Note: Descriptions are shown in the official language in which they were submitted.
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ASSAYS FOR AGONISTS, AGONISTS AND INVERSE AGONISTS OF MELANIN CONCENTRATING
HORMONE
(MCH) BINDING TO THE SOMATOSTATIN-LIKE RECEPTOR (SLC-I)
Field of the Invention
The present invention is directed to assay methods that can be used to
determine
whether a test compound can be used to modulate the binding of MCH to the SLC-
1 receptor.
Compounds identified as being effective modulators have potential use as
therapeutic agents
in treating obesity and eating disorders.
Background of the Invention
A. Melanin Concentrating Hormone
1o Melanin concentrating hormone (MCH) is a cyclic peptide that was first
isolated from
fish over 15 years ago. In mammals, MCH gene expression is localized to the
ventral aspect
of the zona inserta and the lateral hypothalamic area (Breton, et al., Mol.
Cell. Nezzrosci.
4:271-283 (1993)). The latter region of the brain is associated with the
control of behaviors
such as eating and drinking, with arousal and with motor activity (Baker,
Trends Endocrinol.
Metab. 5:120-126 (1994)). Although the biological activity of MCH in mammals
has not
been fully defined, recent work has indicated that it promotes eating and
weight gain
(U.S. 5,849,708). Thus, MCH and its agonists have been proposed as a treatment
for anorexia
nervosa and weight loss due to AIDS, renal disease, or chemotherapy.
Similarly, antagonists
of MCH can be used as a treatment for obesity and other disorders
characterized by
2o compulsive eating and excessive body weight.
Although MCH has been known for over two decades, its specific receptor has
not
been structurally characterized and cloned. This has limited the ability to
search for
therapeutic agents that act by mimicking or inhibiting MCH.
B. G Protein-Coupled Receptors
G protein coupled receptors (GPCRs) constitute a family of proteins sharing a
common structural organization characterized by an extracellular N-terminal
end, seven
hydrophobic alpha helices putatively constituting transmembrane domains and an
intracellular
C-terminal domain. GPCRs bind a wide variety of ligands that trigger
intracellular signals
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2
through the activation of transducing G proteins (Carom et al., Rec. Prog.
Horm. Res. 48:277-
290 (1993); Freedman, et al., Rec. Prog. Horm. Res. 51:319-353 (1996)).
More than 300 GPCRs have been cloned thus far and it is generally assumed that
there
exist well over 1,000 such receptors. Roughly 50-60% of all clinically
relevant drugs act by
modulating the functions of various GPCRs (Gudermann, et al., J. Mol. lhled.
73:51-63
(1995)). Many of the clinically relevant receptors are located in the central
nervous system.
Among the GPCRs that have been identified and cloned is a gene that encodes a
protein homologous to the receptors of the somatostatin family. Kolakowski
called this
receptor SLC-1 and described the structure of the gene as it exists in humans
(FEBS Lett.
398:253-258, (1996)). A rat counterpart of SLC-1 was found to be essentially
identical and
was described by Lakaye, et al. (Biochim. Biophys. Acta. 1401:216-220 (1998)).
Based upon
the location of cells expressing SLC-1 mRNA, it was proposed that the receptor
plays a role
in functions such as emotion, memory and sensory perception. However, the
endogenous
ligand of this receptor has not previously been identified.
Summary of the Invention
The present invention is based upon the discovery that MCH serves as a ligand
for the
SLC-1 receptor. Recombinant cells expressing either rat or human SLC-1 can be
used in
conjunction with MCH in screening assays designed to identify agonists and
antagonists.
Thus, in its first aspect, the invention is directed to a method of assaying a
test compound for
2o its ability to bind to the SLC-1 receptor. This is accomplished by
incubating cells expressing
the receptor gene with MCH and test compound. The extent to which the binding
of MCH is
displaced is then determined. Radioligand assays or enzyme-linked
immunosorbent assays
may be performed in which either MCH or the test compound is detectably
labeled. Although
any cell expressing SLC-1 may be used, a recombinant cell expressing a
heterologous SLC-1
gene from either the rat or human is preferred. The term "heterologous" as
used herein refers
to any SLC-1 gene transfected into a cell, i.e., the term refers to any non-
endogenous SLC-1.
