U.S. patent application number 10/096777 was filed with the patent office on 2003-09-11 for therapeutic compositions and methods relating to prolactin releasing peptide (prrp).
This patent application is currently assigned to Regents of the University of California. Invention is credited to Civelli, Olivier, Lin, Steven.
Application Number | 20030171270 10/096777 |
Document ID | / |
Family ID | 24239899 |
Filed Date | 2003-09-11 |
United States Patent
Application |
20030171270 |
Kind Code |
A1 |
Civelli, Olivier ; et
al. |
September 11, 2003 |
Therapeutic compositions and methods relating to prolactin
releasing peptide (PrRP)
Abstract
The invention provides a substantially pure Prolactin Releasing
Peptide (PrRP) functional analog which suppresses absence seizures
in a mammal, and related pharmaceutical compositions. The invention
also provides a method of controlling absence seizures in a mammal,
by administering to a mammal susceptible to absence seizures an
effective amount of PrRP or a PrRP functional analog. Also provided
are methods of identifying a compound that modulates AMPA receptor
signaling in a mammal, by providing a compound that is a PrRP or
PrRP functional analog, and determining the ability of the compound
to modulate AMPA receptor signaling. The invention also provides
methods of identifying a compound for controlling absence seizures
in a mammal, by providing a compound that is a PrRP or PrRP
functional analog, and determining the ability of the compound to
control absence seizures in a mammal. Also provided are
pharmaceutical compositions for controlling absence seizures in a
mamma. The compositions and related methods contain a compound
identified by the methods of the invention as a compound that
modulates AMPA receptor signaling or as a compound that controls
absence seizures.
Inventors: |
Civelli, Olivier; (Irvine,
CA) ; Lin, Steven; (Upland, CA) |
Correspondence
Address: |
CAMPBELL & FLORES LLP
4370 LA JOLLA VILLAGE DRIVE
7TH FLOOR
SAN DIEGO
CA
92122
US
|
Assignee: |
Regents of the University of
California
|
Family ID: |
24239899 |
Appl. No.: |
10/096777 |
Filed: |
March 12, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10096777 |
Mar 12, 2002 |
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09560915 |
Apr 28, 2000 |
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6383764 |
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Current U.S.
Class: |
435/7.8 ;
514/11.5; 514/17.7; 514/255.04; 514/557; 530/399 |
Current CPC
Class: |
A61K 31/00 20130101;
A61P 25/00 20180101; G01N 33/74 20130101 |
Class at
Publication: |
514/12 ; 530/399;
514/255.04; 514/557 |
International
Class: |
A61K 038/22; A61K
031/496; A61K 031/19 |
Goverment Interests
[0001] This invention was made in part with U.S. Government support
under Grant No. NIH MH60231. The U.S. Government may have certain
rights in this invention.
Claims
What is claimed is:
1. A substantially pure Prolactin Releasing Peptide (PrRP)
functional analog, which suppresses absence seizures in a
mammal.
2. The PrRP functional analog of claim 1, which is a peptidomimetic
of a peptide comprising the amino acid sequence set forth as SEQ ID
NO: 23.
3. A pharmaceutical composition, comprising the PrRP functional
analog of claim 1 and a pharmaceutically acceptable medium.
4. The pharmaceutical composition of claim 3, further comprising a
second compound which suppresses absence seizures.
5. The pharmaceutical composition of claim 4, wherein said second
compound is selected from the group consisting of valproate,
ethosuximade, flunarizine, trimethadione and lamotrigine.
6. A pharmaceutical composition, comprising PrRP and a second
compound which suppresses absence seizures.
7. The pharmaceutical composition of claim 6, wherein said second
compound is selected from the group consisting of valproate,
ethosuximade, flunarizine, trimethadione and lamotrigine.
8. A method of controlling absence seizures in a mammal, comprising
administering to a mammal susceptible to absence seizures an
effective amount of PrRP or a PrRP functional analog.
9. The method of claim 8, wherein said PrRP comprises the amino
acid sequence set forth as SEQ ID NO: 23.
10. The method of claim 8, wherein said PrRP comprises the amino
acid sequence set forth as SEQ ID NO: 18.
11. The method of claim 8, wherein said PrRP comprises the amino
acid sequence set forth as SEQ ID NO: 15.
12. The method of claim 8, wherein said PrRP functional analog is a
peptidomimetic of a peptide comprising the amino acid sequence set
forth as SEQ ID NO: 23.
13. The method of claim 8, further comprising administering to said
mammal a second compound which suppresses absence seizures.
14. The method of claim 13, wherein said second compound is
selected from the group consisting of valproate, ethosuximade,
flunarizine, trimethadione and lamotrigine.
15. A method of identifying a compound that modulates AMPA receptor
signaling in a mammal, comprising: (a) providing a compound that is
a PrRP or PrRP functional analog; (b) determining the ability of
said compound to modulate AMPA receptor signaling.
16. The method of claim 15, wherein step (a) comprises contacting a
PrRP receptor with one or more candidate compounds under conditions
wherein PrRP promotes a predetermined signal, identifying a
compound that promotes production of said predetermined signal, and
providing said compound.
17. The method of claim 16, wherein said predetermined signal is
selected from the group consisting of calcium ion mobilization and
arachadonic acid metabolite release.
18. The method of claim 16, wherein said PrRP receptor is
GPR10.
19. The method of claim 16, wherein said one or more candidate
compounds comprises greater than about 100 compounds.
20. The method of claim 15, wherein step (a) comprises contacting a
PrRP receptor with one or more candidate compounds under conditions
wherein PrRP binds said PrRP receptor, identifying a compound that
binds said PrRP receptor, and providing said compound.
21. The method of claim 20, wherein said PrRP receptor is
GPR10.
22. The method of claim 20, wherein said one or more candidate
compounds comprises greater than about 100 compounds.
23. The method of claim 15, wherein step (a) comprises contacting a
PrRP receptor with one or more candidate compounds under conditions
wherein PrRP promotes interaction of PrRP receptor with an AMPA
receptor associated protein, identifying a compound that promotes
said interaction, and providing said compound.
24. The method of claim 23, wherein said AMPA receptor associated
protein is selected from the group consisting of GRIP, GRIP2 and
PICK1.
25. The method of claim 15, wherein step (b) comprises contacting a
thalamic preparation with said compound, and determining AMPA
receptor mediated oscillatory activity in said preparation.
26. The method of claim 15, wherein step (b) comprises contacting a
cell with said compound, and determining AMPA receptor mediated
currents in said cell.
27. The method of claim 15, wherein step (b) comprises contacting a
cell with said compound, and determining AMPA receptor mediated ion
influx into said cell.
28. The method of claim 27, wherein said ion influx is determined
using an automated fluorometric assay.
29. A pharmaceutical composition for controlling absence seizures
in a mammal, comprising a compound identified by the method of
claim 15 as a compound that suppresses AMPA receptor signaling, and
a pharmaceutically acceptable medium.
30. A method of controlling absence seizures in a mammal,
comprising administering to a mammal susceptible to absence
seizures an effective amount of the pharmaceutical composition of
claim 29.
31. A method of identifying a compound for controlling absence
seizures in a mammal, comprising: (a) providing a compound that is
a PrRP or PrRP functional analog; (b) determining the ability of
said compound to control absence seizures in a mammal.
32. The method of claim 31, wherein step (a) comprises contacting a
PrRP receptor with one or more candidate compounds under conditions
wherein PrRP promotes a predetermined signal, identifying a
compound that promotes production of said predetermined signal, and
providing said compound.
33. The method of claim 32, wherein said predetermined signal is
selected from the group consisting of calcium ion mobilization and
arachadonic acid metabolite release.
34. The method of claim 32, wherein said PrRP receptor is
GPR10.
35. The method of claim 32, wherein said one or more candidate
compounds comprises greater than about 100 compounds.
36. The method of claim 31, wherein step (a) comprises contacting a
PrRP receptor with one or more candidate compounds under conditions
wherein PrRP binds said PrRP receptor, identifying a compound that
binds said PrRP receptor, and providing said compound.
37. The method of claim 36, wherein said PrRP receptor is
GPR10.
38. The method of claim 36, wherein said one or more candidate
compounds comprises greater than about 100 compounds.
39. The method of claim 31, wherein step (a) comprises contacting a
PrRP receptor with one or more candidate compounds under conditions
wherein PrRP promotes interaction of PrRP receptor with an AMPA
receptor associated protein, identifying a compound that promotes
said interaction, and providing said compound.
40. The method of claim 39, wherein said AMPA receptor associated
protein is selected from the group consisting of GRIP, GRIP2 and
PICK1.
41. The method of claim 31, wherein step (b) comprises
administering said compound to a mammal susceptible to absence
seizures, and determining seizure activity in said mammal.
42. The method of claim 41, wherein said compound is administered
to a mammal selected from the group consisting of a human, a
non-human primate, a rat and a mouse.
43. A pharmaceutical composition for controlling absence seizures
in a mammal, comprising a compound identified by the method of
claim 31 as a compound that controls absence seizures, and a
pharmaceutically acceptable medium.
44. A method of controlling absence seizures in a mammal,
comprising administering to a mammal susceptible to absence
seizures an effective amount of the pharmaceutical composition of
claim 43.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the field of
medicine and, more specifically, to therapeutic compositions and
methods relating to Prolactin Releasing Peptide (PrRP).
[0004] 2. Background Information
[0005] Epilepsy is a common condition, estimated to affect from 40
to 100 million people worldwide, and from 2 to 2.5 million
Americans. Of the several clinically recognized forms of epilepsy,
juvenile myoclonus epilepsy (JME) accounts for about 10% to 30% of
the cases, and childhood absence epilepsy (CAE) accounts for a
further 5% to 15%. Both JME and CAE are associated with a form of
seizures called "generalized absence seizures" or "petit mal
seizures."
[0006] Absence seizures are generalized non-convulsive seizures
characterized by a brief period of unresponsiveness to
environmental stimuli and cessation of activity, that can occur as
frequently as several hundred times a day, primarily during quiet
wakefulness, inattention and the transition between sleep and
waking. In patients with absence seizures, generalized tonic-clonic
seizures (GTCS) or "grand mal seizures" occasionally develop.
[0007] Currently available drugs to control absence seizures are
often associated with adverse side effects, including
gastrointestinal symptoms, tremors, sedation, temporary hair loss,
dizziness, incoordination, rashes, and drug interaction
complications. More seriously, potentially fatal hepatic and
hematopoietic complications, as well as teratogenicity (e.g. neural
tube birth defects), have been associated with absence seizure
medications. Additionally, while currently available drugs are
effective in many individuals, certain individuals are resistant to
all known treatments.
[0008] Thus, there exists a need to identify new therapeutic agents
that can be used to control absence seizures, which will
significantly improve the quality of life of patients suffering
from this disorder. Such drugs will likely also be effective in
ameliorating conditions associated with parts of the brain
responsible for absence seizures, or in diseases that share the
underlying biochemical pathway of absence seizures. However, in
order to rapidly screen for new drugs for controlling absence
seizures, or to rationally design such drugs, it is necessary to
first understand the biochemical mechanism that underlies absence
seizures, and to provide appropriate assay systems for testing for
new drugs. The present invention satisfies these needs and provides
related advantages as well.
SUMMARY OF THE INVENTION
[0009] The invention provides a substantially pure Prolactin
Releasing Peptide (PrRP) functional analog which suppresses absence
seizures in a mammal, and pharmaceutical compositions containing
the PrRP functional analog.
[0010] The invention also provides a method of controlling absence
seizures in a mammal, by administering to a mammal susceptible to
absence seizures an effective amount of PrRP or a PrRP functional
analog.
[0011] Also provided are methods of identifying a compound that
modulates AMPA receptor signaling in a mammal. The methods are
practiced by providing a compound that is a PrRP or PrRP functional
analog, and determining the ability of the compound to modulate
AMPA receptor signaling.
