From the Centre National de la Recherche Scientifique, UPR 9023, 141 rue de la Cardonille, 34094 Montpellier, Cedex 5, France
Received for publication, October 24, 2002, and in revised form, November 8, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
To better understand G-protein-coupled receptor
(GPCRs) signaling, cellular and animal physiology, as well as gene
therapy, a new tool has recently been proposed. It consists of GPCR
mutants that are insensitive to endogenous ligands but sensitive to
synthetic ligands. These GPCRs are called receptor activated solely by
synthetic ligands (RASSL). Only two examples of such engineered
receptors have been described so far: one Gi-coupled
(opioid receptors) and one Gs-coupled
( G protein-coupled receptors
(GPCRs)1 are the most
numerous and the most diverse type of receptors (1, 2). They transduce messengers as different as odorants, nucleotides, nucleosides, peptides, lipids, or proteins. Eight families of GPCRs are classified according their sequences, their binding domains to ligands, and their
ability to activate similar sets of G-proteins. The heterotrimeric ( The concept that modified GPCRs could be used as tools to better
understand GPCR-controlled signal transduction pathways in a given cell
or organ or for gene therapy has recently been proposed (5-7). Several
types of modified GPCRs have been developed to prepare such tools. The
first, engineered by Conklin and collaborators (8), were named
RASSL for "receptor activated solely by synthetic ligands." The
idea is to engineer receptors that would be insensitive to their
endogenous ligand(s) but can be fully activated by synthetic ligands.
Among the GPCRs, only the More recently, a second type of mutant GPCR the "therapeutic
receptor-effector complex" (or "TREC") was proposed to be a
biotechnological tool to study GPCR signal transduction (9). The
In this report, we describe a third example of RASSL type receptors: a
mutant serotonin receptor, the 5-HT4 receptor.
5-HT4 receptors are Gs-coupled receptors
expressed in the gastrointestinal tract, human and pig atria, urinary
bladder, adrenal medulla, and central nervous system including limbic
areas (olfactory tubercles, limbic system, basal ganglia) (10). Over
the past 10 years, the pharmacology and structure of 5-HT4
receptors have been extensively studied (10-12). One of the
interesting characteristics of this receptor is its ability to be
activated by a wide range of compounds from very different chemical
classes (13). Some are used in clinic to treat gastroparesis, dyspepsia
gastro-esophageal reflux, or irritable bowel syndromes. The
5-HT4 agonists are related to tryptamines like 5-HT, to
carbazimidamides (HTF-919 or Zelmac), benzamides (metoclopramide or
PrimperanTM, cisapride or "PrepulsidTM,"
benzoates (SL 10302), benzimidazolones (BIMU8), or aryl ketones (for
reviews, see Refs. 10 and 13).
The demonstration that tryptamines and benzamides have different
pharmacophores (14) prompted us to generate mutant 5-HT4 receptors with the aim of disrupting the 5-HT recognition site, keeping
the recognition site for synthetic 5-HT4 agonists.
Construction of Mutant m5-HT4(a) Receptor
cDNA--
The mutant was generated by exchanging the endogenous
residue Asp100 to Ala in the m5-HT4(a)R
cDNA sequence with the QuikChange site-directed mutagenesis kit
(Stratagene). The sense primer used was: D100A, 5'-ACC TCT CTG
GCT GTC CTA CTC ACC-3'.
Cell Culture and Transfection--
The cDNAs, subcloned into
the pRK5 vector, were introduced into COS-7 cells by transfection. In
summary, cells were trypsinized, centrifuged, and resuspended in EP
Buffer (50 mM K2HPO4, 20 mM CH3CO2 K, 20 mM KOH,
26.7 mM MgSO4, pH 7.4) with 25-2000 ng of receptor cDNA. The total amount of cDNA was kept constant at 15 µg per transfection with pRK5 vector. After 15 min at room
temperature, 300 µl of cell suspension (107 cells) were
transferred to a 0.4-cm electroporation receptacle (Bio-Rad, Ivry sur
Seine, France) and pulsed with a gene pulser apparatus (setting 1000 microfarads, 280 V). Cells were diluted in Dulbecco's modified
Eagle's medium (DMEM; 106 cells/ml) containing 10%
dialyzed and decomplemented fetal bovine serum (dFBS) and plated on
15-cm Falcon Petri dishes or into 12-well clusters at the desired density.
