Short communication
Partial agonist efficacy of EMD386088, a 5-HT 6 receptor ligand, in functional in vitro assays
Magdalena Jastrzêbska-Wiêsek1, Agata Siwek1, Grzegorz Kazek1, Barbara Nawieœniak1, Anna Partyka1, Monika Marcinkowska1, Marcin Ko³aczkowski1,2, Anna Weso³owska1
1Jagiellonian University Medical College, Medyczna 9, PL 30-688 Kraków, Poland
2Adamed Ltd., Pieñków 149, PL 05-152 Czosnów k/Warszawy, Poland Correspondence: Anna Weso³owska, e-mail: awesolowska@cm-uj.krakow.pl
Abstract:
Background: Over recent years, the 5-hydroxytryptamine6(5-HT6) receptor has emerged as a promising molecular target which in- teracts with several central nervous system acting drugs. In animal models, both agonists and antagonists of this receptor exhibit equivalent potency and efficacy as potential antidepressants, anxiolytics and anti-obesity or anti-dementia drugs. EMD386088 (5- chloro-2-methyl-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole hydrochloride) has been described as a high affinity 5-HT6receptor ligand with a full agonist activity and with moderate affinity for 5-HT3sites.
Methods: We have extended these data by broadening its profile for other, not yet tested, monoaminergic, GABAA, opioid µ recep- tors and serotonin transporter (SERT) and we have conducted functional in vitro assays; i.e., measurement of cAMP by homogene- ous TR-FRET immunoassay and HTRF method made by CEREP as well as aequorin-based calcium flux assay.
Results: In two in vitro models based on cAMP formation, maximal efficacy values for EMD386088 were 65 and 31%, for in house and CEREP experiments, respectively. In a model based on calcium response, the studied compound showed 46% of maximal sero- tonin (5-HT) signal. EMD386088 antagonizes 5-HT response in increasing concentrations from 10–9to 10–6M.
Conclusions: The present in vitro findings confirm that EMD386088 is a selective 5-HT6receptor ligand with moderate affinity for 5-HT3sites only and it behaves as a potent partial agonist of 5-HT6receptor with varying levels of agonist intrinsic activity, depend- ing on a method employed. In view of these results, caution is recommended in the interpretation of pharmacological in vivo studies with EMD386088.
Key words:
EMD386088, 5-HT6agonist, cAMP immunoassay, aequorin-based calcium flux assay, in vitro
Introduction
The 5-hydroxytryptamine6(5-HT6) receptor was iden- tified as one of the large family of serotonin (5-HT) receptors which stimulation leads to an increase in adenylyl cyclase activity [4, 13, 19, 20, 22, 25]. Its ex- clusive localization in the central nervous system and high affinity for many antipsychotic and antidepres-
sant drugs [13, 17, 22] have promoted much interest in 5-HT6 receptor as an attractive target for central nervous system therapeutic agents, with the possibil- ity of relatively few peripheral side effects [7, 33].
Much research has focused so far on identification of 5-HT6receptor antagonists, which have been reported to play an important role in cognitive functions, feed- ing behavior and mood control [7, 16, 29, 30].
In contrast to 5-HT6receptor antagonists, the iden- tification of selective 5-HT6agonists has proven to be much more difficult. The first full 5-HT6 receptor agonist reported, 2-ethyl-5-methoxy-N,N-dimethyl- tryptamine (EMDT), showed a 16-nM affinity but poor selectivity over other 5-HT receptors [28], mak- ing it of little use for pharmacological studies. More recently, several research groups have identified selective, high-affinity 5-HT6receptor agonists [5, 12, 15, 23, 24]. One of them is 5-chloro-2-methyl- 3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole hydro- chloride (EMD386088), now commercially available, described by Mattsson et al. [15] as a high affinity (IC50 = 7.4 nM) full 5-HT6 receptor agonist (EC50 = 1.0 nM). It also displays selectivity over other sero- tonin receptors (IC50 values are 110, 180, 240, 450, 620, 660 and 3000 nM for 5-HT1D, 5-HT1B, 5-HT2A, 5-HT2C, 5-HT4, 5-HT1Aand 5-HT7receptors, respec- tively) and shows moderate affinity at 5-HT3 ones (IC50= 34 nM) [15].