The invention is also encompasses methods of determining if a test compound is
an
agonist, antagonist, or inverse agonist of MCH binding based upon a functional
assay. One
way to carry out such assays is to incubate a cell expressing SLC-1 with the
test compound
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and to then determine whether intracellular adenyl cyclase activity or
intracellular calcium
concentration changes. Results should typically be compared with those
obtained when
incubations are performed in a similar manner but in the absence of test
compound. In
general, functional assays of this type will be performed in conjunction with
binding assays of
the sort described above. The preferred cell for use in the assays is a
recombinant cell that has
been transformed with a heterologous SLC-1 gene. Test compounds that act as
agonists
should produce an increase or decrease in adenyl cyclase activity or increase
in intracellular
levels of calcium. Inverse agonists may reduce adenyl cyclase activity or
intracellular calcium
levels, particularly if assays are performed in the presence of a fixed amount
of MCH.
to Antagonists, should block the binding of MCH to receptor but not produce
the opposite
reponse in terms of adenyl cyclase activity or intracellular calcium that is
the hallmark of an
inverse agonist.
Detailed Description of the Invention
The present invention is directed to assays that can be used to screen
compounds for
their ability to modulate the binding of MCH to the SLC-1 receptor. Any form
of MCH that
has been reported may be used, but the preferred peptide is 19 amino acids in
length and has
the sequence: Asp-Phe-Asp-Met-Leu-Arg-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-
Trp
Gln-Val (SEQ ID NO:1). The peptide assumes a cyclic conformation as the result
of a
disulfide between the two cysteines. This peptide may be obtained commercially
(Sigma,
2o St. Louis, MO) or can be synthesized using standard methodology well known
in the art. The
peptide may be detectably labeled with radioisotopes such as ''-5I or,
alternatively, fluorescent
or chemiluminescent labels can be incorporated. Also, the peptide can be
joined to enzymes
that are readily detectable such as horseradish peroxidase.
The SLC-1 receptor may be cloned from human cells using the procedure
described by
Kolakowski, et al. (FEBS Lett. 398:253-258 (1996)) or from rat cells using the
procedure
described by Lakaye, et al. (Biochim. Biophys. Acta. 1401:216-220 (1998)). The
Examples
section provides a detailed description of a procedure that may be used in
cloning SLC-1
which, is also referred to herein as clone 1-18. Once obtained, the SLC-1
sequence should be
incorporated into an expression vector with a promoter active in mammalian
cells (Sambrook,
3o et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Press (1989)).
Examples of promoters that may be used include that of the mouse
metallothionein I gene
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(Hamer, et al., J. Mol. Appl. Gen. 1:273-288 (1982)); the immediate-early and
TK promoter of
herpes virus (Yao, et al., J. Virol. 69:6249-6258 (1995); McKnight, Cell
31:355-365 (1982));
the SV 40 early promoter (Benoist, et al., Nature 290:304-310 (1981)); and,
the CMV
promoter (Boshart, et al., Ce1141:521-530 (1985)). Vectors may also include
enhancers and
other regulatory elements.
Once expression vectors have been constructed, they can be introduced into a
mammalian cell line by methods such as calcium phosphate precipitation,
microinjection,
electroporation, liposomal transfer, viral transfer or particle mediated gene
transfer. Although
other mammalian cells may be used, HEK-293 cells have been found to give
successful
1o results and a procedure for expressing SLC-1 in these cells is described in
the Examples
section. Standard procedures for selecting cells and for assaying them for the
expression of
SLC-1 (e.g., by Northern analysis) may be performed.
Once the MCH peptide and cells producing the SLC-1 receptor have been
obtained,
assays may be performed to determine whether test compounds have any effect on
binding. A
wide variety of different types of assays can be performed using standard
methods well
known in the art. For example, in radioligand binding assays, cells expressing
SLC-1 are
incubated with MCH and with a compound being tested for binding activity. The
preferred
source of SLC-1 is recombinantly transformed HEK-293 cells. Other cells may
also be used
provided they do not express other proteins that strongly bind MCH. This can
easily be
2o determined by performing binding assays on cells transformed with SLC-l and
comparing the
results obtained with those obtained using their untransformed counterparts.
