JPET #138271 Transposition of Three Amino Acids Transforms the

50 Pages·2008·0.41 MB·English

JPET Fast Forward. Published on April 22, 2008 as DOI: 10.1124/jpet.108.138271
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JPET #138271
Transposition of Three Amino Acids Transforms the Human Metabotropic
Glutamate Receptor (mGluR)-3 Positive Allosteric Modulation Site to
mGluR2, and additional Characterization of the mGluR2 Positive Allosteric
Modulation Site
Blake A. Rowe, Hervé Schaffhauser, Sylvia Morales, Laura S. Lubbers, Celine
Bonnefous, Theodore M. Kamenecka, Jeffrey McQuiston and Lorrie P. Daggett.
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Merck Research Laboratories, West Point, PA, USA (B.A.R., H.S., L.S.L.); Merck n
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Research Laboratories, San Diego, CA, USA (S.M., C.B., T.M.K., J.M., L.P.D.) d fro
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Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.
JPET Fast Forward. Published on April 22, 2008 as DOI: 10.1124/jpet.108.138271
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JPET #138271
Running title: A three amino acid change transforms mGluR3 potentiator.
Corresponding author: Blake A. Rowe
770 Sumneytown Pike, P.O. Box 4
West Point, PA. 19486-0004
WP26-354
Phone: [phone], FAX: [phone]
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email: [email protected] n
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Number of text pages: 42 jp
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Number of tables: 4 tjo
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Number of figures: 8 ls
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References: 40 t A
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Number of words: J
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Abstract: 175 ls
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Introduction: 708 eb
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Discussion: 1354 1
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Recommended section: Neuropharmacology
Abbreviations: ANOVA, analysis of variance; C-, carboxyl; DCG-IV, (2S,2'R,3')-
dicarboxycyclopropyl)glycine; GPT, pyridoxal phosphate and pyruvate; GPCRs, G-protein-
coupled receptors; HBS, HEBES-buffered saline; hmGluR, human metabotropic glutamate
receptor; IP , inositol monophosphate; IP , inositol 1,4,5-trisphosphate; MRLSD-650, 2-[(6, 7-
1 3
JPET Fast Forward. Published on April 22, 2008 as DOI: 10.1124/jpet.108.138271
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dichloro-2-cyclopentyl-2-methyl-1-oxo-2, 3-dihydro-1H-inden-5-yl) oxy]-N-[4-(1H-tetraazol-5-yl)
phenyl] acetamide; MRLSD-230, 3'-((2-cyclopentyl-6,7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5-
yloxy)methyl)biphenyl-4-carboxylic acid; MRLSD-041, 6,7-dichloro-2-propyl-5-(3-((pyridin-4-
ylthio)methyl)benzyloxy)-2,3-dihydro-1H-inden-1-one; MRLSD-973, 2,2,2-trifluoro-N-(pyrimidin-5-
ylmethyl)-N-(3-(2-(trifluoromethyl) benzyl)phenyl) ethanesulfonamide; MRLSD-064, 1-(4-((3'-(1H-
tetrazol-5-yl)biphenyl-3-yl)methoxy)-2-hydroxy-3-methylphenyl)-3,3-dimethylbutan-1-one;
MRLSD-726, 1-(4-((3'-(1H-tetrazol-5-yl)biphenyl-3-yl)methoxy)-3-bromo-2-hydroxyphenyl)-3-
methylbutan-1-one; MRLSD-438, 1-(4-(3-(3-(2H-tetrazol-5-yl)phenoxy) benzyloxy)-3-bromo-2-
D
hydroxyphenyl)-3-methylbutan-1-one; MRLSD-667, 3'-((4-(3,3-dimethylbutanoyl)-3-hydroxy-2- o
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methylphenoxy) methyl)biphenyl-3-carboxylic acid; NT, nucleotides; PI, phosphoinositide; TM, a
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JPET Fast Forward. Published on April 22, 2008 as DOI: 10.1124/jpet.108.138271
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JPET #138271
Abstract
Glutamate is a major neurotransmitter in the CNS, and abnormal glutamate
neurotransmission has been implicated in many neurological disorders including
schizophrenia, Alzheimer’s disease, Parkinson's disease, addiction, anxiety,
depression, epilepsy and pain. Metabotropic glutamate receptors (mGluRs)
activate intracellular signaling cascades in a G-protein-dependent manner, which
offer the opportunity for developing drugs that regulate glutamate
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neurotransmission in a functionally selective manner. In the present study, we lo
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further characterize the human-mGluR2 (hmGluR2) potentiator binding site by fro
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showing that the substitution of the three amino acids found to be required for et.a
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hmGluR2 potentiation, specifically S688, G689 and N735 with the homologous tjo
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hmGluR3 amino acids, inactivates the positive allosteric modulator activity of .o
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several structurally unique mGluR2 potentiators. Based on the characterization t A
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of the hmGluR2 potentiator binding site, we developed a novel scintillation J
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proximity assay (SPA) that was able to discriminate between compounds that ls o
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were hmGluR2 specific potentiators, and those that were active on both bru
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hmGluR2 and hmGluR3. In addition, we substituted S688, G689 and N735 into 1, 2
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hmGluR3 and created an active hmGluR2 allosteric modulation site on the
hmGluR3 receptor.