Recently, several groups reported conflicting re- sults for 5-HT6receptor ligands using various animal models. They have demonstrated that both 5-HT6re- ceptor agonist and antagonist compounds showed pro-cognitive [4], anti-obesity [7] and antidepres- sant/anxiolytic [8, 26, 31, 32] activity. It is still un- clear why both 5-HT6 receptor agonists and antago- nists show similar behavioral responses in animal as- says. Although the specificity of such ligands was certainly investigated during developmental stages, there is always the possibility of additional mecha- nisms by which a compound may act. Therefore, in vitro binding studies of EMD386088 by broadening its profile for other, not yet tested, monoaminergic, GABAA, opioid µ receptors and serotonin transporter (SERT) as well as functional assays are the subjects of this communication.
Materials and Methods
Materials
5-Chloro-2-methyl-3-(1,2,3,6-tetrahydro-4-pyridinyl)- 1H-indole hydrochloride (EMD386088) was synthe- sized by Adamed Ltd. (Pieñków, Poland). g-Irradiated recombinant 1321N1 cells expressing the human 5-HT6 receptor and recombinant CHO-K1 cells ex- pressing mitochondrially-targeted aequorin, Ga16and
the human 5-HT6receptor were obtained from Perkin El- mer. 4-Iodo-N-[4-methoxy-3-(4-methyl-piperazin-1-yl)- phenyl]-benzenesulfonamide (hydrochloride, SB258585) was purchased from Tocris, UK.
Radioligand binding assay
Binding experiments were conducted in 96-well mi- croplates in a total volume of 200 µl of appropriate buffers. Reaction mix included 50 µl solution of a tested compound, 50 µl of radioligand and 150 µl of diluted membranes (15 µg protein per well) or tis- sue suspensions were transferred to microplate. Spe- cific assay conditions for each receptor are shown in Table 1. Cell membranes expressing recombinant hu- man 5-HT2A, 5-HT6, 5-HT7 and D3 receptors were used. 5-HT1Areceptors were prepared from rat hippo- campal tissue and rat cortex tissue was used for re- maining receptors. The radioactivity was measured in MicroBeta TriLux 1450 liquid scintillation counter (PerkinElmer, USA).
Measurement of cAMP by homogeneous TR-FRET immunoassay
Activation and inhibition of adenylyl cyclase activity was monitored using g-irradiated recombinant 1321N1 cells expressing the human 5-HT6 receptor (PerkinElemer). Thawed cells were re-suspended in stimulation buffer (HBSS, 5 mM HEPES, 0.5 IBMX, 0.1% BSA, pH 7.4) at 50,000 cells/ml. Ten microliters of cells suspension was added to 10 µl tested com- pounds loaded on half area 96-well microplate. An antagonist response experiment was performed with 22 nM of 5-HT as a reference agonist. Agonist and antagonist were added simultaneously. Cell stimula- tion was performed for 30 min at room temperature.
After incubation, cAMP measurements were per- formed with homogeneous TR-FRET immunoassay using the LANCE Ultra cAMP Kit (PerkinElmer).
Ten microliters Eu-cAMP Tracer Working Solution and 10 µl ULight-anti-cAMP Tracer Working Solu- tion were added, mixed and incubated for 1 h.
TR-FRET signal was read on an EnVision microplate reader (PerkinElmer).
EMD386088 was also tested in an additional cellu- lar functional assays conducted by CEREP (Le Bois l’Eveque, 86600 Celle L’Evescault, France). Func- tional effect was tested on CHO cells expressed hu- man 5-HT6 receptor; cAMP was assayed in HTRF
In vitro partial agonist activity of 5-HT6ligand
Magdalena Jastrzêbska-Wiêsek et al.
method, according to CEREP standard assay proto- cols (see for details: http://www.cerep.fr/cerep/users/
pages/ProductsServices/GPCRPlatform.asp).
Aequorin-based calcium flux assay
Cellular aequorin-based functional assay was per- formed with g-irradiated recombinant CHO-K1 cells expressing mitochondrially-targeted aequorin, Ga16and the human 5-HT6 receptor (PerkinElmer). Assay was executed according to the standard protocol provided by manufacturer. After thawing cells were transferred to assay buffer (DMEM/HAM’s F12 with 0.1%
protease-free BSA) and centrifuged. Cell pellet was re- suspended in assay buffer and coelenterazine h was added at a final concentration of 5 µM. Cell suspension
was incubated at 21°C protected from light with con- stant agitation for 12 h and then diluted with assay buffer to a concentration of 500,000 cells/ml. After 1 h incubation, 50 µl cell suspension was dispended using automatic injector built in microplate reader (POLARstar Omega, BMG Labtech) into white half area 96-well microplate preloaded with tested com- pounds. Immediately light emission generated follow- ing calcium mobilization was recorded for 45 s.
