Note: Descriptions are shown in the official language in which they were submitted.
CA 02255548 1998-11-14
ASSAY METHODS AND COMPOSITIONS USEFUL FOR MEASURING
RECEPTOR LIGAND BINDING
Field of the Invention
This invention is in the field of molecular biology, particularly as applied
to
pharmaceuticals. More particularly, the invention relates to methods and
compositions for detecting receptor ligand binding and to their use in drug
discovery.
Background of the Invention
A wide variety of cell surface receptors have been implicated in disease
processes, and
are accordingly targets of drug discovery programs, which seek to identify
ligands for
these receptors. To facilitate the disovery of such ligands, cells that
express these
receptors and report the presence of bound ligands have been developed; for
instance,
assays which assess ligand binding by monitoring alteration in the level of a
second
messenger, such as cAMP, cGMP or inositoltriphosphate (IP3) are established
and
have been automated, allowing them to be used in high-throughput and ultra
high-
throughput screening of chemical libraries for ligands. In one approach, the
effect of
ligand binding at the receptor is revealed by a reporter gene product whose
expression
is driven by second messengers stimulated upon ligand binding at the receptor.
Typically, the reporter gene codes for a readily detectable protein product,
for
example, CAT or luciferase (see for example US 5,436,128 and US 5,401,629). A
related system has been developed in which ligand binding at a target receptor
is
reported by the formation of a pigment protein (see US 5,462,856). Although
expression of a reporter gene can be stimulated rapidly in systems of this
type,
detection of that response is delayed until the expression product of the
reporter gene
is formed.
It is an object of the present invention to provide an alternative system to
identify
receptor ligands rapidly and efficiently.
CA 02255548 1998-11-14
Summarx of the Invention
To assess the receptor binding properties of chemical compounds, there is
provided
by the present invention a cell-based system in which binding of a ligand to
the
receptor target is reported rapidly and conveniently by assessing ion flux
across the
cell membrane. This is achieved using cells that produce both the target
receptor and
an ion channel protein, as well as a second messenger system that allows the
flow of
ions through the channel protein to be gated in response to a ligand binding
event at
the target receptor. Detection of altered ion channel activity, particularly
altered ion
flow, thereby reports the presence of a target receptor ligand, in the rapid
manner
useful for accelerated drug discovery.
In one aspect, the invention therefore provides a method for identifying
receptor
ligands, which comprises the steps of obtaining a cell useful to screen for
receptor
ligands, the cell expressing a receptor target and an ion channel wherein
gating of the
ion channel is influenced by the level of a second messenger, incubating a
candidate
ligand under receptor binding conditions, such that binding of a ligand to the
receptor
results in an alteration in that second messenger, and then determining if a
change in
the activity of the ion channel has occurred. In embodiments of the invention,
the
method is adapted to allow for the identification of ligands that are agonists
at the
receptor target, and ligands that are antagonists at the receptor target.
In a related aspect, the invention provides a cell that is genetically adapted
to produce
a receptor target and an ion channel, at least one and preferably both of
which is
encoded by a heterologous nucleic acid, wherein binding of a ligand to the
receptor
protein alters the intracellular concentration of a secondary messenger, this
alteration
modulating ion flow across the ion channel when a ligand is bound by the
receptor
target.
In preferred aspects, the ion channel is a cyclic nucleotide gated channel. In
further
preferred aspects, the receptor target is a G-protein coupled receptor target.
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CA 02255548 1998-11-14
In a related further aspect, the invention provides a method for screening
chemical
compounds in a multiplexed fashion to identify G-protein coupled receptor
ligands,
comprising the steps of:
(a) obtaining a mixed culture of cells in which each cell is adapted
genetically to
produce (i) a G-protein coupled receptor target, (ii) a cyclic nucleotide
gated
ion channel protein, and (iii) a second messenger system through which ion
flow through cyclic nucleotide gated channel is modulated in response to
ligand interaction with the G-protein coupled receptor target; wherein said
mixed culture of cells includes a first cell type that produces a first type
of G-
protein coupled receptor and a second cell type that produces a second type of
G-protein coupled receptor different from said first type of G-protein coupled
receptor;
(b) incubating the mixed culture of cells with at least one G-protein coupled
receptor ligand candidate; and
(c) determining the effect of the receptor ligand candidate on ion channel
activity, preferably by determining ion flux through the channel.