[0012] In one method of identifying a compound that modulates AMPA
receptor signaling in a mammal, a compound that is a PrRP or PrRP
functional analog is provided by contacting a PrRP receptor with
one or more candidate compounds under conditions wherein PrRP
promotes a predetermined signal, identifying a compound that
promotes production of the predetermined signal, and providing the
compound. In an alternative method, a compound that is a PrRP or
PrRP functional analog is provided by contacting a PrRP receptor
with one or more candidate compounds under conditions wherein PrRP
binds the PrRP receptor, identifying a compound that binds the PrRP
receptor, and providing the compound.
[0013] In one method of identifying a compound that modulates AMPA
receptor signaling in a mammal, the ability of the PrRP or PrRP
functional analog to modulate AMPA receptor signaling is determined
by contacting a thalamic preparation with the compound, and
determining AMPA receptor mediated oscillatory activity in the
preparation. In an alternative method, the ability of the PrRP or
PrRP functional analog to modulate AMPA receptor signaling is
determined by contacting a cell with the compound, and determining
AMPA receptor mediated currents in the cell. In a further
alternative method, the ability of the PrRP or PrRP functional
analog to modulate AMPA receptor signaling is determined by
contacting a cell with the compound, and determining AMPA receptor
mediated ion influx into the cell.
[0014] Also provided are pharmaceutical compositions for
controlling absence seizures in a mammal, containing a compound
identified by the methods of the invention as a compound that
suppresses AMPA receptor signaling. Further provided are methods of
controlling absence seizures in a mammal by administering to a
mammal susceptible to absence seizures an effective amount of such
pharmaceutical compositions.
[0015] The invention also provides methods of identifying a
compound for controlling absence seizures in a mammal. The methods
are practiced by providing a compound that is a PrRP or PrRP
functional analog, and determining the ability of the compound to
control absence seizures in a mammal.
[0016] In one method of identifying a compound for controlling
absence seizures in a mammal, a compound that is a PrRP or PrRP
functional analog is provided by contacting a PrRP receptor with
one or more candidate compounds under conditions wherein PrRP
promotes a predetermined signal, identifying a compound that
promotes production of the predetermined signal, and providing the
compound. In an alternative method, a compound that is a PrRP or
PrRP functional analog is provided by contacting a PrRP receptor
with one or more candidate compounds under conditions wherein PrRP
binds the PrRP receptor, identifying a compound that binds the PrRP
receptor, and providing the compound.
[0017] In one method of identifying a compound for controlling
absence seizures in a mammal, the ability of the PrRP or PrRP
functional analog to control absence seizures is practiced by
administering the compound to a mammal susceptible to absence
seizures, and determining seizure activity in the mammal.
[0018] Also provided are pharmaceutical compositions for
controlling absence seizures in a mammal, containing a compound
identified by the methods of the invention as a compound for
controlling absence seizures. Further provided are methods of
controlling absence seizures in a mammal by administering to a
mammal susceptible to absence seizures an effective amount of such
pharmaceutical compositions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGS. 1A and 1B show expression of GPR10 RNA on coronal and
horizontal sections, respectively, of adult rat brains.
Abbreviations are RTN: reticular thalamic nucleus; Hyp:
Hypothalamic nuclei; SA: Shell, nucleus accumbens.
[0020] FIG. 1C shows expression of GPR10 RNA within the RTN.
[0021] FIG. 1D shows labeling of GABAnergic neurons within the
section of the RTN in FIG. 1C.
[0022] FIGS. 1E and 1F show higher magnification views of FIGS. 1C
and 1D, respectively.
[0023] FIG. 2A shows an alignment of the amino acid sequence of the
cytoplasmic tail of GPR10 (SEQ ID NO: 1) with the cytoplasmic tails
of the AMPA receptor subunits GluR2 (SEQ ID NO: 2) and GluR3 (SEQ
ID NO: 3).
[0024] FIG. 2B, top, shows an analysis of co-immunoprecipitation of
GRIP with Flag-tagged GPR10 from transiently co-transfected HEK
293T cells. FIG. 2B, bottom, shows G RIP expression in the crude
lysate.
[0025] FIG. 2C shows the C-terminal sequences of Flag GPR10 WT (SEQ
ID NO: 4) and its various mutations having the following sequence
identifiers: del6 (SEQ ID NO: 5); .DELTA.LC (SEQ ID NO: 6); T365A
(SEQ ID NO: 7); V366A (SEQ ID NO: 8); S367A (SEQ ID NO: 9); V368A
(SEQ ID NO: 10); V369A (SEQ ID NO: 11); and I370A (SEQ ID NO:
12).
[0026] FIG. 2D, top, shows an analysis of co-immunoprecipitation of
GRIP with Flag-tagged GPR10 mutants from transiently co-transfected
HEK 293T cells. FIG. 2D, middle, shows GRIP expression in the crude
lysate. FIG. 2D, shows GPR10 expression in the
co-immunoprecipitated samples.
[0027] FIG. 2E shows an analysis of co-immunoprecipitation of
various 6X-His tagged GRIP PDZ domain polypeptides with wild-type
or C-terminally deleted (del6) forms of Flag-tagged GPR10.
[0028] FIG. 2F, top, shows an analysis of co-immunoprecipition of
transiently transfected GRIP, myc-tagged ABP or myc-tagged PSD95
with Flag-tagged GPR10 from HEK 293T cells stably expressing
wild-type or del6 forms of Flag-tagged GPR10. FIG. 2F, bottom,
shows expression of the indicated proteins in crude lysates.
[0029] FIG. 3A shows an analysis of co-immunoprecipitation of
myc-tagged PICK1 and Flag-tagged GPR10 from cotransfected HEK 293T
cells.
[0030] FIG. 3B shows the expression pattern of GPR10 in COS7 cells
transfected with GPR10 alone.
[0031] FIG. 3C shows the expression pattern of PICK1 in COS7 cells
transfected with PICK1 alone.
[0032] FIG. 3D shows cytoplasmic clusters of GPR10 and PICK1 in
COS7 cells co-transfected with GPR10 and PICK1.
[0033] FIG. 3E shows cell surface clusters of GPR10 and PICK1 in
COS7 cells co-transfected with GPR10 and PICK1.
[0034] FIG. 3F shows the expression pattern of a GPR10 mutant
(del6) and PICK1 in COS7 cells co-transfected with GPR10 (del6) and
PICK1.
[0035] FIG. 3G shows the expression pattern of GPR10 and a PICK1
mutant (KD/AA) in COS7 cells co-transfected with GPR10 and PICK1
(KD/AA).
[0036] FIG. 4A shows the effect of PrRP on AMPA receptor-mediated
thalamic oscillatory activity.
[0037] FIG. 4B shows the effect of PrRP on NMDA receptor-mediated
thalamic oscillatory activity.
[0038] FIG. 4C shows the concentration dependent effect of PrRP on
thalamic oscillatory activity. *, p<0.01; ** p<0.001, paired
Student's t-Test.
[0039] FIG. 5 shows seizure frequency in GAERS rats (n=6),
expressed as cumulative duration of spike and wave discharges (SWD)
at 20 min intervals after injection of aCSF control (FIG. 5A) or
injection of various PrRP concentrations (FIGS. 5B-D).
"REF"=reference duration of spontaneous seizure activity before
injection.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention relates to the determination that PrRP
modulates the activity of the reticular thalamic nucleus (RTN), a
region of the brain implicated in sleep rhythms, attention
processing and absence seizures, through a functional interaction
between the PrRP receptor (GPR10), and
Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
receptors. The inventors have determined that PrRP specifically
reduces AMPA receptor mediated oscillatory activity in the RTN, and
effectively suppresses absence seizures in mammals.
[0041] Thus, based on the determination of an important
pharmacological role of PrRP, and its underlying molecular
mechanism, the invention provides compounds and related therapeutic
methods for suppressing absence seizures in mammals. The compounds
and therapeutic methods of the invention can thus be used in the
therapy of epilepsies and other diseases associated with absence
seizures. Additionally, the invention provides methods of rapidly
screening for compounds that modulate AMPA receptor signaling and
compounds that control absence seizures. The compounds so
identified will be useful in the control of absence seizures, as
well as in the prevention and treatment of conditions associated
with tissues in which GPR10 is expressed, or in which known
anti-epileptic drugs are effective.
[0042] In one aspect, the invention provides a method of
controlling absence seizures. The method involves administering to
a mammal susceptible to absence seizures an effective amount of
PrRP or a functional analog thereof. As used herein, the term
"mammal susceptible to absence seizures," refers to a human,
veterinary animal or laboratory animal (e.g. non-human primate,
rodent, feline or canine) that exhibits, can be induced to exhibit,
or is at high risk of developing, absence seizures.
[0043] Absence seizures are brief attacks of impaired consciousness
that can be distinguished from other forms of seizures both by
their distinct electroencephalographic (EEG) patterns and by their
response to pharmacological agents. By EEG, absence seizures are
associated with bilateral synchronous and regular spike and wave
discharges (SWD) with a frequency of 2.5 to 4 c/s, which start and
end abruptly. Pharmacologically, absence seizures generally respond
to the drugs ethosuximade, valproate and trimethadione, but are
worsened by anti-convulsants such as carbamazepine and phenytoine
which are effective in treating convulsive seizures.
[0044] In humans, several clinically recognized epilepsy syndromes,
including childhood absence epilepsy, juvenile absence epilepsy,
juvenile myoclonic epilepsy, myoclonic absence epilepsy, and eyelid
myoclonia with absences, are associated with absence seizures.
Epileptic syndromes are commonly classified according to the
International Classification of Epileptic Syndromes proposed by the
International League against Epilepsy in 1989.
[0045] Epidemiological studies have identified several predictive
factors for the development of absence seizures, including past
history of either febrile convulsions or generalized tonic-clonic
seizures (GTCS), and family history of epilepsy or febrile
convulsions (see, for example, Covanis et al., Seizure 1:281-289
(1992)). Because of the clear genetic predisposition for absence
epilepsies, those skilled in the art understand that it will also
be possible to determine susceptibility to absence seizures by
genetic or biochemical profile.
[0046] Accordingly, a human "susceptible to absence seizures," can
be a human exhibiting absence seizures, such as a human diagnosed
with an epilepsy syndrome characterized by absence seizures, or a
human considered to be at high risk of developing absence
seizures.
[0047] A variety of non-human mammals that exhibit, or can be
induced to exhibit, behavioral, electrographic and pharmacological
characteristics of absence seizures in humans are known in the art.
Such mammals are also considered herein to be "mammals susceptible
to absence seizures." For example, Genetic Absence Epilepsy Rats
from Strasbourg, or GAERS, exhibit behavioral and EEG patterns
during spike and wave discharges (SWD) that are similar to those
observed in humans during absence seizures (see Danober et al.,
Prog. Neurobiol. 55:27-57 (1998)). Other genetic models of absence
epilepsy include the lethargic (lh/lh) mutant mouse (see Hosford et
al., Epilepsia 38:408-414 (1997)), the WAG/Rij strain of rats (see
Coenan et al., Epilepsy Res. 12:75-86 (1992)), and the tremor
(tm/tm) mutant rat (Hanaya et al., Epilepsia 36:938-942 (1995)),
which have been successfully used to predict or confirm the effects
of a variety of anti-epileptic drugs in controlling absence
seizures in humans.
[0048] Other relevant mammalian models of absence seizures in
humans include pharmacological models, in which absence seizures
are induced in laboratory animals, such as rodents, cats and
primates, by administration of pentylenetetrazol, penicillin,
gamma-hydroxybutyrate or GABA agonists (for a review, see Snead,
Epilepsia 29:361-368 (1988)). Additionally, absence seizures can be
induced in primates by thalamic stimulation (see, for example,
David et al., J. Pharmacol. Methods 7:219-229 (1982)).