Determination of Cyclic AMP (cAMP) Production in Intact
Cells--
Six hours after transfection, the surrounding cell
medium was exchanged for DMEM without dFBS with 2 µCi of
[3H]adenine/ml to label the ATP pool and incubated
overnight (16 h). cAMP accumulation was measured, as described
previously (15).
Membrane Preparation and Radioligand Binding
Assay--
Membranes were prepared from transiently transfected cells
plated on 15-cm dishes and grown in DMEM with 10% dFBS for 6 h, followed by incubation for 20 h in DMEM without dFBS. The cells were washed twice in PBS, scraped with a rubber policeman, harvested in
PBS, and centrifuged at 4 °C (200 × g for 4 min).
The pellet was resuspended in buffer containing 10 mM
HEPES, pH 7.4, 5 mM EGTA, 1 mM EDTA, and 0.32 M sucrose and homogenized 10 times with a glass-Teflon
potter at 4 °C. The homogenate was centrifuged at 20,000 × g for 20 min. The membrane pellet was resuspended in 50 mM HEPES, pH 7.4 (5 mg of protein in 1 ml of solution) and stored at Data Analysis--
Competition and saturation experiments were
analyzed by non-linear regression curves using the computer program
LIGAND (17). Saturation experiments were also analyzed according to
Scatchard. IC50 values required to displace 50% of
[3H]GR 113808 binding sites were converted to
KD values, according to the equation
KD = IC50/1 + S/KDS (18), where S is [3H]GR 113808 concentration, and
KDS is the equilibrium constant of
[3H]GR 113808.
Using Kaleidagraph software, the dose-response curves were fitted
according to the following equation.
Drugs--
The following drugs were used: GR 113308 (1-[2-(methylsulfonylamino)ethyl]-4-piperidinyl
1-methyl-indole-3-carboxylate), HTF- 919 (5-methoxy-indole-3-carboxaldehyde 4-pentyl-iminosemicarbazone), BIMU8
(endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2-oxo-3-isopropyl-2,3-dihydro-1H benzimidazole-1-carboxamide), (S)-zacopride
((S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-4-amino-5-chloro-2-methoxy-benzamide monohydrochloride), cisapride
(cis-N-[1-[3-(4-fluorophenoxy)propyl]-3-methoxy-4-piperidinyl]-4-amino-5-chloro-2-methoxy-benzamide, metoclopramide
(N-(2-dimethylamino)ethyl)-4-amino-5-chloro-2-methoxybenzamide), renzapride
((±)-endo-N-(1-azabi-cyclo[3.3.1]non-4-yl)-4-amino-5-chloro-2-methoxybenzamide mo- nohydrochloride), SB 204070 ((1-butyl-4-piperidinyl)methyl 8-amino-7-chloro-1,4-benzodioxane-5-carboxylate), ML 10302 (2-(1-piperidinyl)ethyl-4-amino-5-chloro-2-methoxybenzoate), ML
10375 (2-(cis-3,5-dimethyl-1-piperidinyl)ethyl4-amino-5-chloro-2-methoxybenzoate), RS 67333 (1-(4-amino-5-chloro-2-methoxy-phenyl)-3-(1-butyl-4-piperidinyl)-1-propanone), RS 100350 (N-[2-[4-[3-(8-amino-7-chloro-1,4-benzodioxan-5-yl)-3-oxopropyl]-1-piperidinyl]ethyl]-4-methoxybenzenesulfonamide), RS 124523 (1-(8-amino-7-chloro-1,4-benzodioxan-5-yl)-3-[1-[3-(4-fluorophenyl)propyl]-4-piperidinyl]-1-propanone), RS 47431 (1-(4-amino-5-chloro-2-methoxy-phenyl)-3-[1-[3-(4-methoxyphenyl)propyl]-4-piperidinyl]-1-propanone), and RS 100235 (1-(8-amino- 7-chloro-1,4-benzodioxan-5-yl)-3-[1-[3-(3,4-dimethoxyphenyl)propyl]-4-piperidinyl]-1-propanone).