In a further aspect, the present invention provides, for use in the
multiplexed method
of the present invention, a mixed culture of cells in which each cell is
adapted
genetically to produce (i) a G-protein coupled receptor target, (ii) a cyclic
nucleotide
gated ion channel protein, and (iii) a second messenger system through which
ion
flow through cyclic nucleotide gated channel is modulated in response to
ligand
interaction with the G-protein coupled receptor target; wherein said mixed
culture of
cells includes a first cell type as defined above, and a second cell type that
produces a
species of G-protein coupled receptor different from the G-protein coupled
receptor
species produced by the first cell type.
In another of its aspects, cells of the invention are utilitized in a ligand
screening
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CA 02255548 1998-11-14
10
protocol that expands the amount of information available from the screen.
Particularly,
the screening method comprises the steps of
obtaining a cell of the present invention that produces a target receptor and
elicits a
reference response to a reference agonist,
forming a first mixture by incubating said cell with a candidate ligand under
conditions
capable of generating a detectable response;
detecting a first response from the first mixture;
supplementing the first mixture with said reference agonist, to form a second
mixture;
detecting a second response from the second mixture; and then
identifying the candidate ligand either (1) as a candidate agonist of said
receptor, in the
case where a first response is detected, or (2) as a candidate antagonist in
the case
where the second response signal is weaker than the reference response, or (3)
as a
candidate potentiator of said receptor in the case where the second response
is stronger
than the reference response.
Embodiments of the invention are described in greater detail with reference to
the
accompanying drawings in which:
Brief Reference to the Drawines:
Figure 1 illustrates the fluorescence response of CNG channel-producing cells
(filled
shapes) and control cells (open shapes) to incubation with either a cyclic
nucleotide
(8Br-cGMP) or forskolin;
Figure 2 illustrates the effect on calcium influx of increasing concentrations
of the
cyclic nucleotides 8Br-cGMP (panel A) and 8Br-cAMP(panel B) as well as
forskolin
(panel C) in CNG channel producing cells of Figure 1;
Figure 3 illustrates the specific fluorescence response of cells stably
producing both
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CA 02255548 1998-11-14
CNG channel and SHT6 receptor, to SHT6 receptor selective agonists (5-HT, 5-CT
and 5-MeOT, panel A) and to SHT6 receptor selective antagonists (clozapine and
methiothepin, panel B);
Figure 4 illustrates the specific fluorescence response of cells stably
producing both
CNG channel and D 1 receptor, to D 1 receptor agonists (dopamine, ADTN, and
SKF38393, panel A) and to D1 receptor antagonists (flupentixol and SCH23390,
panel B);
Figure 5 illustrates specific fluorescence response of cells stably producing
both CNG
channel and D 1 receptor, to D 1 receptor agonists as a control (panel A) and
to D 1
receptor agonists when incubated 5 minutes after treatment with D 1 receptor
antagonist (panel B);
Figure 6 illustrates the specific fluorescence responses of a mixed culture
containing
two cell lines producing either CNG channel loaded with Fura-Red (panels A and
B)
or CNG channel and D1 receptor loaded with Fluo-3 (panels C and D) Response of
cells loaded with Fluo-3 was measured as an increase in fluorescence, while
response
of cells loaded with Fura Red was measured as a decrease in fluorescence;
Figure 7 illustrates the specific fluorescence responses to reference
compounds,
including D 1 and SHT6 receptor reference agonists, of a mixed culture
containing
two cell lines producing either CNG channel and SHT6 receptor loaded with Fura
Red, or CNG channel and D1 receptor loaded with Fluo-3; and
Figure 8 illustrates the identification of D 1 and SHT6 reference antagonists
in a
mixed culture containing two cell lines producing either CNG channel and SHT6
receptor or CNG channel and D1 receptor loaded with Fura Red and Fluo-3
respectively.
Figures 9 and 10 provide illustrative results that can be obtained when the
"tandem"
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CA 02255548 1998-11-14
screening protocol is performed, as described in more detail in Example 6.
Detailed Descr~tion of the Invention
According to the present invention, there are created and exploited cells that
produce
both a receptor target and an ion channel, and which further have a second
messenger
system through which binding activity at the receptor target results in
detectable
activity at the ion channel. The invention is applicable to cells expressing a
wide
variety of receptor targets, and a wide variety of ion channels.
The term "receptor target" is used herein with reference to protein molecules
that
occur on the surface of cells which interact with the extracellular
environment, and
transmit or transduce that external information in a manner that ultimately
modulates
the intracellular environment. Cell surface-localized receptors are membrane
spanning proteins that bind extracellular signalling molecules and transmit
the signal
via signal transduction pathways to effect a cellular response. Cell surface
receptors
bind circulating signal molecules, such as growth factors and hormones, as the
initiating step in the induction of numerous second messenger pathways.