Data analysis
Radioligand binding data were analyzed using itera- tive curve fitting routines (GraphPAD/Prism, Version 3.0 – San Diego, CA, USA). The logIC50 estimated from the radioligand binding data was used to obtain Ki
(nonspecific) conditions
5-HT1A [3H]8-OH-DPAT 10 µM serotonin
50 mM Tris-HCl pH 7.7 37°C, 20 min 80 320.4 ± 41.0 nM 563.6 ± 54.0 nM
5-HT2A [3H]-ketanserin 1 µM ketanserin
50 mM Tris-HCl pH 7.4, 4 mM CaCl2
27°C, 60 min 80
5-HT6 [3H]-LSD 10 µM
methiothepine
50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.5 mM EDTA
37°C, 60 min 27 8.00 ± 0.07a
5-HT7 [3H]-LSD 10 µM
methiothepine
50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM EDTA
30°C, 60 min 74
SERT [3H]citalopram 1 µM imipramine
50 mM Tris-HCl pH 7.7, 150 mM NaCl, 5 mM KCl,
24°C, 60 min 86 4.75 ± 2.0 µM 9.95 ± 4.1 µM
a1 [3H]prazosin 10 µM
phentolamine
50 mM Tris-HCl pH 7.6 30°C, 30 min 76 463.2 ± 41.0 nM 991.5 ± 54.3 nM
a2 [3H]clonidine 10 µM clonidine
50 mM Tris-HCl pH 7.6 30°C, 25 min 248.2 ± 12.9 nM 413.6 ± 21.5 nM
b1 [3H]CGP-12177 1 µM propranolol 50 mM Tris-HCl pH 7.6 37°C, 60 min 1.1 ± 0.1 µM 4.2 ± 0.3 µM D2 [3H]methylspiperone 10 µM
(+)-butaclamol
NHME 30°C, 60 min 64
D3 [3H]methylspiperone 1 µM chlorpromazine
50 mM Tris-HCl pH 7.4, 120 mM NaCl
24°C, 60 min 100 µ [3H]dihydromorphine 0.1 µM
levallorphan
50 mM Tris-HCl pH 7.7 24°C, 30 min 13.2 ± 0.4 µM 25.9 ± 2.4 µM
GABAA [3H]muscimol 100 µM GABA
50 mM Tris-HCl pH 7.4 0–4°C, 10 min no affinity in a concentration of 10–4M
X/T% - percent of total binding was calculated for EMD386088 in screening test, which was determined in a concentration of 1.66 ´ 107M;
apKi value
value by applying Cheng-Prusoff equation. The con- centrations of an analyzed compound EMD386088 ranged from 10–10to 10–5M. Percent of total binding was calculated for EMD386088 in screening test, which was determined in a concentration of 1.66
× 10–7 M. IC50 and EC50 were determined by non- linear regression analysis using GraphPad Prism soft- ware. The Emaxvalues are relative to a maximally ef- fective concentration of 5-HT (1 µM). Data are pre- sented as the mean values ± standard error of the mean (SEM) from two to three independent experi- ments performed in duplicates.
value by applying Cheng-Prusoff equation. The con- centrations of an analyzed compound EMD386088 ranged from 10–10to 10–5M. Percent of total binding was calculated for EMD386088 in screening test, which was determined in a concentration of 1.66
× 10–7 M. IC50 and EC50 were determined by non- linear regression analysis using GraphPad Prism soft- ware. The Emaxvalues are relative to a maximally ef- fective concentration of 5-HT (1 µM). Data are pre- sented as the mean values ± standard error of the mean (SEM) from two to three independent experi- ments performed in duplicates.
Results and Discussion
The binding affinities of EMD386088 are summa- rized in Table 1. The present data indicate that EMD386088 displays high affinity for 5-HT6 recep- tor, with a high degree of selectivity compared with all the other 5-HT receptor subtypes as well as the other tested monoaminergic, GABAA, opioid µ recep- tors and SERT. In a receptor binding study carried out on EMD386088 by CEREP, its high interaction with 5-HT6 sites was additionally endorsed (Tab. 2). The results obtained in 5-HT6 receptor functional assays do not agree with the previously reported by Mattsson et al. [15] full agonist activity for EMD386088. In ho- mogenous TR-FRET immunoassay, EMD386088 in- creased cAMP formation with a maximal response of 65% relative to that induced by 5-HT (Tab. 2, Fig.
1A). Since its maximal effect was significantly lower than that of 5-HT, the data reveal that EMD386088 behaves as a partial agonist under these conditions.