First, we wanted to render the receptor unresponsive to
5-HT, but responsive to synthetic 5-HT4 agonists. Based on
Strader's work on Indeed, the mutation of Asp100(3.32) to alanine in the
5-HT4 receptor totally abolished the 5-HT stimulation of
cAMP accumulation (11, 12), as well as the stimulation by other
tryptamines (5-CT or 5-MeOT) or by a substituted indole
carbazimidamide (HTF-919) (Fig.
1, A and B) (Table
I). Compared with 5-HT, HTF-919 has a
guanidine function instead of a protonated amine in the indole side
chain (20). Surprisingly, when we mutated other well conserved residues
within the biogenic amine receptor family and likely to be
involved in the 5-HT binding pocket
(Ser197(5.43), Trp272(6.48), and
Phe275(6.51)), none were able to totally suppress the 5-HT
or HTF-919 responses. 5-HT has a 50-500-fold reduced affinity for
F275A, W272A, and S197A, with no change in the maximal 5-HT
stimulation (12).
2-adrenergic receptors). Here, we describe the
first RASSL related to serotonin receptors (D100(3.32)A
Gs-coupled 5-HT4 receptor or
5-HT4-RASSL). 5-HT4-RASSL is generated by a single mutation, is totally insensitive to serotonin (5-HT), and still
responds to synthetic ligands. These ligands have affinities in the
range of nanomolar concentrations for the mutant receptor and exhibit
full efficacy. More interestingly, two synthetic ligands behave as
antagonists on the wild type but as agonists on the 5-HT4-RASSL.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,
,
) G proteins are divided into four families based on the nature of their G
subunits (G
i, G
s,
G
q, G
12-13). Each one couples to a
distinct class of receptors and signals through a specific pathway. A
series of pathologies have been found to be related to the mutation of
GPCRs. These changes lead either to a loss or a gain of function, such
as blindness, diabetes insipidus, and hypo- or hyperthyroidism
(3). Depressed GPCR signal transduction is related to numerous complex
diseases. Heart failure and asthma are associated with a decrease
in the Gs-signaling pathway, whereas an increase
in Gi signaling is a potential cause of dilated cardiomyopathy).
opioid receptors have been modified so far by Conklin's group (8) to produce two opioid receptor-RASSLs, Ro1 and Ro2. Ro1 was constructed by substituting the
second extracellular loop of the
opioid receptor with the corresponding portion of the human
opioid receptor. This
substitution induced a lower affinity for the endogenous peptide
ligands (including dynorphin) without significantly reducing the
response to
synthetic ligands, like spiradoline (8). The
specificity of Ro1 for the synthetic ligands was further enhanced in
Ro2 by substituting glutamine for Glu297 in Ro1.
2-adrenergic receptor mutated in 19 positions and fused
with G
s was not activated by
-adrenergic agonists,
but only by a non-biogenic amine agonist, L156870, although with
relatively low potency.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
80 °C until use. Saturation experiments were performed using the specific 5-HT4 receptor radioligand
[3H]GR 113808 at height concentrations ranging from 0.048 to 0.51 nM. The 5-HT4 receptor binding site
density was estimated with [3H]GR 113808 at a saturating
concentration (0.5 nM), as described previously (16). 5-HT
(5 × 10
5 M) or RS 100235 (10
7 M), a 5-HT4 receptor
antagonist, was used to determine nonspecific binding. Protein
concentration in the samples was determined with the Bio-Rad protein assay.
where EC50 (or EC50inv) is the
concentration of agonist (or inverse agonist) that evokes a
half-maximal response, ymax and ymin correspond to the maximal and minimal
responses, respectively, and nH is the Hill coefficient. Data were
compared using the Stat-View Student program (Abacus Concepts,
Berkeley, CA) with t tests.