Receptors
are classified on the basis of the particular type of pathway that is induced.
Included
among these classes of receptors are those that bind growth factors and have
intrinsic
tyrosine kinase activity, such as the heparin binding growth factor (HBGF)
receptors,
and those that couple to effector proteins through guanine nucleotide binding
regulatory proteins, which are referred to as G protein coupled receptors
(GPCR) and
G proteins, respectively.
The term " ion channel" refers to membrane spanning proteins that permit
controlled
entry of various ions into cells from the extracellular fluid. They function
as gated
pores in the cell membrane and permit the flow of ions down electrical or
chemical
gradients. Ion channels are classified on the basis of the ion that enters the
cell via
the channel. The modulation of transmembrane ion transport is often the
primary
event in the coupling of extracellular signals to intracellular events. Ion
fluxes play
essential roles in stimulus-mitosis, stimulus-contraction (see, Curran et al.
(1986)
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CA 02255548 1998-11-14
Proc Natl. Acad. Sci. USA 83:8521-8524). For example, the voltage-gating of
calcium ions mediates the coupling of membrane depolarizing stimuli to
transcriptional activation of c-fos gene. Elevation of intracellular calcium
activates a
calmodulin/calmodulin kinase system, which induces c-fos expression.
An ion channel that gates ions in response to cytosolic levels of a second
messenger
triggered by target receptor ligand binding is utilized in the present system.
In a
preferred aspect of the invention, the present system exploits, and the cells
therefore
produce, an ion channel that is gated by a cyclic nucleotide such as cyclic
AMP
(CAMP) and cyclic GMP (cGMP) (for general review see Zagotta, An. Rev.
Neurosci. (1996) 19: 235-263). In their natural environment, cyclic nucleotide-
gated
channels (CNG channels) act as biological signal transducers, converting
sensory input,
such as light and smell, to electrical signals for processing by the central
nervous
system. In the olfactory system, for instance, odorant molecules bind to
receptors in the
olfactory epithelium which are positively linked via a second messenger
system, i.e. are
linked positively via a G-protein to adenylyl cyclase (AC). Stimulation of AC
leads to
increases in intracellular levels of the cAMP, which directly binds to and
activates CNG
channels located in the plasma epithelium. These CNG channels are canon
nonselective
channels, fluxing both monovalent canons such as Na+, and divalent cations
including
Caz+ and Mg2+ the opening of which leads to cellular depolarization and
ultimately
neurotransmitter release.
Suitable for use in the present system are any of the CNG channels that have
been
identified in a variety of tissues (for a review, see Biel et al, Trends
Cardiovase Med,
6(8):274, 1996. In one embodiment of the invention, the CNG channel is a
retinal CNG
channel. The retinal CNG channels typically are much more sensitive to
activation by
cGMP than cAMP. Specific retinal CNG channels useful in the present system
include
the cloned human retinal CNG described by Dhallan et al. (1992), J. Neruo
Sci., 112:
3248-3256, and species homologs thereof.
In other, more preferred embodiments of the invention, the CNG channel is an
olfactory
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CA 02255548 1998-11-14
CNG channel. The olfactory CNG channels are activated with high potency and
efficacy by both cAMP and cGMP and are significantly more permeable to Ca2+
than
are retinal CNG channels. Suitable for use in the present system are the
olfactory CNG
channels cloned from a number of species including cow (Lydwig et al. (1990),
FEBS,
270: 24-29), rat (Dhallan et al. (1990). Nature, 347: 184-187), catfish
(Goulding et al.
(1992), Neuron, 8: 45-58) and mouse (Ruiz et al. (1996), J. Mol. Cell.
Cardiol., 28:
1453-1461 ) .
The CNG channels are formed structurally as heterodimers of a.- and (3-
subunits
(Dhallan et al. (1990), Nature, 347: 184-187). The a-subunit, when exogenously
expressed in Xenopus oocytes or a human embryonic kidney cell line (HEK293),
forms
functional CNG channels, while the (3-subunit does not (Bradley et al. (1994),
PNAS,
91: 8890-8894). However, co-expression of the a- and (3-subunits results in
formation
of heteromeric CNG channels with ion permeability, pharmacology and cyclic
nucleotide selectivity different from the homomeric channel formed by the a-
subunit
alone, and more similar to the properties seen in the native channel (Bradley
et al.
(1994), PNAS, 91: 8890-8894).