[0049] As used herein, the term "controlling," in relation to
absence seizures, refers to a reduction in the frequency, duration,
number or intensity of absence seizures in a treated mammal, as
compared with the frequency, duration, number or intensity of
absence seizures expected or observed without treatment. A
determination of whether absence seizures are "controlled" by
treatment can be made, for example, by direct observation, by
self-reporting, or by examining on an EEG readout the frequency,
duration, number or intensity or duration of spike and wave
discharges (SWD)(see, for example, Example IV, below).
[0050] An amount of a pharmaceutical composition effective to
control absence seizures is an amount effective to reduce the
determined parameter (e.g. frequency, duration, number or intensity
of absence seizures or SWD) by at least 10%. Preferably, the
determined parameter will be reduced by at least 20%, more
preferably at least 50%, such as at least 80%, in at least some
treated mammals. Accordingly, a treatment that controls absence
seizures will be useful in improving the quality of life in the
treated mammals. Further description of effective amounts,
formulations and routes of administration of the pharmaceutical
compositions useful in the methods of the invention is provided
below.
[0051] PrRP was originally identified as a peptide having the
physiological role of promoting the release of prolactin, a hormone
involved in mammary development and lactation, from the anterior
pituitary (Hinuma et al., Nature 393:272-276 (1998)). As used
herein, the term "PrRP" refers to a peptide having identity with at
least 5 residues of the native sequence of a mammalian
prolactin-releasing peptide (PrRP), and which binds a "PrRP
receptor" with an affinity (Kd) of about 10.sup.-5 M or less. A
PrRP of the invention can thus have identity with at least 5, 6, 7,
8, 9, 10, 15, 20 or more contiguous or non-contiguous amino acid
residues of a native PrRP. Preferably, a PrRP of the invention
binds a PrRP receptor with a Kd of about 10.sup.-6 M or less, more
preferably about 10.sup.-7 M or less, most preferably about
10.sup.-8 M or less, including about 10.sup.-9 M or less, such as
10.sup.-10 M or less.
[0052] Mature, native PrRP peptides exist in at least two forms, a
31 amino acid peptide (PrRP-31) and a 20 amino acid peptide
(PrRP-20), which are amidated at the carboxy-terminus. PrRP-31 and
PrRP-20 are derived from a longer preproprotein. The purification
of PrRP-31 and PrRP-20 from bovine hypothalamus, the cloning of
PrRP preproprotein from bovine, rat and human, the characterization
of PrRP-31 and PrRP-20 as peptides having prolactin-releasing
activity towards rat anterior pituitary cells in vitro, and the
importance of the C-terminal amide for PrRP activity, are described
in Hinuma et al., Nature 393:272-276 (1998).
[0053] The amino acid sequences of PrRP-31 from bovine, rat and
human are as follows:
1 Bovine: SRAHQHSMEIRTPDINPAWYAGRGIRPVGRF (SEQ ID NO:13) Rat:
SRAHQHSMETRTPDTNPAWYTGRGIRPVGRF (SEQ ID NO:14) Human:
SRTHRHSMEIRTPDINPAWYASRGI- RPVGRF (SEQ ID NO:15)
[0054] The amino acid sequences of PrRP-20 from bovine, rat and
human, which contain the C-terminal 20 amino acids of PrRP-31, are
as follows:
2 Bovine: TPDINPAWYAGRGIRPVGRF (SEQ ID NO:16) Rat:
TPDINPAWYTGRGIRPVGRF (SEQ ID NO:17) Human: TPDINPAWYASRGIRPVGRF
(SEQ ID NO:13)
[0055] The term "PrRP" is intended to encompass PrRP-31 and PrRP-20
from bovine, rat and human, having the amino acid sequences shown
above, as well as PrRP-31 and PrRP-20 from other mammalian species,
including, for example, non-human primates, mouse, rabbit, porcine,
ovine, canine and feline species. The sequences of PrRP from other
mammalian species can be readily determined by those skilled in the
art, for example either by purifying PrRP from hypothalamic
extracts, or by cloning PrRP preproproteins, following the methods
described in Hinuma et al., Nature 393:272-276 (1998). Because of
the high degree of identity between bovine, rat and human
sequences, it is expected that PrRP from other mammalian species
will be substantially similar in structure and function to the
known PrRP sequences.
[0056] The term "PrRP" is also intended to encompass peptides that
are longer or shorter than PrRP-31 or PrRP-20, so long as they have
identity with at least 5 residues of the native sequence of a
mammalian prolactin-releasing peptide (PrRP), and can bind the PrRP
receptor GPR10 with an affinity (Kd) of less than about 10.sup.-5
M. Thus, the term "PrRP" encompasses peptides that have one or
several amino acid additions or deletions compared with the amino
acid sequence of a PrRP-31 or PrRP-20. Those skilled in the art
recognize that such modifications can be desirable in order to
enhance the bioactivity, bioavailability or stability of the PrRP,
or to facilitate its synthesis or purification.
[0057] The term "PrRP" is further intended to encompass peptides
having identity with at least 5 residues of the native sequence of
a mammalian prolactin-releasing peptide (PrRP), which bind a PrRP
receptor with an affinity (Kd) of about 10.sup.-5 M or less, and
which have one or several minor modifications to the native PrRP
sequence. Contemplated modifications include chemical or enzymatic
modifications (e.g. acylation, phosphorylation, glycosylation,
etc.), and substitutions of one or several amino acids to a native
PrRP sequence. Those skilled in the art recognize that such
modifications can be desirable in order to enhance the bioactivity,
bioavailability or stability of the PrRP, or to facilitate its
synthesis or purification.
[0058] Contemplated amino acid substitutions to the native sequence
of a PrRP include conservative changes, wherein a substituted amino
acid has similar structural or chemical properties (e.g.,
replacement of an apolar amino acid with another apolar amino acid;
replacement of a charged amino acid with a similarly charged amino
acid, etc.). Those skilled in the art also recognize that
nonconservative changes (e.g., replacement of an uncharged polar
amino acid with an apolar amino acid; replacement of a charged
amino acid with an uncharged polar amino acid, etc.) can be made
without affecting the function of PrRP. Furthermore, non-linear
variants of a PrRP sequence, including branched sequences and
cyclic sequences, and variants that contain one or more D-amino
acid residues in place of their L-amino acid counterparts, can be
made without affecting the function of PrRP.
[0059] In particular, the term "PrRP" is intended to encompass
peptides having minor modifications to the native PrRP sequence
that serve to increase its penetration through the blood-brain
barrier (BBB). For a review of strategies for increasing
bioavailability of peptides and peptide drugs in the brain, and of
methods for determining the permeability of peptides through the
BBB using in vitro and in vivo assays, see Engleton et al.,
Peptides 9:1431-1439 (1997).
[0060] Strategies that have been successfully used to increase the
permeability of other neuropeptides through the BBB are
particularly contemplated. For example, modifying the opioid
peptide analgesic Met-enkephalin with D-penicillamine at two
positions, forming a disulfide bridge that conformationally
constrains the peptide, dramatically increases its stability
towards BBB endothelial cell proteases and its BBB permeability.
Likewise, linking two enkephalin peptides, each containing a
D-amino acid residue at the second position, with a hydrazide
bridge, results in a metabolically stable peptide with improved
brain penetration. Additionally, halogenation of an enkephalin
peptide has been shown to increase its BBB permeability. Similar
modifications to PrRP peptides are likewise expected to be
advantageous.
[0061] Additional modifications to a PrRP peptide that can increase
its BBB penetration include conjugating the peptide to a lipophilic
moiety, such as a lipophilic amino acid or methyldihydropyridine.
PrRP peptide can also be conjugated to a transporter, such as the
monoclonal antibody OX26 which recognizes the transferrin receptor,
or cationized albumin which utilizes the adsorptive mediated
endocytosis pathway, so as to increase its BBB penetration.
[0062] Those skilled in the art can determine which residues and
which regions of a native PrRP sequence are likely to be tolerant
of modification and still retain the ability to bind PrRP receptor
with high affinity. For example, amino acid substitutions, or
chemical or enzymatic modifications, at residues that are less well
conserved between species are more likely to be tolerated than
substitutions at highly conserved residues. Accordingly, an
alignment can be performed among PrRP sequences of various species
to determine residues and regions in which modifications are likely
to be tolerated.
[0063] Additional guidance for determining residues and regions of
PrRP likely to be tolerant of modification is provided by studies
of PrRP fragments and variants. For example, based on the
observation that PrRP-20 has similar ability to transduce signals
through the PrRP receptor as PrRP-31 (see, for example, Hinuma et
al., Nature 393:272-276 (1998)), it is likely that the N-terminus
of PrRP is highly tolerant of the modifications described
herein.
[0064] In particular, as described in Roland et al., Endocrinology
140:5736-5745 (1999), a peptide designated PrRP(25-31), consisting
of the C-terminal seven amino acids of PrRP (IRPVGRF, SEQ ID NO:
23) binds GPR10 with an apparent affinity of 200 nM, compared with
an affinity of about 1 nM for PrRP-31 or PrRP-20, and mobilizes
calcium in CHOK1 cells transfected with GPR10. Thus, a peptide
consisting of, or comprising, the amino acid sequence designated
SEQ ID NO: 23 is encompassed by the term "PrRP."
[0065] Alanine scanning mutagenesis of PrRP(25-31) indicates that
variants with substitutions of Ile25, Pro27, Val28, or Phe31 retain
the ability to bind GPR10 with an affinity of about 10.sup.-6 M.
Thus, a PrRP can consist of, or comprise, the amino acid sequences
XRPVGRF (SEQ ID NO: 19), IRXVGRF (SEQ ID NO: 20), IRPXGRF (SEQ ID
NO: 21), IRPVGRX (SEQ ID NO: 22), where "X" is any amino acid,
preferably a non-polar amino acid, more preferably alanine.
Substitutions of Arg26 or Gly29 were shown to substantially reduce
binding affinity of PrRP(25-31) for GPR10, and substitution of
Arg30 completely eliminated binding. Substitution of either Arg26
or Arg30 with lysine or citrulline also completely eliminated
binding. More generally, a PrRP peptide can be considered to
consist of, or comprise, the amino acid sequence XRXXGRX, so long
as it retains PrRP receptor binding activity.
[0066] In the modified PrRP sequences described above, the effect
of amino acid substitutions on calcium signaling was commensurate
with the effect on binding to GPR10 (see Roland et al.,
Endocrinology 140:5736-5745 (1999)). Accordingly, in view of the
disclosure herein, it is predictable that a peptide considered to
be a "PrRP" by GPR10 binding criteria will also be functionally
active in mediating G-protein coupled signaling through PrRP
receptor, inhibiting AMPA mediated signaling in whole cell
preparations, inhibiting oscillatory activity in RTN preparations,
suppressing absence seizures in susceptible-mammals, and preventing
or treating neurological and psychiatric disorders in which PrRP-31
or PrRP-20 are effective. Thus, as described further below, a
peptide having a modified PrRP sequence can be assayed by any of
these functional criteria to confirm that it is a PrRP.
[0067] The PrRP peptides of the invention can be prepared in
substantially purified form using either conventional peptide
synthetic methods (see, for example, Roland et al., Endocrinology
140:5736-5745 (1999)), or using conventional biochemical
purification methods, starting either from tissues containing PrRP
or from recombinant sources (see, for example, Hinuma et al.,
Nature 393:272-276 (1998)).
[0068] In methods of controlling absence seizures, and for certain
other therapeutic applications, it may be preferable to use a PrRP
functional analog rather than a PrRP peptide. For example, a PrRP
functional analog can be more stable, more active, or have higher
inherent ability to penetrate the BBB than a PrRP. As used herein,
the term "PrRP functional analog" refers to a molecule that binds
the PrRP receptor GPR10 with an affinity (Kd) of about 10.sup.-5 M
or less, and which is not encompassed within the definition of a
"PrRP," as set forth above. Preferably, a PrRP functional analog
will bind a PrRP receptor with a Kd of about 10.sup.-6 M or less,
more preferably about 10.sup.-7 M or less, most preferably about
10.sup.-8 M or less, including about 10.sup.-9 M or less, such as
about 10.sup.-10 M or less.