JPET Fast Forward. Published on April 22, 2008 as DOI: 10.1124/jpet.108.138271
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JPET #138271
Introduction
Glutamate is the primary excitatory neurotransmitter in the mammalian central
nervous system and its activity is mediated by two classes of receptors:
ionotropic glutamate receptors (iGluRs), which are multimeric ion channels
responsible for fast synaptic transmission, and metabotropic glutamate receptors
(mGluRs), which are 7-transmembrane receptors that couple to G-proteins to
modulate slower synaptic transmission through intracellular second messengers.
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Because iGluRs are expressed by nearly all types of neurons and mediate fast lo
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excitatory neurotransmission throughout the brain, direct pharmacological fro
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manipulation of this group of receptors has been difficult because inhibition could et.a
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produce widespread disruption of brain function, which could lead to negative tjo
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side effects. On the other hand, mGluRs which activate intracellular signaling .o
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cascades offer an opportunity for developing drugs that regulate glutamate t A
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neurotransmission in a functionally selective manner. J
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mGluRs belong to the G-protein coupled receptors (GPCRs) super family and ls o
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have eight known subtypes (mGluR1-8). These receptors divide into three bru
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groups based upon sequence homology, intracellular messengers and , 2
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pharmacology. Group I receptors (mGluR1, 5) activate phospholipase C via G
q
proteins and initiate an inositol triphosphate/diacylglycerol (IP /DAG) second
3
messenger cascade. Group II (mGluR2, 3) and Group III (mGluR4, 6, 7, 8)
receptors inhibit adenylate cyclase via G proteins (Schoepp, 1994; Conn and
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Pin, 1997; Schoepp, 2001).
JPET Fast Forward. Published on April 22, 2008 as DOI: 10.1124/jpet.108.138271
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mGluR2 and mGluR3 are located primarily presynaptically in the
hippocampus, cortex, striatum, thalamus and amygdala (Ohishi et al., 1993a).
Activation of these receptors has been shown to decrease synaptic transmission
and glutamate release in the hippocampus (Macek et al., 1996). The distribution
and prominent functions of group II mGluRs in neuronal excitability and synaptic
transmission suggests that modulation of these GPCRs is a promising strategy
for the treatment of neurological and neuropsychiatric disorders, such as anxiety,
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schizophrenia and pain (Conn and Pin, 1997; Chavez-Noriega et al., 2002; n
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Varney and Gereau, 2002; Lorrain et al., 2003). Furthermore, mGluR mediated d fro
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glutamate activity has been shown to influence calcium and potassium ion jp
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channels, modulate NMDA and AMPA receptor currents, and GABA release (Lea tjo
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and Faden, 2003). The ability of mGluRs to modulate neuroprotective and ls
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neurotoxic systems suggests that some mGluRs play a role in neuronal injury t A
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following trauma. Indeed, studies have shown group II and III mGluRs to have a J
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neuroprotective role (for review see Lea and Faden, 2003). ls
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Several lines of evidence suggest that positive allosteric modulators of the eb
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mGluRs may offer advantages over classical GPCR agonists by overcoming 1
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receptor desensitization which often occurs after repeated dosing of agonists
(Pin et al., 2001; May and Christopoulos, 2003). Also, in contrast to full agonists,
potentiators can be designed to be devoid of any agonist activity or act as weak
partial agonists. However, when agonist is present, they can significantly
increase potency and/or efficacy of the agonist. Development of potentiators
could also be useful in disease states in which the level of the endogenous ligand
JPET Fast Forward. Published on April 22, 2008 as DOI: 10.1124/jpet.108.138271
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is attenuated. Patients administered a potentiator could achieve normal agonist
responses. Due to the high level of conservation of the agonist binding site of
mGluRs, developing subtype-specific agonists has proven to be difficult. To date
no agonist has been reported to discriminate between mGluR2 and mGluR3.