(Eq. 1)
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-adrenergic receptors and on many other biogenic
amine receptors (19), the key interaction between 5-HT4
receptors and 5-HT should obviously occur at the highly conserved Asp
residue found in TM-III within the biogenic amine receptor
family. In the 5-HT4 receptor this Asp is at position 100 in TM-III (Asp100(3.32)). This Asp residue is believed to
interact with the positively charged nitrogen of the neurotransmitter
protonated amine.
View larger version (36K):
[in a new window]
Fig. 1.
Agonists structurally related to serotonin
fail to stimulate D100(3.32)A RASSL. A and B,
concentration-response curves for the wild-type and the D100(3.32)A
receptors exposed to the tryptamine derivatives (5-MeOT, 5-CT, HTF-919,
and 5-HT) on cAMP production in COS-7 cells transiently expressing
either 5-HT4(a) or mutant D100(3.32)A receptors at a level
of 1500 ± 230 and 1450 ± 160 fmol/mg of protein,
respectively. cAMP production was measured for 15 min and expressed as
a percentage of cAMP formation in mock-transfected cells taken as
control. In mock-transfected cells, the percentage conversion of
[3H]ATP to [3H]cAMP was equal to 0.12 ± 0.04%. Data are the means ± S.E. values of four experiments
performed in triplicate. C, competition binding performed
for 5-HT in presence of 0.24 nM [3H]GR
113808. The assays were carried out on membranes (8 µg of protein)
derived from COS-7 cells expressing similar levels of WT or D100A
receptors (3500 fmol/mg of protein). Results are expressed as a
percentage of the specific binding in the absence of a competing
ligand. Data are the means ± S.E. values of three experiments
performed in triplicate. D, Scatchard analysis of saturation
experiments of [3H]GR 113808 binding (0.048-0.51
nM) to 5-HT4(a) and 5-HT4
D100(3.32)A receptors expressed in COS-7 cells. Membranes (10 µg of protein) were derived from COS-7 cells, and RS 100235 (10 µM) was used to determine nonspecific binding.
5-HT4 receptor drug efficiency on WT and D100(3.32)A mutant
5-HT4 receptor
Interestingly, and in contrast to other biogenic amine receptors (9), the mutation of the conserved Asp100(3.32) only moderately affected the specific 5-HT4R radioligand, [3H]GR 113808 (KD = 0.17 ± 0.08 nM and 0.38 ± 0.09 nM at WT and D100(3.32)A receptors, respectively) (Fig. 1D). Using [3H]GR 113808 binding, we verified that 5-HT was unable to bind the 5-HT4 D100(3.32)A receptor (Fig 1C). The binding of [3H]GR 113808 was the first indication that ligands, which include the basic nitrogen of the aromatic ring side chain, in a structured ring, associated with an increase in the distance between this basic nitrogen and the main aromatic ring of the compound, suppressed the requirement of the Asp100(3.32) carboxylic group for ligand binding. This was confirmed when we screened most of the non-tryptamine 5-HT4 receptor agonists for their ability to activate the 5-HT4 D100(3.32)A receptor.
BIMU8, a specific 5-HT4 agonist structurally very
different from 5-HT, belonging to the azabicycloalyl benzymidazolone
class (21), was found to be a potent agonist. As shown in Fig.
2, this compound remained fully active
and even showed higher efficacy and potency on the D100A mutant than on
the WT (EC50 values for cAMP stimulation were 4 ± 1.5 and 1 ± 0.5 nM for WT and D100A, respectively) (Fig.
2A). The affinity of BIMU8 for the 5-HT4
receptor was also slightly better on the D100A mutant than on the WT.
