U.S. patent application number 10/300473 was filed with the patent office on 2003-06-19 for chimeric receptors and methods for identifying compounds active at metabotropic glutamate receptors and the use of such compounds in the treatment of neurological disorders and diseases.
This patent application is currently assigned to NPS Pharmaceuticals. Invention is credited to Fuller, Forrest H., Hammerland, Lance G., Krapcho, Karen J., Storjohann, Laura L., Stormann, Thomas M..
Application Number | 20030113873 10/300473 |
Document ID | / |
Family ID | 21696494 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030113873 |
Kind Code |
A1 |
Fuller, Forrest H. ; et
al. |
June 19, 2003 |
Chimeric receptors and methods for identifying compounds active at
metabotropic glutamate receptors and the use of such compounds in
the treatment of neurological disorders and diseases
Abstract
The present invention provides chimeric receptors. The chimeric
receptors comprise at least one region homologous to a region of a
metabotropic glutamate receptor and at least one region homologous
to a region of a calcium receptor. The invention also includes
methods of preparing such chimeric receptors, and methods of using
such receptors to identify and characterize compounds which
modulate the activity of metabotropic glutamate receptors or
calcium receptors. The invention also relates to compounds and
methods for modulating metabotropic glutamate receptor activity and
binding to metabotropic glutamate receptors. Modulation of
metabotropic glutamate receptor activity can be used for different
purposes such as treating neurological disorders and diseases,
inducing an analgesic effect, cognition enhancement, and inducing a
muscle-relaxant effect.
Inventors: |
Fuller, Forrest H.; (La
Jolla, CA) ; Hammerland, Lance G.; (Bountiful,
UT) ; Krapcho, Karen J.; (Salt Lake City, UT)
; Storjohann, Laura L.; (Salt Lake City, UT) ;
Stormann, Thomas M.; (Salt Lake City, UT) |
Correspondence
Address: |
NPS PHARMACEUTICALS, INC. C/O FOLEY & LARDNER
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Assignee: |
NPS Pharmaceuticals
|
Family ID: |
21696494 |
Appl. No.: |
10/300473 |
Filed: |
November 19, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10300473 |
Nov 19, 2002 |
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09435897 |
Nov 8, 1999 |
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6534289 |
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09435897 |
Nov 8, 1999 |
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08687289 |
Jul 25, 1996 |
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5981195 |
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60001526 |
Jul 26, 1995 |
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Current U.S.
Class: |
435/69.7 ;
435/325; 435/455; 435/7.2 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 25/04 20180101; A61P 25/00 20180101; A01K 2217/05 20130101;
C07K 14/70571 20130101; A61K 38/00 20130101; C07K 2319/00 20130101;
C07K 14/705 20130101 |
Class at
Publication: |
435/69.7 ;
435/7.2; 435/455; 435/325 |
International
Class: |
G01N 033/53; G01N
033/567; C12P 021/04; C12N 005/06; C12N 015/85 |
Claims
What is claimed is:
1. A method of screening for a compound that binds to a
metabotropic glutamate receptor or a calcium receptor, comprising
the steps of: a. preparing a nucleic acid sequence encoding a
fragment of a receptor, b. inserting said sequence into a
replicable expression vector capable of expressing said fragment in
a host cell, c. transforming a host cell with the vector of (b), d.
recovering the fragment from said host cell, e. introducing said
fragment and said compound into an acceptable medium, and f.
monitoring the binding of the compound to the fragment by
physically detectable means.
2. The method of claim 1, wherein said receptor is a metabotropic
glutamate receptor.
3. The method of claim 2, wherein said fragment comprises an
extracellular domain of said metabotropic glutamate receptor.
4. The method of claim 2, wherein said fragment comprises a seven
transmembrane domain of said metabotropic glutamate receptor.
5. The method of claim 2 wherein said fragment comprises a seven
transmembrane domain and a cytoplasmic tail domain of a
metabotropic glutamate receptor.
6. The method of claim 1 wherein said receptor is a calcium
receptor.
7. The method of claim 6 wherein said fragment comprises an
extracellular domain of said calcium receptor.
8. The method of claim 6 wherein said fragment comprises a seven
transmembrane domain of said calcium receptor.
9. The method of claim 6 wherein said fragment comprises a seven
transmembrane domain and a cytoplasmic tail domain of said calcium
receptor.
10. A method of screening for a compound that binds to or modulates
a metabotropic glutamate receptor or a calcium receptor, comprising
the steps of: a. preparing a nucleic acid sequence encoding a
fragment of a receptor, b. inserting said sequence into a
replicable expression vector capable of expressing said fragment in
a host cell, c. transforming a host cell with the vector of (b), d.
introducing said transformed host cell and said compound into an
acceptable medium, and e. monitoring the effect of said compound on
said host cell.
11. The method of claim 10, wherein said fragment comprises the
seven transmembrane domain and cytoplasmic tail domain of a
metabotropic glutamate receptor.
12. The method of claim 10, wherein said fragment comprises the
seven transmembrane domain and cytoplasmic tail domain of a calcium
receptor.
13. A method of screening for a compound that binds to or modulates
a receptor, comprising the steps of: a. preparing a nucleic acid
sequence encoding a first fragment comprising a fragment of a first
receptor, b. inserting the sequence into a replicable expression
vector capable of expressing said first fragment in a host cell, c.
transforming a host cell with the vector of (b), d. recovering the
first fragment from the host cell, e. preparing a nucleic acid
sequence encoding a second fragment comprising a fragment of a
second receptor, f. inserting the sequence of (e) into a replicable
expression vector capable of expressing said second fragment in a
host cell, g. transforming a host cell with the vector of (f), h.
recovering the second fragment from the host cell of (g), and i.
introducing said first fragment and said second fragment and said
compound into an acceptable medium, and j. monitoring the binding
and/or modulation of the compound by physically detectable
means.
14. The method of claim 13, wherein said first fragment comprises
the extracellular domain of a metabotropic glutamate receptor, and
said second fragment comprises the seven transmembrane domain and
the cytoplasmic tail domain of a calcium receptor.
15. The method of claim 13, wherein said first fragment comprises
the extracellular domain and the seven transmembrane domain of a
metabotropic glutamate receptor, and said second fragment comprises
the cytoplasmic tail domain of a calcium receptor.
16. The method of claim 13 whereinsaid first fragment comprises the
extracellular domain of a calcium receptor, and said second
fragment comprises the seven transmembrane domain and the
cytoplasmic tail domain of a metabotropic glutamate receptor.
17. The method of claim 13 wherein said first fragment comprises
the extracellular domain of a calcium receptor, andsaid second
fragment comprises the seven transmembrane domain of a metabotropic
glutamate receptor and the cytoplasmic tail domain of a calcium
receptor.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This is a divisional application of U.S. Ser. No.
09/435,897, which is a divisional application of U.S. Ser. No.
08/687,289 filed Jul. 25, 1996, now U.S. Pat. No. 5,981,195, which
was based on Provisional Application, Forrest H. Fuller et al.,
U.S. Ser. No. 60/001,526, filed Jul. 26, 1995, entitled CHIMERIC
RECEPTORS AND METHODS FOR IDENTIFYING COMPOUNDS ACTIVE AT
METABOTROPIC GLUTAMATE RECEPTORS AND THE USE OF SUCH COMPOUNDS IN
THE TREATMENT OF NEUROLOGICAL DISORDERS AND DISEASES, which are
incorporated herein by reference in their entireties including
drawings.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to chimeric receptors
containing one or more regions homologous to a metabotropic
glutamate receptor and one or more regions homologous to a calcium
receptor.
[0003] The following description provides a summary of information
relevant to the present invention. It is not an admission that any
of the information provided herein is prior art to the presently
claimed invention, nor that any of the publications specifically or
implicitly referenced are prior art to that invention.
[0004] Glutamate is the major excitatory neurotransmitter in the
mammalian brain. Glutamate produces its effects on central neurons
by binding to and thereby activating cell surface receptors. These
receptors have been subdivided into two major classes, the
ionotropic and metabotropic glutamate receptors, based on the
structural features of the receptor proteins, the means by which
the receptors transduce signals into the cell, and pharmacological
profiles.
[0005] The ionotropic glutamate receptors (iGluRs) are ligand-gated
ion channels that, upon binding glutamate, open to allow the
selective influx of certain monovalent and divalent cations,
thereby depolarizing the cell membrane. In addition, certain iGluRs
with relatively high calcium permeability can activate a variety of
calcium-dependent intracellular processes. These receptors are
multisubunit protein complexes that may be homomeric or heteromeric
in nature. The various iGluR subunits all share common structural
motifs, including a relatively large amino-terminal extracellular
domain (ECD), followed by a multiple transmembrane domain (TMD)
comprising two membranespanning regions (TMs), a second smaller
intracellular loop, and a third TM, before terminating with an
intracellular carboxy-terminal domain (CT). Historically the iGluRs
were first subdivided pharmacologically into three classes based on
preferential activation by the agonists
alpha-amino-3-hydroxy-5-methyl-is- oxazole-4-propionic acid (AMPA),
kainate (KA), and N-methyl-D-aspartate (NMDA). Later, molecular
cloning studies coupled with additional pharmacological studies
revealed a greater diversity of iGluRs, in that multiple subtypes
of AMPA, KA and NMDA receptors are expressed in the mammalian CNS
(Hollman and Heinemann, Ann. Rev. Neurosci. 7:31, 1994).
[0006] The metabotropic glutamate receptors (mGluRs) are G
protein-coupled receptors capable of activating a variety of
intracellular second messenger systems following the binding of
glutamate or other potent agonists including quisqualate and
1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) (Schoepp et
al., Trends Pharmacol. Sci. 11:508, 1990; Schoepp and Conn, Trends
Pharmacol. Sci. 14:13, 1993).
[0007] Activation of different metabotropic glutamate receptor
subtypes in situ elicits one or more of the following responses:
activation of phospholipase C, increases in phosphoinositide (PI)
hydrolysis, intracellular calcium release, activation of
phospholipase D, activation or inhibition of adenylyl cyclase,
increases and decreases in the formation of cyclic adenosine
monophosphate (cAMP), activation of guanylyl cyclase, increases in
the formation of cyclic guanosine monophosphate (cGMP), activation
of phospholipase A.sub.2, increases in arachidonic acid release,
and increases or decreases in the activity of voltage- and
ligand-gated ion channels (Schoepp and Conn, Trends Pharmacol. Sci.
14:13, 1993; Schoepp, Neurochem. Int. 24:439, 1994; Pin and
Duvoisin, Neuropharmacology 34:1, 1995).
[0008] Thus far, eight distinct mGluR subtypes have been isolated
via molecular cloning, and named mGluR1 to mGluR8 according to the
order in which they were discovered (Nakanishi, Neuron 13:1031,
1994, Pin and Duvoisin, Neuropharmacology 34:1, 1995; Knopfel et
al., J. Med. Chem. 38:1417, 1995). Further diversity occurs through
the expression of alternatively spliced forms of certain mGluR
subtypes (Pin et al., PNAS 89:10331, 1992; Minakami et al., BBRC
199:1136, 1994). All of the mGluRs are structurally similar, in
that they are single subunit membrane proteins possessing a large
amino-terminal extracellular domain (ECD) followed by seven
putative transmembrane domain (7TMD) comprising seven putative
membrane spanning helices connected by three intracellular and
three extracellular loops, and an intracellular carboxy-terminal
domain of variable length (cytoplasmic tail) (CT) (see, Schematic
FIG. 1a).
[0009] The eight mGluRs have been subdivided into three groups
based on amino acid sequence identities, the second messenger
systems they utilize, and pharmacological characteristics
(Nakanishi, Neuron 13:1031, 1994; Pine and Duvoisin,
Neuropharmacology 34:1, 1995; Knopfel et al., J. Med. Chem.
38:1417, 1995). The amino acid identity between mGluRs within a
given group is approximately 70% but drops to about 40% between
mGluRs in different groups. For mGluRs in the same group, this
relatedness is roughly paralleled by similarities in signal
transduction mechanisms and pharmacological characteristics.
[0010] The Group I mGluRs comprise mGluR1, mGluR5 and their
alternatively spliced variants. The binding of agonists to these
receptors results in the activation of phospholipase C and the
subsequent mobilization of intracellular calcium. For example,
Xenopus oocytes expressing recombinant mGluR1 receptors have been
utilized to demonstrate this effect indirectly by
electrophysiological means (Masu et al., Nature 349:760, 1991; Pin
et al., PNAS 89:10331, 1992). Similar results were achieved with
oocytes expressing recombinant mGluR5 receptors (Abe et al., J.
Biol. Chem. 267:13361, 1992; Minakami et al., BBRC 199:1136, 1994).
Alternatively, agonist activation of recombinant mGluR1 receptors
expressed in Chinese hamster ovary (CHO) cells stimulated PI
hydrolysis, cAMP formation, and arachidonic acid release as
measured by standard biochemical assays (Aramori and Nakanishi,
Neuron 8:757, 1992). In comparison, activation of mGluR5 receptors
expressed in CHO cells stimulated PI hydrolysis and subsequent
intracellular calcium transients but no stimulation of cAMP
formation or arachidonic acid release was observed (Abe et al., J.
Biol. Chem. 267:13361, 1992). The agonist potency profile for Group
I mGluRs is quisqualate>glutamate=ibotenate&-
gt;(2S,1'S,2'S)-2-carboxycyclopropyl)glycine
(L-CCG-I)>(1S,3R)-1-aminoc- yclopentane-1,3-dicarboxylic acid
(ACPD). Quisqualate is relatively selective for Group I receptors,
as compared to Group II and Group III mGluRs, but it also potently
activates ionotropic AMPA receptors (Pin and Duvoisin,
Neuropharmacology, 34:1, Knopfel et al., J. Med. Chem. 38:1417,
1995).
[0011] The Group II mGluRs include mGluR2 and mGluR3. Activation of
these receptors as expressed in CHO cells inhibits adenylyl cyclase
activity via the inhibitory G protein, G.sub.i, in a pertussis
toxin-sensitive fashion (Tanabe et al., Neuron 8:169, 1992; Tanabe
et al., Neurosci. 13:1372, 1993). The agonist potency profile for
Group II receptors is
L-CCG-I>glutamate>ACPD>ibotenate>quisqualate.
Preliminary studies suggest that L-CCG-I and
(2S,11'R,2'R,3'R)-2-(2,3-dicarboxycyclop- ropyl)glycine (DCG-IV)
are both relatively selective agonists for the Group II receptors
(Knopfel et al., J. Med. Chem. 38:1417, 1995).
[0012] The Group III mGluRs include mGluR4, mGluR6, mGluR7 and
mGluR8. Like the Group II receptors these mGluRs are negatively
coupled to adenylate cyclase to inhibit intracellular cAMP
accumulation in a pertussis toxin-sensitive fashion when expressed
in CHO cells (Tanabe et al., J. Neurosci. 13:1372, 1993; Nakajima
et al., J. Biol. Chem. 268:11868, 1993; Okamoto et al., J. Biol.
Chem. 269:1231, 1994; Duvoisin et al., J. Neurosci. 15:3075, 1995).
As a group, their agonist potency profile is
(S)-2-amino-4-phosphonobutyric acid (L-AP4)>glutamate>AC-
PD>quisqualate, but mGluR8 may differ slightly with glutamate
being more potent than L-AP4 (Knopfel et al., J. Med. Chem.
38:1417, 1995; Duvoisin et al., J. Neurosci. 15:3075, 1995). Both
L-AP4 and (S)-serine-O-phosphate (L-SOP) are relatively selective
agonists for the Group III receptors.
[0013] Finally, the eight mGluR subtypes have unique patterns of
expression within the mammalian CNS that in many instances are
overlapping (Masu et al., Nature 349:760, 1991; Martin et al.,
Neuron 9:259, 1992; Ohishi et al., Neurosci. 53:1009, 1993; Tanabe
et al., J. Neurosci. 13:1372; Ohishi et al., Neuron 13:55, 1994,
Abe et al., J. Biol. Chem. 267:13361, 1992; Nakajima et al., J.
Biol. Chem. 268:11868, 1993; Okamoto et al., J. Biol. Chem.
269:1231, 1994; Duvoisin et al., J. Neurosci. 15:3075, 1995). As a
result certain neurons may express only one particular mGluR
subtype, while other neurons may express multiple subtypes that may
be localized to similar and/or different locations on the cell
(i.e., postsynaptic dendrites and/or cell bodies versus presynaptic
axon terminals). Therefore, the functional consequences of mGluR
activation on a given neuron will depend on the particular mGluRs
being expressed; the receptors' affinities for glutamate and the
concentrations of glutamate the cell is exposed to; the signal
transduction pathways activated by the receptors; and the locations
of the receptors on the cell. A further level of complexity may be
introduced by multiple interactions between mGluR expressing
neurons in a given brain region. As a result of these complexities,
and the lack of subtype-specific mGluR agonists and antagonists,
the roles of particular mGluRs in physiological and
pathophysiological processes affecting neuronal function are not
well defined. Still, work with the available agonists and
antagonists have yielded some general insights about the Group I
mGluRs as compared to the Group II and Group III mGluRs.
[0014] Attempts at elucidating the physiological roles of Group I
mGluRs suggest that activation of these receptors elicits neuronal
excitation. Various studies have demonstrated that ACPD can produce
postsynaptic excitation upon application to neurons in the
hippocampus, cerebral cortex, cerebellum, and thalamus as well as
other brain regions. Evidence indicates that this excitation is due
to direct activation of postsynaptic mGluRs, but it has also been
suggested to be mediated by activation of presynaptic mGluRs
resulting in increased neurotransmitter release (Baskys, Trends
Pharmacol. Sci. 15:92, 1992; Schoepp, Neurochem. Int. 24:439, 1994;
Pin and Duvoisin, Neuropharmacology 34:1). Pharmacological
experiments implicate Group I mGluRs as the mediators of this
excitation. The effect of ACPD can be reproduced by low
concentrations of quisqualate in the presence of iGluR antagonists
(Hu and Storm, Brain Res. 568:339, 1991; Greene et al. Eur. J.
Pharmacol. 226:279, 1992), and two phenylglycine compounds known to
activate mGluR1, (S)-3-hydroxyphenylglycine ((S)-3HPG) and
(S)-3,5-dihydroxyphenylglycine ((S)-DHPG), also produce the
excitation (Watkins and Collingridge, Trends Pharmacol. Sci.
15:333, 1994). In addition, the excitation can be blocked by
(S)-4-carboxyphenylglycine ((S)-4CPG),
(S)-4-carboxy-3-hydroxyphenylgl- ycine ((S)-4C3HPG) and
(+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG), compounds known
to be mGluR1 antagonists (Eaton et al., Eur. J. Pharmacol. 244:195,
1993; Watkins and Collingridge, Trends Pharmacol. Sci. 15:333,
1994).
[0015] Other studies examining the physiological roles of mGluRs
indicate that activation of presynaptic mGluRs can block both
excitatory and inhibitory synaptic transmission by inhibiting
neurotransmitter release (Pin and Duvoisin, Neuropharmacology
34:1). Presynaptic blockade of excitatory synaptic transmission by
ACPD has been observed on neurons in the visual cortex, cerebellum,
hippocampus, striatum and amygdala (Pin et al., Curr. Drugs:
Neurodegenerative Disorders 1:111, 1993), while similar blockade of
inhibitory synaptic transmission has been demonstrated in the
striatum and olfactory bulb (Calabresi et al., Neurosci. Lett.
139:41, 1992; Hayashi et al., Nature 366:687, 1993). Multiple
pieces of evidence suggest that Group II mGluRs mediate this
presynaptic inhibition. Group II mGluRs are strongly coupled to
inhibition of adenylyl cyclase, like alpha.sub.2-adrenergic and
SHT.sub.1A-serotonergic receptors which are known to mediate
presynaptic inhibition of neurotransmitter release in other
neurons. The inhibitory effects of ACPD can also be mimicked by
L-CCG-I and DCG-IV, which are selective agonists at Group II mGluRs
(Hayashi et al., Nature 366:687, 1993; Jane et al., Br. J.
Pharmacol. 112:809, 1994). Moreover, it has been demonstrated that
activation of mGluR2 can strongly inhibit presynaptic, N-type
calcium channel activity when the receptor is expressed in
sympathetic neurons (Ikeda et al., Neuron 14:1029, 1995), and
inactivation of these channels is known to inhibit neurotransmitter
release. Finally, it has been observed that L-CCG-I, at
concentrations selective for Group II mGluRs, inhibits the
depolarization-evoked release of .sup.3H-aspartate from rat
striatal slices (Lombardi et al., Br. J. Pharmacol. 110: 1407,
1993). Evidence for physiological effects of Group II mGluR
activation at the postsynaptic level is limited. However, one study
suggests that postsynaptic actions of L-CCG-I can inhibit NMDA
receptor activation in cultured mesencephalic neurons (Ambrosini et
al., Mol. Pharmacol. 47:1057, 1995).