However, as described in this paper and previously, several mGluR2 specific
positive allosteric modulators have been identified (Johnson et al., 2003;
Schaffhauser et al., 2003; Hu et al., 2004; Pinkerton et al., 2004a; Pinkerton et
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al., 2004b; Bonnefous et al., 2005; Pinkerton et al., 2005). n
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The present study had three main goals. First, we wanted to provide d fro
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additional pharmacological characterizations of a series of novel and structurally jp
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distinct mGluR2 potentiators. We tested two indanones (Pinkerton et al., 2005), tjo
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a biphenyl-indanone (Bonnefous et al., 2005), and a pyrimidine methyl alinine ls
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(Hu et al., 2004). Second, we wanted to determine if this diverse set of mGluR2 t A
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allosteric modulators also binds to the known mGluR2 potentiator site defined by J
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amino acids S688, G689 and N735 (Schaffhauser et al., 2003) or determine if als
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another novel potentiator site exist. Third we wanted to develop a high- eb
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throughput transfection system and dual scintillation proximity assay (SPA) using 1
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hmGluR2-WT and the hmGluR2_L688, V689 and D735 expressing cells. With these 3
assays, we could easily identify hmGluR2 specific potentiators (i.e. positive only
in the hmGluR2-WT assay) versus allosteric modulators of both hmGluR2 and
hmGluR3 (i.e. active in both hmGluR2-WT and the hmGluR2_L688, V689 and D735
assays).
JPET Fast Forward. Published on April 22, 2008 as DOI: 10.1124/jpet.108.138271
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Methods
Materials: Glutamate, guanosine 5’-diphosphate (GDP), guanosine 5’[γ-thio]-
triphosphate (GTPγS), glutamate pyruvate transaminase (GPT), coenzyme
pyridoxal phosphate, and pyruvate were obtained from Sigma Chemical (St
Louis, MO). [35S]-GTPγS (1250 Ci/mmol), Unifilter GF/B plates and Microscint 20
were purchased from Perkin-Elmer Life Science (Boston, MA). (2S,2’R,3’R)-2-
(2’,3’)-dicarboxycyclopropyl)glycine (DCG-IV) was purchased from Tocris- D
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Cookson Inc. (Ellisville, Missouri). Male Sprague-Dawley rats (250-300g) were loa
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purchased from Harlan (Indianapolis, IN). The pcDNA3.0, pcDNA3.1-based fro
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mammalian expression vectors were purchased from In-Vitrogen (San Diego, t.a
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CA). A QuickChange site-directed mutagenesis kit was obtained from u
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Stratagene (San Diego, CA) and an Effectene kit was purchased from Qiagen rg
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(Valencia, CA). BioCoat Fibronectin 24-well tissue culture plates were obtained S
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from BD Biosciences (Bedford, MA). Dowex-1-X8 (200-300 mesh in the formate ou
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form) was obtained from BioRad (Hercules, CA). Myo [2-3H]-inositol (S.A. 18 o
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Ci/mmol TRK 911) and RNA Binding YSi SPA Beads (RPQ0625) were ru
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purchased from GE Healthcare-Amersham (Piscataway, NJ). Protease inhibitor , 2
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cocktail was obtained from Roche Diagnostics (Indianapolis, IN). Picoplate 96-
well Topcount plates and TopSeal were purchased from Packard Biosciences
Inc. (Prospect, CT). Compounds: MRLSD-650, MRLSD-230, MRLSD-041 and
MRLSD-973 (figure 1 A-D) have described previously (Hu et al., 2004;
Bonnefous et al., 2005; Pinkerton et al., 2005), and MRLSD-064, MRLSD-726,
MRLSD-438 and MRLSD-667 (figure 1 E-H) are novel (patents pending:
JPET Fast Forward. Published on April 22, 2008 as DOI: 10.1124/jpet.108.138271
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WO2004/018386; WO2006/014918; WO2006/015158 and WO2006/091496). All
MRLSD-compounds were synthesized at Merck Research Laboratories.