KD values for BIMU8, measured by competition with
the [3H]GR 113808 radioligand, were 30 ± 11 nM and 6.5 ± 3 nM for WT and D100A,
respectively (Fig. 2B). Similarly, the benzamides bearing the 2-methoxy-4-amino-5-chloro substitution (renzapride, S-zacopride, or cisapride) were equi-effective on the WT and the D100(3.32)A mutant
(Table I) with nanomolar affinity. A slight decrease in their potency
on the mutant D100A receptor was observed. These data suggest that the
D100(3.32)A mutant could still be activated as long as the agonist
could bind the receptor. Furthermore, this mutation had no effect on
the receptor expression level.
|
Two drugs were antagonists on WT and agonists at the D100(3.32)A mutant.
Benzoate derivatives bearing the
2-methoxy-4-amino-5-chloro-substitution (ML 10302) (22) and the related
aryl ketones (RS 67333, RS 124523, RS 100350, RS 47431) (13) were
partial agonists on 5-HT4 WT receptors. As shown in Table
I, they were more efficacious on 5-HT4 D100(3.32)A
receptors than on WT. Another benzoate derivative, reported to be
either a highly specific 5-HT4 antagonist (ML 10375) (23)
or an inverse agonist on the 5-HT4 receptor (24), was indeed an antagonist of WT 5-HT4 receptors (Fig.
3C), which became a full
agonist on the 5-HT4 D100(3.32)A mutant (EC50 = 1 ± 0.6 nM) (Fig. 3, A and C).
This encouraged us to screen the putative agonist properties of a
series of 5-HT4 antagonists. We found that the known GR
113808 antagonist (Fig. 3C) used for 5-HT4
receptor studies (25) was a highly potent (EC50 = 0.17 ± 0.04 nM) and efficacious agonist on D100(3.32)A receptor
(Fig. 3B).
|
Compared with the previously described 2-adrenergic
Gs-coupled RASSL (9), the 5-HT4 RASSL has the
advantage of being stimulated by synthetic compounds of much higher
affinity (micromolar for
2-adrenergic RASSL and
nanomolar for 5-HT4 RASSL).
The structure-activity relationships and the structural analyses of the 5-HT4 receptor ligands used in this study are consistent with a recent report on comparative receptor mapping of 5-HT4 binding sites (26). The authors proposed structural insights to assist the design of selective 5-HT4 receptor ligands. The structural features that define the 5-HT4 ligands are an aromatic moiety, a coplanar carbonyl, carboxyl or ketone function, and a basic nitrogen atom. The substitute of the basic nitrogen must be voluminous (as in all the active drugs acting on the 5-HT4 RASSL) to interact in an hydrophobic pocket of the receptor. The size of the substitute improves selectivity and potency. One of our hypotheses is that the basic nitrogen of these drugs interacts within the hydrophobic pocket, with a negative charge, but not with Asp(3.32). In contrast, this Asp is necessary for the binding of the protonated primary amine of 5-HT derivatives. The affinity of the voluminous substituted ligands for the 5-HT4 RASSL (structures in Table I) would depend of the interactions between their hydrophobic rings and the hydrophobic pocket, defined by highly conserved aromatic residues in TMVI and VII (Trp(6.48), Phe(6.51), and Tyr(7.43)) (11, 27, 28).
The above results indicate that the D100(3.32)A Gs-coupled 5-HT4 receptor described here is the first RASSL related to serotonin receptors and that it has unique properties not found in two previously described RASSL receptors (5, 7).
RASSL is generated by a single mutation and is completely insensitive to its endogenous agonist: 5-HT. RASSL can be stimulated by numerous synthetic agonists from different chemical classes.
Many of these synthetic ligands have a nanomolar affinity for the 5-HT4 RASSL, and some of them can be administered orally such as cisapride or metoclopramide. Cisapride has been used for years to treat dyspepsia and gastro-esophageal reflux and has only been removed from the market because of one second effect (arrhythmia probably caused by action on K+ channels) (29, 30). However, other benzamides are being developed and will certainly be used in humans. Interestingly, RS 67333 was found to be another potent agonist of 5-HT4 RASSL. This compound possesses a great ability to cross the blood-brain barrier.