Embodiments of the present invention therefore embrace CNG channels that are
homomeric CNG channels consisting only of functional alpha subunits, as well
as
heterodimeric CNG channels that incorporate both alpha and beta subunits.
In further embodiments of the invention, the CNG channel is one that retains
the
characteristic function of gating ion in response cyclic nucleotide binding,
but is altered
structurally to modify such properties as ion permeability and cyclic
nucleotide binding
affinity. Mutation studies have identified the molecular basis of ion
selectivity, including
Ca2+ permeability, cyclic nucleotide selectivity, and modulation of channel
function by
Ca+/calmodulin and transition metal divalent canons. Thus, much is known about
the
molecular basis of channel properties which can be exploited in the
optimization of these
proteins for specific purposes. For example, Warnum et al. (1995) Neuron,
15(3):
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CA 02255548 1998-11-14
619-625, have demonstrated that the cyclic nucleotide selectivity was
significantly
altered by the substitution of a nonpolar residue for an aspartic acid residue
in the
cyclic nucleotide binding domain. Still other such modifications to CNG
channels are
received by Biel et al, supra. Thus, the ability of a cyclic nucleotide to
gate the ion
channel can be manipulated while retaining functional properties useful in the
present
system.
In its preferred form, the present assay system exploits cells that produce
ion channels
that regulate ion flow in response to altered cytosolic levels of a cyclic
nucleotide,
such as cAMP and cGMP. These CNG channels are capable of reporting activity at
any receptor target that is coupled to a second messenger system that
modulates
cytosolic levels of a cyclic nucleotide. Receptor targets that are naturally
coupled to
such a second messenger system are the G-protein coupled receptors, and the
present
system is accordingly well suited for identifying ligands of G-protein coupled
receptors.
Thus, the present invention provides, in a preferred aspect, a method for
screening
chemical compounds to identify ligands for G-protein coupled receptors,
comprising
the steps of:
(a) obtaining a culture of cells that produce (i) a G-protein coupled receptor
target,
(ii) a cyclic nucleotide gated ion channel, and (iii) a second messenger
system through
which ion flow through cyclic nucleotide gated channel is modulated in
response to
ligand interaction with the G-protein coupled receptor target;
(b) incubating the culture of cells with a chemical compound that is a target
G-protein
coupled receptor ligand candidate; and
(c) determining the effect of the receptor ligand candidate on ion channel
activity.
The term "G-protein coupled receptor" or "GPCR" refers to a diverse class of
9
CA 02255548 1998-11-14
receptors that mediate signal transduction via an intracellular second
messenger
system involving binding to G proteins. Briefly, in this second messenger
system,
signal transduction is initiated via ligand binding to the GPCR, which
stimulates
binding of the receptor to the G protein. Interaction between the receptor and
G
protein releases GDP, which is specifically bound to the G protein, and
permits the
binding of GTP, which activates the G protein. Activated G protein dissociates
from
the receptor and, depending on the type of G protein, activates or deactivates
an
effector protein such as adenyl cyclase or guanyl cyclase. Thus, in the second
messenger system associated with GPCRs, the effector protein regulates the
intracellular levels of specific intracellular messengers (secondary
messengers)
including cyclic nucleotide such as cAMP and cGMP, as well as
inositoltriphosphate
(IP3) and diacyl glycerol (DAG).
GPCRs, which are glycoproteins, are known to share certain structural
similarities
and homologies (see, e.g., Gilman, A.G., Ann. Rev. Biochem. 56: 615-649
(1987),
Strader, C.D. et al. FASEB Journal 3: 1825-1832 (1989), Kobilka, B.K., et al.
Nature 329:75-79 (1985) and Young et al. Cell 45: 711-719 (1986)). Among the G
protein-coupled receptors that have been identified and cloned are the
muscarinic
receptors (Hulme et al. (1990), Annu. Rev. Pharmacol. Toxicol., 30: 633-673),
the
adenosine receptors (Olah et al. (1995), Annu. Rev. Pharmacol. Toxicol., 35:
581-
606), the adrenergic receptors (Hieble et al. (1995), J. Med. Chem., 38: 3415-
3444),
the lysophosphatidic acid receptor (Thompson et al. (1996), Mol. Pharmacol.,
45:
718-723), the NPY receptors (Wan et al. (1995), Life Sci., 56: 1055-1064) and
the
dopamine receptors (Strange et al. (1996), Adv. Drug Res., 28: 313-351). GPCRs
share a conserved structural motif. The general and common structural features
of
the G protein-coupled receptors are the existence of seven hydrophobic
stretches of
about 20-25 amino acids each surrounded by eight hydrophilic regions of
variable
length. It has been postulated that each of the seven hydrophobic regions
forms a
membrane-spanning alpha helix and the intervening hydrophilic regions form
alternately intracellularly and extracellularly exposed loops. The third
cytosolic loop
formed between the transmembrane domains is known to be principally
responsible
CA 02255548 1998-11-14
for the selective interaction with G proteins.