Assays may be performed using either intact cells or with membranes prepared
from
the cells (see e.g., Wang, et al., Proc. Natl. Acad. Sci. U.S.A. 90:10230-
10234 (1993)). As
suggested above, the membranes, or cells, are incubated with MCH and with a
preparation of
the compound being tested. After binding is complete, receptor is separated
from the solution
containing ligand and test compound, e.g., by filtration, and the amount of
binding that has
occurred is determined. Preferably, the ligand used is detectably labeled with
a radioisotope
such as '25I. However, if desired, other types of labels can also be used.
Among the most
commonly used fluorescent labeling compounds are fluorescein, isothiocynate,
rhodamine,
3o phycoerythrin, phycocyanin, allophycocyanin o-phthaldehyde and
fluorescamine. Useful
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chemiluminescent compounds include luminol, isoluminol, theromatic of
acridinium ester,
imidazole, acridinium salt, and oxalate ester.
Nonspecific binding may be determined by carrying out the binding reaction in
the
presence of a large excess of unlabeled ligand. For example, labeled MCH may
be incubated
5 with receptor and test compound in the presence of a thousandfold excess of
unlabeled MCH.
Nonspecific binding should be subtracted from total binding, i.e., binding in
the absence of
unlabeled ligand, to arrive at the specific binding for each sample tested.
Other steps such as
washing, stirring, shaking, filtering and the like may be included in the
assays as necessary.
Typically, wash steps are included after the separation of membrane-bound
ligand from ligand
1o remaining in solution and prior to quantitation of the amount of li.gand
bound, e.g., by
counting radioactive isotope. The specific binding obtained in the presence of
test compound
is compared with that obtained in the presence of labeled ligand alone to
determine the extent
to which the test compound has displaced receptor binding.
In performing binding assays, care must be taken to avoid artifacts which may
make it
appear that a test compound is interacting with receptor when, in fact,
binding is being
inhibited by some other mechanism. For example, the compound being tested
should be in a
buffer which does not itself substantially inhibit the binding of MCH and
should, preferably,
be tested at several different concentrations. Preparations of test compound
should also be
examined for proteolytic activity and it is desirable that antiproteases be
included in assays.
2o Finally, it is highly desirable that compounds identified as displacing the
binding of MCH be
reexamined in a concentration range sufficient to perform a Scatchard analysis
on the results.
This type of analysis is well known in the art and can be used for determining
the affinity of a
test compound for receptor (see e.g., Ausubel, et al., Current Protocols and
Molecular
Biology, 11.2.1-11.2.19 (1993); Laboratory Techniques in Biochemistry and
Molecular
Biology, Work, et al., Ed. N.Y. (1978)). Computer programs may be used to help
in the
analysis of results (e.g., Munson, P., Methods Enrymol. 92:543-577 (1983)).
Depending upon their effect on the activity of the receptor, agents that
inhibit the
binding of MCH to receptor may be either agonists or antagonists. Activation
of receptor may
be monitored using a number of different methods. For example, adenyl cyclase
assays may
3o be performed by growing cells in wells of a microtiter plate and then
incubating the wells in
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the presence or absence of test compound. cAMP may then be extracted in
ethanol,
lyophilized and resuspended in assay buffer. Assay of cAMP thus recovered can
be carned
out using any method for determining cAMP concentration. Typically, adenyl
cyclase assays
will be performed separately from binding assays, but it may also be possible
to perform
binding and adenyl cyclase assays on a single preparation of cells.
Activation of receptor may also be determined based upon a measurement of
intracellular calcium concentration. For example, transformed HEK-293 cells
may be grown
on glass cover slides to confluence. After rinsing, they may be incubated in
the presence of an
agent such as Fluo-3 or FURA-2 AM (Molecular Probe F-1221). After rinsing and
further
1o incubation, calcium displacement may be measured using a photometer, Other
types of assays
for determining intracellular calcium concentrations are well known in the art
and may also be
employed.