Similarly, in CEREP’s experiment a maximal re- sponse to EMD386088 was Emax= 31% of that ob- served with 5-HT (Tab. 2). In order to confirm partial
agonist activity of EMD386088, its effect was com- pared with that of a selective 5-HT6receptor antago- nist SB-258585. As is presented in Figure 1B, SB-258585 in a concentration-dependent manner at- tenuated cAMP formation while the ability of EMD- 386088 to antagonize the studied effect was partially pronounced. Hence, the curves for EMD386088 and SB258585 do not overlap each other. Moreover, in the presence of 5-HT (22 nM), EMD386088, in accor- dance with increasing concentrations from 10–9 to 10–6 M, first potentiated the effect of 5-HT, then at- tenuated it, shifting the 5-HT concentration-response curve (Fig. 1C).
For comparative purposes, an additional in vitro model based on calcium response has been employed.
The obtained results appear to be comparable, since the maximal stimulation in this system by EMD- 386088 was 46% relative to that of 5-HT (Tab. 2, Fig.
1D). Fig. 1E presents that in this model a partial ago- nist effect observed for EMD386088 was less ex- pressed than using a different method; i.e., homoge- nous TR-FRET immunoassay.
Our data on EMD386088 binding affinity are in line with the results obtained by Mattsson et al. [15]
confirming its high affinity for 5-HT6sites. In the ma- jority of studies, labeled agonists [2, 13, 17, 21] or an- tagonists [9, 10] have been employed to determine drug affinities at 5-HT6 receptors. In this regard, all above-mentioned experiments applied [3H]-LSD. De- spite the different cell lines used (HEK by Mattsson et al. [15], CHO by CEREP, HEK293 in house), the ob- tained results concerning 5-HT6 receptor affinity for EMD386088 are comparable. Furthermore, we have also extended receptor profile for EMD386088, pre- sented for the first time by Mattsson et al. [15], con- firming that this compound is a selective 5-HT6ligand with a moderate affinity for 5-HT3sites only.
Different functional in vitro assays have been em- ployed and all the obtained results indicate that
In vitro partial agonist activity of 5-HT6ligand
Magdalena Jastrzêbska-Wiêsek et al.
Tab. 2. Summary of data from in vitro assays
Binding TR-FRET immunoassay CEREP Aequorin-based
calcium flux assay pKi CEREP pKi pEC50 Emax pIC50/pKi pEC50 Emax pIC50/pKi pEC50 Emax pIC50/pKi
5-HT 7.19 ± 0.24 7.19 8.81 ± 0.03 100 ± 0.2 1.23 7.47 1.04 7.85 ± 0.05 103 ± 4 1.09 EMD386088 8.00 ± 0.07 9.02 ± 0.05 8.31 ± 0.07 65 ± 6.0 1.04 8.96 ± 0.16 31 ± 2 0.99 8.05 ± 0.07 46 ± 2 1.01
EMD386088 possesses partial agonist properties at 5-HT6receptors. These findings did not confirm the data presented by Mattsson et al. [15], who described EMD386088 as a full agonist with an EC50 value of 1.0 nM. The reason for this discrepancy is difficult to explain and seems to be multifaceted. The methodo- logical differences are the most visible; first of all, cell lines tested. We used recombinant 1321N1 and CHO-K1 in assays employed in-house; i.e., homoge- neous TR-FRET immunoassay and aequorin-based calcium flux assay, respectively. In CEREP’s experi- ment human recombinant CHO cells were used and Mattsson et al. [15] conducted research on cloned
h5-HT6receptors stably expressed in BHK cells. All those cells may vary in 5-HT6receptor density which was not indicated by the authors. It is well known that agonist activity is more pronounced when the level of 5-HT6receptor expression is higher. Moreover, in the in vivo experiments, different isoforms of adenylyl cyclase and different downstreams of G-protein acti- vation may be running, hence all these conditions must be taken into account when considering results of in vivo tests carried out with EMD386088.