[0016] Physiological studies have demonstrated that L-AP4 can also
inhibit excitatory synaptic transmission on a variety of CNS
neurons. Included are neurons in the cortex, hippocampus, amygdala,
olfactory bulb and spinal cord (Koerner and Johnson, Excitatory
Amino Acid Receptors; Design of Agonists and Antagonists p. 308,
1992; Pin et al., Curr. Drugs: Neurodegenerative Disorders 1:111,
1993). The accumulated evidence indicates that the inhibition is
mediated by activation of presynaptic mGluRs. Since the effects of
L-AP4 can be mimicked by L-SOP, and these two agonists are
selective for Group III mGluRs, members of this mGluR group are
implicated as the mediators of the presynaptic inhibition (Schoepp,
Neurochem. Int. 24:439, 1994; Pin and Duvoisin, Neuropharmacology
34:1). In olfactory bulb neurons it has been demonstrated that
L-AP4 activation of mGluRs inhibits presynaptic calcium currents
(Trombley and Westbrook, J. Neurosci. 12:2043, 1992). It is
therefore likely that the mechanism of presynaptic inhibition
produced by activation of Group III mGluRs is similar to that for
Group II mGluRs, i.e., blockade of N-type calcium channels and
inhibition of neurotransmitter release. L-AP4 is also known to act
postsynaptically to hyperpolarize ON bipolar cells in the retina.
It has been suggested that this action may be due to activation of
a mGluR, which is coupled to the cGMP phosphodiesterase in these
cells (Schoepp, Neurochem. Int. 24:439, 1994; Pin and Duvoisin,
Neuropharmacology 34:1).
[0017] Metabotropic glutamate receptor activation studies using
agonists, antagonists and recombinant vertebrate cell lines
expressing mGluRs have been used to evaluate the cellular effects
of the stimulation and the inhibition of different metabotropic
glutamate receptors. For example, agonist stimulation of mGluR1
expressed in Xenopus oocytes demonstrated coupling of receptor
activation to mobilization of intracellular calcium as assessed
indirectly using electrophysiology techniques (Masu et al, Nature
349:760-765, 1991). Agonist stimulation of mGluR1 expressed in CHO
cells stimulated PI hydrolysis, cAMP formation and arachidonic acid
release (Aramori and Nakanishi, Neuron 8:757-765, 1992). Agonist
stimulation of mGluR5 expressed in CHO cells also stimulated PI
hydrolysis which was shown to be associated with a transient
increase in cytosolic calcium as assessed by loading cells with the
fluorescent calcium chelator fura-2 (Abe et al., J. Biol. Chem.
267:13361-13368, 1992). Agonist-induced activation of mGluR1 and
mGluR5 induced PI hydrolysis in CHO cells was not antagonized by
AP3 and AP4, which are both antagonists of glutamate-stimulated PI
hydrolysis in situ (Nicoletti et al., Proc. Natl. Acad. Sci. USA
833:1931-1935, 1986; Schoepp and Johnson, J. Neurochem. 53:273-278,
1989). Agonist stimulation of CHO cells expressing mGluR2 (Tanabe
et al., Neuron 8:169-179, 1992) or mGluR7 (Okamoto et al., J. Biol.
Chem. 269:1231-1236, 1994) resulted in receptor-mediated inhibition
of cAMP formation and also confirmed the ligand specificity
previously observed in situ. Studies using agonists were also
carried out in conjunction with site-directed mutagenesis to reveal
specific amino acids playing important roles in glutamate binding
(O'Hara et al., Neuron 11:41-52, 1993).
[0018] Metabotropic glutamate receptors (mGluRs) have been
implicated in a variety of neurological pathologies including
stroke, head trauma, spinal cord injury, epilepsy, ischemia,
hypoglycemia, anoxia, and neurodegenerative diseases such as
Alzheimer's disease (Schoepp and Conn, Trends Pharmacol. Sci.
14:13, 1993; Cunningham et al., Life Sci. 54: 135, 1994; Pin et
al., Neuropharmacology 34:1, 1995; Knopfel et al., J. Med. Chem.
38:1417, 1995;). A role for metabotropic glutamate receptors in
nociception and analgesia has also been demonstrated (Meller et
al., Neuroreport 4:879, 1993). Metabotropic glutamate receptors
have also been shown to be required for the induction of
hippocampal long-term potentiation and cerebellar long-term
depression (Bashir et al., Nature 363:347, 1993; Bortolotto et al.,
Nature 368:740, 1994; Aiba et. al. Cell 79: 365 and Cell 79: 377,
1994).
[0019] Metabotropic glutamate receptor agonists have been reported
to have effects on various physiological activities. For example,
trans-ACPD was reported to possess both proconvulsant and
anticonvulsant effects (Zheng and Gallagher, Neurosci. Lett.
125:147, 1991; Sacaan and Schoepp, Neurosci. Lett. 139:77, 1992;
Taschenberger et al., Neuroreport 3:629, 1992; Sheardown,
Neuroreport 3:916, 1992), and neuroprotective effects in vitro and
in vivo (Pizzi et al., J. Neurochem. 61:683, 1993; Koh et al.,
Proc. Natl. Acad. Sci. USA 88:9431, 1991; Birrell et al.,
Neuropharmacol. 32:1351, 1993; Siliprandi et al., Eur. J.
Pharmacol. 219:173, 1992; Chiamulera et al., Eur. J. Pharmacol.
216:335, 1992). The metabotropic glutamate receptor antagonist
L-AP3 was shown to protect against hypoxic injury in vitro (Opitz
and Reymann, Neuroreport 2:455, 1991). A subsequent study reported
that trans-ACPD produced neuroprotection which was antagonized by
L-AP3 (Opitz and Reymann, Neuropharmacol. 32:103, 1993). (5)-4C3HPG
was shown to protect against audiogenic seizures in DBA/2 mice
(Thomasen et al., J. Neurochem. 62:2492, 1994). Other modulatory
effects expected of metabotropic glutamate receptor modulators
include synaptic transmission, neuronal death, neuronal
development, synaptic plasticity, spatial learning, olfactory
memory, central control of cardiac activity, waking, control of
movements, and control of vestibulo ocular reflex (for reviews, see
Nakanishi, Neuron 13:1031-37, 1994; Pin et al., Neuropharmacology
34:1, 1995; Knopfel et al., J. Med. Chem. 38:1417, 1995).
[0020] The structures of mGluR-active molecules currently known in
the art are limited to amino acids which appear to act by binding
at the glutamate binding site (Pin, et al, Neuropharmacology 34:1,
1995; Knopfel et al., J. Med. Chem. 38:1418). This limits the range
of pharmacological properties and potential therapeutic utilities
of such compounds. Furthermore, the range of pharmacological
specificities associated with these mGluRactive molecules does not
allow for complete discrimination between different subtypes of
metabotropic glutamate receptors (Pin et al., Neuropharmacology
34:1, 1995 and Knopfel et al., J. Med. Chem. 38:1418). Rapid
progress in the field of mGluR-active molecules cannot be made
until more potent and more selective mGluR agonists, antagonists
and modulators are discovered (Pin et al., Neuropharmacology 34:1,
1995; Knopfel et al., J. Med. Chem. 38:1418). Indeed, no
mGluR-active molecules are presently under clinical development.
High throughput functional screening of compounds and compound
libraries using cell lines expressing individual mGluRs represents
an important approach to identifying such novel compounds (Knopfel
et al., J. Med. Chem. 38:1418).
[0021] Several laboratories have constructed cell lines expressing
metabotropic glutamate receptors which appear to function
appropriately (Abe et al., J. Biol. Chem. 267:13361, 1992; Tanabe
et al., Neuron 8:169, 1992; Aramori and Nakanishi, Neuron 8:757,
1992, Nakanishi, Science 258:597, 1992; Thomsen et al., Brain Res.
619:22, 1992; Thomsen et al., Eur. J. Pharmacol. 227:361, 1992;
O'Hara et al., Neuron 11:41, 1993; Nakjima et al., J. Biol. Chem.
268:11868, 1993; Tanabe et al., J. Neurosci. 13:1372, 1993;
Saugstad et al., Mol. Pharmacol. 45:367, 1994; Okamoto et al., J.
Biol. Chem. 269:1231, 1994; Gabellini et al., Neurochem. Int.
24:533, 1994; Lin et al., Soc. Neurosci. Abstr. 20:468, 1994; Flor
et al., Soc. Neurosci. Abstr. 20:468, 1994; Flor et al.,
Neuropharmacology 34:149, 1994). Other reports have noted that
expression of functional mGluR expressing cell lines is not
predictable. For example, Tanabe et al., (Neuron 8:169, 1992) were
unable to demonstrate functional expression of mGluR3 and mGluR4,
and noted difficulty obtaining expression of native mGluR1 in CHO
cells. Gabellini et al., (Neurochem. Int. 24:533, 1994) also noted
difficulties with mGluR1 expression in HEK 293 cells and it is
possible that some of these difficulties may be due to
desensitization characteristics of these receptors. Furthermore,
screening methodologies useful for identification of compounds
active at Class I mGluRs are not readily amenable to identification
of compounds active at class II and III mGluRs and vice versa due
to the differences in second messenger coupling. Finally, mGluRs
have been noted to rapidly desensitize upon agonist stimulation
which may adversely affect the viability of cell lines expressing
these receptors and makes the use of native mGluRs for screening
difficult.
[0022] Different G-protein coupled receptors exhibit differential
ligand affinities and coupling to second messengers. G-protein
coupled receptors all have a similar structure: an N-terminal
extracellular domain (ECD), a seven-transmembrane domain (7TMD)
comprising seven membrane spanning helices and therefore defining
three intracellular and three extracellular loops, and a
cytoplasmic tail (CT), but differ in the exact sequences comprising
each region. These sequence differences are thought to provide the
specificity of receptor interactions with ligands of different
chemical compositions and receptor interaction with different
G-proteins. Construction of chimeric receptors in which small
peptide segments from related receptors are exchanged using
recombinant DNA techniques has proven a useful technique to assess
the participation of different sequence regions in determining this
specificity. For example, exchanging the third intracellular loops
between various adrenergic, muscarinic acetylcholine and
angiotensin receptors results in conversion of G-protein coupling
specificity. Thus, receptors whose activation normally results in
inhibition or activation of adenylate cyclase can be converted to
receptors with the same or similar ligand binding properties but
whose activation leads to stimulation of phospholipase-C and vice
versa (Kobilka et al., Science 240:1310, 1988; Wess et al., FEBS
Lett. 258:133, 1989; Cotecchia et al., Proc. Nat'l. Acad. Sci.
U.S.A. 87:2896, 1990; Lechleiter et al., EMBO J. 9:4381, 1990; Wess
et al., Mol. Pharmacol. 38:517, 1990; Wong et al., J. Biol. Chem.
265:6219, 1990; Cotecchia et al., J. Biol. Chem. 267:1633, 1992;
Wang et al., J. Biol. Chem. 270:16677, 1995). In these receptors
which share the third intracellular loop plays an important role in
determining the specificity of G-protein coupling. While such
experiments indicate that the third intracellular loop plays an
important role in determining the specificity of G protein coupling
in these related receptors, they have failed to identify any
specific amino acid sequence motif which is responsible. In
addition, the third intracellular loop has been shown to be at
least partly responsible for desensitization of such receptors
(Okamoto et al., Cell 67:723, 1991; Liggett et al., J. Biol. Chem.
267:4740, 1992).
[0023] Metabotropic glutamate receptors are related to other
G-protein coupled receptors in overall topology, but not in
specific amino acid sequence. An unusual feature of mGluRs is their
very large ECDs (ca. 600 amino acids). In many other G-protein
coupled receptors, ligand binding takes place within the 7TMD.
However, the large ECD of each mGluR is thought to provide the
ligand binding determinants (Nakanishi, Science 258:597, 1992;
O'Hara et al., Neuron 11:41, 1993; Shigemoto et al., Neuron
12:1245, 1994). Chimeric mGluRs in which the ECDs of mGluRs with
different ligand affinities and different G-protein coupling are
exchanged have been used to demonstrate that the ECD of mGluRs
defines ligand specificity but not G-protein specificity (Takahashi
et al., J. Bio. Chem. 268:19341, 1993). Also unlike other G-protein
coupled receptors in which the third intracellular loop is variable
in size and sequence, the third intracellular loops of mGluRs are
small and extremely well conserved (Brown E. M. et al., Nature
366:575, 1993). Chimeric mGluRs have been prepared in which the
second intracellular loops and/or cytoplasmic tails were exchanged
(Pin et al., EMBO J. 13:342). These experiments lead the
investigators to conclude that unlike most other G-protein coupled
receptors, "both the C-terminal end of the second intracellular
loop and the segment located downstream of the seventh
transmembrane domain are necessary for the specific activation of
phospholipase-C by mGluR1c" and to suggest that the second
intracellular loop of mGluRs plays the role of the third
intracellular loop of other G-protein coupled receptors.
[0024] Naturally occurring mRNA splice variants have been noted to
produce prostaglandin E3 (EP3) receptors with essentially identical
ligand binding properties but which preferentially activate
different second messenger pathways (differential G-protein
coupling) and which exhibit different desensitization properties
(Namba et al., Nature 365:166, 1993; Sgimoto et al., J. Biol. Chem.
268:2712, 1993; Negishi et al., J. Biol. Chem. 268:9517, 1993).
These variant receptor isoforms differ only in their cytoplasmic
tails. The isoforms with the longer tails couple efficiently to
phospholipase-C while those with the shorter tails do not. However,
analyses of naturally occurring mRNA splice variants of mGluR1 and
mGluR5 have indicated that their long cytoplasmic tails may not be
directly involved in G protein coupling (Pin et al., Proc. Nat'l.
Acad. Sci. U.S.A. 89:10331, 1992; Joly et al., J. Neuroscience
15:3970, 1995). In fact, Pin et al., (supra) have stated that "The
very long C-terminal domain found only in PLC-coupled mGluRs
(mGluR1 and 5) is, however, probably not involved in the specific
interaction with PLC-activating G proteins."
[0025] Recently, calcium receptor has been described (Brown E. M.
et al., Nature 366:575, 1993; Riccardi D., et al., Proc. Nat'l.
Acad. Sci. USA 92:131-135, 1995; Garrett J. E., et al., J. Biol.
Chem. 31:12919-12925, 1995). This CaR is the only known receptor
which exhibits significant sequence homology with mGluRs except for
other mGluRs. The CaR exhibits about .about.25% sequence homology
(amino acid identities) to any one mGluR while mGluRs are >40%
homologous (amino acid identities) to one another. The CaR is
structurally related to mGluRs having a large ECD which has been
implicated in receptor function and probable ligand binding (Brown
E. M. et al., Nature 366:575, 1993; Pollak, M. R., et al., Cell
75:1297-1303, 1993). This similarity of structure does not confer
close similarity in ligand binding specificity since the native
ligand for the CaR is the inorganic ion, Ca.sup.2+, and glutamate
does not modulate CaR activity. The CaR also has a large
cytoplasmic tail and is coupled to the stimulation of
phospholipase-C. Thus, the CaR is structurally and functionally
more related to mGluR1 and 5 than to other mGluRs. Pin et al.,
(EMBO J. 13:342, 1994) have noted that certain amino acids are
conserved within the intracellular loops of mGluRs which couple to
phospholipase-C and different amino acids are conserved in these
same positions within the intracellular loops of mGluRs which
couple to the inhibition of adenylate cyclase. Intracellular loops
1 and 3 are the most highly conserved sequences between mGluRs and
the CaR (Brown E. M. et al., Nature 366:575, 1993), but only about
half of these particular amino acids are found in the corresponding
position of the CaR and only one of these is actually the amino
acid predicted for a receptor which couples to phospholipase-C.
Thus, sequence conservation between CaRs and mGluRs appears to be
consistent mostly with conservation of structural domains involved
in ligand binding and G-protein coupling and does not provide
evidence for specific sequence motifs within intracellular regions
predictive of G-protein coupling specificity. Cell lines expressing
CaRs have been obtained and their use to identify compounds which
modulate the activity of CaRs disclosed (pending U.S. patent
application U.S. Ser. No. 08/353,784, filed Dec. 9, 1994, hereby
incorporated by reference herein).
[0026] An ideal screening procedure for identifying molecules
specifically affecting the activity of different mGluRs would
provide cell lines expressing each functional mGluR in such a
manner that each was coupled to the same second messenger system
and amenable to high throughput screening.
[0027] None of the references mentioned herein are admitted to be
prior art to the claims.
SUMMARY OF THE INVENTION
[0028] The present invention concerns (1) chimeric receptor
proteins having sequences from metabotropic glutamate receptors and
calcium receptors, and fragments of metabotropic glutamate
receptors, calcium receptors, and chimeric receptors; (2) nucleic
acids encoding such chimeric receptor proteins and fragments; (3)
uses of such receptor proteins, fragments and nucleic acids; (4)
cell lines expressing such nucleic acids; (5) methods of screening
for compounds that bind to or modulate the activity of metabotropic
glutamate receptors or calcium receptors using such chimeric
receptor proteins and fragments; (6) compounds for modulating
metabotropic glutamate receptors or calcium receptors identified by
such methods of screening; (7) methods for modulating metabotropic
glutamate receptors or calcium receptors utilizing such compounds;
and (8) methods of treating neurological disorders using such
compounds.
[0029] A preferred use of the compounds and methods of the present
invention is to screen for compounds which modulate metabotropic
glutamate receptor activity and to use such compounds to aid in the
treatment of neurological diseases or disorders.
[0030] As described in the Background of the Invention above,
metabotropic glutamate receptors and calcium receptors have similar
structures. Both types of receptors have an extracellular domain
(ECD), a seven transmembrane domain (7TMD) and an intracellular
cytoplasmic tail (CT). Thus, in the chimeric receptors of the
present invention, a portion of the sequence of the receptor is the
same as or homologous to a portion of the sequence of an mGluR and
a portion of the sequence is the same as or homologous to a portion
of the sequence of a CaR. For example, the chimeric receptor can
consist of the ECD of an mGluR and the 7TMD and CT of a CaR.
Likewise, a chimeric receptor may include the ECD and 7TMD of an
mGluR and the CT of a CaR. Other combinations of mGluR and CaR
domains or portions of domains may also be constructed and
utilized. These chimeric receptors are of interest, in part,
because they allow the coupling of certain functional aspects of an
mGluR with certain functional aspects of a CaR. Thus, experiments
have shown that ligands known in the art which are agonists or
antagonists on a native mGluR also exhibit such activities on
chimeric receptors in which the extracellular domain is from the
mGluR. Similarly, experiments have shown that ligands known in the
art which modulate mGluRs act on chimeric receptors in which the
extracellular domain and the 7TMD are from an mGluR. In both of
these cases, it is expected that other ligands which modulate mGluR
activity will also act on these types of chimeric receptors.
[0031] The use of mGluRs for screening for mGluR active compounds
has been complicated by a number of factors including a rapid
desensitization of the receptor upon ligand binding/activation and
difficulties in stably expressing the receptors in recombinant
vertebrate cells (see, for example, FIG. 8B and also published PCT
Patent Application). Certain of the chimeric receptors of the
present invention can be utilized to overcome these technical
difficulties and provide much improved screening methods by
utilizing the more robust aspects of calcium receptors. For
example, by coupling the 7TMD and the CT of the CaR with the
extracellular domain of an mGluR, or the CT of the CaR to the ECD
and 7TMD of the mGluR, the mGluR extracellular domain has the
benefit of the Gq coupling property of a CaR as well as the
improved property of a lack of rapid densensitization (see, for
example, FIG. 8C). Thus, such a chimeric receptor has the ligand
binding and activation properties similar to those of a native
mGluR but having the improved second messenger coupling similar to
a CaR. Therefore, the chimeric receptor simplifies and enables
efficient, practical, and reproducible functional screens to
identify mGluR active molecules.
[0032] For these novel chimeric receptors, not only is the
combination of mGluR and CaR sequences in a chimeric receptor
novel, but also the successful coupling of the activities is
unexpected. Previously, such coupling had only been accomplished
using portions of receptors with closely related sequences. In this
case the sequence identity between metabotropic glutamate receptors
and calcium receptors is only about 19-25%, and the two types of
receptors share only a 25-30% sequence similarity (Brown et al.,
Nature 366:575, 1993).
[0033] It is recognized that the three domains described above are
made up of subdomains, for example, ligand binding sites and G
protein coupling sites. Therefore, for some applications it is not
necessary to include in a chimeric receptor a complete domain from
a particular receptor in order to have the desired activity. For
example, only the ligand binding site from an mGluR can be
incorporated in a chimeric receptor in which most or all of the
remainder of the sequence is homologous to a CaR. Likewise, in a
chimeric receptor, one of the cytoplasmic loops of the 7TMD can be
homologous to a loop sequence of an mGluR and substantially the
remainder of the sequence of the receptor can be homologous to a
CaR, or conversely, one of the cytoplasmic loops can be homologous
to a loop sequence of a CaR and substantially the remainder of the
sequence of the receptor can be homologous to an mGluR.
[0034] Thus, in a first aspect the invention features a composition
including a chimeric receptor which has an extracellular domain, a
seven transmembrane domain, and an intracellular cytoplasmic tail
domain. The chimeric receptor has a sequence of at least 6
contiguous amino which is homologous to a sequence of a
metabotropic glutamate receptor and a sequence of at least 6
contiguous amino acids which is homologous to a sequence of a
calcium receptor.
[0035] In preferred embodiments, at least one domain is homologous
to a domain of a metabotropic glutamate receptor, or at least one
domain is homologous to a domain of a calcium receptor. In
particular, this includes chimeric receptors having a domain
homologous to a metabotropic glutamate receptor and a domain
homologous to a calcium receptor.
