate 3 and to some
extent the corresponding urea 4 retained activity. However, the
carbonyl oxygen was critical, since the corresponding
sulfonamide 5 as well as analogues with a N-unsubstituted or
N-alkylated piperidine were inactive. The ethyl group in
B
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Table 2. Inhibition of B0AT2 ([3H]Proline Uptake) by
Desloratadine Analogues Bearing Different Substituents at
the Piperidine Moiety
Table 3. Inhibition of B0AT2 ([3H]Proline Uptake) by
Desloratadine-N-carboxylic Esters
a
B0AT2 activity was measured by uptake of 80 nM [3H]proline by
eGFP-B0AT2-expressing HEK293 cells within 10 min in the presence
of 0.01−80 μM loratadine analogues.
a
B0AT2 activity was measured by uptake of 80 nM [3H]proline by
eGFP-B0AT2-expressing HEK293 cells within 10 min in the presence
of 0.01−80 μM loratadine analogues. Measurements at higher
concentrations were precluded by cellular toxicity.
Table 4. Inhibition of B0AT2 ([3H]Proline Uptake) by N-
Acylated Analogues of Desloratadine
b
a
B0AT2 activity was measured by uptake of 80 nM [3H]proline by
eGFP-B0AT2-expressing HEK293 cells within 10 min in the presence
of 0.01−80 μM loratadine analogues.
Figure 3. Synthesis of desloratadine analogues. Reagents and
conditions: (a) (RCO)2O, DIPEA, CH2Cl2; (b) RCOCl, DIPEA,
CH2Cl2; (c) RSO2Cl, DIPEA, CH2Cl2; (d) RNCO, DIPEA, CH2Cl2;
(e) MeI, NaH, THF, 0 °C to rt; (f) (1) Boc-amino acid, HATU,
DIPEA, CH2Cl2; (2) 20% TFA.
with very bulky groups such as Boc or Cbz were inactive.
Finally, we introduced polar substituents at the piperidine
moiety (Table 5). The methyl ether 24 as well as the amino
acid derivatives 25−28 retained activity for B0AT2.
loratadine contributed to inhibition of B0AT2 since the smaller
methyl analogue 7 was less potent (Table 3; compare also 2
with 15 and 16 in Table 4). Longer alkyl chains were tolerated,
as were unsaturated moieties and α, β, or γ branches, especially
in the amide series (Table 4). However, carbamate analogues
To support a future structural optimization of B0AT2
inhibitors, we aimed to identify differences in the structure−
activity relationship profile of loratadine analogues toward
B0AT2 and the original target, the H1 receptor. The first
indication for different binding requirements of these two
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Table 5. Inhibition of B0AT2 ([3H]Proline Uptake) by
Desloratadine Derivatives Bearing Polar Substituents at the
Piperidine Moiety
target for mood or metabolic disorders. The purpose of this
work was to assess the chemical tractability of B0AT2. The
identification of loratadine from a chemical library of 3133
chemically distinct compounds corresponds to a confirmed hit
rate of 0.03%. This result shows that druglike ligands for B0AT2
can be easily found, which is in line with the high druggability
of other closely related SLC6A family members such as SERT,
NET (norepinephrine transporter), DAT, or GlyT1 (glycine
transporter 1).
Loratadine and its analogues identified here represent the
first well-defined inhibitors for B0AT2. So far, the most potent
inhibitor seems to be BCH (2-aminobicyclo[2.2.1]heptane-2-
carboxylic acid), which inhibited B0AT2-mediated [3H]proline
uptake by 80% at a concentration of 5 mM4 while not being a
substrate.5
As famously stated by Sir James Black, “The most fruitful
basis for the discovery of a new drug is to start with an old
drug.” 20 Loratadine might therefore represent a high-quality
chemical starting point for the development of improved
B0AT2 inhibitors. However, several issues have to be
considered for a further optimization: (i) Loratadine is a
second-generation antihistamine and poorly penetrates into the
brain. To reach the B0AT2 expressed in the brain, the blood−
brain barrier permeability will have to be increased. (ii) The
potency for B0AT2 inhibition has to be increased substantially.
In this regard, the ligand efficiency of 0.28 of loratadine for
B0AT2 is moderate and would have to be improved. (iii)
Loratadine is 10-fold more potent for its original target, the H1
receptor. Inhibition of H1R is associated with undesired side
effects, and therefore, selectivity for B0AT2 has to be achieved.