[0069] The invention thus provides PrRP functional analogs. The
PrRP functional analogs of the invention will generally act as PrRP
receptor agonists, and thus be able to mediate the same biochemical
and pharmacological effects (e.g. signal transduction through the
PrRP receptor, reduction of AMPA receptor activity, suppression of
absence seizures in mammals) as PrRP. However, a PrRP functional
analog identified by the methods described herein can alternatively
act as a PrRP receptor antagonist, and thus inhibit signaling
through GPR10, prevent the suppression of AMPA receptor mediated
activity, or both. Such antagonists can advantageously be used in
therapeutic applications where a reduction in PrRP receptor
signaling is desired, including in the treatment of sleep and
attention disorders. PrRP functional analogs of the invention,
which are themselves not appropriate for therapeutic use, can
advantageously be used to optimize the design of effective
therapeutic compounds, or used in the screening methods described
herein as competitors.
[0070] A PrRP functional analog can be a naturally occurring
macromolecule, such as a peptide, nucleic acid, carbohydrate,
lipid, or any combination thereof. A PrRP functional analog also
can be a partially or completely synthetic derivative, analog or
mimetic of such a macromolecule, or a small organic or inorganic
molecule prepared partly or completely by synthetic chemistry
methods. A PrRP functional analog can be identified starting either
by rational design based on the corresponding peptide, by
functional screening assays, or by a combination of these
methods.
[0071] PrRP functional analogs include peptidomimetics of PrRP,
such as peptidomimetics of a peptide containing, or consisting of,
the amino acid sequence set forth as SEQ ID NO: 23. As used herein,
the term "peptidomimetic" refers to a non-peptide agent that is a
topological analog of the corresponding peptide. Those skilled in
the art understand that the identified ability of PrRP-31, PrRP-20,
PrRP(25-31) and of certain single amino acid variants of
PrRP(25-31) to bind PrRP receptor with high affinity, provides
sufficient structural and functional information to rationally
design peptidomimetics of PrRP.
[0072] Such a peptidomimetic can, for example, retain some or all
of the functional groups of the amino acids shown to be
functionally important in the C-terminus of PrRP (such as the
3-guanylpropyl radical of Arg26 and Arg30, the hydrogen of Gly29,
etc.). A peptidomimetic of PrRP can also, for example, consist
partially or completely of a non-peptide backbone used in the art
in the design of other peptidomimetics, such as a glucose scaffold,
a pyrrolidinone scaffold, a steroidal scaffold, a benzodiazepine
scaffold, or the like.
[0073] Methods of rationally designing peptidomimetics of peptides,
including neuropeptides, are known in the art. For example, the
rational design of three peptidomimetics based on the sulfated
8-mer peptide CCK26-33, and of two peptidomimetics based on the
11-mer peptide Substance P, and related peptidomimetic design
principles, are described in Horwell, Trends Biotechnol. 13:132-134
(1995).
[0074] Individual, rationally designed peptidomimetics of PrRP
peptides can be assayed for their ability to bind the PrRP
receptor, or to induce signaling through the PrRP receptor, or
both, using one or more of the assays described herein. Similarly,
a plurality of peptidomimetic compounds, such as variants of a
peptidomimetic lead compound, or a plurality of other compounds,
can be assayed simultaneously or sequentially using the binding,
signaling and pharmacological assays described herein.
[0075] A candidate compound can be assayed to determine whether it
is a PrRP or PrRP functional analog either by a signaling assay, a
binding assay, or both. The number of different compounds to screen
in a particular assay can be determined by those skilled in the
art, and can be 2 or more, such as 5, 10, 15, 20, 50 or 100 or more
different compounds. For certain applications, such as when a
library of random compounds is to be screened, and for automated
procedures, it may be desirable to screen 10.sup.3 or more
compounds, such as 10.sup.5 or more compounds, including 10.sup.7
or more compounds.
[0076] Methods for producing large libraries of chemical compounds,
including simple or complex organic molecules, metal-containing
compounds, carbohydrates, peptides, proteins, peptidomimetics,
glycoproteins, lipoproteins, nucleic acids, antibodies, and the
like, are well known in the art and are described, for example, in
Huse, U.S. Pat. No. 5,264,563; Francis et al., Curr. Opin. Chem.
Biol. 2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371
(1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med.
Res. Rev. 15:481-496 (1995); and the like. Libraries containing
large numbers of natural and synthetic compounds also can be
obtained from commercial sources.
[0077] In one embodiment, a signaling assay can be performed to
determine whether a candidate compound is a PrRP or a PrRP
functional analog. In such an assay, a PrRP receptor is contacted
with one or more candidate compounds under conditions wherein the
PrRP receptor produces a predetermined signal in response to PrRP,
and a compound is identified that alters production of the
predetermined signal. The candidate compound can be tested at a
range of concentrations to establish the concentration where
half-maximal signaling occurs; such a concentration is generally
similar to the dissociation constant (Kd) for PrRP receptor
binding.
[0078] As used herein, the term "PrRP receptor," is intended to
refer to a mammalian seven-transmembrane-domain G-protein coupled
receptor, variously designated in the art "GPR10"(Marchese et al.,
Genomics 29:335-344 (1995)), "hGR3"(Hinuma et al., Nature
393:272-276 (1998)) or "UHR-1"(Welch et al., Biochem. Biophys. Res.
Commun. 209:606-613 (1995)). A "PrRP receptor" can have minor
modifications to the native mammalian sequence, so long as the
minor modifications do not significantly alter its ability to bind
PrRP, interact with AMPA receptor associated molecules, signal
through a G-protein coupled signal transduction pathway, or
modulate AMPA receptor signaling, depending on the particular
application of the PrRP receptor in the methods of the
invention.
[0079] The PrRP receptor to be contacted in the methods of the
invention can be naturally expressed in a tissue, cell or extract.
Alternatively, where it is desired to increase the PrRP receptor
concentration, or to express PrRP receptor in host cells where it
is not normally expressed, including mammalian, yeast and bacterial
cells, the PrRP receptor can be recombinantly expressed. Methods of
recombinantly expressing PrRP receptor, either transiently or
stably, in a variety of host cells, are well known in the art (see,
for example, Hinuma et al., Nature 393:272-276 (1998) and Roland et
al., Endocrinology 140:5736-5745 (1999)).
[0080] As used herein, the term "predetermined signal" refers to a
readout, detectable by any analytical means, that is a qualitative
or quantitative indication of activation of G protein-dependent
signal transduction through PrRP receptor. The term "G protein"
refers to a class of heterotrimeric GTP binding proteins, with
subunits designated G.alpha., G.beta. and G.gamma., that couple to
seven-transmembrane cell surface receptors to transduce a variety
of extracellular stimuli, including light, neurotransmitters,
hormones and odorants to various intracellular effector proteins. G
proteins are present in both eukaryotic and prokaryotic organisms,
including mammals, other vertebrates, Drosophila and yeast.
[0081] As described in Hinuma et al., Nature 393:272-276 (1998),
contacting PrRP receptor with PrRP leads to activation of
arachidonic acid metabolite release in mammalian cells
recombinantly expressing PrRP receptor. Therefore, an exemplary
predetermined signal that is a qualitative or quantitative
indication of activation of G protein-dependent signal transduction
through PrRP receptor is arachadonic acid metabolite release.
Similarly, as described in Roland et al., Endocrinology
140:5736-5745 (1999), contacting PrRP receptor with PrRP leads to
calcium mobilization in mammalian cells recombinantly expressing
PrRP receptor, which can be measured, for example, using the
calcium indicator fluo-3 and a fluorescence monitoring system.
[0082] If desired, a predetermined signal other than arachadonic
acid metabolite release or Ca.sup.2+ influx can be used as the
readout in the methods of the invention. The specificity of a G
protein for cell-surface receptors is determined by the C-terminal
five amino acids of the G.alpha. subunit. The nucleotide sequences
and signal transduction pathways of different classes and
subclasses of G.alpha. subunits in a variety of eukaryotic and
prokaryotic organisms are well known in the art. Thus, any
convenient G-protein mediated signal transduction pathway can be
assayed by preparing a chimeric G.alpha. containing the C-terminal
residues of a G.alpha. that couples to PrRP receptor, such as
G.alpha.q, with the remainder of the protein corresponding to a
G.alpha. that couples to the signal transduction pathway it is
desired to assay.
[0083] Methods of recombinantly expressing chimeric G.alpha.
proteins, and their use in G-protein signaling assays, are known in
the art and are described, for example, in, and Saito et al.,
Nature 400:265-269 (1999), and Coward et al., Anal. Biochem.
270:2424-248 (1999)).
[0084] Signaling through G proteins can lead to increased or
decreased production or liberation of second messengers, including,
for example, arachidonic acid, acetylcholine, diacylglycerol, cGMP,
cAMP, inositol phosphate and ions; altered cell membrane potential;
GTP hydrolysis; influx or efflux of amino acids; increased or
decreased phosphorylation of intracellular proteins; or activation
of transcription. Thus, by using a chimeric G.alpha. subunit that
binds PrRP receptor and couples to a desired signal transduction
pathway in the methods of the invention, those skilled in the art
can assay any convenient G protein mediated predetermined signal in
response to PrRP and PrRP functional analogs.
[0085] Various assays, including high throughput automated
screening assays, to identify alterations in G protein coupled
signal transduction pathways are well known in the art. Various
screening assay that measure Ca.sup.++, cAMP, voltage changes and
gene expression are reviewed, for example, in Gonzalez et al.,
Curr. Opin. in Biotech. 9:624-631 (1998); Jayawickreme et al.,
Curr. Opin. Biotech. 8:629-634 (1997); and Coward et al., Anal.
Biochem. 270:2424-248 (1999). Yeast cell-based bioassays for
high-throughput screening of drug targets for G protein coupled
receptors are described, for example, in Pausch, Trends in Biotech.
15:487-494 (1997). A variety of cell-based expression systems,
including bacterial, yeast, baculovirus/insect systems and
mammalian cells, useful for detecting G protein coupled receptor
agonists and antagonists are reviewed, for example, in Tate et al.,
Trends in Biotech. 14:426-430 (1996).
[0086] Assays to detect and measure G protein-coupled signal
transduction can involve first contacting the isolated cell or
membrane with a detectable indicator. A detectable indicator can be
any molecule that exhibits a detectable difference in a physical or
chemical property in the presence of the substance being measured,
such as a color change. Calcium indicators, pH indicators, and
metal ion indicators, and assays for using these indicators to
detect and measure selected signal transduction pathways are
described, for example, in Haugland, Molecular Probes Handbook of
Fluorescent Probes and Research Chemicals, Sets 20-23 and 25
(1992-94). For example, calcium indicators and their use are well
known in the art, and include compounds like Fluo-3 AM, Fura-2,
Indo-1, FURA RED, CALCIUM GREEN, CALCIUM ORANGE, CALCIUM CRIMSON,
BTC, OREGON GREEN BAPTA, which are available from Molecular Probes,
Inc., Eugene Oreg., and described, for example, in U.S. Pat. Nos.
5,453,517, 5,501,980 and 4,849,362.
[0087] Assays to determine changes in gene expression in response
to a PrRP or PrRP functional analog can involve first transducing
cells with a promoter-reporter nucleic acid construct such that a
protein such as .beta.-lactamase, luciferase, green fluorescent
protein or .beta.-galactosidase will be expressed in response to
contacting PrRP receptor with a PrRP or PrRP functional analog.
Such assays and reporter systems are well known in the art.