Antagonist compounds (ML 10375 and GR 113808), which are highly
specific on the native receptor, exhibit agonist properties on the
5-HT4 RASSL. GR 113808 is also able to cross the
blood-brain barrier. ML 10375 and GR 113808 will be particularly
interesting for in vivo stimulation of 5-HT4
RASSL. Indeed, with these compounds, native 5-HT4 receptors can be kept
silent, while generating a Gs signal by activating
5-HT4 RASSL, expressed in a given tissue at a given
developmental time, by bioengineering in animals and possibly in humans.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: UPR CNRS 9023, 141 rue de la Cardonille, 34094 Montpellier, Cedex 5, France. Tel.: 33-4-67-14-29-30; Fax: 33-4-67-54-24-32; E-mail: bockaert@ccipe. montp.inserm.fr.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.C200588200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GPCR, G-protein-coupled receptor; RASSL, receptor activated solely by synthetic ligands; DMEM, Dulbecco's modified Eagle's medium; dFBS, dialyzed fetal bovine serum; PBS, phosphate-buffered saline; TM, transmembrane domain.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Bockaert, J., Claeysen, S., Becamel, C., Pinloche, S., and Dumuis, A. (2002) Int. Rev. Cytol. 212, 63-132[Medline] [Order article via Infotrieve] |
2. |
Bockaert, J.,
and Pin, J. P.
(1999)
EMBO J.
18,
1723-1729 |
3. | Spiegel, A. M. (1996) Annu. Rev. Physiol. 58, 143-170[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Redfern, C. H.,
Degtyarev, M. Y.,
Kwa, A. T.,
Salomonis, N.,
Cotte, N.,
Nanevicz, T.,
Fidelman, N.,
Desai, K.,
Vranizan, K.,
Lee, E. K.,
Coward, P.,
Shah, N.,
Warrington, J. A.,
Fishman, G. I.,
Bernstein, D.,
Baker, A. J.,
and Conklin, B. R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4826-4831 |
5. | Coward, P., Chan, S. D., Wada, H. G., Humphries, G. M., and Conklin, B. R. (1999) Anal. Biochem. 270, 242-248[CrossRef][Medline] [Order article via Infotrieve] |
6. | Scearce-Levie, K., Coward, P., Redfern, C. H., and Conklin, B. R. (2001) Trends Pharmacol. Sci. 22, 414-420[CrossRef][Medline] [Order article via Infotrieve] |
7. | Scearce-Levie, K., Coward, P., Redfern, C. H., and Conklin, B. R. (2002) Methods Enzymol. 343, 232-248[Medline] [Order article via Infotrieve] |
8. |
Coward, P.,
Wada, H. G.,
Falk, M. S.,
Chan, S. D.,
Meng, F.,
Akil, H.,
and Conklin, B. R.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
352-357 |
9. |
Small, K. M.,
Brown, K. M.,
Forbes, S. L.,
and Liggett, S. B.
(2001)
J. Biol. Chem.
276,
31596-31601 |
10. | Bockaert, J., Fagni, L., and Dumuis, A. (1997) in Handbook of Experimental Pharmacology: Serotoninergic Neurons and 5-HT Receptors in the CNS (Baumgarten, H. G. , and Göthert, M., eds), Vol. 129 , pp. 439-465, Spinger-Verlag, Berlin |
11. |
Mialet, J.,
Dahmoune, Y.,
Lezoualc'h, F.,
Berque-Bestel, I.,
Eftekhari, P.,
Hoebeke, J.,
Sicsic, S.,
Langlois, M.,
and Fischmeister, R.
(2000)
Br. J. Pharmacol.
130,
527-538 |
12. |
Joubert, L.,
Claeysen, S.,
Sebben, M.,
Bessis, A. S.,
Clark, R. D.,
Martin, R. S.,
Bockaert, J.,
and Dumuis, A.