(iv) Loratadine is rapidly metabolized in vivo to yield
desloratadine, resulting in a substantially reduced inhibition of
B0AT2. Thus, the metabolic stability has to be improved. (v)
The tricyclic scaffold of loratadine is known to promiscuously
bind to many proteins, and this seems to also apply to
loratadine itself (see below). Therefore, selectivity against other
loratadine targets has to be addressed, possibly by deviating
from the tricyclic scaffold during an optimization of B0AT2
inhibitors.
For a future optimization, the substituent at the piperidine
moiety of loratadine appears to be rather variable beyond the
carbonyl group and does not seem to contribute substantially to
the affinity for B0AT2. Structural modification in this position,
however, harbors the potential to improve the metabolic
stability and, in combination with variations of the A ring in
loratadine, to tune the selectivity for B0AT2. The R4 position
and especially the R3 position of the A ring of loratadine seem
to be suited for the introduction of substituents. Taken
together, the initial SAR analysis suggests that the binding
pockets of B0AT2 and of H1R are sufficiently different to allow
the development of B0AT2-selective ligands (Figure 4).
Loratadine analogues have also been described as inhibitors
of 5-lipoxygenase,17 platelet-activating factor (PAF),21,22
farnesyl protein transferase (FPT),23 and K 2p18.1 channels.24
However, the SAR for these biological targets clearly diverges
from the SAR for B0AT2. For example, the prototypic PAF
antagonist 15 (Sch-37370) and the dual PAF/H1R antagonist
rupatadine are much more effective for inhibition of PAF-
induced platelet aggregation compared to loratadine,21,22 while
they are much less active at B0AT2. For FPT an order of
affinities of 23 < 22 < 16 ≈ 2 < 15 was reported,23 whereas for
B0AT2 the order was 2 ≈ 22 < 23 < 16 < 15. Thus, also for
PAF and for FPT, a dissociation of activities vs B0AT2 seems
a
B0AT2 activity was measured by uptake of 80 nM [3H]proline by
eGFP-B0AT2-expressing HEK293 cells within 10 min in the presence
of 0.01−80 μM loratadine analogues.
proteins was provided by desloratadine. This active metabolite
of loratadine binds with approximately 100-fold higher affinity
than the parent compound to the H1R,17,18 but is only a very
weak inhibitor of B0AT2 (Table 2). To examine whether the
activity ratio can be shifted in favor of B0AT2, we determined
the affinity of selected B0AT2 inhibitors for the human H1R
(Table 6). Modifications at the 3-position and especially at the
Table 6. Ki Values of Selected B0AT2 Antagonists at hH1R,
Determined in the [3H]Mepyramine Competition Binding
Assay
a
compound
Ki
SEM (nM)
loratadine
311 46
2
171 12
1274 182
153 19
10
18
19
20
22
28
289
147
8
8
441 44
4647 619
1955 186
>10000
3-methoxyloratadine
2-(methoxycarbonyl)loratadine
a
Determined on membrane preparations of Sf9 cells expressing the
human H1R. Data represent mean values SEM, determined in three
independent experiments performed in triplicate.
2-position significantly reduced the binding to H1R compared
to that of loratadine (loratadine vs 3-methoxyloratadine or 2-
(methoxycarbonyl)loratadine, p < 0.05), while only minimally
affecting B0AT2 inhibition. By analogy with doxepine,19
loratadine is suggested to bind in a pocket between TM III,
TM V, and TM VI. The drop in H1R affinity for 3-
methoxyloratadine and 2-(methoxycarbonyl)loratadine can be
explained by insufficient space in the binding pocket of H1R.
Branched substituents at the piperidine ring (e.g., 10 or 28)
also seem to be tolerated by B0AT2 but much less so by H1R.
DISCUSSION
■
B0AT2, an amino acid transporter of the solute carrier
transporter family, has recently gained attention as a potential
D
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the SPECS Natural Product Library (400 compounds), the NIH
clinical collection (464 compounds, DPI), the NIMH library (271
compounds, RTI), and 103 individually ordered bioactive substances,
comprising 3133 different, nonredundant compounds in total.