[0036] Also in preferred embodiments, the chimeric receptor has two
domains from a metabotropic glutamate receptor and one domain from
a calcium receptor, or two domains from a calcium receptor and one
domain from a metabotropic glutamate receptor. This includes each
of the possible combinations of the three domains. For example, in
a more preferred embodiment, the chimeric receptor has one domain
homologous to the extracellular domain of a metabotropic glutamate
receptor, one domain homologous to the seven transmembrane domain
of a metabotropic glutamate receptor, and one domain homologous to
the intracellular cytoplasmic tail domain of a calcium
receptor.
[0037] In other preferred embodiments, the chimeric receptor has at
least one cytoplasmic loop of the seven transmembrane domain which
is homologous to a cytoplasmic loop of a metabotropic glutamate
receptor. Similarly, in other preferred embodiments, the chimeric
receptor has at least one cytoplasmic loop homologous to a
cytoplasmic loop of a calcium receptor.
[0038] Also in other preferred embodiments, the chimeric receptor
has a sequence of at least 6 contiguous amino acids which is
homologous to an amino acid sequence of a calcium receptor, and the
rest of the sequence of the chimeric receptor is homologous to an
amino acid sequence of a metabotropic glutamate receptor. In other
embodiments, the sequence homologous to an amino acid sequence of a
calcium receptor may beneficially be longer, for example at least
12, 18, 24, 30, 36, or more amino acids in length.
[0039] Similarly, in other preferred embodiments, the chimeric
receptor has a sequence of at least 6 contiguous amino acids which
is homologous to an amino acid sequence of a metabotropic glutamate
receptor, and the rest of the sequence of the chimeric receptor is
homologous to an amino acid sequence of a calcium receptor. In
other embodiments, the sequence homologous to an amino acid
sequence of a metabotropic glutamate receptor may beneficially be
longer, for example at least 12, 18, 24, 30, 36, or more amino
acids in length.
[0040] In a related aspect, the invention provides a composition
which includes an isolated, enriched, or purified nucleic acid
molecule which codes for a chimeric receptor as described for the
aspect above. In particular, this includes nucleic acid coding for
a chimeric receptor having a sequence of at least 6 contiguous
amino acids which is homologous to an amino acid sequence of a
calcium receptor and a sequence of at least 6 contiguous amino
acids which is homologous to an amino acid sequence of a
metabotropic glutamate receptor. Similarly to the above aspect, in
particular embodiments the chimeric receptor sequence homologous to
an amino acid sequence from a calcium receptor and/or a
metabotropic glutamate receptor may beneficially be longer, for
example, at least 12, 18, 24, 30, 36, or more amino acids in
length.
[0041] In preferred embodiments, the chimeric receptor has a domain
homologous to a domain of a metabotropic glutamate receptor, and/or
a domain homologous to a calcium receptor. In more preferred
embodiments, the chimeric receptor has two domains homologous to
domains of a metabotropic glutamate receptor and a domain
homologous to a domain of a calcium receptor, or two domains
homologous to domains of a calcium receptor and a domain homologous
to a domain of a metabotropic glutamate receptor.
[0042] In another related aspect, the nucleic acid encoding a
chimeric receptor, as described above, is present in a replicable
expression vector. Thus, the vector can include nucleic acid
sequences coding for any of the chimeric receptors described.
[0043] Also in a related aspect, the invention provides a
recombinant host cell transformed with a replicable expression
vector as described above.
[0044] The invention also features a process for the production of
a chimeric receptor; the process involves growing, under suitable
nutrient conditions, procaryotic or eucaryotic host cells
transformed or transfected with a replicable expression vector
containing a nucleic acid sequence coding for a chimeric receptor
as described above, in a manner allowing expression of the chimeric
receptor.
[0045] By "isolated" in reference to a nucleic acid is meant the
nucleic acid is present in a form (i.e., its association with other
molecules) other than found in nature. For example, isolated
receptor nucleic acid is separated from one or more nucleic acids
which are present on the same chromosome. Preferably, the isolated
nucleic acid is separated from at least 90% of the other nucleic
acids present on the same chromosome. Preferably, the nucleic acid
is provided as a substantially purified preparation representing at
least 75%, more preferably 85%, most preferably 95% of the total
nucleic acids present in the preparation.
[0046] Another example of an isolated nucleic acid is recombinant
nucleic acid. Preferably, recombinant nucleic acid contains nucleic
acid encoding a chimeric metabotropic glutamate receptor or
metabotropic glutamate receptor fragment cloned in an expression
vector. An expression vector contains the necessary elements for
expressing a cloned nucleic acid sequence to produce a polypeptide.
An expression vector contains a promoter region (which directs the
initiation of RNA transcription) as well as the DNA sequences
which, when transcribed into RNA, will signal synthesis initiation.
"Expression vector" includes vectors which are capable of
expressing DNA sequences contained therein, i.e., the coding
sequences are operably linked to other sequences capable of
effecting their expression. It is implied, although not always
explicitly stated, that these expression vectors must be replicable
in the host organisms either as episomes or as an integral part of
the chromosomal DNA.
[0047] A useful, but not a necessary, element of an effective
expression vector is a marker encoding sequence--i.e., a sequence
encoding a protein which results in a phenotypic property (e.g.
tetracycline resistance) of the cells containing the protein which
permits those cells to be readily identified. In sum, "expression
vector" is given a functional definition, and any DNA sequence
which is capable of effecting expression of a specified contained
DNA code is included in this term, as it is applied to the
specified sequence. As at present, such vectors are frequently in
the form of plasmids, thus "plasmid" and "expression vector" are
often used interchangeably. However, the invention is intended to
include such other forms of expression vectors, including viral
vectors, which serve equivalent functions and which may, from time
to time become known in the art. Recombinant nucleic acids may
contain nucleic acids encoding for a chimeric metabotropic
glutamate receptor, receptor fragment, or chimeric metabotropic
glutamate receptor derivative, under the control of its genomic
regulatory elements, or under the control of exogenous regulatory
elements including an exogenous promoter. By "exogenous" is meant a
promoter that is not normally coupled in vivo transcriptionally to
the coding sequence for the metabotropic glutamate receptor or
calcium receptor.
[0048] The invention also provides methods of screening for
compounds which bind to and/or modulate the activity of a
metabotropic glutamate receptor and/or a calcium receptor. These
methods utilize chimeric receptors as described above or nucleic
acid sequence encoding such chimeric receptors. Such chimeric
receptors provide useful combinations of characteristics from the
two types of receptors, such as combining the binding
characteristics from a metabotropic glutamate receptor with the
cellular signaling characteristics from a calcium receptor.
[0049] Thus, in another aspect the invention provides a method of
screening for a compound that binds to or modulates the activity of
a metabotropic glutamate receptor. The method involves preparing a
chimeric receptor having an extracellular domain, a seven
transmembrane domain, and an intracellular cytoplasmic tail domain,
in which at least one domain is homologous to a domain of a
metabotropic glutamate receptor and at least one domain is
homologous to a domain of a calcium receptor. The chimeric receptor
and a test compound are introduced into an acceptable medium. The
binding of a test compound to the chimeric receptor, or the
modulation of the chimeric receptor by the compound, is monitored
by physically detectable means to identify those compounds which
bind to or modulate the activity of a metabotropic glutamate
receptor.
[0050] In a preferred embodiment the extracellular domain of the
chimeric receptor is homologous to the extracellular domain of a
metabotropic glutamate receptor. Also in preferred embodiments, the
chimeric receptor has two domains homologous to domains of a
metabotropic glutamate receptor and a domain homologous to a domain
of a calcium receptor, or two domains homologous to domains of a
calcium receptor and a domain homologous to a domain of a
metabotropic glutamate receptor.
[0051] In another aspect the invention provides a method of
screening for a compound which binds to or modulates the activity
of a metabotropic glutamate receptor, utilizing a nucleic acid
coding for a chimeric receptor. This method involves preparing a
nucleic acid sequence encoding a chimeric receptor which has an
extracellular domain, a seven transmembrane domain and an
intracellular cytoplasmic tail domain, in which the chimeric
receptor has a sequence of at least six contiguous amino acids
which is homologous to a sequence of amino acids of a calcium
receptor and a sequence of at least six contiguous amino acids
which is homologous to a sequence of amino acids of a metabotropic
glutamate receptor. The nucleic acid sequence is inserted into a
replicable expression vector capable of expressing the chimeric
receptor in a host cell. A suitable host cell is transformed with
this vector and the transformed host cell and a test compound are
introduced into an acceptable medium. Identification of binding or
modulation by the test compound is performed by monitoring the
effect of the compound on the cell.
[0052] In a preferred embodiment the chimeric receptor has at least
one domain homologous to a domain of metabotropic glutamate
receptor and/or at least one domain homologous to a domain of a
calcium receptor. In particular this includes preferred embodiments
in which the chimeric receptor has an extracellular domain
homologous to an extracellular domain of a metabotropic glutamate
receptor and/or a seven transmembrane domain of a metabotropic
glutamate receptor. In particular embodiments, the chimeric
receptor has two domains homologous to domains of a metabotropic
glutamate receptor and a domain homologous to a domain of a calcium
receptor, or two domains homologous to domains of a calcium
receptor and a domain homologous to a domain of a metabotropic
glutamate receptor.
[0053] Also in a preferred embodiment the chimeric receptor has at
least one cytoplasmic loop of the seven transmembrane domain which
is homologous to a cytoplasmic loop of a calcium receptor; in
particular embodiments the sequence of the remainder of the
chimeric receptor is homologous to the sequence of a metabotropic
glutamate receptor.
[0054] In another preferred embodiment the chimeric receptor has a
sequence of at least six contiguous amino acids which is homologous
to a sequence of amino acids of a calcium receptor and the
remainder of the amino acids sequence of a chimeric receptor is
homologous to an amino acid sequence of a metabotropic glutamate
receptor. In yet another preferred embodiment the chimeric receptor
has at least one cytoplasmic loop of the seven transmembrane domain
which is homologous to a cytoplasmic loop of a metabotropic
glutamate receptor.
[0055] In still another preferred embodiment the host cell is a
eucaryotic cell.
[0056] In the context of the methods of this invention, "monitoring
the effect" of a compound on a host cell refers to determining the
effects of the compound on one or more cellular processes or on the
level of activity of one or more cellular components, or by
detection of an interaction between the compound and a cellular
component.
[0057] The invention also provides methods of screening for
compounds that bind to or modulate a metabotropic glutamate
receptor or calcium receptor using fragments of such receptors.
Such fragments can, for example, be chosen to include a sequence
which has been shown to be important in activation of the
receptor's signal pathway.
[0058] Thus, in another aspect the invention features a method of
screening for a compound that binds to a metabotropic glutamate
receptor or a calcium receptor, by preparing a nucleic acid
encoding a fragment of such a receptor, inserting the sequence into
a replicable expression vector which can express that fragment in a
host cell, transforming a suitable host cell with a vector,
recovering the fragment from the host cell, introducing the
fragment in a test compound into an acceptable medium and
monitoring the binding of the compound to the fragment by
physically detectable means.
[0059] In a preferred embodiment the fragment is a fragment of a
metabotropic glutamate receptor; in a more preferred embodiment the
fragment includes the extracellular domain of that receptor.
[0060] In another preferred embodiment the fragment includes the
seven transmembrane domain of a metabotropic glutamate receptor. In
a more preferred embodiment the fragment includes both the seven
transmembrane domain and the cytoplasmic tail domain of a
metabotropic glutamate receptor.
[0061] Similarly in another preferred embodiment the fragment is a
fragment of a calcium receptor, preferably including the
extracellular domain over the seven transmembrane domain of that
receptor. In a more preferred embodiment the fragment includes the
seven transmembrane domain and cytoplasmic tail domain of the
calcium receptor.
[0062] Certain receptor fragments are able to activate one or more
cellular responses in a manner similar to the receptor from which
the fragment was derived. Therefore, in a related aspect, the
invention provides a method of screening for a compound that binds
to or modulates a metabotropic glutamate receptor or a calcium
receptor by preparing a nucleic acid sequence encoding a fragment
of such a receptor, inserting that sequence into a replicable
expression vector, transforming a host cell with that vector,
introducing the host cell and a test compound into an acceptable
medium, and monitoring the effect of the compound on the host
cell.
[0063] For certain receptors it is possible to utilize fragments of
two different receptors to screen for compounds which bind to or
modulate a receptor. The method involves preparing a nucleic acid
encoding a fragment of a first receptor, inserting the sequence
into a replicable expression vector capable of expressing that
fragment in a host cell, transforming a suitable host cell with a
vector, and recovering the first fragment from the host cell. A
fragment of a second receptor is prepared in a similar manner. The
two fragments and a test compound are introduced into an acceptable
medium and the binding and/or modulation by the compound is
monitored by physically detectable means.
[0064] In preferred embodiments a fragment is from a metabotropic
glutamate receptor and a fragment is from a calcium receptor. In
particular preferred embodiments the first fragment includes the
extracellular domain of a metabotropic glutamate receptor and the
second fragment includes the seven transmembrane domain and
cytoplasmic tail domain of a calcium receptor, or the first
fragment includes the extracellular domain and the seven
transmembrane domain of a metabotropic glutamate receptor and the
second fragment includes the cytoplasmic tail domain of a calcium
receptor.
[0065] In another particular embodiment the first fragment includes
the extracellular domain of a calcium receptor and the second
fragment includes the seven transmembrane domain and the
cytoplasmic tail domain of a metabotropic glutamate receptor. In
still another particular preferred embodiment, the first fragment
includes the extracellular domain of a calcium receptor and the
second fragment includes the seven transmembrane domain of a
metabotropic glutamate receptor and the cytoplasmic tail domain of
a calcium receptor.
[0066] Certain compounds can be identified which modulate the
activity of both a metabotropic glutamate receptor and of a calcium
receptor. Thus, this invention also provides a method for screening
for such compounds by preparing a nucleic acid sequence encoding a
chimeric receptor which includes a domain homologous to a domain of
a metabotropic glutamate receptor and a domain homologous to a
domain of a calcium receptor. The sequence is inserted in a
replicable expression vector capable of expressing the receptor in
a host cell; a suitable host cell is transformed with the vector
and the transformed host cell and a test compound are introduced
into an acceptable medium. The binding or modulation by the
compound is observed by monitoring the effect of a compound on the
host cell.
[0067] The invention also provides methods for determining the site
of action of a compound active on a metabotropic glutamate receptor
or on a calcium receptor. The methods involve preparing a nucleic
acid sequence which encodes a chimeric receptor. In two related
aspects, a chimeric receptor has at least a six amino acid sequence
which is homologous to a sequence of amino acids of a calcium
receptor and the remainder of the amino acid sequence is homologous
to an amino acid sequence of a metabotropic glutamate receptor, or
the chimeric receptor has at least a six amino acid sequence which
is homologous to a sequence of amino acids of a metabotropic
glutamate receptor and the remainder of amino acid sequence is
homologous to a sequence of a calcium receptor. In these aspects,
the nucleic acid sequence is inserted into a replicable expression
vector which is capable of expressing the receptor in a host cell.
The vector is transformed into a suitable host cell and the
transformed host cell in the compound are introduced into an
acceptable medium. The effect of the compound on the host cell is
monitored; thus if a compound is active on a receptor through an
interaction at the sequence of at least six amino acids from the
corresponding receptor, the chimeric receptor will be activated and
the cellular effects can be observed. On the other hand if the
compound does not interact with the at least six amino acid
sequence, thereby activating the receptor, the corresponding
cellular effects will not be observed.
[0068] Thus, "site of action" refers to the location(s) on the
receptor which are involved in interaction with a natural ligand
for that receptor, or with another compound of interest. For
example, for a compound which modulates the activity of a
metabotropic glutamate receptor by binding to the receptor, the
site of action can include amino acid sequences associated with
binding of the compound to the receptor, but may also include other
sequences. Such other sequences can, for example, include sequences
whose secondary or tertiary structure is altered in response to the
binding of the compound.
[0069] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments, and from the claims.
[0070] Additional aspects and embodiments will be apparent from the
following Detailed Description and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0071] FIGS. 1 A-F is a schematic illustration of the various
chimeras described herein, illustrating the extracellular domains,
7-transmembrane domains, and intracellular cytoplasmic tail domains
of the chimeras.
[0072] FIGS. 2 (A-H) is a representation of the nucleotide sequence
(SEQ ID NO: 1) and corresponding amino acid sequence (SEQ ID NO: 5)
of pmGluR1/CaR, described in Example 2.
[0073] FIGS. 3 (A-H) is a representation of the nucleotide sequence
(SEQ ID NO: 2) and corresponding amino acid sequence (SEQ ID NO: 6)
of pCaR/R.sub.1, described in Example 3.
[0074] FIGS. 4 (A-G) is a representation of the nucleotide sequence
(SEQ ID NO: 3) and corresponding amino acid sequence (SEQ ID NO: 7)
of pratCH3, described in Example 4.
[0075] FIGS. 5 (A-G) is a representation of the nucleotide sequence
(SEQ ID NO: 4) and corresponding amino acid sequence (SEQ ID NO: 8)
of phCH4, described in example 4.
[0076] FIG. 6 is a graphical representation showing the activation
of mGluR1/CaR by the mGluR1 agonists quisqualate and 1-glutamate as
measured by C1- currents generated in response to the release of
intracellular Ca2+ in the oocyte.
[0077] FIG. 7 is a graphical representation of activation of CaR
and CaR/R1 chimera by increasing extracellular calcium. Response
amplitudes (C1- currents in response to increases in intracellular
Ca2+) are shown. The data shows that CaR/R1 is activated by
extracellular Ca2+ in a manner similar to CaR.
[0078] FIGS. 8 A-C is a graphical representation showing that
extracellular glutamate elicits oscillatory increases in C1-
current in Xenopus oocytes injected with A) ratmGluR1 RNA, B) human
CaR RNA, and C) ratCH3 RNA. However, when oocytes are repeatedly
supplied with agonist, the rat mGluR1 receptor desensitizes and
does not activate the release of intracellular Ca2+. RatCH3, which
encodes the cytoplasmic tail of the CaR does not desensitize like
the native rat mGluR1 and is thus amenable to repeated challenges
with compounds.
[0079] FIG. 9 is a graphical representation showing increases in
intracellular calcium induced by extracellular calcium in fura-2
loaded stably transfected HEK293 cells expressing pCEPCaR/R1.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0080] It will be readily apparent to one skilled in the art that
various substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
Definitions
[0081] The following is a list of some of the definitions used in
the present disclosure. These definitions are to be understood in
light of the entire disclosure provided herein.
[0082] By "adjunct in general anesthesia" is meant a compound used
in conjunction with an anesthetic agent which decreases the ability
to perceive pain associated with the loss of consciousness produced
by the anesthetic agent.
[0083] By "allodynia" is meant pain due to a stimulus that does not
normally provoke pain.
[0084] By "analgesic" is meant a compound capable of relieving pain
by altering perception of nociceptive stimuli without producing
anesthesia resulting in the loss of consciousness.
[0085] By "analgesic activity" is meant the ability to reduce pain
in response to a stimulus that would normally be painful.
[0086] By "anticonvulsant activity" is meant efficacy in reducing
convulsions such as those produced by simple partial seizures,
complex partial seizures, status epilepticus, and trauma-induced
seizures such as occur following head injury, including head
surgery.
[0087] By "binds to or modulates" is meant that the agent may both
bind and modulate the activity of a receptor, or the agent may
either bind to or modulate the activity of a receptor.
[0088] By "causalgia" is meant a painful disorder associated with
injury of peripheral nerves.
[0089] By "central pain" is meant pain associated with a lesion of
the central nervous system.
[0090] By "cognition-enhancement activity" is meant the ability to
improve the acquisition of memory or the performance of a learned
task. Also by "cognition-enhancement activity" is meant the ability
to improve normal rational thought processes and reasoning.
[0091] By "cognition enhancer" is meant a compound capable of
improving learning and memory.
[0092] By "efficacy" is meant that a statistically significant
level of the desired activity is detectable with a chosen compound;
by "significant" is meant a statistical significance at the
p<0.05 level.
[0093] By "homologous" is meant a functional equivalent to the
domain, the amino acid sequence, or the nucleic acid sequence,
having similar nucleic acid and/or amino acid sequence and
retaining, to some extent, one or more activities of the related
receptor. Homologous domains or sequences of receptors have at
least 50% sequence similarity, preferably 70%, more preferably 90%,
even more preferably 95% sequence similarity to the related
receptor. "Sequence similarity" refers to "homology" observed
between amino acid sequences in two different polypeptides,
irrespective of polypeptide origin. Thus, homologous includes
situations in which the nucleic acid and/or amino acid sequences
are the same. In related phrases, reference to a sequence,
sub-domain, or domain being "from a metabotropic glutamate
receptor" or "of a metabotropic glutamate receptor" means that the
portion is the same as or homologous to a portion of a metabotropic
glutamate receptor; like references to portions being "from a
calcium receptor" or "of a calcium receptor" also indicate the
portions are the same as or homologous to portions of a calcium
receptor. These phrases can be used in reference to amino acid
sequences and/or nucleic sequences.
[0094] The ability of the homologous domain or sequence to retain
some activity can be measured using techniques described herein.
Such homologous domains may also be derivatives. Derivatives
include modification occurring during or after translation, for
example, by phosphorylation, glycosylation, crosslinking,
acylation, proteolytic cleavage, linkage to an antibody molecule,
membrane molecule or other ligand (see Ferguson et al., 1988, Ann.