(2002)
J. Biol. Chem.
277,
25502-25511 |
13. | Clark, R. D. (1998) in 5-HT4 Receptors in the Brain and Periphery (Eglen, R. M., ed) , pp. 1-48, Springer-Verlag, Berlin |
14. | Buchheit, K. H., Gamse, R., Giger, R., Hoyer, D., Klein, F., Klöppner, E., Pfannküche, H. J., and Mattes, H. (1995) J. Med. Chem. 38, 2326-2330[Medline] [Order article via Infotrieve] |
15. | Dumuis, A., Bouhelal, R., Sebben, M., Cory, R., and Bockaert, J. (1988) Mol. Pharmacol. 34, 880-887[Abstract] |
16. | Ansanay, H., Sebben, M., Bockaert, J., and Dumuis, A. (1996) Eur. J. Pharmacol. 298, 165-174[CrossRef][Medline] [Order article via Infotrieve] |
17. | Munson, P. J., and Rodbard, D. (1980) Anal. Biochem. 107, 220-239[Medline] [Order article via Infotrieve] |
18. | Cheng, Y., and Prusoff, W. H. (1973) Biochem. Pharmacol 22, 3099-3108[CrossRef][Medline] [Order article via Infotrieve] |
19. | Strader, C., Fong, T., Tota, M., Underwood, D., and Dixon, R. (1994) Annu. Rev. Biochem. 63, 101-132[CrossRef][Medline] [Order article via Infotrieve] |
20. | Buchheit, K., Gamse, R., Giger, R., Hoyer, D., Klein, F., Klöppner, E., Pfannfucke, H., and Mattes, H. (1995) J. Med. Chem. 38, 2331-2338[Medline] [Order article via Infotrieve] |
21. | Dumuis, A., Sebben, M., Monferini, E., Nicola, M., Ladinsky, H., and Bockaert, J. (1991) Naunyn-Schmiedeberg's Arch. Pharmacol. 343, 245-251[Medline] [Order article via Infotrieve] |
22. | Langlois, M., Zhang, L., Yang, D., Brémont, B., Shen, S., Manara, L., and Croci, T. (1994) Bioorg. Med. Chem. Lett. 4, 1433-1436[CrossRef] |
23. | Yang, D., Soulier, J. L., Sicsic, S., Mathe-Allainmat, M., Bremont, B., Croci, T., Cardamone, R., Aureggi, G., and Langlois, M. (1997) J. Med. Chem. 40, 608-621[CrossRef][Medline] [Order article via Infotrieve] |
24. | Blondel, O., Gastineau, M., Langlois, M., and Fischmeister, R. (1998) Br. J. Pharmacol. 125, 595-597[Abstract] |
25. | Grossman, C. J., Kilpatrick, G. J., and Bunce, K. T. (1993) Br. J. Pharmacol. 109, 618-624[Abstract] |
26. | Lopez-Rodriguez, M. L., Murcia, M., Benhamu, B., Viso, A., Campillo, M., and Pardo, L. (2001) Bioorg. Med. Chem. Lett. 11, 2807-2811[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Roth, B. L.,
Shoham, M.,
Choudhary, M. S.,
and Khan, N.
(1997)
Mol. Pharmacol.
52,
259-266 |
28. | Lopez-Rodriguez, M. L., Benhamu, B., Viso, A., Morcillo, M. J., Murcia, M., Orenzanz, L., Alfaro, M. J., and Martin, M. I. (1999) Bioorg. Med. Chem. 7, 2271-2281[CrossRef][Medline] [Order article via Infotrieve] |
29. | Kii, Y., and Ito, T. (1997) J. Cardiovasc. Pharmacol. 29, 670-675[CrossRef][Medline] [Order article via Infotrieve] |
30. | Kaumann, A. J., Lynham, J. A., and Brown, A. M. (1996) Naunyn-Schmiedeberg's Arch. Pharmacol. 353, 592-595[Medline] [Order article via Infotrieve] |