Membrane preparations. Membranes were isolated from hmGluR2 and
hmGluR3-expressing stable cell lines as previously described (Schaffhauser et
al., 2003). Briefly, confluent cells were washed into PBS and harvested by
centrifugation (200g). The resulting cell pellet was 1) homogenized in buffer A
(20 mM HEPES and 10 mM EDTA, pH 7.4) and centrifuged at 40,000g (20 min
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at 4°C), 2) the resulting pellet was washed 1X in buffer A then 1X in buffer B (20 n
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mM HEPES and 0.1 mM EDTA, pH 7.4) and 3) re-suspended in buffer B. d fro
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Membranes were stored at –80°C; protein concentration ~1 mg/ml. jp
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Whole rat brain membranes were isolated as previously described tjo
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(Schaffhauser et al, 2003). Briefly, rats were decapitated and whole brains were ls
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homogenized in 6 volumes (w/v) of 10% sucrose at 4°C using a glass-Teflon t A
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homogenizer. The homogenate was centrifuged (1000g, 10 min), supernatant J
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was removed and centrifuged again (40,000g, 20 min at 4°C). The pellet was re- ls
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suspended in 25 ml water and centrifuged (8,000g,10 min). The supernatant and eb
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the buffy coat layer were removed and centrifuged (40,000g, 20 min at 4°C). The 1
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pellet was re-suspended in buffer C (5 mM HEPES-KOH, pH 7.4), freeze-thawed
twice and then centrifuged at 40,000g for 20 min. The resulting pellet was re-
suspended in buffer C and stored at –80°C at a protein concentration of ~3
mg/ml. Protein measurements were determined with the Bio-Rad DC kit (Bio-
Rad, Hercules, CA).
JPET Fast Forward. Published on April 22, 2008 as DOI: 10.1124/jpet.108.138271
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[35S]-GTPγS binding assay. This assay was performed as described in
Schaffhauser et al. (2003). Briefly, mGluR2, mGluR3 or rat brain membranes
were thawed, further processed then re-suspended in assay buffer (20 mM
HEPES (pH 7.4), 100 mM NaCl, 3 mM MgCl ) to a final protein concentration of
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0.5 mg/ml (hmGluR2 and hmGluR3 expressing membranes) or 0.1 mg/ml (rat
brain). In a Beckmann 96-well plate, test compounds were added (300 pM-10
µM) along with 5 µM GDP, membrane (10 µg/well rat brain and 50 µg for
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recombinant cells) and 0.05 nM [35S]-GTPγS (total volume= 0.5 ml). Plates were n
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incubated at 30°C for 1 hour then the assay was terminated by rapid filtration fro
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(Unifilter GF/B plate with 96-well cell harvester (Brandel Gaithersburg, MD)). e
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Plates were then rinsed, dried and Microscint 20 added to each well, followed by tjo
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counting in a Topcount scintillation counter (Perkin-Elmer, Boston MA). ls.o
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Cloning of wild-type hmGluR2 and hmGluR3 cDNAs. Cloning of wild-type t A
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(WT) hmGluR2 cDNA (Flor et al., 1995; Schaffhauser et al., 2003) and WT J
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hmGluR3 cDNA were performed as described previously (Varney et al., 1998; ls
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Schaffhauser et al., 2003) and then cloned into pcDNA 3.0-based mammalian bru
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expression vectors. , 2
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Construction of chimeric and point mutations of hmGluR2/3 and
hmGluR3/2 cDNAs. The mGluR2 and mGluR3 chimeric receptor cDNAs were
generated by site-directed mutagenesis from WT cDNAs to introduce single or
multiple mutations as previously described (Schaffhauser, 2003). All of the
single- and multiple-point mutations of hmGluR2/3 receptor cDNAs were