Rev. Biochem. 57:285-320).
[0095] Specific types of derivatives also include amino acid
alterations such as deletions, substitutions, additions, and amino
acid modifications. A "deletion" refers to the absence of one or
more amino acid residue(s) in the related polypeptide. An
"addition" refers to the presence of one or more amino acid
residue(s) in the related polypeptide. Additions and deletions to a
polypeptide may be at the amino terminus, the carboxy terminus,
and/or internal. Amino acid "modification" refers to the alteration
of a naturally occurring amino acid to produce a non-naturally
occurring amino acid. A "substitution" refers to the replacement of
one or more amino acid residue(s) by another amino acid residue(s)
in the polypeptide. Derivatives can contain different combinations
of alterations including more than one alteration and different
types of alterations.
[0096] While the effect of an amino acid change varies depending
upon factors such as phosphorylation, glycosylation, intra-chain
linkages, tertiary structure, and the role of the amino acid in the
active site or a possible allosteric site, it is generally
preferred that the substituted amino acid is from the same group as
the amino acid being replaced. To some extent the following groups
contain amino acids which are interchangeable: the basic amino
acids lysine, arginine, and histidine; the acidic amino acids
aspartic and glutamic acids; the neutral polar amino acids serine,
threonine, cysteine, glutamate, asparagine and, to a lesser extent,
methionine; the nonpolar aliphatic amino acids glycine, alanine,
valine, isoleucine, and leucine (however, because of size, glycine
and alanine are more closely related and valine, isoleucine and
leucine are more closely related); and the aromatic amino acids
phenylalanine, tryptophan, and tyrosine. In addition, although
classified in different categories, alanine, glycine, and serine
seem to be interchangeable to some extent, and cysteine
additionally fits into this group, or may be classified with the
polar neutral amino acids.
[0097] While proline is a nonpolar neutral amino acid, its
replacement represents difficulties because of its effects on
conformation. Thus, substitutions by or for proline are not
preferred, except when the same or similar confornational results
can be obtained. The conformation conferring properties of proline
residues may be obtained if one or more of these is substituted by
hydroxyproline (Hyp).
[0098] Examples of modified amino acids include the following:
altered neutral nonpolar amino acids such as--amino acids of the
formula H.sub.2N(CH.sub.2).sub.nCOOH where n is 2-6, sarcosine
(Sar), t-butylalanine (t-BuAla), t-butylglycine (t-BuGly), N-methyl
isoleucine (N-MeIle), and norleucine (Nleu); altered neutral
aromatic amino acids such as phenylglycine; altered polar, but
neutral amino acids such as citrulline (Cit) and methionine
sulfoxide (MSO); altered neutral and nonpolar amino acids such as
cyclohexyl alanine (Cha); altered acidic amino acids such as
cysteic acid (Cya); and altered basic Ramino acids such as omithine
(Orn).
[0099] Preferred derivatives have one or more amino acid
alteration(s) which do not significantly affect the receptor
activity of the related receptor protein. In regions of the
receptor protein not necessary for receptor activity amino acids
may be deleted, added or substituted with less risk of affecting
activity. In regions required for receptor activity, amino acid
alterations are less preferred as there is a greater risk of
affecting receptor activity. Such alterations should be
conservative alterations. For example, one or more amino acid
residues within the sequence can be substituted by another amino
acid of a similar polarity which acts as a functional
equivalent.
[0100] Conserved regions tend to be more important for protein
activity than nonconserved regions. Standard procedures can be used
to determine the conserved and nonconserved regions important of
receptor activity using in vitro mutagenesis techniques or deletion
analyses and measuring receptor activity as described by the
present disclosure.
[0101] Derivatives can be produced using standard chemical
techniques and recombinant nucleic acid techniques. Modifications
to a specific polypeptide may be deliberate, as through
site-directed mutagenesis and amino acid substitution during
solidphase synthesis, or may be accidental such as through
mutations in hosts which produce the polypeptide. Polypeptides
including derivatives can be obtained using standard techniques
such as those described in Section I.G.2. supra, and by Sambrook et
al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989).
For example, Chapter 15 of Sambrook describes procedures for
site-directed mutagenesis of cloned DNA.
[0102] By "hyperalgesia" is meant an increased response to a
stimulus that is normally painful.
[0103] By "minimal" is meant that any side effect of the drug is
tolerated by an average individual, and thus that the drug can be
used for therapy of the target disease or disorders. Such side
effects are well known in the art. Preferably, minimal side effects
are those which would be regarded by the FDA as tolerable for drug
approval for a target disease or disorder.
[0104] By "modulate" is meant to cause an increase or decrease in
an activity of a cellular receptor.
[0105] By "modulator" is meant a compound which modulates a
receptor, including agonists, antagonists, allosteric modulators,
and the like. Preferably, the modulator binds to the receptor.
[0106] By "muscle relaxant" is meant a compound that reduces
muscular tension.
[0107] By "neuralgia" is meant pain in the distribution ofa nerve
or nerves.
[0108] By "neurodegenerative disease" is meant a neurological
disease affecting cells of the central nervous system resulting in
the progressive decrease in the ability of cells of the nervous
system to function properly. Examples of neurodegenerative diseases
include Alzheimer's disease, Huntington's disease, and Parkinson's
disease.
[0109] By "neurological disorder or disease" is meant a disorder or
disease of the nervous system. Examples of neurological disorders
and diseases include global and focal ischemic and hemorrhagic
stroke, head trauma, spinal cord injury, hypoxia-induced nerve cell
damage as in cardiac arrest or neonatal distress, and epilepsy.
[0110] By "neuroprotectant activity" is meant efficacy in treatment
of the neurological disorders or diseases.
[0111] By "physically detectable means" is meant any means known to
those of ordinary skill in the art to detect binding to or
modulation of mGluR or CaR receptors, including the binding and
screening methods described herein. Thus, for example, such means
can include spectroscopic methods, chromatographic methods,
competitive binding assays, and assays of a particular cellular
function, as well as other techniques.
[0112] By "potent" is meant that the compound has an EC.sub.50
value (concentration which produces a half-maximal activation), or
IC.sub.50 (concentration which produces half-maximal inhibition),
or K.sub.d (concentration which produces half-maximal binding) at a
metabotropic glutamate receptor, with regard to one or more
receptor activities, of less than 100 .mu.M, more preferably less
than 10 .mu.M, and even more preferably less than 1 .mu.M.
[0113] By "selective" is meant that the compound activates,
inhibits activation and/or binds to a metabotropic glutamate
receptor at a lower concentration than that at which the compound
activates, inhibits activation and/or binds to an ionotropic
glutamate receptor. Preferably, the concentration difference is a
10-fold, more preferably 50-fold, and even more preferably
100-fold.
[0114] By "therapeutically effective amount" is meant an amount of
a compound which produces the desired therapeutic effect in a
patient. For example, in reference to a disease or disorder, it is
the amount which reduces to some extent one or more symptoms of the
disease or disorder, and returns to normal, either partially or
completely, physiological or biochemical parameters associated or
causative of the disease or disorder. When used to therapeutically
treat a patient it is an amount expected to be between 0.1 mg/kg to
100 mg/kg, preferably less than 50 mg/kg, more preferably less than
10 mg/kg, more preferably less than 1 mg/kg. Preferably, the amount
provides an effective concentration at a metabotropic glutamate
receptor of about 1 nM to 10 .mu.M of the compound. The amount of
compound depend on its EC.sub.50 (IC.sub.50 in the case of an
antagonist) and on the age, size, and disease associated with the
patient.
[0115] Techniques
[0116] 1. Chimeric Receptors and General Approach to Uses
[0117] As indicated in the Summary above, this invention concerns
chimeric receptors, which include portions of both metabotropic
glutamate receptor and calcium receptor proteins. It also is
concerned with fragments of metabotropic glutamate receptors and
calcium receptors. Related aspects include nucleic acids encoding
such chimeric receptors and fragments, uses of such receptors,
fragments and nucleic acids, and cell lines expressing such nucleic
acids. The uses disclosed include methods of screening for
compounds that bind to or modulate the activity of metabotropic
glutamate receptors or calcium receptors using such chimeric
receptors and fragments. The invention also includes compounds for
modulating metabotropic glutamate receptors or calcium receptors
identified by such methods of screening, and methods for treating
certain disorders or for modulating metabotropic glutamate
receptors or calcium receptors utilizing such compounds.
[0118] Experiments carried out on several distinct G-protein
coupled receptors have suggested the general principle that
G-protein coupling specificity and receptor desensitization are
determined primarily by amino acid sequences which are
intracellular (i.e., sequences within one or more of the three
cytoplasmic loops and/or the intracellular cytoplasmic tail).
Recent experiments in which chimeric receptors were formed by
combining distinct protein segments from different metabotropic
glutamate receptors (mGlurs), suggest that, in these receptors,
ligand binding specificity is determined by the extracellular
domain.
[0119] Thus, preferred embodiments of the present invention include
chimeric receptors consisting of the extracellular domain (ECD) of
an mGluR and the seven-transmembrane domain (7TMD) and the
intracellular cytoplasmic tail (CT) of a calcium receptor (CaR)
that responds to mGluR-active molecules by signal transduction
analogous to that observed when CaR-active molecules act on a
CaR.
[0120] Similarly, in other preferred embodiments, the invention
includes chimeric receptors in which the intracellular cytoplasmic
C-terminal tail domain of a chosen mGluR is replaced by the
C-terminal tail of a calcium receptor. The C-terminal tail
encompasses the cytoplasmic region which follows the seventh
transmembrane region.
[0121] Preferred embodiments of the invention also include chimeric
receptors in which the peptide sequences encompassing all or some
of the cytoplasmic loop domains (between the first and second, the
third and fourth, and the fifth and sixth transmembrane regions) of
an mGluR have been replaced similarly with corresponding peptide
sequences from one or more CaRs. In particular such embodiments
include chimeric receptors having the ECD of an mGluR, the 7TMD of
an mGluR, and the C-terminal tail of a calcium receptor, except
that one or more sub-domains of the 7-TMD are substituted with
sequences from a CaR. This specifically includes receptors in which
one or more of the cytoplasmic loops of the 7TMD are replaced with
sequences from a CaR. Such substitution of cytoplasmic loops may be
done singly or in any combination. In general, using techniques
known to those skilled in the art, such target "domains" and
"sub-domains" may be "swapped" individually or in combination.
[0122] These chimeric receptors are unknown in the art and their
function is unexpected because functional chimeric receptors had
previously been successfully constructed only by combining portions
of much more closely related receptors. Indeed, the sequence
identity between metabotropic glutamate receptors and calcium
receptors is only about 19-25%, and the two types of receptors
share only about 25-30% sequence similarity (Brown E. M. et al.,
Nature 366:575, 1993).
[0123] Experiments have shown that ligands known in the art which
are agonists or antagonists on the native mGluRs also exhibit such
activities on the chimeric receptors in which the extracellular
domain is from an mGluR. Other ligands which bind to the ECD and
modulate the activity of mGluRs, for example, agonists,
antagonists, allosteric modulators and the like, are also predicted
to act on such chimeric receptors. Experiments have also shown that
ligands known in the art which modulate mGluRs act on the chimeric
receptors in which the ECD and 7TMD are from an mGluR. Other
ligands which modulate mGluR activity are also predicted to act on
this type of chimeric receptors regardless of whether they bind the
ECD or 7TMD of mGluRs.
[0124] The chimeric receptors are linked to intracellular or second
messenger functions in a similar fashion to the linkage known for
non-modified calcium receptors. For example, as is the case for
CaRs, the chimeric receptors are also coupled through a
G-protein(s) to the activation of phospholipase C, to the
generation of inositol phosphates and/or to the release of calcium
ions from intracellular stores. Although the mGluRs rapidly
desensitize upon ligand binding/activation, the CaRs do not,
allowing for more efficient high-throughput screening of compounds
active at the CaR and stable receptor expression in recombinant
cell lines. Importantly, the chimeric mGluR/CaR receptors do not
rapidly desensitize upon ligand binding/activation and can be
therefore efficiently used for high throughput screening. In
addition, the chimeric receptors can be functionally expressed in
stable cell lines.
[0125] Cells expressing such chimeric receptors can be prepared and
used in functional assays to identify compounds which modulate
activities of selected mGluRs. For example, increases in
intracellular calcium levels resulting from receptor activation can
be monitored by use of fluorescent calcium chelating dyes.
Functional assays have been described for identifying molecules
active at calcium receptors (see for example, published PCT patent
application "Calcium Receptor-Active Molecules," PCT No. US93/01642
(WO94/18959), published September 1994 hereby incorporated by
reference herein in its entirety).
[0126] An increasingly common practice in modem drug discovery is
the use of various target-site-specific assays to identify specific
molecules with activities of interest. These assays select drug
lead molecules from large collections or libraries of molecules
(e.g., combinatorial libraries, proprietary compound libraries held
by large drug companies, etc.). Drug lead molecules are "selected"
when they bind to pharmacological targets of interest and thus
potentially modify the activities of these targets. The assays can
be of many types including direct binding displacement assays or
indirect functional assays. In order to successfully develop and
use an assay to isolate lead therapeutic compounds, the target
molecule (e.g., receptor) must first be identified and isolated.
Many functional assays have been described in the literature for
identifying molecules active at various receptors and these provide
unique advantages over binding assays. It is not necessary to know,
a priori, which ligands modulate the activity of the receptor in
vivo, nor is it necessary to know the exact physiological function
of the receptor. Compounds identified in functional assays and in
subsequent medicinal chemistry efforts can be used as experimental
test compounds to obtain such knowledge.
[0127] While eight distinct mGluRs are currently known, their
discrete functions remain largely undetailed. Nevertheless,
molecules active at mGluRs are sought by pharmaceutical companies
because these receptors are found in the central nervous system and
are known to be involved in the regulation of processes related to
memory, motor functions, pain sensation, neurodegeneration and the
like. Thus, compounds which modulate mGluRs may be useful in the
treatment of disorders or diseases affecting memory, cognition, and
motor function (e.g., in seizures) as well as in the treatment of
pain and neurodegenerative disorders (e.g., stroke, Alzheimers
disease and the like).
[0128] Screens to identify molecules active at mGluRs can be
constructed using cloned mGluRs themselves. However, functional
screens using native mGluRs are problematic. First, most mGluRs are
coupled through G.sub.i proteins and this limits their use in
functional assays because G.sub.i proteins are linked to inhibition
of adenylate cyclase and changes in adenylate cyclase are not
easily measured in high throughput functional screens designed to
select drug lead molecules from large compound libraries.
[0129] Receptors which couple through other G-proteins to
activation of phospholipase C (e.g., G.sub.q-coupled receptors) do
not suffer this drawback, so it was initially thought that mGluR1
and mGluR5 could find utility in functional assays because these
two mGluRs are coupled through Gq-protein(s) to measurable
intracellular functions (e.g., activation of phospholipase C,
generation of inositol phosphates and the release of calcium ions
from intracellular stores).
[0130] A second limitation is presented here, however, because
these particular mGluRs rapidly desensitize upon agonist binding.
That is, the functional response disappears rapidly and cannot
quickly be recovered (see for example FIG. 8a). Furthermore, it has
not always been possible to obtain fully functional stable cell
lines expressing mGluRs regardless of the G-protein to which they
couple (Tanabe et al., 1992, Neuron 8:169-179; Gabellini et al.,
1994, Neurochem Int. 24:533-539). Thus, nontrivial technical
difficulties must be overcome in order to use native mGluRs in an
optimal manner in high throughput functional screening assays.
[0131] The invention described herein overcomes these technical
difficulties and provides a much improved screening method by
utilizing the more robust aspects of the calcium receptors which do
not rapidly desensitize upon ligand binding/activation and can be
expressed stably in recombinant vertebrate cells (see for example,
FIG. 8b and see also published PCT patent application "Calcium
Receptor-Active Molecules," PCT No. US93/01642 (WO94/18959),
published September 1994, hereby incorporated by reference herein).
Thus, for example, by coupling the 7TMD and the CT of the CaR to
the extracellular domain of mGluR, or the CT of the CaR to the ECD
and 7TMD of the mGluR, the mGluR extracellular domain has the
benefit of the Gq coupling property of a CaR, as well as the
improved property of a lack of rapid desensitization (see, for
example, FIG. 8c). Thus, the present invention provides chimeric
receptors with ligand binding and activation properties similar to
those of the native mGluRs, but with improved second messenger
coupling similar to CaRs.
[0132] Thus, since the chimeric receptors simplify and enable,
efficient, practical and reproducible functional screens to
identify mGluR-active molecules, compositions and methods of the
present invention are useful for the identification of molecules
which modulate mGluR activity or calcium receptor activity. These
can, for example, include agonists, antagonists, allosteric
modulators, and the like. For example, chimeric receptors
constructed to screen compounds active at metabotropic glutamate
receptors may employ the signaling properties of certain domains of
a calcium receptor. Such a chimeric receptor would take advantage
of certain unique properties associated with the agonist-induced
coupling of the calcium receptor to G-proteins which activate
phospholipase C and mobilize intracellular calcium. These
properties include, for example, the lack of ligand induced
down-regulation/desensitization which is associated with ligand
activation of metabotropic glutamate receptors. Thus the superior
signaling properties of the calcium receptor can be transferred to
metabotropic glutamate receptors which normally do not couple to
G-proteins that activate phospholipase C and mobilize intracellular
calcium such as those which couple to G.sub.i.
[0133] In certain embodiments, recombinant cells expressing such
chimeric receptors are used in screening methods. The cells will
obtain properties, such as those indicated above, which facilitate
their use in high-throughput functional assays, and thus provide a
more efficient method of screening for compounds which bind to or
modulate metabotropic glutamate receptor activity.
[0134] Generally, useful chimeric receptors include portions of
mGluRs and CaRs, such that the portions confer a desired binding,
signal coupling, or other functional characteristic to the chimeric
receptor. The length of a sequence from a particular receptor can
be of different sizes in different applications. In addition, the
sequence of a portion from a particular receptor may be identical
to the corresponding sequence in the mGluR or CaR, or it may be a
homologous sequence, which retains the relevant function of the
mGluR or CaR sequence. Therefore, chimeric receptors of this
invention have an extracellular domain, a seven transmembrane
domain, and an intracellular cytoplasmic tail domain. These
chimeric receptors have a contiguous sequence of at least 6 amino
acids which is homologous to a sequence from an mGluR, and a
contiguous sequence of at least 6 amino acids which is homologous
to a sequence from a CaR. However, in many cases, the sequences
from the mGluR and/or the CaR may be longer than 6 amino acids.
Thus, either or both of such sequences may be at least 12, 18, 24,
30, 36, or more amino acids in length.
[0135] The portions from the mGluR and the CaR will usually not be
the same length. Thus, for example, the sequence from one of those
types of receptor may be of a length as indicated above (e.g., et
at least 6, 12, 18, 24, 30, 36, or more amino acids), while the
rest of the sequence of the chimeric receptor is the same as or
homologous to a sequence from the other type of receptor.
[0136] In certain embodiments, the portion from at least one
receptor type is a subdomain. In this context, "subdomain" refers
to a sequence of amino acids which is less than the entire sequence
of amino acids for a domain. Examples of subdomains include, but
are not limited to, ligand binding domains. Other examples include
one of the cytoplasmic loops or regions of the seven transmembrane
domain. Therefore, in certain cases, a chimeric receptor has an
extracellular domain, a seven transmembrane domain, and an
intracellular cytoplasmic tail domain, which include subdomains. In
one example of such chimeric receptors, at least one subdomain is
homologous to a subdomain of a calcium receptor and the remaining
subdomains and domains are homologous to subdomains and domains of
a metabotropic glutamate receptor. In another example, at least one
subdomain is homologous to a subdomain of a metabotropic glutamate
receptor and the remaining subdomains and domains are homologous to
subdomains and domains of a calcium receptor.
[0137] In a more specific example, the seven transmembrane domain
of a chimeric receptor includes three cytoplasmic loops; at least
one cytoplasmic loop is homologous to a cytoplasmic loop of a
metabotropic glutamate receptor; or at least one cytoplasmic loop
is homologous to a cytoplasmic loop of a calcium receptor. In
another specific example, the extracellular domain is homologous to
the extracellular domain of a metabotropic glutamate receptor, the
seven transmembrane domain is homologous to the seven transmembrane
domain of a metabotropic glutamate receptor except that one or more
of the cytoplasmic loops of the seven transmembrane domain is
homologous to a cytoplasmic loop(s) of a calcium receptor, and the
cytoplasmic tail is homologous to the cytoplasmic tail of a calcium
receptor. Thus, any of cytoplasmic loops 1, 2, and 3 may be
replaced, either singly or in any combination, with a cytoplasmic
loop(s) of a calcium receptor.