[0088] An assay to determine whether a candidate compound is a PrRP
or a PrRP functional analog is performed under conditions in which
contacting the receptor with a known PrRP, such as PrRP-31 or
PrRP-20, would produce a predetermined signal. If desired, the
assay can be performed in the presence of a known PrRP. Preferably,
the PrRP concentration will be within 10-fold of the EC.sub.50.
Thus, an agonist that competes with PrRP for signaling through the
PrRP receptor, or indirectly potentiates the signaling activity of
PrRP, can be readily identified. Likewise, an antagonist that
prevents PrRP from binding the PrRP receptor, or indirectly
decreases the signaling activity of PrRP, can also be
identified.
[0089] As described in Example II, below, functional interaction of
PrRP with GPR10 results in the association of GPR10 through its
C-terminus with AMPA receptor associated molecules. Thus, a further
signaling assay for identifying a PrRP or PrRP functional analog
consists of contacting a PrRP receptor with a candidate compound
under conditions wherein PrRP promotes interaction of PrRP receptor
with an AMPA receptor associated protein, and determining the
ability of the candidate compound to promote the interaction of the
PrRP receptor with the AMPA receptor associated protein. A
candidate compound that promotes the interaction of PrRP receptor
with an AMPA receptor associated protein is characterized as a PrRP
or PrRP functional analog.
[0090] Exemplary AMPA receptor associated molecules include PICK1
(see Xia et al., Neuron 22:179-187 (1999)), GRIP1 (Dong et al., J.
Neurosci. 19:6930-6941 (1999)), and GRIP2/ABP (Dong et al., J.
Neurosci. 19:6930-6941 (1999); Srivista et al., Neuron 21:581-591
(1998)), which are PDZ domain containing proteins, and other
proteins that similarly interact with the GluR2 or GluR3 subunits
of AMPA receptors.
[0091] Methods of determining the interaction between PrRP receptor
and an AMPA receptor associated protein, and suitable compositions
for practicing the methods, are described in Example II, below. For
example, a cell, such as a mammalian, yeast or bacterial cell, can
be cotransfected with a nucleic acid expression construct directing
the expression of PrRP receptor, and a nucleic acid molecule
expression construct directing the expression of AMPA receptor
associated protein, and the cell contacted with a candidate
compound. Interaction between the PrRP receptor and AMPA receptor
associated protein following such contacting can be determined, for
example, by co-immunoprecipitation of the two proteins, or by
intracellular or surface clustering of the two proteins. Nucleic
acid expression constructs and suitable host cells for expressing
PrRP receptor and AMPA receptor associated proteins, and
immunological reagents and methods suitable for detecting such
interactions, are known in the art.
[0092] A candidate compound can alternatively or additionally be
assayed to determine whether it is a PrRP or PrRP functional analog
by a PrRP receptor binding assay. If desired, a binding assay can
be followed by a signaling assay, to determine whether the
identified compound is a PrRP receptor agonist or antagonist.
Receptor binding assays, including high-throughput automated
binding assays, are well known in the art, and any suitable direct
or competitive binding assay can be used. Exemplary high-throughput
receptor binding assays are described, for example, in
Mellentin-Micelotti et al., Anal. Biochem. 272:P182-190 (1999);
Zuck et al., Proc. Natl. Acad. Sci. USA 96:11122-11127 (1999); and
Zhang et al., Anal. Biochem. 268;134-142 (1999). The assay format
can employ a cell, cell membrane, or artificial membrane system, so
long as the PrRP receptor is in a suitable conformation for binding
PrRP with a similarly affinity and specificity as a PrRP receptor
expressed on the surface of a mammalian cell.
[0093] Contemplated binding assays can involve detectably labeling
a candidate compound, or competing an unlabeled candidate compound
with a detectably labeled PrRP. A detectable label can be, for
example, a radioisotope, fluorochrome, ferromagnetic substance, or
luminescent substance. Exemplary radiolabels useful for labeling
compounds include .sup.125I, .sup.14C and .sup.3H. Methods of
detectably labeling organic molecules, either by incorporating
labeled amino acids into the compound during synthesis, or by
derivatizing the compound after synthesis, are known in the
art.
[0094] In the binding and signaling assays described above,
appropriate conditions for determining whether a compound is a PrRP
or PrRP functional analog are those in which a control PrRP
exhibits the binding or signaling property. The control assay can
be performed before, after or simultaneously with the test
assay.
[0095] The invention also provides methods of identifying compounds
that modulate AMPA receptor signaling, including compounds that
suppress AMPA receptor signaling and compounds that enhance AMPA
receptor signaling. Such compounds can be used, for example, as
therapeutic compounds for controlling absence seizures, as well as
in the prevention and treatment of conditions associated with
tissues in which GPR10 is expressed. Such compounds can also be
used, for example, in the design and development of compounds which
themselves can be used as therapeutics, or for further analysis of
biochemical pathways.
[0096] The method consists of providing one or more compounds that
are PrRPs or PrRP functional analogs, and determining the ability
of the compound to modulate AMPA receptor signaling. The one or
more compounds that are PrRPs or PrRP functional analogs can be
identified, isolated or prepared by the methods and criteria set
forth above.
[0097] Assays for determining AMPA receptor signaling can either
directly measure AMPA receptor electrophysiological activity in a
cell or tissue, or measure a biochemical or physiological property
that is correlated with AMPA receptor activity. Appropriate assays
and conditions for determining whether a compound modulates AMPA
receptor signaling are those in which a control PrRP modulates AMPA
receptor signaling. The control assay can be performed before,
after or simultaneously with the test assay, depending on the
particular assay. Such assays are known in the art or described
herein, and include both manual and high-throughput automated
assays.
[0098] A method of determining whether a PrRP or PrRP functional
analog modulates AMPA receptor electrophysiological activity can
involve determining AMPA receptor-mediated oscillatory activity in
a tissue, such as a neural tissue, that expresses both PrRP
receptors and AMPA receptors. Example III, below, describes
exemplary conditions for determining AMPA receptor-driven
oscillatory activity in a thalamic preparation. Application of PrRP
reduced AMPA receptor mediated oscillatory activity, in a
dose-dependent manner. Accordingly, an assay of thalamic
oscillatory activity can be used to determine whether a compound
modulates AMPA receptor signaling.
[0099] A further method of determining whether a PrRP or PrRP
functional analog modulates AMPA receptor electrophysiological
activity can involve an assay of the electrophysiological
properties of a single cell or cell population which normally
expresses (e.g. RTN neurons), or which recombinantly expresses,
functional PrRP receptors and AMPA receptors. Methods of
transiently or stably transfecting cells with AMPA receptors are
well known in the art and are described, for example, in Hall et
al., J. Neurochem. 68:625-630 (1997), and in Hennegriff et al., J.
Neurochem. 68:2424-2434 (1997).
[0100] Example 5, below, and Smith et al., J. Neuroscience
20:2073-2085 (2000), describe exemplary conditions for determining
AMPA receptor mediated electrophysiological recordings from whole
cells. In brief, the method involves detecting AMPA receptor
mediated currents using whole cell patch clamp recordings in the
presence of an AMPA agonist. The modulatory effect of a test
compound on the AMPA receptor mediated currents can thus be
determined. Such assays can be performed in the presence of a drug
such as cyclothiazide to reduce AMPA receptor densensitization.
[0101] Alternatively, or additionally, a method of determining
whether a PrRP or PrRP functional analog modulates AMPA receptor
signaling activity can involve an assay of AMPA receptor-mediated
second messenger responses in cells expressing functional PrRP
receptors and AMPA receptors. Such assays are advantageous in that
they are readily amenable to automation, using methods known in the
art, allowing rapid and high-throughput screening of compounds.
[0102] Example 6, below, describes exemplary conditions for
determining AMPA receptor mediated calcium ion or sodium ion influx
into cells in response to a compound that modulates AMPA receptor
signaling. In brief, the method involves detecting AMPA receptor
mediated ion influx using fluorescent ion indicators and either
microscopic visualization, or an automated fluorometric imaging
plate reader (FLIPR). The modulatory effect of a test compound on
AMPA receptor mediated ion influx can thus be determined.
[0103] The invention also provides methods of identifying compounds
for controlling absence seizures. The method consists of providing
a compound that is a PrRP or PrRP functional analog, and
determining the ability of the compound to control absence seizures
in a mammal. Optionally, the compound can be a compound determined
to suppress AMPA receptor mediated signaling by any of the assays
described herein.
[0104] Assays for determining whether a compound controls absence
seizures in a mammal are known in the art. For example, as
described in Example IV, below, absence seizure activity can be
determined in a mammalian model of absence epilepsy, the GAERS, in
which spontaneous spike-and-wake discharges are evidenced by EEG
recordings. Administration of PrRP decreased seizure activity in
the GAERS, in a dose-dependent manner. Accordingly, an in vivo
assay in a mammal susceptible to absence seizures, including a
rodent, non-human primate, or human, can be used to identify a
compound for controlling absence seizures.
[0105] It is expected that the PrRP and PrRP functional analog
compounds and therapeutic compositions of the invention will have
beneficial activities apart from, or in addition to, controlling
absence seizures. As described herein, high levels of GPR10
expression have been observed in a number of discrete locations in
the brain and peripheral tissues (see Example I and Table 2). In
particular, GPR10 is expressed at high levels in the GABAergic
neurons of the RTN. The GABAergic neurons of the RTN change their
firing patterns in response to sleep and wake states. During
periods of EEG-synchronized, deep sleep, RTN neurons generate
rhythmic, high-frequency bursts of action potentials, while during
waking and REM sleep, these neurons generate sequences of tonic
action potential activity (for a review, see McCormick et al.,
Annu. Rev. Neurosci., 20:185-215 (1997)). Accordingly, it is
contemplated that PrRP and PrRP functional analogs, including
functional analogs that act as antagonists of the PrRP receptor,
will be effective in preventing or ameliorating sleep disorders and
attention disorders by modulating signaling through the GABAergic
neurons of the RTN. Attention disorders are well known in the art
and include, for example, attention deficit hyperactivity disorder,
affective disorders, and disorders of memory.
[0106] A variety of sleep disorders are also well known in the art
and are described, for example, in Diagnostic and Statistical
Manual of Mental Disorders, 4th Edition (1994), published by the
American Psychiatric Association. The most common sleep disorder is
primary insomnia, or a difficulty in initiating or maintaining
sleep, which affects a large percentage of the population at some
point in their lives. Other common sleep disorders include
hypersomnia, or excessive daytime sleepiness, narcolepsy, which is
characterized by sudden and irresistible bouts of sleep, and sleep
apnea, which is a temporary cessation of breathing during
sleep.
[0107] As described herein, GPR10 is also expressed in the Area
Postrema (AP), Bed nucleus stria terminalis (BST), Central nucleus
amygdala (CeA), hypothalamic nucleus (Hypo), Superior colliculus
(SC), and Shell, nucleus accumbens (SNAc) of the brain, as well as
in peripheral tissues including the Adrenal medulla (AdM) and
uterus. Accordingly, it is contemplated that PrRP and PrRP
functional analogs, including analogs that act as antagonists of
the PrRP receptor, will be effective in preventing, ameliorating or
modulating conditions associated with these regions of the brain
and periphery, as shown in Table 1, below.
3TABLE 1 THERAPEUTIC POTENTIAL OF PrRP Therapeutic Potential GPR10
Localization Stress-induced anorexia Hypo, BST, CeA Stress-induced
hypertensive crisis NTS, AP Anxiety BST, CeA, Hypo Excessive fear
response CeA Posttraumatic Stress disorder BST, CeA, Hypo, AP
Nicotine induced cardiac NTS, AP arrhythmias Nicotine induced
coronary spasms NTS, AP Pheochromocytoma AdM Insomnia RTN Excessive
somnolence RTN Petit mal (absence) seizure RTN Visual processing
and attention SC deficits Drug addiction SNAc Inducing labor Uterus
Birth control Uterus
[0108] It is known in the art that currently available drugs for
controlling absence seizures are effective in the prevention and
treatment of a variety of neurologic and psychiatric conditions.