Compounds were stored as 1 μL stocks (2.5 mM in DMSO) in
384-well storage plates (Abgene, no. 1056) at −20 °C and were freshly
dissolved immediately before screening. The purity of all tested
compounds was >95% as assessed by analytical HPLC.
Cloning and Cell Culture. The cDNA of the long form of
SLC6A15 (IRAKp961L15168Q; ImaGenes) was inserted into the
pEGFP-C1 vector (Clontech) using the restriction sites BglII and SalI.
The eGFP tag was initially chosen to detect localization of
overexpressed B0AT2 at the plasma membrane and to allow
comparisons of differnet B0AT2 constructs.13 A stable SLC6A15-
overexpressing HEK293 cell line was generated using the selection
marker Geneticin.
HEK293 cells stably expressing the transporters hSERT and hDAT
were kindly provided by Blakely et al.25 HEK293 cell lines stably
expressing hGAT1, mGAT2, mGAT3, and mGAT4 were generated as
described.26 The plasmid NTT4-B0AT2 (where the carboxyl terminus
of NTT4 was replaced by the analogous region of B0AT2) was kindly
provided by the Reimer laboratory.27 This plasmid was used to
generate HEK293 cells stably expressing the NTT4-B0AT2 chimera.
HEK293 cells overexpressing the different transporters were
cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco)
containing 10% fetal calf serum (FCS), 500 units/mL penicillin, and
500 μg/mL streptomycin at 37 °C in a humidified incubator (5%
CO2).
Figure 4. Correlation of B0AT2 inhibition and histamine H1 receptor
binding data of selected loratadine analogues. IC50 values for B0AT2,
determined in a [3H]proline uptake assay, are plotted against Ki values
for binding to hH1R, determined in a [3H]mepyramine competition
assay. Loratadine analogues with reduced H1R binding are highlighted
in green.
Uptake Assays. The [3H]proline uptake assay was performed in
384-well plates (Corning 3846) in a total assay volume of 50 μL. A
total of 10 000 HEK-eGFP-hSLC6A15 cells were plated per well in
plates precoated with poly-D-lysine (PDL) and incubated overnight at
37 °C in a humidified incubator (5% CO2). After 24 h, the medium
was removed with six washing steps (ELx405, Biotek) and replaced by
assay buffer (4.7 mM KCl, 1.2 mM MgSO4, 2.2 mM CaCl2, 0.4 mM
KH2PO4, 10 mM D-(+)-glucose, 10 mM HEPES, 120 mM NaCl, pH
7.4),27 with 20 μL of assay buffer remaining in the well after the last
wash. For screening, 2.5 mM compound stock in DMSO was diluted
to 1:25 in assay buffer. For IC50 determinations, 10 mM compound
stock in DMSO was serially diluted in DMSO, and the different
concentrations were then dissolved in assay buffer. A 10 μL volume of
compound in 4 % DMSO was added (BioMek 2000, Beckman) and
the resulting solution incubated at room temperature (rt) for 10 min.
The final DMSO concentration in the assay was 0.8 %. Afterward, 20
μL of 125 nM [3H]proline (L-[2,3,4,5-3H]proline, NET483,
PerkinElmer, >75 Ci (2.8 TBq)/mmol) was dispensed (Microdrop,
Thermo Scientific), and the cells were incubated for another 10 min at
rt. The uptake reaction was stopped by nine washing steps performed
with assay buffer. After washing, 15 μL of buffer remained in the wells.
The cells were lysed with 1 M NaOH, and the radioactive signal was
amplified with scintillation cocktail (PerkinElmer). The plates were
incubated for 6 h in the dark, and the radioactivity was quantified using
a Wallac Microbeta (PerkinElmer) by counting for 2 min per well. In
the primary screening, compounds that reduced [3H]proline by more
than 30 % were declared a hit.