[0138] In other cases, the chimeric receptor has a domain which has
a sequence which is the same as or homologous to the sequence of a
domain of an mGluR, or a CaR, or preferably, at least one domain
from each of an mGluR and a CaR. More preferably, the chimeric
receptor has two domains from one receptor type and one domain from
the other receptor type. The compositions of certain preferred
embodiments of such chimeric receptors are described below:
[0139] A. A composition comprising a chimeric receptor having:
[0140] 1. one domain homologous to the extracellular domain of a
calcium receptor, one domain homologous to the seven transmembrane
domain of a metabotropic glutamate receptor, and one domain
homologous to the intracellular cytoplasmic tail domain of a
metabotropic glutamate receptor; or
[0141] 2. one domain homologous to an extracellular domain of a
metabotropic glutamate receptor, one domain homologous to the seven
transmembrane domain of a calcium receptor, and one domain
homologous to the intracellular cytoplasmic tail domain of a
calcium receptor; or
[0142] 3. one domain homologous to an extracellular domain of a
metabotropic glutamate receptor, one domain homologous to the seven
transmembrane domain of a calcium receptor, and one domain
homologous to the intracellular cytoplasmic tail domain of a
metabotropic glutamate receptor; or
[0143] 4. one domain homologous to the extracellular domain of a
calcium receptor, one domain homologous to the seven transmembrane
domain of a metabotropic glutamate receptor, and one domain
homologous to the intracellular cytoplasmic tail domain of a
calcium receptor; or
[0144] 5. one domain homologous to the extracellular domain of a
calcium receptor, one domain homologous to the seven transmembrane
domain of a calcium receptor, and one domain homologous to the
intracellular cytoplasmic tail domain of a metabotropic glutamate
receptor; or
[0145] 6. one domain homologous to the extracellular domain of a
metabotropic glutamate receptor, one domain homologous to the seven
transmembrane domain of a metabotropic glutamate receptor, and one
domain homologous to the intracellular cytoplasmic tail domain of a
calcium receptor; or
[0146] 7. one domain homologous to the extracellular domain of a
metabotropic glutamate receptor, one domain homologous to the seven
transmembrane domain of a metabotropic glutamate receptor except
that one or more cytoplasmic loops are replaced with a cytoplasmic
loop(s) homologous to a cytoplasmic loop(s) of a calcium receptor,
and one domain homologous to the intracellular cytoplasmic tail
domain of a calcium receptor.
[0147] B. Nucleic Acids Encoding Chimeric Receptors
[0148] Compositions which include isolated nucleic acid molecules
which code for chimeric receptors as described above are also
useful in this invention. Such nucleic acid molecules can be
isolated, purified, or enriched. Preferably, the nucleic acid is
provided as a substantially purified preparation representing at
least 75%, more preferably 85%, most preferably 95% of the total
nucleic acids present in the preparation.
[0149] Such nucleic acid molecules may also be present in a
replicable expression vector. The replicable expression vector can
be transformed into a suitable host cell to provide a recombinant
host cell. Using such transformed host cells, the invention also
provides a process for the production of a chimeric receptor, which
includes growing, under suitable nutrient conditions, procaryotic
or eucaryotic host cells transformed or transfected with a
replicable expression vector comprising the nucleic acid molecule
in a manner allowing expression of said chimeric receptor.
[0150] Uses of nucleic acids encoding chimeric receptors or
receptor fragments include one or more of the following: producing
receptor proteins which can be used, for example, for structure
determination, to assay a molecule's activity on a receptor, to
screen for molecules useful as therapeutics and to obtain
antibodies binding to the receptor. The chimeras of the present
invention are useful for identifying compounds active at either
calcium receptors or metabotropic glutamate receptors, or both.
Also, the fragments of the present invention are useful for
identifying compounds which bind to or modulate either calcium
receptors or metabotropic glutamate receptors, or both.
[0151] Thus, the invention also provides, for example, an isolated
nucleic acid encoding an extracellular domain of a metabotropic
glutamate receptor that is substantially free of the seven
transmembrane domain and intracellular cytoplasmic tail domain of
that metabotropic glutamate receptor. Similarly, the isolated
nucleic acid can encode a metabotropic glutamate receptor that is
substantially free of at least one membrane spanning domain
portion. In another example, an isolated nucleic acid can encode a
metabotropic glutamate receptor that is substantially free of the
extracellular domain of that metabotropic glutamate receptor.
[0152] C. Metabotropic Glutamate Receptor Fragments and Calcium
Receptor Fragments
[0153] Receptor fragments are portions of metabotropic glutamate
receptors or of calcium receptors. Receptor fragments preferably
bind to one or more binding agents which bind to a full-length
receptor. Binding agents include ligands, such as glutamate,
quisqualate, agonists and antagonists, and antibodies which bind to
the receptor. Fragments have different uses such as to select other
molecules able to bind to a receptor.
[0154] Fragments can be generated using standard techniques such as
expression of cloned partial sequences of receptor DNA and
proteolytic cleavage of a receptor protein. Proteins are
specifically cleaved by proteolytic enzymes, such as trypsin,
chymotrypsin or pepsin. Each of these enzymes is specific for the
type of peptide bond it attacks. Trypsin catalyzes the hydrolysis
of peptide bonds whose carbonyl group is from a basic amino acid,
usually arginine or lysine. Pepsin and chymotrypsin catalyze the
hydrolysis of peptide bonds from aromatic amino acids, particularly
tryptophan, tyrosine and phenylalanine.
[0155] Alternate sets of cleaved protein fragments are generated by
preventing cleavage at a site which is susceptible to a proteolytic
enzyme. For example, reaction of the amino group of lysine with
ethyltrifluorothioacetate in mildly basic solution yields a blocked
amino acid residue whose adjacent peptide bond is no longer
susceptible to hydrolysis by trypsin. Goldberger et al.,
Biochemistry 1:401, 1962). Treatment of such a polypeptide with
trypsin thus cleaves only at the arginyl residues.
[0156] Polypeptides also can be modified to create peptide linkages
that are susceptible to proteolytic enzyme-catalyzed hydrolysis.
For example, alkylation of cysteine residues with haloethylamines
yields peptide linkages that are hydrolyzed by trypsin. (Lindley,
Nature 178:647, 1956).
[0157] In addition, chemical reagents that cleave polypeptide
chains at specific residues can be used. (Witcop, Adv. Protein
Chem. 16:221, 1961). For example, cyanogen bromide cleaves
polypeptides at methionine residues. (Gross & Witkip, J. Am.
Chem. Soc. 83: 1510, 1961).
[0158] Thus, by treating a metabotropic glutamate receptor, or
fragments thereof with various combinations of modifiers,
proteolytic enzymes and/or chemical reagents, numerous discrete
overlapping peptides of varying sizes are generated. These peptide
fragments can be isolated and purified from such digests by
chromatographic methods. Alternatively, fragments can be
synthesized using an appropriate solid-state synthetic
procedure.
[0159] Fragments may be selected to have desirable biological
activities. For example, a fragment may include just a ligand
binding site. Such fragments are readily identified by those of
ordinary skill in the art using routine methods to detect specific
binding to the fragment. For example, in the case of a metabotropic
glutamate receptor, nucleic acid encoding a receptor fragment can
be expressed to produce the polypeptide fragment which is then
contacted with a receptor ligand under appropriate association
conditions to determine whether the ligand binds to the fragment.
Such fragments are useful in screening assays for agonists and
antagonists of glutamate, and for therapeutic effects where it is
useful to remove glutamate from serum, or other bodily tissues.
[0160] Other useful fragments include those having only the
external portion, membrane-spanning portion, or intracellular
portion of the receptor. These portions are readily identified by
comparison of the amino acid sequence of the receptor with those of
known receptors, or by other standard methodology. These fragments
are useful for forming chimeric receptors with fragments of other
receptors to create a receptor with an intracellular portion which
performs a desired function within that cell, and an extracellular
portion which causes that cell to respond to the presence of
glutamate, or those agonists or antagonists described herein.
Chimeric receptor genes when appropriately formulated are useful in
genetic therapies for a variety of diseases involving dysfunction
of receptors or where modulation of receptor function provides a
desirable effect in the patient.
[0161] Additionally, chimeric receptors can be constructed such
that the intracellular domain is coupled to a desired enzymatic
process which can be readily detected by calorimetric, radiometric,
luminometric, spectrophotometric or fluorimetric assays and is
activated by interaction of the extracellular portion with its
native ligand (e.g., glutamate) or agonist and/or antagonists of
the invention. Cells expressing such chimeric receptors can be used
to facilitate screening of metabotropic glutamate receptor agonists
and antagonists, and in some cases inorganic ion receptor agonists
and antagonists.
[0162] Thus, this invention also provides fragments, or purified
polypeptides of calcium receptors, metabotropic glutamate
receptors, or chimeric receptors including calcium receptor
sequences and metabotropic glutamate receptor sequences. The
fragments may be used to screen for compounds that are active at
either metabotropic glutamate or calcium receptors. For example, a
fragment including the extracellular domain of a calcium receptor
or a metabotropic glutamate receptor may be used in a soluble
receptor binding assay to identify which molecules in a
combinatorial library can bind the receptor within the region
assayed. Such "binding" molecules may be predicted to affect the
function of the receptor. Preferred receptor fragments include
those having functional receptor activity, a binding site, epitope
for antibody recognition (typically at least six amino acids),
and/or a site which binds a metabotropic glutamate receptor
agonist, antagonist or modulator. Other preferred receptor
fragments include those having only an extracellular portion, a
transmembrane portion, an intracellular portion, and/or a multiple
transmembrane portion (e.g., seven transmembrane portion). Such
receptor fragments have various uses such as being used to obtain
antibodies to a particular region and being used to form chimeric
receptors and fragments of other receptors to create a new receptor
having unique properties.
[0163] The purified polypeptides or fragments preferably have at
least six contiguous amino acids of a metabotropic glutamate
receptor or calcium receptor or chimeric receptor. By "purified" in
reference to a polypeptide is meant that the polypeptide is in a
form (i.e., its association with other molecules) distinct from
naturally occurring polypeptide. Preferably, the polypeptide is
provided as a substantially purified preparation representing at
least 75%, more preferably 85%, most preferably 95%, of the total
protein in the preparation.
[0164] In many applications, it is preferable that the purified
polypeptide or fragment have more than 6 contiguous amino acids
from the metabotropic glutamate receptor or calcium receptor or
chimeric receptor. For example, the purified polypeptide can have
at least 12, 18, 14, 30, or 36 contiguous amino acids of the
"parent" receptor.
[0165] Other fragments may be prepared which include only the seven
transmembrane domain and the cytoplasmic tail domain of calcium
receptors, metabotropic glutamate receptors, or chimeric receptors.
Such fragments may be useful, for example, in functional assays to
screen for compounds whose site of action is at the seven
transmembrane domain. As indicated above, the invention provides
methods of screening for a compound that binds to a receptor, which
utilizes receptor fragments. In one example, the method includes
the steps of: preparing a nucleic acid sequence encoding a fragment
of a receptor; inserting the sequence into a replicable expression
vector capable of expressing said fragment in a host cell;
transforming a host cell with the vector; recovering the fragment
from the host cell; introducing fragment and a test compound into
an acceptable medium; and monitoring the binding of the compound to
the fragment by physically detectable means. In cases in which the
receptor is a metabotropic glutamate receptor, the fragment
preferably includes an extracellular domain of the metabotropic
glutamate receptor, or a seven transmembrane domain of the
metabotropic glutamate receptor, or a seven transmembrane domain
and a cytoplasmic tail domain of a metabotropic glutamate receptor.
In cases in which the receptor is a calcium receptor, the fragment
preferably includes an extracellular domain of the calcium
receptor, a seven transmembrane domain of the calcium receptor, or
a seven transmembrane domain and a cytoplasmic tail domain of a
calcium receptor.
[0166] Certain fragments of metabotropic glutamate receptors and
calcium receptors retain the functions of activating one or more of
the cellular responses normally activated by the "parent" receptor
when contacted with a compound which interacts. Thus, for example,
a cellular expressed fragment which includes the 7TMD and CT of an
mGluR or a CaR, but do not include the ECD, may activate a cellular
response(s) when contacted with a compound which interacts with the
7TMD. Thus, incorporation of such fragments in a cell-based method
of screening for compounds which bind to or modulate a metabotropic
glutamate receptor or calcium receptor, such as that described
herein for chimeric receptors, is useful to identify active
compounds which interact with the fragment rather that the deleted
sequence.
[0167] Isolated fragments of calcium receptors, metabotropic
glutamate receptors, or chimeric receptors comprising calcium
receptor sequences and metabotropic glutamate receptor sequences
may be combined in an in vitro functional assay to screen for
compounds active at either receptor. Such an in vitro assay, for
example, may include a fragment having the extracellular domain of
one receptor and a fragment having the seven transmembrane domain
and the cytoplasmic tail domain of the other receptor, where the
extracellular domain will complement the seven
transmembrane/cytoplasmic tail domain fragment in vitro. In this
way functional chimeric receptors which are useful in a screening
assay may be prepared without the need for recombination of the
nucleic acids encoding them. Instead, these functional chimeric
receptors may be achieved by combining, in vitro, portions of
different receptors.
[0168] Such combinations of fragments provide methods of screening
for compounds which bind to or modulate a receptor. An example of
such a method includes the steps of: preparing a nucleic acid
sequence encoding a first fragment which is a fragment of a first
receptor; inserting the sequence into a replicable expression
vector capable of expressing that fragment in a host cell;
transforming a host cell with the vector; recovering the fragment
from the host cell; preparing a nucleic acid sequence encoding a
second fragment which is a fragment of a second receptor; inserting
the sequence into a replicable expression vector capable of
expressing the second fragment in a host cell; transforming a host
cell with the vector; recovering the second fragment from the host
cell, introducing both the first fragment and the second fragment
into an acceptable medium, and monitoring the binding and
modulation of the compound by physically detectable means.
[0169] In particular preferred examples, the first fragment
includes the extracellular domain of a metabotropic glutamate
receptor and the second fragment includes the seven transmembrane
domain and the cytoplasmic tail domain of a calcium receptor; the
first fragment includes the extracellular domain of a calcium
receptor and the second fragment includes the seven transmembrane
domain and the cytoplasmic tail domain of a metabotropic glutamate
receptor; or the first fragment includes the extracellular domain
of a calcium receptor and the second fragment includes the seven
transmembrane domain of a metabotropic glutamate receptor and the
cytoplasmic tail domain of a calcium receptor.
[0170] D. Screening Procedures to Identify Compounds which Modulate
Metabotropic Glutamate Receptor Activities Using Chimeric
Receptors
[0171] The mGluR agonist and antagonist compounds described in the
scientific literature are related to the endogenous agonist,
glutamate (for reviews see: Cockcroft et al., Neurochem. Int.
23:583-594, 1993; Schoepp and Conn, TIPS 14:13-20, 1993; Hollmann
and Heinemann, Annu. Rev. Neurosci. 17:31-108, 1994). Such agonist
and antagonist compounds have an acidic moiety, usually a
carboxylic acid, but sometimes a phosphatidic acid. Presumably
then, such compounds bind mGluRs at the same site as the amino
acid, glutamate. This has been confirmed for
methylcarboxyphenylglycine, which was shown to be a competitive
antagonist of glutamate (Eaton et al., Eur. J. Pharm.--Mol. Pharm.
Sect. 244:195-197, 1993). It can be assumed that compounds active
at mGluRs, lacking negative charges, and not resembling the amino
acid glutamate, may not act at the glutamate binding site.
[0172] Compounds targeted to the metabotropic glutamate receptor
have several uses including diagnostic uses and therapeutic use.
The syntheses of many of the compounds is described by Nemeth et
al., entitled "Calcium Receptor Active Molecule" International
Publication Number WO 93/04373, hereby incorporated by reference
herein. Those compounds binding to a metabotropic glutamate
receptor and those compounds efficacious in modulating metabotropic
receptor glutamate activity can be identified using the procedures
described herein. Those compounds which can selectively bind to the
metabotropic glutamate receptor can be used diagnostically to
determine the presence of the metabotropic glutamate receptor
versus other glutamate receptors.
[0173] The following is a description of procedures which can be
used to obtain compounds modulating metabotropic glutamate receptor
activity. Various screening procedures can be carried out to assess
the ability of a compound to modulate activity of chimeric
receptors of the invention by measuring its ability to have one or
more activities of a metabotropic glutamate receptor modulating
agent or a calcium receptor modulating agent. In cells expressing
chimeric receptors of the invention, such activities include the
effects on intracellular calcium, inositol phosphates and cyclic
AMP.
[0174] Measuring [Ca.sup.2+].sub.i with fura-2 provides a very
rapid means of screening new organic molecules for activity. In a
single afternoon, 10-15 compounds (or molecule types) can be
examined and their ability to mobilize or inhibit mobilization of
intracellular Ca.sup.2+ can be assessed by a single experiment. The
sensitivity of observed increases in [Ca.sup.2+].sub.i to
depression by PMA can also be assessed.
[0175] For example, recombinant cells expressing chimeric receptors
of the invention loaded with fura-2 are initially suspended in
buffer containing 0.5 mM CaCl.sub.2. A test substance is added to
the cuvette in a small volume (5-15 .mu.l) and changes in the
fluorescence signal are measured. Cumulative increases in the
concentration of the test substance are made in the cuvette until
some predetermined concentration is achieved or no further changes
in fluorescence are noted. If no changes in fluorescence are noted,
the molecule is considered inactive and no further testing is
performed.
[0176] In the initial studies, molecules may be tested at
concentrations as high as 5 or 10 mM. As more potent molecules
became known, the ceiling concentration was lowered. For example,
newer molecules are tested at concentrations no greater than 500
.mu.M. If no changes in fluorescence are noted at this
concentration, the molecule can be considered inactive.
[0177] Molecules causing increases in [Ca.sup.2+].sub.i are
subjected to additional testing. Two characteristics of a molecule
which can be considered in screening for a positive modulating
agent of a chimeric receptor of the invention are the mobilization
of intracellular Ca.sup.2+ and sensitivity to PKC activators.
[0178] A single preparation of cells can provide data on
[Ca.sup.2+].sub.i cyclic AMP levels, IP.sub.3 and other
intracellular messengers. A typical procedure is to load cells with
fura-2 and then divide the cell suspension in two; most of the
cells are used for measurement of [Ca.sup.2+].sub.i and the
remainder are incubated with molecules to assess their effects on
cyclic AMP.
[0179] Measurements of inositol phosphates are a time-consuming
aspect of the screening. However, ion-exchange columns eluted with
chloride (rather than formate) provide a very rapid means of
screening for IP.sub.3 formation, since rotary evaporation (which
takes around 30 hours) is not required. This method allows
processing of nearly 100 samples in a single afternoon by a single
experimenter. Those molecules that prove interesting, as assessed
by measurements of [Ca.sup.2+].sub.i, cyclic AMP, and IP.sub.3 can
be subjected to a more rigorous analysis by examining formation of
various inositol phosphates and assessing their isomeric form by
HPLC.
[0180] The following is illustrative of methods useful in these
screening procedures.
[0181] i. Measurement of Cyclic AMP
[0182] This section describes measuring cyclic AMP levels. Cells
were incubated as above and at the end of the incubation, a 0.15-ml
sample was taken and transferred to 0.85 ml of hot (70 C) water and
heated at this temperature for 5-10 minutes. The tubes were
subsequently frozen and thawed several times and the cellular
debris sedimented by centrifugation. Portions of the supernatant
were acetylated and cyclic AMP concentrations determined by
radioimmunoassay.
[0183] ii. Measurement of Inositol Phosphate Formation
[0184] This section describes procedures measuring inositol
phosphate formation. Membrane phospholipids were labeled by
incubating parathyroid cells with 4 .mu.Ci/ml .sup.3H-myo-inositol
for 20-24 hours. Cells were then washed and resuspended in PCB
containing 0.5 mM CaCl.sub.2 and 0.1% BSA. Incubations were
performed in microfuge tubes in the absence or presence of various
concentrations of organic polycation for different times. Reactions
were terminated by the addition of 1 ml chloroform-methanol-12 N
HCl (200:100:1; v/v/v). Aqueous phytic acid hydrolysate (200 .mu.l;
25 .mu.g phosphate/tube). The tubes were centrifuged and 600 .mu.l
of the aqueous phase was diluted into 10 ml water.
[0185] Inositol phosphates were separated by ion-exchange
chromatography using AG1-X8 in either the chloride- or
formate-form. When only IP.sub.3 levels were to be determined, the
chloride-form was used, whereas the formate form was used to
resolve the major inositol phosphates (IP.sub.3, IP.sub.2, and
IP.sub.1). For determination of just IP.sub.3, the diluted sample
was applied to the chloride-form column and the column was washed
with 10 ml 30 mM HCl followed by 6 ml 90 mM HCl and the IP.sub.3
was eluted with 3 ml 500 mM HCl. The last eluate was diluted and
counted. For determination of all major inositol phosphates, the
diluted sample was applied to the formate-form column and IP.sub.1,
IP.sub.2, and IP.sub.3 eluted sequentially by increasing
concentrations of formate buffer. The eluted samples from the
formate columns were rotary evaporated, the residues brought up in
cocktail, and counted.
[0186] The isomeric forms of IP.sub.3 were evaluated by HPLC. The
reactions were terminated by the addition of 1 ml 0.45 M perchloric
acid and stored on ice for 10 minutes. Following centrifugation,
the supernatant was adjusted to pH 7-8 with NaHCO.sub.3. The
extract was then applied to a Partisil SAX anion-exchange column
and eluted with a linear gradient of ammonium formate. The various
fractions were then desalted with Dowex followed by rotary
evaporation prior to liquid scintillation counting in a Packard
Tri-carb 1500 LSC.
[0187] For all inositol phosphate separation methods, appropriate
controls using authentic standards were used to determine if
organic polycations interfered with the separation. If so, the
samples were treated with cation-exchange resin to remove the
offending molecule prior to separation of inositol phosphates.