For example, valproate, one of the most commonly used medications
for controlling absence seizures, is also useful in the treatment
of bipolar and schizoaffective disorders, depression, anxiety,
alcohol withdrawal and dependence, agitation associated with
dementia, impulsive aggression, neuropathic pain, and for the
prophylactic treatment of migraine (see, for example, Loscher,
Prog. Neurobiol. 58:31-59 (1999), and Davis et al., J. Clin.
Psychopharmacol. 20:1S-17S (2000)). Thus, the PrRP and PrRP analogs
and compositions of the invention can be used to treat conditions
in which other anti-absence seizures drugs are effective.
[0109] The PrRP compounds and compositions of the invention can be
formulated and administered by those skilled in the art in a manner
and in an amount appropriate for the condition to be treated; the
weight, gender, age and health of the individual; the biochemical
nature, bioactivity, bioavailability and side effects of the
particular compound; and in a manner compatible with concurrent
treatment regimens. An appropriate amount and formulation for
controlling absence seizures in humans can be extrapolated based on
the activity of the compound in the assays described herein. An
appropriate amount and formulation for use in humans for other
indications can be extrapolated from credible animal models known
in the art of the particular disorder.
[0110] The total amount of compound can be administered as a single
dose or by infusion over a relatively short period of time, or can
be administered in multiple doses administered over a more
prolonged period of time. Additionally, the compounds can be
administered in slow-release matrices, which can be implanted for
systemic delivery or at the site of the target tissue. Contemplated
matrices useful for controlled release of therapeutic compounds are
well known in the art, and include materials such as DepoFoam.TM.,
biopolymers, micropumps, and the like.
[0111] The compounds and compositions of the invention can be
administered to the subject by any number of routes known in the
art including, for example, intravenously, intramuscularly,
subcutaneously, intraorbitally, intracapsularly, intraperitoneally,
intracisternally, intra-articularly, intracerebrally, orally,
intravaginally, rectally, topically, intranasally, or
transdermally. A preferred route for humans is oral
administration.
[0112] PrRP or a PrRP functional analog can be administered to a
subject as a pharmaceutical composition comprising the compound and
a pharmaceutically acceptable carrier. Those skilled in the art
understand that the choice of a pharmaceutically acceptable carrier
depends on the route of administration of the compound and on its
particular physical and chemical characteristics. Pharmaceutically
acceptable carriers are well known in the art and include sterile
aqueous solvents such as physiologically buffered saline, and other
solvents or vehicles such as glycols, glycerol, oils such as olive
oil and injectable organic esters.
[0113] A pharmaceutically acceptable carrier can contain
physiologically acceptable compounds that stabilize the compound,
increase its solubility, or increase its absorption. Such
physiologically acceptable compounds include carbohydrates such as
glucose, sucrose or dextrans; antioxidants, such as ascorbic acid
or glutathione; chelating agents; and low molecular weight
proteins.
[0114] For applications that require the compounds and compositions
to cross the blood-brain barrier, formulations that increase the
lipophilicity of the compound are particularly desirable. For
example, the compounds of the invention can be incorporated into
liposomes (Gregoriadis, Liposome Technology, Vols. I to III, 2nd
ed. (CRC Press, Boca Raton Fla. (1993)). Liposomes, which consist
of phospholipids or other lipids, are nontoxic, physiologically
acceptable and metabolizable carriers that are relatively simple to
make and administer.
[0115] The compounds of the invention can also be prepared as
nanoparticles. Adsorbing peptide compounds onto the surface of
nanoparticles has proven effective in delivering peptide drugs to
the brain (see Kreuter et al., Brain Res. 674:171-174 (1995)).
Exemplary nanoparticles are colloidal polymer particles of
poly-butylcyanoacrylate with PrRP adsorbed onto the surface and
then coated with polysorbate 80.
[0116] In current absence seizure treatment regimes, more than one
compound is often administered to an individual for maximal seizure
control. Thus, for use in controlling absence seizures, PrRP and
its functional analogs can advantageously be formulated with a
second compound that controls absence seizures. Such compounds
include, for example, valproate, ethosuximade, flunarizine,
trimethadione and lamotrigine. Contemplated methods of controlling
absence seizures include administering the compounds and
compositions of the invention alone, in combination with, or in
sequence with, such other compounds.
[0117] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
Localization of GPR10 in the Rat Brain
[0118] This example shows the distribution of GPR10 mRNA in the
brain, and particularly shows that GPR10 is highly expressed the
GABAnergic neurons of the RTN.
[0119] Previous studies using Northern blot analysis have
demonstrated that the receptor for PrRP, GPR10 is highly expressed
in the pituitary, but absent in the brain. In order to determine
the expression pattern of GPR10 in the brain, the more sensitive
approach of in situ hybridization analysis was used.
[0120] In situ hybridization was performed essentially as described
in Winzer-Serhan et al., Brain Res. Brain Res. Protoc. 3:229-241
(1999), and Winzer-Serhan et al., J. Comp. Neural. 386:540-554
(1997). Briefly, adult Sprague-Dawley rat brains were quickly
removed and placed in methylbutane cooled to -20.degree. C. for 1
minute. Twenty micron frozen sections were thaw-mounted onto
poly-L-lysine coated glass slides, fixed in 4% paraformaldehyde in
0.1 M PBS pH 7.4, dessicated and stored at -20.degree. C. until
prehybridization. Pretreated sections were incubated overnight at
60.degree. C. in hybridization buffer (50% formamide, 10% dextran
sulfate, 500 .mu.g/ml tRNA, 10 mM DTT, 0.3 M NaCl, 10 mM Tris pH
8.0, 1 mM EDTA pH 8.0) with .sup.35S-labeled GPR10 sense and
antisense cRNA probes (10.sup.7 cpm/ml) . Sections were washed,
dehydrated in graded ethanol, and opposed to B-max film with
.sup.14C standards of known radioactivity. Slides were dipped in
liquid Kodak NT2B emulsion and exposed for 4 weeks at 4.degree. C.
Developed sections were cresyl violet stained, coverslipped, and
viewed under dark field microscopy. Adjacent sections were also
labeled with S35-labeled sense GPR10 cRNA as control. The control
sections showed no specific staining.
[0121] By in situ hybridization analysis, GPR10 was shown to be
prominently expressed in the reticular thalamic nucleus (RTN), as
well as in a few other discrete locations of the forebrain,
midbrain and brainstem, as shown in FIGS. 1A and 1B and Table 2. In
situ hybridization analysis of GPR10 expression has also been
presented in Roland et al., Endocrinology 140:5736-5745 (1999).
[0122] By Northern blot (NB) analysis of a human tissue mRNA blot
(obtained from Clontech), GPR10 was also shown to be expressed in
the uterus.
4TABLE 2 GPR10 CENTRAL AND PERIPHERAL DISTRIBUTION Forebrain Shell
accumbens + + Cortex + Lateral septal nucleus + +
Ventricular/Ependymal lining + + Bed nucleus stria terminalis +
Medial preoptic area + + Medial preoptic nucleus + + +
Paraventricular nucleus, parvicellular + + + division, hypothalamus
Periventricular nucleus, hypothalamus + + + Ventrolateral
hypothalamus + + Lateral hypothalamus + Ventomedial hypothalamus +
Lateral hypothalamus + Central nucleus, amygdala + + Reticular
thalamic nucleus + + + + Dorsal premammillary nucleus + Ventral
premammillary nucleus + + Supramammillary nucleus + Midbrain
Nucleus of superior colliculus + + Brainstem Area postrema + + +
Nucleus tractus solitarius + + + Periphery Adrenal medulla + +
Uterus NB
[0123] The RTN plays an important role in the gating of sensory
information into the cortex, in generating sleep spindles, a
hallmark of slow wave sleep, and is implicated in the formation of
absence seizure activity (reviewed in Danober et al., Prog.
Neurobiol. 55:27-57 (1998) and McCormick et al., Annu. Rev.
Neurosci. 20:185-215 (1997)). The RTN consists of predominantly
GABAergic neurons, the activities of which are known to silence
thalamocortical activity during spike-wave (absence) seizures,
possibly contributing to the loss of consciousness during these
states (see Steriade et al., Cereb Cortex 7:583-604 (1997) and Liu
et al., Brain Res. 545:1-7 (1991)).
[0124] To determine whether GPR10 is expressed on the GABAergic
neurons of the RTN, in situ double labeling using digoxigenin-GAD
and .sup.35S-GPR10 probes was performed. For GAD (glutamic acid
decarboxylase) double, labeling, the samples were treated as
described above, except hybridization included both .sup.35S
labeled GPR10 cRNA and digoxigenin labeled GAD cRNA (E. Jones,
University of California, Irvine). Subsequent incubation with
alkaline phosphatase conjugated anti-digoxigenin antibody, washes,
and substrate development were preformed according to
manufacturer's instructions (Genius kit, Roche). Emulsion dipped
slides were not cresyl violet stained. Double labeling was detected
by viewing GAD positivity with light microscopy and GPR10 labeling
under dark field.
[0125] The distribution of GPR10 expresssion observed corresponded
with the "shell" pattern seen for GAD labeling (see FIGS. 1C and
1D). Examining the labeled sections under higher magnification
revealed that GPR10 expression overlaps with the GAD labeling of
GABAergic neurons of the RTN (see FIGS. 1E and 1F).
[0126] Based on expression of GPR10 in the GABAnergic neurons of
the RTN, it was predicted that PrRP modulates GABAergic output in
the RTN.
EXAMPLE II
Interaction of GPR10 with AMPA Receptor-Interacting Proteins
[0127] This example shows that GPR10 interacts with GRIP-like
proteins through its cytoplasmic tail, and forms clusters with
PICK1.
[0128] An analysis of GPR10 sequence revealed that its
carboxy-terminal tail contained a sequence motif of 4 amino acids
(-SVVI) (SEQ ID NO: 24) similar to those found in GluR2 and GluR3
subunits of AMPA receptors (FIG. 2A). The sequence -SVXI (X=any
amino acid) has been shown to be critical for the binding of AMPA
receptors to GRIP (Dong et al., Nature 386:279-284 (1997)), ABP
(AMPA binding protein, also designated GRIP2) (Srivastava et al.,
Neuron 21:581-591 (1998); Dong et al., J. Neurosci. 19:6930-6941
(1999)), and PICK1 (Xia et al., Neuron 22:179-187 (1999)). GRIP,
ABP and PICK1 are PDZ domain proteins which have been shown to be
important for the proper targeting and scaffolding of AMPA
receptors to the postsynaptic density (Craven et al., Cell
83:495-498 (1998); O'Brien et al., Curr. Opin. Neurobiol. 8:364-369
(1998)).
[0129] To determine whether GPR10 interacts with these proteins,
incremental amounts of Flag-tagged GPR10 cDNA, or Flag-tagged GPR10
cDNA with C-terminal mutations, were transiently co-transfected by
calcium phosphate transfection with fixed amounts of cDNA encoding
GRIP, myc-tagged ABP , myc-tagged PSD95, or myc-tagged PICK1 in HEK
293T cells. Flag-tag and C-terminal mutations were introduced by
PCR into GPR10 cDNA in pcDNA (Brian O'Dowd, U. Toronto), and
sequences were confirmed by dideoxy cycle sequencing containing
deaza-dGTP on an ALF-Express automated sequencer (Pharmacia).
Myc-PICK1 was generated by PCR using PICK-1 Flag (Jeff Staudinger,
GlaxoWellcome) as the template. GRIP cDNA in pRK/CMV was obtained
from Richard Huganir (Johns Hopkins), and PSD-95 myc was obtained
from Morgan Sheng (Harvard University).
[0130] Forty-eight hours after transfection, cells were washed once
in PBS and lysed with IP buffer (1% triton X-100, 25 mM Tris, pH
7.4, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1 mM phenylmethylsulphonyl
fluoride (PMSF), aprotinin, leupeptin, and bacitracin).