Assays that measure the intrinsic activity of the receptor, such as those
based upon
inositol phosphate measurement, may be used in order to determine the activity
of inverse
agonists. Unlike antagonists which block the activity of agonists but produce
no activity of
their own, inverse agonists produce a biological response diametrically
opposed to the
response produced by an agonist. For example, if an agonist promoted an
increase in
intracellular calcium, an inverse agonist would decrease intracellular calium
levels.
The radioligand and cell activation assays discussed above merely provide
examples
of the types of assays that can be used for determining whether a particular
test compound
alters the binding of MCH to the SLC-1 receptor and acts as an agonist or
antagonist. There
are many variations on these assays that are compatible with the present
invention. Such
assays may involve the use of labeled antibodies as a means for detecting MCH
that has
bound to receptor or may take the form of the fluorescent imaging plate reader
assays
described in the Examples section herein.
Examples
I. Methods
Preparation of Clone 1-18
A PCR-based strategy was used to clone the rat 1-18 gene (SLC-1). Rat spinal
cord
3o mRNA was isolated using the FastTrackO kit (InVitrogen, San Diego, Ca). The
templates for
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PCR amplification were synthesized using GeneAmp RNA PCR kits (N808-0017
Perkin
Elmer) with 200 ng of the rat spinal dorsal horn polyA+ RNA and were amplified
using the
following primers:
TM3-5: 5'-G(C or T)G(A or C)(C or G)(A or G)(C or G)(C or T)ITIGA(C or T)
CGCTA-3' (SEQ ID N0:2)
TM7-5: 5'-AAGC(C or T)(A or G)TA(G or T)AI(A or C or G)AI(A or C)GG(A or
G)TT-3' (SEQ ID N0:3).
The reaction mixture contained 200 pmoles of each of the TM3-5 and TM7-5
primers and
2.5 units of Taq DNA polymerase in 50 mM KCI, 1.5 mM MgCI" 10 mM Tris(HCl),
200 mM
1o dNTPs, pH 9Ø The reaction tubes were heated at 95 °C for one
minute and subjected to 39
cycles of denaturation (95 °C / 1 min), annealing (42 °C / 1
min)and extension (72 °C / 1 min).
The amplified fragments were analyzed and size fractionated on a 1 % agarose
gel. Fragments
between 500 by and 800 by were excised from the gel, purified using the
Sephaglas
BandPrepO kit from Pharmacia (cat# 27-9285-O1), and subcloned into the pGEM-T
vector
from Promega (cat# A3600). Recombinant pGEM-T clones were selected randomly
and
plasmid DNA was prepared using the alkaline lysis method starting with 2 ml of
bacterial
culture. The Sanger dideoxy nucleotide chain termination method was used to
sequence the
DNA from these clones, with the T7 sequencing kit from Pharmacia (cat# 27-1682-
Ol). The
insert DNA fragment of the clone pGEMT-1-18 was excised from the vector using
Pst I and
2o Sac II, isolated from an agarose gel and labeled with 32P by random primed
synthesis using the
Ready-To-Go0 DNA labeling kit (cat#27-9251-O1)from Pharmacia. This probe was
used to
screen a rat brain stem-spinal cord cDNA library in 1 ZAP II (Stratagene, cat#
936521). The
filters were incubated with the probe for 18 hours at 65°C in 2x SSC,
5x Denhardt's solution
and 0.2% SDS. The filters were rinsed twice in O.lx SSC, 0.2% SDS at room
temperature.
The filters were then washed twice for 45 min in O.lx SSC, 0.2% SDS at
65°C, once for 45
min at 65°C in 5 mM EDTA, 0.2% SDS, pH 8.0 and finally rinsed with O.lx
SSC at room
temperature.
Hybridization-positive phages were purified and their inserts rescued by
helper phage
mediated excision to yield plasmid DNA. The insert of plasmid pBS/1-18 was
sequenced
3o progressively with the 1-18-specific primers. To generate a mammalian
expression vector, a 2
Kb Sma I - Xho I fragment from pBS/1-18 was isolated and subcloned into the
Eco RV and
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Xho I sites of pcDNA3 (InVitrogen, San Diego, Ca). This expression vector was
called
pcDNA3-1-18. Plasmid DNA was prepared using the Qiaprep system from Qiagen.