[0188] iii. Use of Lead Molecules
[0189] By systematically measuring the ability of a lead molecule
to mimic or antagonize the effect of a natural ligand, the
importance of different functional groups for agonists and
antagonists can be identified. Of the molecules tested, some are
suitable as drug candidates while others are not necessarily
suitable as drug candidates. The suitability of a molecule as a
drug candidate depends on factors such as efficacy and toxicity.
Such factors can be evaluated using standard techniques. Thus, lead
molecules can be used to demonstrate that the hypothesis underlying
receptor-based therapies is correct and to determine the structural
features that enable the receptor-modulating agents to act on the
receptor and, thereby, to obtain other molecules useful in this
invention.
[0190] The examples described herein demonstrate the general design
of molecules useful as modulators of the activity of mGluRs and
CaRs. The examples also describe screening procedures to obtain
additional molecules, such as the screening of natural product
libraries. Using these procedures, those of ordinary skill in the
art can identify other useful modulators of mGluRs and CaRs.
[0191] Cell lines expressing calcium receptors have been obtained
and methods applicable to their use in high throughput screening to
identify compounds which modulate the activity of calcium receptors
disclosed (See U.S. Ser. No. 08/353,784, filed Dec. 9, 1994, hereby
incorporated by reference herein). Cell lines expressing
metabotropic glutamate receptors have been obtained and methods
applicable to their potential use to identify compounds which
modulate activity of metabotropic glutamate receptors disclosed
(European Patent Publication No. 0 568 384 A1; European Patent
Publication No. 0 569 240 A1; PCT Publication No. WO 94/29449; and
PCT Publication No. WO 92/10583). Thus, recombinant cell-based
assays which use biochemical, spectrophotometric or other physical
measurements to detect the modulation of activity of an expressed
receptor, especially by measuring changes in affected intracellular
messengers, are known to those in the art and can be constructed
such that they are suitable for high throughput functional
screening of compounds and compound libraries. It will be
appreciated by those in the art that each functional assay has
advantages and disadvantages for high throughput screening which
will vary depending on the receptor of interest, the cell lines
employed, the nature of the biochemical and physical measurements
used to detect modulation of receptor function, the nature of the
compound library being screened and various other parameters. An
exceptionally useful and practical method is the use of fluorescent
indicators of intracellular Ca.sup.2+ to detect modulation of the
activity of receptors coupled to phospholipase-C.
[0192] The use of [.sup.3 H]glutamate, or any other compound found
to modulate the mGluR discovered by the methods described herein,
as a lead compound is expected to result in the discovery of other
compounds having similar or more potent activity which in turn can
be used as lead compounds. Lead compounds such as
[.sup.3H]glutamate can be used for molecular modeling using
standard procedures and to screen compound libraries. Radioligand
binding techniques [a radio labeled binding assay] can be used to
identify compounds binding at the glutamate binding site. While
such binding assays are useful for finding new compounds binding to
the glutamate binding site on mGluR's, the current invention
provides for the discovery of novel compounds with unique and
useful activities at mGluR's which can be radio labeled and used
similarly in Radioligand assays to find additional compounds
binding to the new lead defined site. This screening test allows
vast numbers of potentially useful compounds to be screened for
their ability to bind to the glutamate binding site. Other rapid
assays for detection of binding to the glutamate binding site on
metabotropic glutamate receptors can be devised using standard
detection techniques. Other compounds can be identified which act
at the glutamate binding using the procedures described in this
section. A high-throughput assay is first used to screen product
libraries (e.g., natural product libraries and compound files) to
identify compounds with activity at the glutamate (or lead
compound) binding site. These compounds are then utilized as
chemical lead structures for a drug development program targeting
the glutamate or lead compound binding site on metabotropic
glutamate receptors. Routine experiments, including animal studies
can be performed to identify those compounds having the desired
activities.
[0193] The following assay can be utilized as a high-throughput
assay. Rat brain membranes are prepared according to the method of
Williams et al. (Molec. Pharmacol. 36:575, 1989), with the
following alterations: Male Sprague-Dawley rats (Harlan
Laboratories) weighing 100-200 g are sacrificed by decapitation.
The cortex or cerebellum from 20 rats are cleaned and dissected.
The resulting brain tissue is homogenized at 4 C with a polytron
homogenizer at the lowest setting in 300 ml 0.32 M sucrose
containing 5 mM K-EDTA (pH 7.0). The homogenate is centrifuged for
10 min at 1,000.times.g and the supernatant removed and centrifuged
at 30,000.times.g for 30 minutes. The resulting pellet is
resuspended in 250 ml 5 mM K-EDTA (pH 7.0) stirred on ice for 15
minutes, and then centrifuged at 30,000.times.g for 30 minutes. The
pellet is resuspended in 300 ml 5 mM K-EDTA (pH 7.0) and incubated
at 32 C for 30 minutes. The suspension is then centrifuged at
100,000.times.g for 30 minutes. Membranes are washed by
resuspension in 500 ml 5 mM K-EDTA (pH 7.0), incubated at 32 C for
30 minutes, and centrifuged at 100,000.times.g for 30 minutes. The
wash procedure, including the 30-minute incubation, is repeated.
The final pellet is resuspended in 60 ml 5 mM K-EDTA (pH 7.0) and
stored in aliquots at -80 C.
[0194] To perform a binding assay with [.sup.3H]glutamate (as an
example of a lead compound), aliquots of SPMs (synaptic plasma
membranes) are thawed, resuspended in 30 ml of 30 mM EPPS/1 mM
K-EDTA, pH 7.0, and centrifuged at 100,000.times.g for 30 minutes.
SPMs are resuspended in buffer A (30 mM EPPS/1 mM K-EDTA, pH 7.0).
The [.sup.3H]-glutamate is added to this reaction mixture. Binding
assays are carried out in polypropylene test tubes. The final
incubation volume is 500 .mu.l. Nonspecific binding is determined
in the presence of 100 .mu.M nonradioactive glutamate. Duplicate
samples are incubated at 0 C for 1 hour. Assays are terminated by
adding 3 ml of ice-cold buffer A, followed by filtration over
glass-fiber filters (Schleicher & Schuell No. 30) that are
presoaked in 0.33% polyethyleneimine (PEI). The filters are washed
with another 3.times.3 ml of buffer A, and radioactivity is
determined by scintillation counting at an efficiency of 35-40% for
.sup.3H.
[0195] In order to validate the above assay, the following
experiments can also be performed:
[0196] (a) The amount of nonspecific binding of the
[.sup.3H]glutamate to the filters is determined by passing 500
.mu.l of buffer A containing various concentrations of
[.sup.3H]glutamate through the presoaked glass-fiber filters. The
filters are washed with another 4.times.3 ml of buffer A, and
radioactivity bound to the filters is determined by scintllation
counting at an efficiency of 35-40% for .sup.3H.
[0197] (b) A saturation curve is constructed by resuspending SPMs
in buffer A. The assay buffer (500 .mu.l) contains 60 .mu.g of
protein. Concentrations of [.sup.3H]glutamate are used, ranging
from 1.0 nM to 400 .mu.M in half-log units. A saturation curve is
constructed from the data, and an apparent K.sub.D value and
B.sub.max value determined by Scatchard analysis (Scatchard, Ann.
N. Y Acad. Sci. 51: 660, 1949). The cooperativity of binding of the
[.sup.3H]glutamate is determined by the construction of a Hill plot
(Hill, J. Physiol. 40:190, 1910).
[0198] (c) The dependence of binding on protein (receptor)
concentration is determined by resuspending SPMs in buffer A. The
assay buffer (500 .mu.l) contains a concentration of [3H]glutamate
equal to its K.sub.D value and increasing concentrations of
protein. The specific binding of [.sup.3H]glutamate should be
linearly related to the amount of protein (receptor) present.
[0199] (d) The time-course of ligand-receptor binding is determined
by resuspending SPMs in buffer A. The assay buffer (500 .mu.l)
contains a concentration of [.sup.3H]glutamate equal to its K.sub.D
value and 100 .mu.g of protein. Duplicate samples are incubated at
0 C for varying lengths of time; the time at which equilibrium is
reached is determined, and this time point is routinely used in all
subsequent assays.
[0200] (e) The pharmacology of the binding site can be analyzed by
competition experiments. In such experiments, the concentration of
[.sup.3H]glutamate and the amount of protein are kept constant,
while the concentration of test (competing) drug is varied. This
assay allows for the determination of an IC.sub.50 and an apparent
K.sub.D for the competing drug (Cheng and Prusoff, J. Biochem.
Pharmacol. 22:3099, 1973). The cooperativity of binding of the
competing drug is determined by Hill plot analysis.
[0201] Specific binding of the [.sup.3H]glutamate represents
binding to the glutamate binding site on metabotropic glutamate
receptors. As such, analogs of glutamate should compete with the
binding of [.sup.3H]glutamate in a competitive fashion, and their
potencies in this assay should correlate with their potencies in a
functional assay of metabotropic glutamate receptor activity (e.g.,
electrophysiological assessment of the activity of cloned
metabotropic glutamate receptors expressed in Xenopus oocytes).
Conversely, compounds which have activity at the sites other that
the glutamate binding site should not displace [.sup.3H]glutamate
binding in a competitive manner. Rather, complex allosteric
modulation of [.sup.3H]glutamate binding, indicative of
noncompetitive interactions, might occur.
[0202] (f) Studies estimating the dissociation kinetics are
performed by measuring the binding of [.sup.3H]glutamate after it
is allowed to come to equilibrium (see (d) above), and a large
excess of nonradioactive competing drug is added to the reaction
mixture. Binding of the [.sup.3H]glutamate is then assayed at
various time intervals. With this assay, the association and
dissociation rates of binding of the [.sup.3H]glutamate are
determined (Titeler, Multiple Dopamine Receptors: Receptor Binding
Studies in Dopamine Pharmacology. Marcel Dekker, Inc., New York,
1983). Additional experiments involve varying the reaction
temperature (0 C to 37 C) in order to understand the temperature
dependence of this parameter.
[0203] The following is one example of a rapid screening assay to
obtain compounds modulating metabotropic glutamate receptor
activity. The screening assay first measures the ability of
compounds to bind to recombinant receptors, or receptor fragments
containing the glutamate binding site. Compounds binding to the
metabotropic glutamate receptor are then tested for their ability
to modulate one or more activities at a metabotropic glutamate
receptor.
[0204] In one procedure, a cDNA or gene clone encoding the chimeric
receptor or fragment of a metabotropic glutamate receptor from a
suitable organism such as a human is obtained using standard
procedures. Distinct fragments of the clone are expressed in an
appropriate expression vector to produce the smallest receptor
polypeptide(s) obtainable able to bind glutamate. In this way, the
polypeptide(s) containing the glutamate binding site is identified.
Such experiments can be facilitated by utilizing a stably
transfected mammalian cell line (e.g., HEK 293 cells) expressing
metabotropic glutamate receptors.
[0205] Alternatively, the metabotropic glutamate receptor can be
chemically reacted with glutamate chemically modified so that amino
acid residues of the metabotropic glutamate receptor which contact
(or are adjacent to) the selected compound are modified and thereby
identifiable. The fragment(s) of the metabotropic glutamate
receptor containing those amino acids which are determined to
interact with glutamate and are sufficient for binding to
glutamate, can then be recombinantly expressed using standard
techniques.
[0206] The recombinant polypeptide(s) having the desired binding
properties can be bound to a solid-phase support using standard
chemical procedures. This solid-phase, or affinity matrix, may then
be contacted with glutamate to demonstrate that this compound can
bind to the column, and to identify conditions by which the
compound may be removed from the solid-phase. This procedure may
then be repeated using a large library of compounds to determine
those compounds which are able to bind to the affinity matrix.
Bound compounds can then can be released in a manner similar to
glutamate. Alternative binding and release conditions may be
utilized to obtain compounds capable of binding under conditions
distinct from those used for glutamate binding (e.g., conditions
which better mimic physiological conditions encountered especially
in pathological states). Compounds binding to the glutamate binding
site can thus be selected from a very large collection of compounds
present in a liquid medium or extract.
[0207] In an alternate method, chimeric receptors are bound to a
column or other solid phase support. Those compounds which are not
competed off by reagents binding to the glutamate binding site on
the receptor can then be identified. Such compounds define
alternative binding sites on the receptor. Such compounds may be
structurally distinct from known compounds and may define chemical
classes of agonists or antagonists which may be useful as
therapeutics agents.
[0208] Modulating metabotropic glutamate receptor activity causes
an increase or decrease in a cellular response which occurs upon
metabotropic glutamate receptor activation. Cellular responses to
metabotropic glutamate receptor activation vary depending upon the
type of metabotropic glutamate receptor activated. Generally,
metabotropic glutamate receptor activation causes one or more of
the following activities: (1) increase in PI hydrolysis; (2)
activation of phospholipase C; (3) increases and decreases in the
formation of cyclic adenosine monophosphate (cAMP); (4) decrease in
the formation of cAMP; (5) changes in ion channel function; (6)
activation of phospholipase D; (7) activation or inhibition of
adenylyl cyclase; (8) activation of guanylyl cyclase; (9) increases
in the formation of cyclic guanosine monophosphate (cGMP); (10)
activation of phospholipase A.sub.2; (11) increases in arachidonic
acid release; (12) increases or decreases in the activity of
voltage- and ligand-gated ion channels; (13) and increase in
intracellular calcium. Inhibition of metabotropic glutamate
receptor activation prevents one or more of these activities from
occurring.
[0209] Activation of a particular metabotropic glutamate receptor
refers to an event which subsequently causes the production of one
or more activities associated with the type of receptor activated.
Activation of mGluR1 can result in one or more of the following
activities: increase in PI hydrolysis, increase in cAMP formation,
increase in intracellular calcium (Ca.sup.2+) and increase in
arachidonic acid formation. Compounds can modulate one or more
metabotropic glutamate receptor activities by acting as an agonist
or antagonist of glutamate binding site activation.
[0210] The chimeric receptors of the present invention provide a
method of screening for compounds active at mGluRs by the detection
of signals produced by CaRs. The chimeric receptors may be used in
the screening procedures described in PCT/US93/01642 (WO94/18959),
which are hereby incorporated by reference herein, including
methods of screening using fura-2, and measurement of cytosolic
Ca.sup.2+ using cell lines expressing calcium receptors and methods
of screening using oocyte expression.
[0211] Active compounds identified by the screening methods
described herein, may be useful as therapeutic molecules to
modulate metabotropic glutamate receptor activity or as a
diagnostic agents to diagnose those patients suffering from a
disease characterized by an abnormal metabotropic glutamate
receptor activity. Preferably the screening methods are used to
identify metabotropic glutamate receptor modulators by screening
potentially useful molecules for an ability to mimic or block an
activity of extracellular glutamate or other metabotropic glutamate
receptor agonists on a cell having a metabotropic glutamate
receptor and determining whether the molecule has an EC.sub.50
IC.sub.50 of less than or equal to 100 .mu.M. More preferably, the
molecules tested for its ability to mimic or block an increase in
[Ca.sup.2+]; elicited by extracellular glutamate or other mGluR
agonists.
[0212] Identification of metabotropic glutamate receptor-modulating
agents is facilitated by using a high-throughput screening system.
High-throughput screening allows a large number of molecules to be
tested. For example, a large number of molecules can be tested
individually using rapid automated techniques or in combination
using a combinatorial library. Individual compounds able to
modulate metabotropic glutamate receptor activity present in a
combinatorial library can be obtained by purifying and retesting
fractions of the combinatorial library. Thus, thousands to millions
of molecules can be screened in a single day. Active molecules can
be used as models to design additional molecules having equivalent
or increased activity. Preferably the identification method uses a
recombinant chimeric metabotropic glutamate receptor. Chimeric
receptors can be introduced into different cells using a vector
encoding a receptor. Preferably, the activity of molecules in
different cells is tested to identify a metabotropic glutamate
receptor agonist or metabotropic glutamate receptor antagonist
molecule which mimics or blocks one or more activities of glutamate
at a first type of metabotropic glutamate receptor but not at a
second type of metabotropic glutamate receptor.
[0213] As indicated above, the present invention provides a novel
method of screening for compounds which modulate metabotropic
glutamate receptor activity, by using a chimeric receptor having
portions of a metabotropic glutamate receptor and portions of a
calcium receptor. In particular receptors of this type, the
signaling process of the calcium receptor portion is used to detect
modulation of mGluR activity, as various compounds are tested for
binding to the mGluR portion. The method of screening can be
conducted in a variety of ways, such as utilizing chimeric
receptors having different portions from the metabotropic glutamate
receptor and calcium receptor. Certain preferred examples are
described below.
[0214] In one example, the method of screening for a compound that
binds to or modulates the activity of a metabotropic glutamate
receptor involves preparing a chimeric receptor having an
extracellular domain, a seven transmembrane domain, and an
intracellular cytoplasmic tail domain. A sequence of at least 6
contiguous amino acids is the same as or homologous to a sequence
from a metabotropic glutamate receptor and a sequence of at least 6
contiguous amino acids is the same as or homologous to a sequence
from a calcium receptor. The chimeric receptor and a test compound
are introduced into a acceptable medium, and the binding of the
test compound to the receptor or the modulation of the receptor by
the test compound is monitored by physically detectable means in
order to identify such binding or modulating compounds. Generally,
acceptable media will include those in which a natural ligand of an
mGluR and/or a CaR will interact with an mGluR or a CaR.
[0215] Often it will be beneficial to use chimeric receptors which
have longer sequences from one or both of the mGluR and the CaR.
For example, the chimeric receptor can have a sequence of at least
12, 18, 24, 30, 36, or more amino acids the same as or homologous a
sequence from one or both of the mGluR or CaR. In one useful
chimeric receptor, one domain is homologous to a domain of a
metabotropic glutamate receptor and at least one domain is
homologous to a domain of a calcium receptor
[0216] In a second example, the method of screening for a compound
which binds to or modulates the activity of a metabotropic
glutamate receptor utilizes a nucleic acid sequence which encodes a
chimeric receptor. The nucleic acid is expressed in a cell, and
binding or modulation by a test compound is observed by monitoring
the effects of the test compound on the cell. Thus, generally the
method includes preparing a nucleic acid sequence encoding a
chimeric receptor. The encoded chimeric receptor has an
extracellular domain, a seven transmembrane domain, and an
intracellular cytoplasmic tail domain. As in the example above, the
chimeric receptor has sequences of at least 6 contiguous amino
acids which are the same as or homologous to sequences from each of
an mGluR and a CaR. Also as indicated above, the sequences from one
or both of the mGluR and the CaR may beneficially be longer in
particular applications, e.g., at least 12, 18, 24, 30, 36, or more
amino acids in length. The nucleic acid sequence is inserted into a
replicable expression vector capable of expressing the chimeric
receptor in a host cell, and a host cell is transformed with the
vector. The transformed host cell and a test compound are
introduced into an acceptable medium and the effect of the compound
on the host cell is monitored (such as be techniques or assays
described above). Preferably, though not necessarily, the host cell
is a eukaryotic cell.
[0217] The amino acid sequences of the chimeric receptor can be
selected in a variety of combinations in particular cases. Thus, a
chimeric receptor can include at least one domain which is
homologous to a domain of a metabotropic glutamate receptor and at
least one domain which is homologous to a domain of a calcium
receptor. A domain(s) of the chimeric receptor can, for example, be
homologous the extracellular domain and/or the seven transmembrane
domain of a metabotropic glutamate receptor.
[0218] Likewise, a chimeric receptor which has three cytoplasmic
loops can have at least one loop homologous to a cytoplasmic loop
of an mGluR, or at least one loop homologous to a cytoplasmic loop
of a CaR, or at least one loop homologous to a cytoplasmic loop of
each of the those receptors.
[0219] Similarly, in other chimeric receptors, there is a portion
of the receptor which is homologous to a sequence of one type of
receptor (CaR or mGluR), while the remainder of the chimeric
receptor is homologous to the other type of receptor (CaR or
mGluR). Thus, the chimeric receptor can have a sequence of at least
6, 12, 18, 24, 30, 36, or more contiguous amino acids which is
homologous to a sequence of one of the receptor types with the
remainder of the sequence of the chimeric receptor homologous to a
sequence from the other receptor type. This further includes cases
in which at least one cytoplasmic loop is from one of the receptor
types, or at least one domain is from one of the receptor
types.
[0220] Other combinations of sequences will also be useful in
particular applications.
[0221] The chimeric metabotropic glutamate/calcium receptors can
also be used to screen for compounds active at both metabotropic
glutamate receptors and calcium receptors. This is particularly
useful for screening for compounds which interact at different
domains or subdomains in an mGluR than in a CaR. Thus, such
chimeras are useful for screening for compounds which, for example,
act within the extracellular domain of a metabotropic glutamate
receptor and also act within the seven transmembrane domain or the
cytoplasmic tail domain of a calcium receptor. Such a chimera would
include the extracellular domain of a metabotropic glutamate
receptor linked to the seven transmembrane domain and cytoplasmic
tail of a calcium receptor.