Immunoprecipitation was performed using 2 .mu.g anti-Flag M2
antibody (Sigma) or 1 .mu.g anti-myc antibody (Roche) followed by
25 .mu.l protein G-agarose (Sigma). Immunoprecipitation was carried
out overnight at 4.degree. C. Immunoprecipitated proteins were
resolved on SDS-PAGE, transferred to PVDF membrane. Western
blotting was performed to detect GRIP using anti-GRIP antibody
(1:1000). ABP-myc, PSD-95-myc, and PICK1-myc were detected using
the monoclonal anti-myc antibody (1:500, Clontech).
[0131] As shown in FIG. 2B, increasing amounts of GRIP were
co-immunoprecipitated with GPR10 as the amount of co-transfected
GPR10 increased. The specificity of this interaction was
demonstrated by introducing mutations at the COOH-terminal tail of
Flag-tagged GPR10 (FIG. 2C). Deletion of the last six amino acids,
as well as substituting the last 4 amino acids with unrelated
sequences completely abolished GRIP co-immunoprecipitation (FIG.
2D). Furthermore, alanine point mutations were introduced in place
of each of the last six amino acids and revealed that
threonine-365, valine-366, and valine-369 were not important for
GRIP interaction, while serine-367, valine-368, and isoleucine-370
were critical (FIG. 2D), consistent with results of similar studies
performed with AMPA receptors (Dong et al., Nature 386:279-284
(1997); Srivastava et al., Neuron 21:581-591 (1998))
[0132] These results indicate that the C-terminal tail of GPR10
contains a sequence that can interact with GRIP or GRIP-like
proteins.
[0133] GRIP is a large cytoplasmic protein containing seven PDZ
domains. A previous study has shown-that GluR2 and GluR3 interact
with the central three PDZ domains of GRIP (domains 4-6) (Dong et
al., Nature 386:279-284 (1997)). To determine whether GPR10
interacts with the same domains, 6X-Histidine tagged GRIP PDZ
domain segments 1-3, 4-6 or 7, generated by PCR, were
co-transfected and immunoprecipitated with Flag-tagged GPR10. As
shown in FIG. 2E, GPR10 interacted with PDZ domains 4-6 of GRIP.
This interaction is specific, since the C-terminally deleted GPR10
mutant (del6) was unable to interact.
[0134] Besides GRIP, other PDZ domain-containing proteins have been
demonstrated to interact with the same C-terminal residues of AMPA
receptors. To determine whether GPR10 interacts specifically with
AMPA receptor interacting proteins, myc-tagged ABP and myc-tagged
PSD95 were transfected into cell lines expressing wild type and
mutant Flag-tagged GPR10. As shown in FIG. 2F, GPR10 interacted
with the AMPA receptor binding protein ABP, but not with PSD95, a
PDZ domain protein important for NMDA receptor trafficking to and
anchoring at glutaminergic synapses (Gomperts, Cell 84:659-662
(1996)).
[0135] PICK1, originally identified as a PKC.alpha. interacting
protein (Staudinger et al., J. Biol. Chem. 272:32019-32024 (1997)),
is another PDZ domain protein recently shown to interact with the
GluR2 subunit of AMPA receptors. Unlike GRIP and ABP, PICK1 is the
only AMPA receptor interacting protein that forms intracellular and
surface aggregates with GluR2 subunits (but not GluR1) in
heterologous cell lines (Xia et al., Neuron 22:179-187 (1999); Dev
et al., Neuropharmacology 38:635-644 (1999)). To determine whether
GPR10 could mimic these characteristics, co-immunoprecipitation
was-performed on cells co-transfected with myc-tagged PICK1 and
Flag-tagged GPR10. As shown in FIG. 3A, immunoprecipitation of
GPR10 caused a concomitant precipitation of PICK1 protein, an
effect which was not observed using the mutant (del6) receptor.
[0136] Immunocytochemistry was also performed as follows. COS7
cells were transfected with LipofectAMINE (Gibco-BRL) using 6 .mu.g
DNA. Twenty-four hours after transfection, cells were trypsinized
and seeded onto poly-D-lysine coated glass coverslips. All
immunocytochemical analysis was done 48 hours after transfection.
Anti-PICK1 antibody (1:500) was incubated overnight at 4.degree.
C., whereas anti-myc monoclonal antibody (1:500, Clontech) was
incubated for 2 hours at room temperature. All chromophore
conjugated secondary antibodies were incubated for 1 hour at room
temperature. Coverslips were mounted on Vectashield mounting media
(Vector laboratories), and visualized at 60X on a Nikon
fluorescence microscope.
[0137] COS7 cells transfected with either GPR10 and PICK1 alone
exhibited diffuse cytoplasmic staining, as shown in FIG. 3B and 3C.
However, cells co-expressing both proteins exhibited intracellular
(FIG. 3D) as well as surface clustering (FIG. 3E) of GPR10 and
PICK1. Such clustering is contingent upon direct protein-protein
interaction between the COOH-terminal residues of GPR10 and the PDZ
domain of PICK1, since deleting the last six residues of GPR10 as
well as mutating critical residues within the PDZ domain of PICK1
(K27D28AA) (Staudinger et al., J. Biol. Chem. 272:32019-32024
(1997)) obliterates clustering (FIGS. 3F and 3G).
[0138] These results strongly support the possibility that
GRIP-like molecules, besides interacting with AMPA receptors at the
postsynaptic density, could help, recruit molecules such as GPR10
and PKC to form a signal transduction complex with AMPA receptors.
The assembly of such proteins in microdomains forms an efficient
network by which activation of one protein could modulate other
proteins in the complex.
[0139] Examples of G protein coupled receptors (GPCRs) affecting
channel function through direct or indirect interactions with PDZ
domain proteins have been reported. For instance, the .beta.2
adrenergic receptor (.beta.2AR) indirectly activates the
Na.sup.+/H.sup.+ exchanger (NHE3) by binding to the PDZ domain
protein NHERF, which normally inhibits NHE3 activity (Hall et al.,
Nature 392:626-630 (1998)). This interaction is mediated through
the COOH-terminal sequence of .beta.2AR in an agonist dependent
manner. Drosophila rhodopsin indirectly affects Ca.sup.++ influx
through a TRP calcium channel via a Gq/PLC/eye PKC
phototransduction cascade organized by the PDZ domain protein inaD
(Tsunoda et al., Nature 388:243-249 (1997)). Finally, the CRF-Rl
receptor has also been shown to interact with PSD95 through an
analogous COOH terminal motif found also in NR2 subunits of NMDA
receptors (Gaudriault et al., Society for Neuroscience Abstract
24:570 (1998)).
EXAMPLE III
Effect of PrRP on Thalamic Oscillatory Activity
[0140] This example shows that PrRP reduces AMPA receptor-mediated,
but not NMDA receptor-mediated, thalamic oscillatory activity.
[0141] Given that GPR10 interacts with AMPA receptor associated
molecules it was postulated that GPR10 receptor activation may
affect AMPA receptor signaling. Such an effect would most likely
influence oscillatory activity produced in the RTN since
glutamatergic inputs are critical for maintaining this network
function (Salt et al., Prog. Neurobiol. 48:55-72 (1996)). To test
this hypothesis, extracellular recordings were made from the RTN in
thalamic slices.
[0142] Horizontal thalamic slices (400 .mu.m) were prepared from
Sprague-Dawley rats (postnatal rats 13-15) using a vibratome
(Leica, VTlOOOS). The slices were transferred to a recording
chamber after at least 1 hr recovery, and were superfused with
artificial cerebrospinal fluid (aCSF) equilibrated with 95%
0.sub.2/5% CO.sub.2 at 0.5 ml/mm. The aCSF contained (in mM): NaCl
126, KC1 2.5, NaH.sub.2PO.sub.4 1.25, CaCl.sub.2 2, MgSO.sub.4
0.63, NaHCO.sub.3 26, and glucose 10. All experiments were carried
out in the presence of 10 .mu.M bicuculline maleate and cytochrome
c (100 .mu.g/ml) at 34.degree. C. A glass electrode filled with 2 M
NaCl was positioned in the nucleus of reticular thalamus (RTN), and
extracellular recording was made in response to stimulation of the
internal capsule (1-50 .mu.A) every 20-30 sec. After establishing
stable oscillatory activity, PrRP was applied for 15-20 min. Paired
Student's t-Test was used for statistical analysis. The data were
digitized at 1-5 kHz with the Neuronal Activity Acquisition Program
(Eclectek Enterprise).
[0143] In the representative experiments shown in FIG. 4, each
block shows 90-100 sweeps of 5 set duration, separated by 20-30
set, and each sweep represents the oscillatory activity in response
to a single stimulation pulse delivered to the internal capsule.
The bar on the left indicates the duration of peptide application.
The traces at the bottom show representative responses before and
in the presence of PrRP, taken at the time points indicated with
arrow heads.
[0144] Under GABA.sub.A receptor blockade and reduced Mg.sup.2+,
stimulation of the internal capsule induced 4-12 spindle-like
discharges oscillating at 3-4 Hz (FIG. 4), as previously reported
(Ulrich et al., Neuron 15,909-918 (1995)). In order to isolate the
AMPA receptor driven-oscillation, slices were equilibrated with the
NMDA receptor antagonist D,L-aminophosphonovaleric acid
(DL-APV).
[0145] Application of PrRP significantly reduced the number of
oscillations (FIG. 4A) and the effect was concentration dependent
(FIG. 4C). Maximum suppression, approximately 40%, was obtained at
10 .mu.M since higher concentrations (20 .mu.M) produced a
comparable degree of inhibition. In the experiments depicted in
FIG. 4C, the number of oscillations in the presence of PrRP was
normalized to that of the pre-peptide response. Columns represent
the mean +/- S.E. of 12 (pre-peptide), 6 (10 .mu.M), 2 (5 .mu.M),
and 4 (1 .mu.M) experiments, respectively.
[0146] It was next investigated whether the suppression of RTN
oscillatory activity by PrRP is the consequence of AMPA receptor
modulation or some general cellular effect. If the latter, thalamic
oscillations driven by NMDA receptors should be similarly reduced
by PrRP. The above experiment was thus repeated in a low Mg.sup.2+
(0.1 mM)-aCSF containing 10 .mu.M of the AMPA receptor antagonist
CNQX. As shown in FIGS. 4B and 4C (right column, n=4), 10 .mu.M
PrRP had no discernible effect on oscillation under these
circumstances.
[0147] In summary, the results described in this example indicate
that PrRP reduces thalamic oscillatory activity by selectively
modulating AMPA receptors.
EXAMPLE IV
Effect of PrRP on Absence Aeizures
[0148] This example shows that PrRP suppresses seizure activity in
GAERS.
[0149] Spindle wave oscillations generated in the brain slice
correlate well with the activity seen during slow wave sleep as
well as during an episode of absence seizure attack (Danober et
al., Prog. Neurobiol. 55:27-57 (1998); McCormick et al., Annu. Rev.
Neurosci. 20:185-215 (1997)). To assay whether the in vitro effects
of PrRP on thalamic oscillations reflect in vivo efficacy, an
animal model of absence seizure that present with spontaneous
spike-and-wave discharges (SWD) was used.
[0150] The GAERS (Genetic Absence Epilepsy Rats from Strasbourg) is
a rat strain that exhibits recurrent seizure activity characterized
by bilateral and synchronous SWD and behavioral arrest (Danober et
al., Prog. Neurobiol. 55:27-57 (1998)). Drugs used to treat absence
seizure in humans have been shown to be effective in suppressing
seizure activity in this model (Danober et al., Prog. Neurobiol.
55:27-57 (1998); Gower et al., Epilepsy Res. 22:207-213
(1995)).
[0151] Electroencephalogram (EEG) recordings and
intracerebroventricular (ICV) injection into GAERS were performed
essentially as described in Liu et al., Brain Res. 545:1-7 (1991).