The uptake assays in HEK-NTT4/B0AT2, HEK-hSERT, HEK-
hDAT, and HEK-hGAT1 cells were performed in 384-well plates
following the protocol for HEK-eGFP-hSLC6A15 using transporter-
specific buffers. For [3H]5HT uptake in HEK-hSERT, the assay buffer
was composed of 5 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 11 mM
D-(+)-glucose, 25 mM HEPES, and 120 mM NaCl, pH 7.4. Before
usage 1 μM pargyline was added.28 For [3H]GABA uptake in HEK-
hGAT1, the assay buffer used was the same as that used for the proline
uptake with 25 mM Tris. For [3H]dopamine uptake in HEK-hDAT,
the assay buffer was composed of 4.7 mM KCl, 1.2 mM MgSO4, 2.2
mM CaCl2, 1.2 mM KH2PO4, 10 mM HEPES, and 120 mM NaCl, pH
7.4.29 A 200 mM concentration of cold proline, 200 μM paroxetine, 40
μM tiagabine, and 50 mM dopamine were used as positive controls in
the SLC6A transporter specificity screens, respectively. GABA uptake
by mGAT2, mGAT3, and mGAT4 was measured as described.26
possible. Within the SLC6 family of transporters, loratadine
discriminates against the monoamine transporters and against
NTT4/SLC6A17, the closest homologue of B0AT2. The latter
is remarkable given the high sequence identity between these
two transporters, especially in the putative active site. An
unexpected finding was the residual inhibitory activity of
loratadine at the GABA transporters, and this should be
addressed during subsequent optimization efforts.
In the absence of direct binding data due to the lack of
radioligands for B0AT2, indirect effects of loratadine analogues
via an upstream regulator of B0AT2 cannot be fully excluded.
However, since inhibition by loratadine was rapid, complete,
and specific for B0AT2 over several closely related SLC6A
transporters, loratadine can be assumed to inhibit B0AT2 by
direct binding. Inhibition of B0AT2 is independent of H1R
inhibition since several structurally distinct antihistaminics such
as mepyramine, chlorpheniramine, ketotifen, and triprolidine
were inactive in the B0AT2 assay.
The discovery of B0AT2 as a new target of loratadine has to
be considered when interpreting biological effects of the latter.
However, in the clinical situation, loradatine and especially the
active metabolite desloratatine have a strong preference for
H1R and only poorly penetrate the brain, where B0AT2 is
expressed. In the clinical situation side effects of loradatine are
therefore unlikely to be caused by B0AT2 inhibition. A better
characterization of B0AT2, ideally with improved B0AT2
inhibitors, will help to address this issue.
EXPERIMENTAL SECTION
■
Chemicals and Chemical Library. Loratadine was from Tocris
(United Kingdom), desloratadine was from AK Scientific (United
States), and commercially available loratadine analogues were obtained
from Toronto Research Chemicals (Canada). All other chemical
reagents were from Aldrich (Germany).
The library of bioactive substances used for screening consisted of
LOPAC (1280 compounds, Sigma-Aldrich), Tocriscreen Total (1120
compounds, Tocris), the Biomol Neurotransmitter Library (694
compounds), the Biomol Natural Product Library (502 compounds),
E
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Histamine H1 Receptor (hH1R) Binding Assay. Preparation of
Compound Stock Solutions. Test compounds (10 mM each) and
loratadine (100 mM) were dissolved in DMSO. The solvent mixture
of the dilution series (≤1 mM) contained 50 % (v/v) DMSO and
50 % (v/v) Millipore water, resulting in a final DMSO concentration
in all assays of 5 % (v/v). Within the [3H]mepyramine competition
binding assays, DMSO concentrations up to 5 % (v/v) were tolerated,
as already shown.30,31 As previously demonstrated for histamine as an
example, DMSO concentrations in the range from 0 % (v/v) to 5 %
(v/v) have no influence on the pKi values.30
Cell Culture and Membrane Preparation. Construction of
baculoviruses was described earlier.30,32 Cell culture, membrane
preparation, and determination of protein concentration were
performed as described previously.30,32
and further stirred overnight. The mixture was concentrated under
reduced pressure and purified by flash chromatography.
Method F: Derivatization of Desloratadine by Using Protected
Amino Acids. Desloratadine (310 mg, 1.0 mmol) was dissolved in
dried CH2Cl2 (15 mL) (solution A). A solution of the corresponding
Boc-protected amino acid (1.1 equiv, 1.1 mmol) and HATU (1.05
equiv, 1.05 mmol) in dried dimethylformamide (5 mL) was activated
by addition of diisopropylethylamine (5 equiv, 5.0 mmol) and stirred
for 2 min (solution B). Solution B was added to solution A, and the
mixture was stirred at rt for 4 h and then concentrated under reduced
pressure. The raw residue was purified by flash chromatography.
Finally, the Boc protecting group was removed by using a solution of
20% TFA in dried CH2Cl2.