[0222] To screen for such compounds, active at both metabotropic
glutamate receptors and calcium receptors, compounds would be
screened according to the various methods of the present invention,
against the chimeric receptor, the calcium receptor, and the
metabotropic glutamate receptor. Compounds active at the seven
transmembrane domain of the calcium receptor portion of the
chimeric receptor should also be active when tested against the
calcium receptor itself. A preferred method of screening for such
compounds is to first screen them according to the methods of the
present invention against a chimeric molecule having the
extracellular domain of the metabotropic glutamate receptor, and
the seven transmembrane and cytoplasmic tail domains of the calcium
receptor and to then screen the positive compounds against both
chimeric molecule having the extracellular and seven transmembrane
domains of the metabotropic glutamate receptor and the cytoplasmic
tail domain of the calcium receptor, and the calcium receptor
itself. Compounds active at both molecules will be positive when
tested against all three chimeric receptors.
[0223] Conversely, a chimera including the extracellular domain of
a calcium receptor linked to the seven transmembrane domain and
cytoplasmic tail of a metabotropic glutamate receptor would be
useful in screening for compounds that act within the extracellular
domain of a calcium receptor and also act within the seven
transmembrane domain or the cytoplasmic tail of a metabotropic
glutamate receptor. Preferably, the chimeric receptor, which
includes the extracellular domain of a calcium receptor and the
seven transmembrane domain and the cytoplasmic tail of a
metabotropic glutamate receptor, is further modified to include
portions of the cytoplasmic tail of a calcium receptor. This more
preferred embodiment would thereby obtain the superior signaling
properties of the calcium receptor while still being useful for
screening for compounds that act at both the calcium receptor and
the metabotropic glutamate receptor.
[0224] Thus in one aspect the invention features a method of
screening for compounds active at both a metabotropic glutamate
receptor and a calcium receptor, by preparing a nucleic acid
sequence encoding a chimeric receptor. The chimeric receptor has an
extracellular domain, a seven transmembrane domain, and an
intracellular cytoplasmic tail domain, and at least one domain is
homologous to a domain of the metabotropic glutamate receptor and
at least one domain is homologous to a domain of a calcium
receptor. The nucleic acid sequence is inserted into a replicable
expression vector capable of expressing said chimeric receptor in a
host cell, and a host cell is transformed with the vector. The
transformed host cell and a test compound are introduced into an
acceptable medium, and the effect of the test compound on the cell
are monitored.
[0225] In general, for each of the above screening methods using
chimeric receptors, the portion of the chimeric receptor homologous
to an mGluR and the portion homologous to a CaR are selected to
provide the binding, modulation, and/or signal coupling
characteristics appropriate for a particular application.
[0226] E. Site of Action
[0227] The chimeric receptor molecules are also useful in methods
for determining the site-of-action of compounds already identified
as metabotropic glutamate receptor or calcium receptor active
compounds. For example, chimeras including the extracellular domain
of a metabotropic glutamate receptor linked to the seven
transmembrane domain and cytoplasmic tail of a calcium receptor, as
well as chimeras including the extracellular domain of a calcium
receptor linked to the seven transmembrane domain and cytoplasmic
tail of a metabotropic glutamate receptor would be useful in
determining the site-of-action of either metabotropic glutamate
receptor or calcium receptor active compounds. Those of ordinary
skill in the art will recognize that these are two examples of
large sequence exchanges and that much smaller sequence exchanges
may also be employed to further refine the determination of the
site-of-action.
[0228] Thus, the invention provides a method of determining the
site-of-action of a metabotropic glutamate receptor active compound
by: preparing a nucleic acid sequence encoding a chimeric receptor
wherein the chimeric receptor comprises at least a 6 amino acid
sequence which is homologous to a sequence of amino acids of a
calcium receptor and the remainder of the amino acid sequence is
homologous to a sequence of amino acids of a metabotropic glutamate
receptor; inserting the sequence into a replicable expression
vector capable of expressing the chimeric receptor in a host cell;
transforming a host cell with the vector; introducing the
transformed host cell and the compound into an acceptable medium;
and monitoring the effect of the compound on the cell.
[0229] As indicated above for methods of screening, in particular
applications it is advantageous to use sequence exchanges of
different sizes. Thus, in other applications, the sequence
homologous to a sequence from a calcium receptor, may for example,
be at least 12, 18, 24, 30, 36, or more amino acids in length.
[0230] Conversely, a method of determining the site-of-action of a
calcium receptor active compound can be performed in the same
manner as described above, but using a nucleic acid encoding a
chimeric receptor which includes at least a 6 amino acid sequence
which is homologous to a sequence of amino acids of a metabotropic
glutamate receptor and the remainder of the amino acid sequence is
homologous to a sequence of amino acids of a calcium receptor. Also
similar to the method above, the sequence homologous to a sequence
from a metabotropic glutamate receptor can be of different lengths
in various applications, for example, at least 12, 18, 24, 30, 36,
or more amino acids in length. .
[0231] F. Modulation of Metabotropic Glutamate Receptor
Activity
[0232] Modulation of metabotropic glutamate receptor activity can
be used to produce different effects such as anticonvulsant
effects, neuroprotectant effects, analgesic effects,
cognition-enhancement effects, and muscle-relaxation effects. Each
of these effects has therapeutic applications. Compounds used
therapeutically should have minimal side effects at therapeutically
effective doses.
[0233] The ability of a compound to modulate metabotropic glutamate
activity can be determined using electrophysiological and
biochemical assays measuring one or more metabotropic glutamate
activities. In general, such assays can be carried out using cells
expressing the metabotropic glutamate receptor(s) of interest, but
the assays can also be carried out using cells expressing a
chimeric receptors of this invention which modulates the cellular
activity which is to be monitored. Examples of such assays include
the electrophysiological assessment of metabotropic glutamate
receptor function in Xenopus oocytes expressing cloned metabotropic
glutamate receptors, the electrophysiological assessment of
metabotropic glutamate receptor function in transfected cell lines
(e.g., CHO cells, HEK 293 cells, etc.) expressing cloned
metabotropic glutamate receptors, the biochemical assessment of PI
hydrolysis and cAMP accumulation in transfected cell lines
expressing cloned metabotropic glutamate receptors, the biochemical
assessment of PI hydrolysis and cAMP accumulation in rat brain
(e.g., hippocampal, cortical, striatal, etc.) slices, fluorimetric
measurements of cytosolic Ca2+ in cultured rat cerebellar granule
cells, and fluorimetric measurements of cytosolic Ca2+ in
transfected cell lines expressing cloned metabotropic glutamate
receptors.
[0234] Prior to therapeutic use in a human, the compounds are
preferably tested in vivo using animal models. Animal studies to
evaluate a compound's effectiveness to treat different diseases or
disorders, or exert an effect such as an analgesic effect, a
cognition023.226173.1 enhancement effect, or a muscle-relaxation
effect, can be carried out using standard techniques.
[0235] G. Novel Agents and Pharmaceutical Compositions
[0236] The chimeric receptors and screening methods described
herein provide metabotropic glutamate receptor-binding agents
(e.g., compounds and pharmaceutical compositions) discovered due to
their ability to bind to a chimeric metabotropic glutamate
receptor. Such binding agents are preferably modulators of a
metabotropic glutamate receptor. Certain of these agent will be
novel compounds identified by the screening methods described
herein. In addition, other such compounds are derived by standard
methodology from such identified compounds when such identified
compounds are used as lead compounds in screening assays based on
analogs of identified active compounds, or in medicinal chemistry
developments using identified compounds as lead compounds.
[0237] Further, by providing novel and efficient screening methods
using chimeric receptors, this invention provides a method for
preparing a pharmaceutical agent active on a metabotropic glutamate
receptor. Without such this efficient method, such agents would not
be identified. The method involves identifying a active agent by
screening using a chimeric receptor of the type described herein in
a screening method as described above. The identified agent or an
analog of that agent is synthesized in an amount sufficient to
administer to a patient in a therapeutically effective amount.
[0238] H. Treatment of Diseases and Disorders
[0239] A preferred use of the compounds and methods of the present
invention is in the treatment of neurological diseases and
disorders. Patients suffering from a neurological disease or
disorder can be diagnosed by standard clinical methodology.
[0240] Neurological diseases or disorders include neuronal
degenerative diseases, glutamate excitotoxicity, global and focal
ischemic and hemorrhagic stroke, head trauma, spinal cord injury,
hypoxia-induced nerve cell damage, and epilepsy. These different
diseases or disorders can be further medically characterized. For
example, neuronal degenerative diseases include Alzheimer's disease
and Parkinson's disease.
[0241] Another preferred use of the present invention is in the
production of other therapeutic effects, such as analgesic effects,
cognition-enhancement effects, or musclerelaxation effects. The
present invention is preferably used to produce one or more of
these effects in a patient in need of such treatment.
[0242] Patients in need of such treatment can be identified by
standard medical techniques. For example, the production of
analgesic activity can be used to treat patients suffering from
clinical conditions of acute and chronic pain including the
following: preemptive preoperative analgesia; peripheral
neuropathies such as occur with diabetes mellitus and multiple
sclerosis; phantom limb pain; causalgia; neuralgias such as occur
with herpes zoster; central pain such as that seen with spinal cord
lesions; hyperalgesia; and allodynia.
[0243] In a method of treating a patient, a therapeutically
effective amount of a compound which in vitro modulates the
activity of a chimeric receptor having at least the extracellular
domain of a metabotropic glutamate receptor is administered to the
patient. Typically, the compound modulates metabotropic glutamate
receptor activity by acting as an agonist or antagonist of
glutamate binding site activation. Preferably, the patient has a
neurological disease or a disorder, preferably the compound has an
effect on a physiological activity. Such physiological activity can
be convulsions, neuroprotection, neuronal death, neuronal
development, central control of cardiac activity, waking, control
of movements and control of vestibo ocular reflex.
[0244] Diseases or disorders which can be treated by modulating
metabotropic glutamate receptor activity include one or more of the
following types: (1) those characterized by abnormal glutamate
homeostasis; (2) those characterized by an abnormal amount of an
extracellular or intracellular messenger whose production can be
affected by metabotropic glutamate receptor activity; (3) those
characterized by an abnormal effect (e.g., a different effect in
kind or magnitude) of an intracellular or extracellular messenger
which can itself be ameliorated by metabotropic glutamate receptor
activity; and (4) other diseases or disorders in which modulation
of metabotropic glutamate receptor activity will exert a beneficial
effect, for example, in diseases or disorders where the production
of an intracellular or extracellular messenger stimulated by
receptor activity compensates for an abnormal amount of a different
messenger.
[0245] The compounds and methods can also be used to produce other
effects such as an analgesic effect, cognition-enhancement effect,
and a muscle-relaxant effect.
[0246] A "patient" refers to a mammal in which modulation of an
metabotropic glutamate receptor will have a beneficial effect.
Patients in need of treatment involving modulation of metabotropic
glutamate receptors can be identified using standard techniques
known to those in the medical profession. Preferably, a patient is
a human having a disease or disorder characterized by one more of
the following: (1) abnormal glutamate receptor activity (2) an
abnormal level of a messenger whose production or secretion is
affected by metabotropic glutamate receptor activity; and (3) an
abnormal level or activity of a messenger whose function is
affected by metabotropic glutamate receptor activity.
[0247] By "therapeutically effective amount" is meant an amount of
an agent which relieves to some extent one or more symptoms of the
disease or disorder in the patient; or returns to normal either
partially or completely one or more physiological or biochemical
parameters associated with or causative of the disease.
[0248] More generally, this invention provides a method for
modulating metabotropic glutamate receptor activity by providing to
a cell having a metabotropic glutamate receptor an amount of a
metabotropic glutamate receptor-modulating molecule sufficient to
either mimic one or more effects of glutamate at the metabotropic
glutamate receptor, or block one or more effects of glutamate at
the metabotropic glutamate receptor. The method can carried out in
vitro or in vivo.
[0249] I. Formulation and Administration
[0250] Active compounds as identified by the methods of this
invention can be utilized as pharmaceutical agents or compositions
to treat different diseases and disorders as described above. In
this context, a pharmacological agent or composition refers to an
agent or composition in a form suitable for administration to a
mammal, preferably a human.
[0251] The optimal formulation and mode of administration of
compounds of the present invention to a patient depend on factors
known in the art such as the particular disease or disorder, the
desired effect, and the type of patient. While the compounds will
typically be used to treat human patients, they may also be used to
treat similar or identical diseases in other vertebrates such as
other primates, farm animals such as swine, cattle and poultry, and
sports animals and pets such as horses, dogs and cats.
[0252] Preferably, the therapeutically effective amount is provided
as a pharmaceutical composition. A pharmacological agent or
composition refers to an agent or composition in a form suitable
for administration into a multicellular organism such as a human.
Suitable forms, in part, depend upon the use or the route of entry,
for example oral, transdermal, or by injection. Such forms should
allow the agent or composition to reach a target cell whether the
target cell is present in a multicellular host or in culture. For
example, pharmacological agents or compositions injected into the
blood stream should be soluble. Other factors are known in the art,
and include considerations such as toxicity and forms which prevent
the agent or composition from exerting its effect.
[0253] The claimed compositions can also be formulated as
pharmaceutically acceptable salts (e.g., acid addition salts)
and/or complexes thereof. Pharmaceutically acceptable salts are
non-toxic salts at the concentration at which they are
administered. The preparation of such salts can facilitate the
pharmacological use by altering the physicalchemical
characteristics of the composition without preventing the
composition from exerting its physiological effect. Examples of
useful alterations in physical properties include lowering the
melting point to facilitate transmucosal administration and
increasing the solubility to facilitate the administration of
higher concentrations of the drug.
[0254] Pharmaceutically acceptable salts include acid addition
salts such as those containing sulfate, hydrochloride, phosphate,
sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate,
ethanesulfonate, benzenesulfonate, p-toluenesulfonate,
cyclohexylsulfamate and quinate. (See e.g., supra. PCT/US92/03736.)
Pharmaceutically acceptable salts can be obtained from acids such
as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic
acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic
acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic
acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic
acid.
[0255] Pharmaceutically acceptable salts can be prepared by
standard techniques. For example, the free base form of a compound
is dissolved in a suitable solvent, such as an aqueous or
aqueous-alcohol solution, containing the appropriate acid and then
isolated by evaporating the solution. In another example, a salt is
prepared by reacting the free base and acid in an organic
solvent.
[0256] Carriers or excipients can also be used to facilitate
administration of the compound. Examples of carriers and excipients
include calcium carbonate, calcium phosphate, various sugars such
as lactose, glucose, or sucrose, or types of starch, cellulose
derivatives, gelatin, vegetable oils, polyethylene glycols and
physiologically compatible solvents. The compositions or
pharmaceutical composition can be administered by different routes
including intravenously, intraperitoneal, subcutaneous, and
intramuscular, orally, topically, or transmucosally.
[0257] The compounds of the invention can be formulated for a
variety of modes of administration, including systemic and topical
or localized administration. Techniques and formulations generally
may be found in Remington's Pharmaceutical Sciences, 18.sup.th
Edition, Mack Publishing Co., Easton, Pa., 1990.
[0258] For systemic administration, oral administration is
preferred. For oral administration, the compounds are formulated
into conventional oral dosage forms such as capsules, tablets and
tonics.
[0259] Alternatively, injection may be used, e.g., intramuscular,
intravenous, intraperitoneal, subcutaneous, intrathecal, or
intracerebroventricular. For injection, the compounds of the
invention are formulated in liquid solutions, preferably in
physiologically compatible buffers such as Hank's solution or
Ringer's solution. Alternatively, the compounds of the invention
are formulated in one or more excipients (e.g., propylene glycol)
that are generally accepted as safe as defined by USP standards. In
addition, the compounds may be formulated in solid form and
redissolved or suspended immediately prior to use. Lyophilized
forms are also included.
[0260] Systemic administration can also be by transmucosal or
transdermal means, or the molecules can be administered orally. For
transmucosal or transdermal administration, penetrants appropriate
to the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art, and include, for
example, for transmucosal administration, bile salts and fusidic
acid derivatives. In addition, detergents may be used to facilitate
permeation. Transmucosal administration may be, for example,
through nasal sprays or using suppositories. For oral
administration, the molecules are formulated into conventional oral
administration dosage forms such as capsules, tablets, and liquid
preparations.
[0261] For topical administration, the compounds of the invention
are formulated into ointments, salves, gels, or creams, as is
generally known in the art.
[0262] The amounts of various compounds to be administered can be
determined by standard procedures. Generally, a therapeutically
effective amount is between about 1 nmole and 3 .mu.mole of the
molecule, preferably 0.1 nmole and 1 .mu.mole depending on its
EC.sub.50 or IC.sub.50 and on the age and size of the patient, and
the disease or disorder associated with the patient. Generally, it
is an amount between about 0.1 and 50 mg/kg, preferably 0.01 and 20
mg/kg of the animal to be treated.
[0263] J. Transgenic Animals
[0264] The invention also provides transgenic, nonhuman mammals
containing a transgene encoding a chimeric receptor, particularly a
chimeric metabotropic glutamate receptor. Transgenic nonhuman
mammals are particularly useful as an in vivo test system for
studying the effects of introducing a chimeric receptor.
Experimental model systems may be used to study the effects in cell
or tissue cultures, in whole animals, or in particular cells or
tissues within whole animals or tissue culture systems. The effects
can be studied over specified time intervals (including during
embryogenesis).
[0265] The present invention provides for experimental model
systems for studying the physiological effects of the receptors.
Model systems can be created having varying degrees of receptor
expression. For example, the nucleic acid encoding a receptor may
be inserted into cells which naturally express the parent
receptors, such that the chimeric gene is expressed at much higher
levels. Also, a recombinant gene may be used to inactivate the
endogenous gene by homologous recombination, and thereby create a
receptor deficient cell, tissue, or animal.
[0266] Inactivation of a gene can be caused, for example, by using
a recombinant gene engineered to contain an insertional mutation
(e.g., the neo gene). The recombinant gene is inserted into the
genome of a recipient cell, tissue or animal, and inactivates
transcription of the receptor. Such a construct may be introduced
into a cell, such as an embryonic stem cell, by techniques such as
transfection, transduction, and injection. Stem cells lacking an
intact receptor sequence may generate transgenic animals deficient
in the receptor.
[0267] Preferred test models are transgenic animals. A transgenic
animal has cells containing DNA which has been artificially
inserted into a cell and inserted into the genome of the animal
which develops from that cell. Preferred transgenic animals are
primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and
cats.
[0268] A variety of methods are available for producing transgenic
animals. For example, DNA can be injected into the pronucleus of a
fertilized egg before fusion of the male and female pronuclei, or
injected into the nucleus of an embryonic cell (e.g., the nucleus
of a two-cell embryo) following the initiation of cell division
(Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442, 1985)).
By way of another example, embryos can be infected with viruses,
especially retroviruses, modified to carry chimeric receptor
nucleotide sequences of the present invention.
[0269] Pluripotent stem cells derived from the inner cell mass of
the embryo and stabilized in culture can be manipulated in culture
to incorporate nucleotide sequences of the invention. A transgenic
animal can be produced from such stem cells through implantation
into a blastocyst that is implanted into a foster mother and
allowed to come to term. Animals suitable for transgenic
experiments can be obtained from standard commercial sources such
as Charles River (Wilmington, Mass.), Taconic (Germantown, N.Y.),
and Harlan Sprague Dawley (Indianapolis, Ind.).
[0270] Methods for the culturing of embryonic stem (ES) cells and
the subsequent production of transgenic animals by the introduction
of DNA into ES cells using methods such as electroporation, calcium
phosphate/DNA precipitation and direct injection also are well
known to those of ordinary skill in the art. See, for example,
Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E.
J. Robertson, ed., IRL Press (1987).
[0271] Procedures for embryo manipulations are well known in the
art. The procedures for manipulation of the rodent embryo and for
microinjection of DNA into the pronucleus of the zygote are well
known to those of ordinary skill in the art (Hogan et al., supra).
Microinjection procedures for fish, amphibian eggs and birds are
detailed in Houdebine and Chourrout (Experientia 47:897-905, 1991).
Other procedures for introduction of DNA into tissues of animals
are described in U.S. Pat. No. 4,945,050 (Sandford et al., Jul. 30,
1990).
[0272] Transfection and isolation of desired clones can be carried
out using standard techniques (e.g., E. J. Robertson, supra). For
example, random gene integration can be carried out by
co-transfecting the nucleic acid with a gene encoding antibiotic
resistance. Alternatively, for example, the gene encoding
antibiotic resistance is physically linked to a nucleic acid
sequence encoding a chimeric receptor of the present invention.
[0273] DNA molecules introduced into ES cells can also be
integrated into the chromosome through the process of homologous
recombination. (Capecchi, Science 244: 1288-1292, 1989). Methods
for positive selection of the recombination event (e.g., neomycin
resistance) and dual positive-negative selection (e.g., neomycin
resistance and gancyclovir resistance) and the subsequent
identification of the desired clones by PCR have been described by
Capecchi, supra and Joyner et al., Nature 338:153-156, 1989), the
teachings of which are incorporated herein.
[0274] The final phase of the procedure is to inject targeted ES
cells into blastocysts and to transfer the blastocysts into
pseudopregnant females. The resulting chimeric animals are bred and
the offspring are analyzed by Southern blotting to identify
individuals that carry the transgene.
[0275] An example describing the preparation of a transgenic mouse
is as follows. Female mice are induced to superovulate and placed
with males. The mated females are sacrificed by CO.sub.2
asphyxiation or cervical dislocation and embryos are recovered from
excised oviducts. Surrounding cumulus cells are removed. Pronuclear
embryos are then washed and stored until the time of injection.