Expression
HEK-293 cells were transfected with a mammalian expression construct coding
for the 1-
18 clone (pcDNA 3.0 vector, Invitrogen) using the Superfect reagent (Qiagen).
A stable
receptor pool of 1-18 was developed by applying a selection marker (G418, 0.6
mg/ml) and
the cells were maintained in this selection medium. The presence of mRNA
specific for clone
1-18 was assessed by Northern blot analysis and by the reverse transcriptase
polymerise chain
reaction (RT-PCR).
Ligands
In order to identify the ligand of clone 1-18, a collection of peptide and non-
peptide
ligands was obtained from commercial sources (Sigma, CalBiochem, American
Peptide
Company, Bachem, RBI). The compounds were dissolved in water/DMSO at 30 iM and
placed in 96 well microplates. A total of 846 compounds (peptides and non-
peptides) were
prepared and tested.
Assay
A functional assay was performed with FLIPR (Fluorescent Imaging Plate Reader,
Molecular Devices) using the fluorescent calcium indicator Fluo-3 (Molecular
Probes) on a 96
well platform. HEK-293 cells, either expressing the receptor or wild type
cells, were loaded
2o with Fluo-3 as follows. Stable HEK-293 clones expressing 1-18 or parental
cells were plated
at a density of 70,000 cells/well in a 96 well plate. On the day of the
experiment, the 1-18
cells were loaded with fluorescent solution (Dulbecco's modified medium with
10% fetal
bovine serum containing 4 ified medium with 10% fetal bovine serum containing
4 Probes)
on a 96 well platform. HEK-293 cells, either expressing the receptor or wild
type cells, were
loaded with Fluo-3 as follows. Stable HEK-2 BSA (pH 7.4). The cells were
analyzed using
the FLIPR system to measure the mobilization of intracellular calcium in
response to different
compounds.
II. Results
HEK-293 cells endogenously express some GPCRs such as bradykinin receptors
which
3o can be used as an internal control for assays. The background signal was
established with all
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of the compounds in the parental HEK-293 cells (non-transfected) using the
FLIPR assay.
HEK-293 cells expressing the clone 1-18 were stimulated with all compounds and
calcium
responses were compared with those in parental HEK-293 cells. Only one
compound, melanin
concentrating hormone (MCH), consistently elicited signals in the transformed
cells but not
the wild type cells. This indicates that MCH is interacting with the
recombinantly expressed
receptor. Confirmation of this conclusion was obtained by the observation of a
dose-response
relationship with MCH in the cells transfected with 1-18, but not in the non-
transfected cells
or in cells transfected with several other different receptors. Thus, it has
been established that
clone 1-18 is, in fact, a specific receptor for MCH and that this receptor can
be used to screen
1o compounds which either mimic the action of MCH (agonists) or antagonize the
action of
MCH (antagonists).
Screening assays can be performed using the FLIPR assay described above.
Alternatively,
MCH can be iodinated and used as a tracer in radioligand binding assays on
whole cells or
membranes. Other assays that can be used include the GTPaS assay, adenylate
cyclase
assays, assays measuring inositol phosphates, and reporter gene assays (e.g.,
those utilizing
luciferase, aqueorin, alkaline phosphatase, etc.).
All references cited herein are fully incorporated by reference. Having now
fully
2o described the invention, it will be understood by those of skill in the art
that the invention may
be performed within a wide and equivalent range of conditions, parameters and
the like,
without affecting the spirit or scope of the invention or any embodiment
thereof.
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SEQUENCE LISTING
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Lembo, Paola
Cao, Jack
Walker, Philippe
Grazzini, Eric
Ahmad, Sultan
<120> Assays for Agonists, Antagonists and Inverse Agonists
of Melanin Concentrating Hormone
<130> mch appl.
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Asp Phe Asp Met Leu Arg Cys Met Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10 15
Trp Gln Val
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