Briefly, six male GAERS (300-400 g) were implanted stereotaxically
(AP=-0.8, ML=1.2, CV=3 mm, with bregma as reference) with a
permanent stainless steel cannula under pentobarbital anesthesia
(40 mg/kg i.p.). All rats were also implanted bilaterally with 4
stainless-steel electrodes at the frontal and parietal cortex and
connected to a microconnector. Both the guide cannulae and EEG
electrodes were anchored to the skull using retaining screws and
dental acrylic cement.
[0152] After one week of recovery, stainless steel injection
cannulae were introduced into the guide cannulae so as to extend
2-4 mm beyond their tips. Injection cannulae were connected to a 10
.mu.l microsyringe driven by a pump. Five microliters saline or
peptides at various concentrations were microinjected over 1 min
through the cannulae while taking EEG recordings continuously
throughout the duration of the experiment, in freely moving
animals. During EEG recordings the rats were carefully watched and
were prevented from falling asleep by gentle sensory stimulation.
Seizure frequency was determined as a cumulative duration of spike
and wave discharges per consecutive 20 minute periods (seconds of
seizure activity per 20 minute recording) after the initial
injection. Statistical analysis was performed using the Wilcoxon
test.
[0153] PrRP was administered intracerebroventricularly into GAERS
rats. EEG was taken throughout the course of the experiment and
time spent in seizures (spike and wave discharges (SWD) duration)
was tabulated for 20 minute intervals from the initial infusion of
either aCSF control or various peptide concentrations. Data from
six animals receiving the same treatment were pooled. In comparison
to aCSF control, PrRP was able to dose-dependently suppress seizure
activity in this animal model (FIG. 5A-5D). At 10 nmol PrRP, the
SWD duration was reduced by half for the first 20 minutes, and
returned to baseline after 40 minutes (FIG. 5B). At higher peptide
concentrations, seizure activity was reduced more significantly
(i.e. by 75% at 50 nmol (FIG. 5C), and by almost 100% at 100 nmol
at 20 minutes (FIG. 5D)). This seizure suppressing activity
remained significant 60 to 80 minutes after peptide injection. No
other mobility or behavioral changes were observed at all peptide
concentrations.
[0154] The results described above demonstrate that PrRP is
effective in suppressing absence seizures.
[0155] In summary, the results described in Examples I-IV
demonstrate that GPR10, the PrRP receptor, is highly expressed in
the GABAergic neurons of the RTN; that GPR10 contains structural
motifs that allow it to interact with PDZ domain-containing
proteins; that application of PrRP specifically reduces AMPA
receptor mediated oscillatory activity; and that PrRP suppresses
absence seizures. These results show a novel role of GPR10 and PrRP
in regulating thalamic networks and implicate GPR10 as a potential
therapeutic target in the treatment of disorders associated with
the RTN, including absence seizures and sleep disorders.
EXAMPLE V
Whole cell Electrophysiological Recordings from RTN Neurons
[0156] This example shows an exemplary method of determining the
ability of a compound to modulate AMPA receptor signaling.
[0157] Horizontal slices (200 .mu.m) are prepared from postnatal
Sprague Dawley rats (P13-P15). After at least 1 hr of incubation at
room temperature in artificial cerebrospinal fluid (aCSF)
containing (in mM): NaCl 126, KCl 2.5, NaH.sub.2PO.sub.4 1.25,
CaCl.sub.2 2, MgSO.sub.4 2, NaHCO.sub.3 26, and glucose 10), slices
are transferred to a recording chamber and submerged in low
Mg.sup.2+-aCSF (0.63 mM) equilibrated with 95% O.sub.2/5% CO2.
Whole cell recording are made from neurons in the nucleus of
reticular thalamus at room temperature in low Mg.sup.2+-aCSF
containing 100 mM DL 2-aminophosphonovaleric acid (APV), 10 mM
bicuculline maleate (BMI), and 100 mg/ml cytochrome c. The flow
rate is 2 ml/min. Patch electrodes are pulled from borosilicate
glass (2-3 MOhm) and filled with a pipette solution containing (in
mM): CsCl 135, MgCl 2, EGTA 10, HEPES 10, ATP 10, and QX314 5 (pH
7.3). Excitatory postsynaptic currents (EPSCs) are recorded in
response to activation of the internal capsule. The holding current
and access resistance are constantly monitored and the holding
potential is maintained at -70 mV throughout the experiment. After
establishing a stable baseline, a compound is applied to the
perfusion line for 15 minutes. The data is digitized and analyzed
offline. Aspects of the method are described in further detail in
Cox et al., J. Neurophysiol. 74:990-1000 (1995).
EXAMPLE VI
Whole Cell Ion Influx Assays
[0158] This example shows a further exemplary method of determining
the ability of a compound to modulate AMPA receptor signaling.
[0159] The method uses cells that express both AMPA receptors and
PrRP receptors, such as cell lines (e.g. HEK 293 cells) transiently
or stably transfected with AMPA receptors and GPR10, or RTN
neurons. A determination is made as to effect of the test compound
on calcium ion or sodium ion influx in response to AMPA receptor
agonists. The method is amenable to either low-throughput (e.g.
light microscopy) or high-throughput (e.g. FLIPR) assays.
[0160] AMPA receptor agonists include, for example, AMPA (e.g.
s-AMPA zwitterion, at a concentration of about 10 .mu.M) or
glutamate (at a concentration of about 10 mM). Optionally, an assay
can be performed in the presence of an AMPA receptor antagonist
(e.g. CNQX at a concentration of about 20 .mu.M), for example to
ensure that any response is dependent on AMPA receptors. Since AMPA
receptors desensitize rapidly upon activation, a drug such as
cyclothiazide can be added, at a concentration of about 100 .mu.M.
The addition of cyclothiazide is expected to reduce the
desensitization rate and potentiate AMPA signals by a factor of
about 200 fold. The use of such antagonists and modulators to
isolate or stabilize AMPA signals are well known in the art.
[0161] To determine whether PrRP or a PrRP functional analog
modulates AMPA receptor signaling, the test compound is added to
the cells, followed by an AMPA receptor agonist. The relative
amplitude change of calcium or sodium influx through the AMPA
receptors is a measure of the ability of the compound to modulate
AMPA receptor signaling.
[0162] In calcium ion influx assays, PrRP can be added to the cells
for a sufficient period (e.g. about 3 to 5 minutes) to desensitize
GPR10-mediated calcium signals and more clearly determine specific
AMPA receptor mediated signals. Calcium channels can be blocked, if
desirable, with calcium channel blockers known in the art. Calcium
ion influx in response to AMPA receptor activation can be
determined using fluorescent sensitive calcium indicators (e.g.
Fluo-3 and the like), by visualizing single cells under light
microscopy. Alternatively, particularly where high-throughput
screening is desired, calcium influx in response to AMPA receptor
activation can be determined using a fluorometric imaging plate
reader (FLIPR), which allows rapid detection of changes in
intracellular calcium levels.
[0163] Likewise, sodium ion influx in response to AMPA receptor
activation can be determined using fluorescent sensitive sodium
indicators (e.g. SBFI and the like, available from Molecular
Probes), either by visualizing single cells under light microscopy
or by adapting FLIPR technology.
[0164] All journal article, reference and patent citations provided
above, in parentheses or otherwise, whether previously stated or
not, are incorporated herein by reference in their entirety.
[0165] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
Sequence CWU 1
1
24 1 29 PRT Homo Sapien 1 Phe Arg Glu Glu Leu Arg Lys Leu Leu Val
Ala Trp Pro Arg Lys Ile 1 5 10 15 Ala Pro His Gly Gln Asn Met Thr
Val Ser Val Val Ile 20 25 2 29 PRT Homo Sapien 2 Asn Pro Ser Ser
Ser Gln Asn Ser Gln Asn Phe Ala Ala Thr Tyr Lys 1 5 10 15 Glu Gly
Tyr Asn Tyr Tyr Gly Ile Glu Ser Val Lys Ile 20 25 3 29 PRT Homo
Sapien 3 Phe Lys Pro Ala Pro Ala Thr Asn Thr Gln Asn Tyr Ala Thr
Tyr Arg 1 5 10 15 Glu Gly Tyr Asn Val Tyr Gly Thr Glu Ser Val Lys
Ile 20 25 4 12 PRT Homo Sapien 4 Pro His Gly Gln Asn Met Thr Val
Ser Val Val Ile 1 5 10 5 6 PRT Artificial Sequence human GPR10
variant 5 Pro His Gly Gln Asn Met 1 5 6 12 PRT Artificial Sequence
human GPR10 variant 6 Pro His Gly Gln Asn Met Thr Val Pro Arg Pro
Ala 1 5 10 7 12 PRT Artificial Sequence human GPR10 variant 7 Pro
His Gly Gln Asn Met Ala Val Ser Val Val Ile 1 5 10 8 12 PRT
Artificial Sequence human GPR10 variant 8 Pro His Gly Gln Asn Met
Thr Ala Ser Val Val Ile 1 5 10 9 12 PRT Artificial Sequence human
GPR10 variant 9 Pro His Gly Gln Asn Met Thr Val Ala Val Val Ile 1 5
10 10 12 PRT Artificial Sequence human GPR10 variant 10 Pro His Gly
Gln Asn Met Thr Val Ser Ala Val Ile 1 5 10 11 12 PRT Artificial
Sequence human GPR10 variant 11 Pro His Gly Gln Asn Met Thr Val Ser
Val Ala Ile 1 5 10 12 12 PRT Artificial Sequence human GPR10
variant 12 Pro His Gly Gln Asn Met Thr Val Ser Val Val Ala 1 5 10
13 31 PRT Bos taurus 13 Ser Arg Ala His Gln His Ser Met Glu Ile Arg
Thr Pro Asp Ile Asn 1 5 10 15 Pro Ala Trp Tyr Ala Gly Arg Gly Ile
Arg Pro Val Gly Arg Phe 20 25 30 14 31 PRT Rattus 14 Ser Arg Ala
His Gln His Ser Met Glu Thr Arg Thr Pro Asp Ile Asn 1 5 10 15 Pro
Ala Trp Tyr Thr Gly Arg Gly Ile Arg Pro Val Gly Arg Phe 20 25 30 15
31 PRT Homo Sapien 15 Ser Arg Thr His Arg His Ser Met Glu Ile Arg
Thr Pro Asp Ile Asn 1 5 10 15 Pro Ala Trp Tyr Ala Ser Arg Gly Ile
Arg Pro Val Gly Arg Phe 20 25 30 16 20 PRT Bos taurus 16 Thr Pro
Asp Ile Asn Pro Ala Trp Tyr Ala Gly Arg Gly Ile Arg Pro 1 5 10 15
Val Gly Arg Phe 20 17 20 PRT Rattus 17 Thr Pro Asp Ile Asn Pro Ala
Trp Tyr Thr Gly Arg Gly Ile Arg Pro 1 5 10 15 Val Gly Arg Phe 20 18
20 PRT Homo Sapien 18 Thr Pro Asp Ile Asn Pro Ala Trp Tyr Ala Ser
Arg Gly Ile Arg Pro 1 5 10 15 Val Gly Arg Phe 20 19 7 PRT
Artificial Sequence human PrRP variant 19 Xaa Arg Pro Val Gly Arg
Phe 1 5 20 7 PRT Artificial Sequence human PrRP variant 20 Ile Arg
Xaa Val Gly Arg Phe 1 5 21 7 PRT Artificial Sequence human PrRP
variant 21 Ile Arg Pro Xaa Gly Arg Phe 1 5 22 7 PRT Artificial
Sequence human PrRP variant 22 Ile Arg Pro Val Gly Arg Xaa 1 5 23 7
PRT Homo Sapien 23 Ile Arg Pro Val Gly Arg Phe 1 5 24 4 PRT Homo
Sapien 24 Ser Val Val Ile 1
* * * * *