[0276] Randomly cycling adult female mice paired with vasectomized
males serve as recipients for implanted embryos. Recipient females
are mated at the same time as donor females and embryos are
transferred surgically to recipient females.
[0277] The procedure for generating transgenic rats is similar to
that of mice. See Hammer et al., Cell 63:1099-1112, 1990).
Procedures for the production of transgenic non-rodent mammals and
other animals are known in art. See, for example, Houdebine and
Chourrout, supra; Pursel et al., Science 244:1281-1288, 1989); and
Simms et al., Bio/Technology 6:179-183, 1988).
[0278] F. Transfected Cell Lines
[0279] Nucleic acid expressing a functional chimeric receptor can
be used to create transfected cell lines which functionally express
a specific chimeric receptor. Such cell lines have a variety of
uses such as being used for high-throughput screening for molecules
able to modulate metabotropic glutamate receptor activity; and
being used to assay binding to a metabotropic glutamate
receptor.
[0280] A variety of cell lines are capable of coupling exogenously
expressed receptors to endogenous functional responses. A number of
these cell lines (e.g., NIH-3T3, HeLa, NG115, CHO, HEK 293 and
COS7) can be tested to confirm that they lack an endogenous
metabotropic glutamate. Those lines lacking a response to external
glutamate can be used to establish stably transfected cell lines
expressing the cloned chimeric receptors of the invention.
[0281] Production of these stable transfectants is accomplished by
transfection of an appropriate cell line with a eukaryotic
expression vector, such as pMSG, in which the coding sequence for
the chimeric metabotropic glutamate receptor cDNA has been cloned
into the multiple cloning site. These expression vectors contain a
promoter region, such as the mouse mammary tumor virus promoter
(MMTV), that drive high-level transcription of cDNAS in a variety
of mammalian cells. In addition, these vectors contain genes for
the selection of cells that stably express the cDNA of interest.
The selectable marker in the PMSG vector encodes an enzyme,
xanthine-guanine phosphoribosyl transferase (XGPRT), that confers
resistance to a metabolic inhibitor that is added to the culture to
kill the nontransfected cells. A variety of expression vectors and
selection schemes are usually assessed to determine the optimal
conditions for the production of metabotropic glutamate
receptor-expressing cell lines for use in high-throughput screening
assays.
[0282] The most effective method for transfection of eukaryotic
cell lines with plasmid DNA varies with the given cell type. The
chimeric receptor expression construct will be introduced into
cultured cells by the appropriate technique, either Ca.sup.2+
phosphate precipitation, DEAE-dextran transfection, lipofection or
electroporation.
[0283] Cells that have stably incorporated or are episomally
maintaining the transfected DNA will be identified by their
resistance to selection media, as described above, and clonal cell
lines will be produced by expansion of resistant colonies. The
expression of the chimeric metabotropic glutamate receptor cDNA by
these cell lines will be assessed by solution hybridization and
Northern blot analysis. Functional expression of the receptor
protein will be determined by measuring the mobilization of
intracellular Ca.sup.2+ in response to externally applied calcium
receptor agonists.
[0284] The following examples illustrate the invention, but do not
limit its scope.
EXAMPLES
[0285] Examples are provided below to illustrate different aspects
and embodiments of the present invention. These examples are not
intended in any way to limit the disclosed invention. Rather, they
illustrate methodologies by which the novel chimeric receptors of
the present invention may be constructed. They also illustrate
methodologies by which compounds may be screened to determine which
compounds bind to or modulate a desired mGluR.
Example 1
phPCaR4.0 and pmGluR1s
[0286] Plasmid phPCaR4.0 (Garrett et al., J. Biol. Chem.,
270:12919, 1995, hereby incorporated by reference herein) was
isolated from E. coli bacterial cells containing the plasmid grown
up in nutrient broth containing 100 ug/ml ampicillin (Boerhringer
Mannheim). This plasmid DNA was used as the source for the DNA
encoding the human calcium receptor which was cloned into the EcoRI
site of vector pBluescript SK (Stratagene) in the T7 orientation.
All restriction enzymes and modification enzymes were purchased
from New England Biolabs unless otherwise noted.
[0287] Plasmid p7-3/6A was assembled in pBluescript SK from two
overlapping subclones of rat mGluR1 obtained from an
oligonucleotide screen of a commercially available rat olfactory
bulb cDNA library (Stratagene). This plasmid DNA was used as the
source of the metabotropic glutamate receptor, mGluR1. It was also
used to screen a commercially available human cerebellar cDNA
library for the human analogue. The human cerebellar library was
screened with a radioactively labeled rat mGluR1 by a method
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Chapter 1, 1989. Positive plaques were rescued using the
manufacturer's protocol and restriction mapped to compare them
against the published human mGluR1 sequence (Eur. Patent
publications 0 569 240 A1 and 0 568 384 A1). Two subclones were
assembled to create a complete human mGluR1.
[0288] Alternatively, the sequence of human mGluR1 may be obtained
from European Publication Nos. 0 569 240 A1 and 0 568 384 A1.
Probes prepared using this sequence may be used to probe human cDNA
libraries to obtain the full length human clone. In addition, the
relevant sequences may be synthesized using the sequence described
therein.
Example 2
pmGluR1/CaR
[0289] Chimeric receptors were constructed using recombinant PCR
and a multi-step cloning strategy. An overview of recombinant PCR
is presented by R. Higuchi in PCR Protocols: A Guide to Methods and
Applications, (1990) Academic Press, Inc. In the first construct
recombinant PCR was used to combine the sequences of mGluR1 and the
CaR across the junction of the extracellular and transmembrane
domains. The first chimera, pR1/CaR. contained the extracellular
domain of mGluR1 and the transmembrane and intracellular region of
the calcium receptor. The chimeric junction was created using three
separate PCR reactions. The first reaction used two primers
specific for rat mGluR1, A4, a 22 mer encoding nucleotides 1146 to
1167, and an antisense primer, oligoB, a 43 mer containing 22 bases
of mGluR1 (nucleotides -1755 to -1776) and 21 bases from the CaR
(nucleotides -1837 to -1857). These primers were used to amplify a
650 bp fragment of rat mGluR1. In a separate PCR reaction, a 500 bp
fragment of the CaR was amplified using hybrid primer C, a 43 mer
which was the complement of oligo B, and D4, an antisense primer
corresponding to nucleotides-2256 to -2279 of the CaR. These two
PCR products were purified from an agarose gel and annealed
together in equal molar ratio in the presence of the external
primers A4 and D4 and the proof-reading DNA polymerase, Pfu
(Stratagene). The 1,100 bp chimeric PCR product was digested with
Nsi I and subcloned into phCar4.0 digested with EcoRV and Nsi I.
The resultant subclone was subsequently digested with Xho I and Sfi
I to remove the extracellular domain of the CaR which was then
replaced with the Xho I-Sfi I fragment of rat mGluR1. The resultant
chimera, pR1/Car was validated by restriction mapping and
double-stranded DNA sequencing with Sequenase Version 2.0 (US
Biochemical). The DNA sequence for pR1/Car (SEQ ID NO: 1) and the
corresponding amino acid sequence (SEQ ID NO: 5) is depicted in
FIG. 2.
Example 3
pCaR/R1
[0290] A second construct, pCaR/R1, was a reciprocal of the chimera
described in example 2 in that it encoded the extracellular domain
of CaR and the transmembrane and intracellular region of mGluR1.
The chimeric junction was created as described above using
recombinant PCR. The first reaction used two primers specific for
CaR, CRSf1, a 22 mer corresponding to nucleotides 862 to 883, and
an antisense primer, CR1794, a 36 mer with 18 bases corresponding
to CaR (nucleotides -1777 to -1794) and 18 bases from mGluR1
(nucleotides -2110 to -2127). These primers were used to amplify a
935 bp fragment of CaR. In a separate PCR reaction, a 360 bp
fragment of mGluR1 was amplified using hybrid primer R12110, a 36
mer containing 18 bases of CaR (nucleotides 1777 to 1794)
covalently attached to 18 bases of mGluR1 (nucleotides 2110 to
2127) and R1Bgl, an antisense primer corresponding to nucleotides
-2451 to -2470 of mGluR1. These two PCR products were purified from
an agarose gel and annealed together in equal molar ratio in the
presence of the external primers CRSf1 and R1Bgl and the
proof-reading DNA polymerase, Pfu (Stratagene). The 1,250 bp
chimeric PCR product was digested with Sfi I and Bgl II and
subcloned into p7/3A digested with the same enzymes. A subclone was
subsequently digested with Sal I and SfiI to remove the
extracellular domain of mGluR1 which was then replaced with the Sal
I-Sfi I fragment of CaR. The resultant chimera, pCaR/R1 was
validated by restriction mapping and double-stranded DNA sequencing
using Sequenase Version 2.0 (US Biochemical). The DNA sequence is
for pCaR/R1 (SEQ ID NO: 2) and the corresponding amino acid
sequence (SEQ ID NO: 6) is depicted in FIG. 3.
Example 4
pratCH3 and phCH4
[0291] These chimeras are a result of swapping the CaR cytoplasmic
tail onto the extracellular and transmembrane domains of either rat
or human mGluR1. Recombinant PCR was used to attach the C-terminal
tail of the CaR onto human mGluR1 (which encodes the rat mGluR1
signal sequence) after nucleotide 2535. The first PCR reaction used
two primers specific for human mGluR1, M-1rev a 24 mer
corresponding to nucleotides 2242 to 2265, and an antisense primer,
CH3R1, a 36 mer composed of 18 bases of hmGluR1 (nucleotides -2518
to -2535) and 18 bases of CaR (nucleotides -2602 to -2619). These
primers were used to amplify a 300 bp fragment of hmGluR1. In a
separate PCR reaction, a 750 bp fragment of the CaR was amplified
using hybrid primer CH3CaR, a 36 mer which is the complement of
oligo CH3R1, and a commercially available T3 primer (Stratagene)
which primes in the Bluescript vector in a region downstream from
the 3' end of the CaR. The two PCR products were purified from an
agarose gel and annealed together in equal molar ratio in the
presence of the external primers M-1 rev and T3 and the
proof-reading DNA polymerase, Pfu (Stratagene). The 1 kb chimeric
PCR product was digested with Nhe I and Not I and subcloned into
phmGluR1 digested with the same enzymes. The resultant chimera,
phCH4 was validated by restriction mapping and double-stranded DNA
sequencing. To detect functional activity in the oocyte assay with
this clone it was necessary to exchange the 5' untranslated region
and the signal sequence from rat mGluR1 with the same region of
this human clone. This was done utilizing a Bsu36I restriction
site. Additionally, an Ace I fragment of rat mGluR1 was subcloned
into phCH4 to create a rat version of this same chimera. This
chimera is referred to as ratCH3. The DNA sequence for pratCh3 (SEQ
ID NO: 3) and the corresponding amino acid sequence (SEQ ID NO:
7)are depicted in FIG. 4. The DNA sequence for phCH44 (SEQ ID NO:
4) and the corresponding amino acid sequence (SEQ ID NO: 8) are
depicted in FIG. 5.
[0292] Using the techniques described in the above-mentioned
examples, we therefore envision the construction, evaluation and
screening utility of other mGluR/CaR chimeras. In this example we
have taken a Group I metabotropic glutamate receptor which, similar
to the calcium receptor, is coupled to the activation of
phospholipase C and mobilization of intracellular calcium, and by
swapping the C-terminal tail, maintained the integrity of the
second messenger system. Additionally, when the CaR tail was added
to mGluR1, the desensitization properties were lost. This
demonstrates the feasibility of changing specific G-protein
coupling of metabotropic glutamate receptors to those of the CaR by
swapping intracellular domains. By example, Group II mGluRs, such
as mGluR2 or mGluR3 which are G.sub.i coupled, could be changed to
Gq coupled receptors. This can be done by exchanging onto these
receptors the C-terminal cytosolic tail of the CaR using the
protocol described in examples 2, 3 and 4. Effective Gq coupling
could be evaluated in the oocyte as described in examples 5 and 6.
Activation of a Group II by L-CCG-I (their most potent agonist),
should induce mobilization of intracellular Ca2+ which will cause
the detectable inward rectifying C1- current measured in the
voltage-clamped oocyte.
[0293] To increase the effectiveness of G-protein binding it may be
useful to swap one or more additional intracellular (cytoplasmic)
loops of the CaR onto the mGluR1. By example, such substitution can
involve any of: intracellular loop 1, intracellular loop 2 and
intracellular loop 3 from a calcium receptor, substituted alone or
in any combination of loops. Such subdomain swapping may be
necessary for the most effective transference of G-protein binding
specificity.
Example 5
In vitro Transcription of RNA
[0294] RNA transcripts encoding the receptors described in examples
1 through 4 were produced by enzymatic transcription from plasmid
templates using T7 polymerase supplied with the mMessage mMachine
.TM.(Ambion). Each plasmid was treated with a restriction enzyme to
make a single cut distal to the 3' end of the cDNA insert to
linearize the template. This DNA was incubated with T7 RNA
polymerase in the presence of GpppG cap nucleotide, rATP, rCTP,
rUTP and rGTP. The synthetic RNA transcript is purified by DNase
treatment of the reaction mix and subsequent alcohol
precipitations. RNA was quantitated by absorbance spectroscopy
(OD.sub.260) and visualized on an ethidium stained 1.2%
formaldehyde gel.
Example 6
Functional Expression in Oocytes
[0295] Oocytes suitable for injection were obtained from adult
female Xenopus laevis toads using procedures described in C. J.
Marcus-Sekura and M. J. M. Hitchcock, Methods in Enzymology, Vol.
152 (1987). Pieces of ovarian lobe were incubated for 30 minutes in
Ca.sup.2+-free Modified Barths Saline (MBS) containing 1.5 mg/ml
collagenase type IA (Worthington). Subsequently, 5 ng of RNA
transcript prepared as described in Example 5, were injected into
each oocyte. Following injection, oocytes were incubated at 16 C in
MBS containing 0.5 mM CaCl.sub.2 for 2-7 days prior to
electrophysiological examination.
[0296] The ability of each chimeric receptor to function was
determined by voltage-recording of current-passing electrodes
across the oocyte membrane in response to glutamate and calcium
receptor agonists. Oocytes were voltage clamped at a holding
potential of -60 mV with an Axoclamp 2A amplifier (Axon
Instruments, Foster City, Calif.) using standard two electrode
voltage-clamp techniques. Currents were recorded on a chart
recorder. The standard control saline was MBS containing 0.3 mM
CaCl.sub.2 and 0.8 MgCl.sub.2. Test substances were applied by
superfusion at a flow rate of about 5 ml/min. All experiments were
done at room temperature. The holding current was stable in a given
oocyte and varied between +10 to -200 nA for different oocytes.
Activation of I.sub.C1 in response to activation of receptors and
subsequent increases in intracellular Ca2+ ([Ca].sub.in) was
quantified by measuring the peak inward current stimulated by
agonist or drug, relative to the holding current at -60 mV.
[0297] FIG. 6 pR1/CaR vs. rat mGluR1 (glutamate and
quisqualate).
[0298] FIG. 7 CaR/R1 vs. hPCar (calcium)
[0299] FIG. 8 pratCH3 vs. rat mGluR1 and CaR (desensitization
traces)
Example 7
Construction of pCEPCaR/R1 from pCaR/R1
[0300] The DNA from plasmid pCaR/R1 was digested and cloned into
the commercially available episomal mammalian expression vector,
pCEP4 (Invitrogen), using the restriction enzymes Kpn I and Not I.
The ligation products were transfected into DH5a cells which had
been made competent for DNA transformation. These cells were plated
on Luria-Bertani Media (LB) plates (described in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 1989)) containing 100 ug/ml
ampicillin. A clone was selected from the colonies which grew. This
clone, pCEPCaR/R1 was characterized by restriction enzyme
digestion.
Example 8
Transfection and Growth of HEK293/pCEPCaR/R1
[0301] Human embryonic kidney cells (293, ATCC, CRL 1573) were
grown in a routine manner. Cells were plated in 10 cm cell-culture
plates in Dulbecco's modified Eagle's medium (D-MEM) containing 10%
fetal calf serum (FCS) and 1.times.Penicillin-Streptomycin (PS,
Life Technologies) so that they would be .about.70% confluent after
an overnight incubation. To prepare DNA for transfection, the
plasmid pCEPCaR/R1 was precipitated with ethanol, rinsed and
resuspended in sterile water at a concentration of 1 ug/ul.
Fourteen micrograms of DNA was incubated with the liposome
formulation LipofectAMINE (Life Technologies) for 20 minutes in
serum-free Opti-MEM.RTM. (Life Technologies). After the room
temperature incubation, 6.8 mls of Opti-MEM.RTM. was added to the
transfection mix. This solution was added to the cells which had
been rinsed with 2.times.5 ml washes of serum-free Opti-MEM.RTM..
The cells and transfection mix were incubated at 37 C for 5 hours
at which time more media and fetal bovine serum were added to bring
the serum concentration to 10%. After an overnight incubation the
media was changed back to D-MEM with 10% FCS and 1.times.PS. After
an additional 24 h incubation, cells were detached with trypsin and
replated in media containing 200 ug/ml hygromycin (Boerhringer
Mannheim). Those cells which grew contained pCEPCaR/R1 which
encodes the hygromycin resistance gene. Individual clones were
recovered and propagated using standard tissue-culture techniques.
Subcultures of both individual clones and pooled stables were
prepared by dissociation into fresh tissue culture media, and
plated into fresh culture dishes at {fraction (1/10)}th the
original volume.
Example 9
HEK293/pCEPCaR/R1 Fura assay
[0302] Measurements of intracellular calcium release in response to
increases in extracellular calcium is quantitated using the Fura
assay (Parks et al. 1989). Stably transfected cells containing
pCEPCaR/R1 are loaded with 2 .mu.M fura-2 acetoxymethylester by
incubation for 20-30 minutes at 37 C in SPF-PCB (126 mM NaCl, 5 mM
KCl, 1 mM MgCl.sub.2, 20 mM HEPES, pH 7.4), containing 1.25 mM
CaCl.sub.2, 1 mg/ml glucose, 0.5% BSA.sup.1. The cells are then
washed 1 to 2 times in SPF-PCB containing 0.5 mM CaCl.sub.2, 0.5%
BSA and resuspended to a density of 4 to 5 million cells/ml and
kept at 22 C in a plastic beaker. For recording fluorescent
signals, the cells are diluted fivefold into a quartz cuvette with
BSA-free 37 C SPF-PCB to achieve a final BSA concentration of 0.1%
(1.2 ml of 37 C BSA-free SPF-PCB+0.3 ml cell suspension).
Measurements of fluorescence are performed at 37 C with constant
stirring using a custom-built spectrofluorimeter (Biomedical
Instrumentation Group, University of Pennsylvania). Excitation and
emission wavelengths are 340 and 510 nm, respectively. To calibrate
fluorescence signals, digitonin (Sigma, St. Louis, Mo.; catalog #D
5628; 50 .mu.g/ml, final) is added to obtain F.sub.max, and the
apparent F.sub.min is determined by adding EGTA (10 mM, final) and
Tris base (pH .about.10, final). Concentrations of released
intracellular Ca.sup.2+ is calculated using a dissociation constant
(Kd) of 224 nM and the equation:
[Ca.sup.2+].sub.i=(F-F.sub.min/F.sub.max-F).times.Kd
[0303] The results are graphically represented in FIG. 9.
Example 10
Recombinant Receptor Binding Assay
[0304] The following is one example of a rapid screening assay to
obtain compounds modulating metabotropic glutamate receptor
activity. The screening assay first measures the ability of
compounds to bind to recombinant chimeric receptors, or receptor
fragments or mGluR, CaR or chimeric receptors. Compounds binding to
such receptors or fragments are then tested for their ability to
modulate one or more activities at a metabotropic glutamate
receptor.
[0305] In one procedure, a cDNA or gene clone encoding a
metabotropic glutamate receptor is obtained. Distinct fragments of
the clone are expressed in an appropriate expression vector to
produce the smallest receptor polypeptide(s) obtainable able to
bind glutamate. Such experiments can be facilitated by utilizing a
stably transfected mammalian cell line (e.g., HEK 293 cells)
expressing the metabotropic glutamate receptor.
[0306] The recombinant polypeptide(s) having the desired binding
properties can be bound to a solid-phase support using standard
chemical procedures. This solid-phase, or affinity matrix, may then
be contacted with glutamate to demonstrate that glutamate can bind
to the column, and to identify conditions by which glutamate may be
removed from the solid-phase. This procedure may then be repeated
using a large library of compounds to determine those compounds
which are able to bind to the affinity matrix. Bound compounds can
then can be released in a manner similar to glutamate. Alternative
binding and release conditions may be utilized to obtain compounds
capable of binding under conditions distinct from those used for
glutamate binding (e.g., conditions which better mimic
physiological conditions encountered especially in pathological
states). Compounds binding to the mGluR can thus be selected from a
very large collection of compounds present in a liquid medium or
extract.
[0307] In an alternate method, chimeric metabotropic
glutamate/calcium receptors are bound to a column or other solid
phase support. Those compounds which are not competed off by
reagents binding to the glutamate binding site on the receptor can
then be identified. Such compounds define alternative binding sites
on the receptor. Such compounds may be structurally distinct from
known compounds and may define chemical classes of agonists or
antagonists which may be useful as therapeutics agents.
[0308] Other embodiments are within the following claims.
* * * * *