[3H]Mepyramine Competition Binding Assays at hH1R. The
competition binding assays at hH1R were performed as described.30,32
In brief, the assays were performed in the presence of 5 nM
[3H]mepyramine. Reaction mixtures, containing binding buffer, ligand
(final concentration in the range from 1 nM to 100 mM), radioligand,
and Sf9 membranes coexpressing hH1R and RGS4, were incubated for
90 min at room temperature and shaken at 250 rpm. Nonspecific
binding was determined in the presence of 10 μM diphenhydramine.30
Filtration through glass microfiber filters (Whatman GF/C), pre-
treated with poly(ethylenimine) (0.3%, w/v), using a Brandel 96
sample harvester separated unbound from membrane-associated
[3H]mepyramine. After three washing steps with binding buffer, for
each well filter pieces were punched and transferred into 96-well
sample plates 1450-401 (PerkinElmer, Rodgau, Germany). Each well
was supplemented with 200 μL of scintillation cocktail (Rotiscint Eco
plus, Roth, Karlsruhe, Germany) and incubated in the dark under
shaking at 200 rpm. Radioactivity was measured with a Micro Beta2
1450 scintillation counter. All ligands were tested in three independent
experiments in triplicate.
ASSOCIATED CONTENT
* Supporting Information
Additional description of the analytical methods, reagents, and
chemical characterization of compounds 1−28. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*E-mail: hausch@mpipsykl.mpg.de. Fax: +49 (89) 30622-610.
■
Present Addresses
∥C.D.: Hoffmann·Eitle, 81925 Munchen, Germany.
̈
⊥D.H.: Hamilton Robotics, 82152 Martinsried, Germany.
Author Contributions
#S.C. and C.D. contributed equally to this work.
Notes
The authors declare no competing financial interest.
Data Analysis. The Ki values were calculated according to Cheng
and Prusoff.33 For calculation of Ki, the KD value of [3H]mepyramine
ACKNOWLEDGMENTS
We thank Ms. C. Dubler (Ludwig-Maximilians-Universitat
Munich) for NMR measurements, Silke Duensing-Kropp for
technical assistance, and Prof. T. Berg (University Leipzig) for
sharing the chemical libraries. This work was funded in part by
the Neuronova gGmbH.
■
(4.49
0.35 nM) was used.32 The Ki values were analyzed by
̈
nonlinear regression and the best fit to sigmoidal dose−response
curves using GraphPad Prism 5.01 (GraphPad Software Inc., San
Diego, CA). All data are the means
SEM of three independent
experiments, performed in triplicate.
Patch-Clamp Experiments. The functionality of the transporter
was also validated by patch-clamp experiments. A total of 50 000 HEK-
eGFP-hSLC6A15 cells were plated in 6 cm plates. The day after, the
cells were washed with extracellular buffer (135 mM NaCl, 3 mM KCl,
1.5 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.2, with NaOH),
and single cells were patched with glass microelectrodes (2−3 MΩ).
Voltage-clamp experiments were performed at a holding potential of
−50 mV. Pipets were filled with intracellular buffer (125 mM
potassium gluconate, 2 mM MgCl2, 1 mM CaCl2, 10 mM NaCl, 10
mM BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic
acid), pH 7.25, with KOH). Proline (10 mM) and loratadine (100
μM) were applied to the cells using a fast superfusion device (PI
Physik Instrumente).
Medicinal Chemistry. Loratadine analogues were prepared by
derivatizing desloratadine by one of the following methods.
Methods A−D: Derivatization of Desloratadine by Using
Anhydrides, Acyl Chlorides, Sulfonyl Chlorides, or Isocyanates.
Desloratadine (310 mg, 1.0 mmol, or 155.4 mg, 0.5 mmol) was
dissolved in anhydrous CH2Cl2 (20 or 10 mL). Diisopropylethylamine
(5 equiv) was added, and the mixture was stirred for 5 min. The
corresponding anhydride, acyl chloride, sulfonyl chloride, or isocyanate
(1.1 equiv) was added, and the mixture was stirred for 2 h, then
concentrated under reduced pressure, and finally purified by flash
chromatography.
ABBREVIATIONS USED
■
DAT, dopamine transporter; NET, norepinephrine transporter;
SERT, serotonin transporter; GAT, GABA transporter; GlyT,
glycine transporter.
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