It is noteworthy that in the case of other 5-HT6ago- nists, i.e., WAY181,187 and WAY208,466 similar dis- crepancies in activity have been observed based on
–11 –10 –9 –8 –7 –6 –5
0 50
log [compound]
Fig. 1. Functional in vitro assays
methodological differences. Both above-mentioned compounds acted as full agonists in stimulating adenylyl cyclase at h5-HT6 receptors expressing in HeLa cells [1, 23, 24], and they only sub-maximally activated Gasand, over a similar concentration range, attenuated its recruitment by 5-HT. In contrast, WAY181,187 and WAY208,466 behaved as partial agonists in a scintillation proximity assay/antibody- immunocapture procedure of coupling to Gasused by Dupuis et al. [6] to evaluate interactions of these com- pounds at rat 5-HT6 receptors stably expressed in HEK293 cells. These contrasting maximal effects for Gas versus camp stimulation were likely related to
“amplification” of signaling, a phenomenon well known in other systems. Irrespective of the explana- tion, EMD386088 may be classified as a partial ago- nist of 5-HT6receptors with varying levels of agonist intrinsic activity depending on the method employed.
It will also be interesting to extent this work by use of other parameters, such as a procedure of coupling to Gas.
Similar discrepancies have been observed for an- other 5-HT6 receptor ligand, compound E-6801 by analyzing its ability to modulate Gs-coupled-cAMP pathway linked to the 5-HT6 receptor in two experi- mental conditions [20]. E-6801 induced cAMP forma- tion in HEK-293F cells stably transfected with a rat 5-HT6 receptor displaying potent partial agonism in the absence of forskolin and a pEC50value similar to its pKivalue as was observed for 5-HT. Interestingly, E-6801 displayed an Emax value indistinguishable to 5-HT when measured in the copresence of forskolin.
In contrast, both at human wild-type and mutant S267K 5-HT6 receptor in Cos-7 cells, compound E- 6801 displayed full agonist efficacy with an Emaxval- ues indistinguishable from 5-HT [20]. The latter data agree with those of Kendall et al. [12], who reported full agonist activity of E-6801 at the rat 5-HT6recep- tor (cAMP measurements on HEK-293F cells that sta- bly expressed rat 5-HT6receptors were performed by using a system based on homogeneous time resolved fluorescence). Another available 5-HT6 receptor ligand ST1936 has been presented as a full agonist of 5-HT6receptors on adenylyl cyclase [3].
A molecular characterization of new ligands of not only 5-HT6 receptors but others as well [14, 27], should be based on several observations as made in different expression systems and using different read-outs [20]. Partial agonism could be observed probably in most systems for such compounds al-
though full agonist properties can be also seen in very efficiently coupled systems.
There is no doubt that all these in vitro model sys- tems are useful to differentiate between functional properties of compounds, but as mentioned above, some caution should be taken to extrapolate findings from such recombinant systems to in vivo integrated systems. Indeed, a compound with partial agonist properties in a recombinant expression system may demonstrate antagonist activity in in vivo integrated systems [11]. As was demonstrated by Kendall et al.
[12], E-6801, described by the authors as a full ago- nist, and EMD386088, administered alone and in low dose combinations with a selective 5-HT6receptor an- tagonist SB-271046, enhanced recognition memory in a novel object discrimination task in rats. Pro- cognitive effects appear analogous after treatment with an agonist and an antagonist of 5-HT6receptors.
This apparently paradoxical effect of 5-HT6 ligands seems to be mediated by different mechanisms. The agonist activates 5-HT6receptors located directly on glutamatergic and/or cholinergic neurons which re- ceive a low tonic serotonergic input under normal conditions. In contrast, a 5-HT6 receptor antagonist may predominantly attenuate active serotonergic in- put to upstream inhibitory GABAergic neurons which disinhibit glutamate and/or acetylcholine release [12, 18]. Thus, both 5-HT6 agonist and antagonist com- pounds may elevate glutamate and/or acetylcholine levels resulting in observed pro-cognitive activity.
Further investigation with EMD386088 on its poten- tial pro-cognitive, antidepressant, anxiolytic and other effects would be worthwhile to expand evidence on the differences in the mechanisms of action of 5-HT6
receptor agonists and antagonists.
Taken together, the obtained data confirm and ex- tend the binding profile of EMD386088, a selective 5-HT6receptor ligand with only moderate affinity for 5-HT3sites. Moreover, these data provide for the first time in vitro picture of partial agonist activity of EMD386088, which should be taken into considera- tion when interpreting results of in vivo pharmacol- ogical studies.
Acknowledgments:
The study was financially supported by Adamed Ltd., NCBiR, Warszawa, Poland (project no. KB/88/12655/ITT1-C/U/08) and Funds for Statutory Activity of Jagiellonian University Medical College, Kraków, Poland (K/ZDS/001293).
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Received: October 15, 2012; in the revised form: February 5, 2013;
accepted: February 18, 2013.
In vitro partial agonist activity of 5-HT6ligand
Magdalena Jastrzêbska-Wiêsek et al.