The variety of GPCRs for which ligands can be identified in accordance with
the
invention is extensive, and includes receptors for the following ligands:
adenosine,
cannabinoid, melanocortin, adrenergic, dopamine, serotonin, histamine,
muscarinic,
bombesin/neuromedin, cholecystokinin, gastrin, tachykinin, opsin, bradykinin,
angiotensin, chemokine, angiotensin, endothelin, neuropeptide Y, calcitonin,
corticotropin releasing factor, CSa, C3a, fMLP, opsin, eicosanoid, FSH,
galanin,
leukotriene, opioid, oxytocin, PAF, vasopressin, glucagon, GLP-1, GLP-2, GIP,
PACAP, VIP, secretin, vasotocin, melatonin, latrotoxin, metabotropic glutamate
and
GABA-B receptors, and pheromones. The method of the present invention can
further
be exploited to identify ligands for GPCR receptor targets that are so-called
"orphan"
receptors. Particularly, many GPCRs have been cloned which show insufficient
homology to previously characterized GPCRs to readily predict their endogenous
ligand.
These novel putative receptors represent a large pool of potential therapeutic
targets for
novel drug discovery.
Furthermore, the method of the present invention can be exploited to identify
receptors that are targets for known ligands for which the receptor is unknown
or has
yet to be identified and cloned. Particularly, cells are constructed in which
DNA
coding for the putative receptor target is incorporated expressibly, in the
manner
exemplified herein, and the present assay system is thereafter utilized to
identify the
receptor-encoding clone that modulates ion channel activity when incubated
with the
known ligand.
For purposes of discovery drugs to treat human medical conditions, it is
herein
preferred that the GPCR targets are human GPCRs.
In embodiments of the invention, the present system is exploited to identify
ligands of
positively coupled GPCRs, in which the G protein is of the Gs type. Examples
of
positively coupled GPCRs are numerous and include the GLP-2 receptor, the SHT6
receptor, and the the D 1 sub-family of dopamine receptors including D 1 and
D5. In
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CA 02255548 1998-11-14
the second messenger system coupled to these receptors, receptor stimulation,
such as
would be caused by binding of an agonist ligand, results ultimately in an
upregulation
of cyclic nucleotide. Ligands having agonist activity at these Gs protein
coupled
receptors will accordingly trigger a readily detectable influx of ion through
the CNG
channel.
The present system can be exploited also to identify ligands at positively
coupled
GPCRs that function as antagonists. This can be achieved, in accordance with
embodiments of the invention, by introducing the candidate antagonist either
together
with, or more preferably prior to introduction of a reference agonist for the
receptor,
and then determining whether the antagonist candidate depresses the effect of
the
reference agonist on ion flux. Reference agonists for a given receptor can be
identified using the procedure just described above.
In other embodiments of the invention, the present system is exploited to
identify
ligands for negatively coupled GPCRs, in which the G protein is of the Gi type
or the
Go type. Examples of negatively coupled GPCRs are numerous and include the edg
receptors, the NPY receptors, members of the D2 subfamily of dopamine
receptors
including D2, D3 and D4, and the 5-HT1 subfamily of 5-HT receptors, including
the
5-HTld receptor. In the second messenger system coupled to these receptors,
receptor/binding does not stimulate cyclic nucleotide production, and hence
does not
cause ion influx through the channel. Ligand binding events at these
negatively
coupled GPCRs can nevertheless be determined on the basis of ion flux as
described
below.
In embodiments of the invention, the present system is exploited to identify
ligands
that are agonists at negatively coupled GPCRs. This is achieved by treating
the cell
to upregulate cyclic nucleotide and thereby stimulate influx of ion through
the CNG
channel. Such stimulation can be achieved by treating the cell to upregulate
cyclic
nucleotide artificially and thereby stimulate the influx of ion. Such
upregulation can
be achieved using effector protein activators. If the effector protein is
adenylate
cyclase a suitable activator is forskolin (Seamon et al. (1981), J. Cyclic
Nucleotide
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CA 02255548 1998-11-14
Res., 7(4): 201-224). In stimulating ion flux this way, ligand candidates can
then be
identified as agonists by their ability to diminish such ion flow relative to
activator
alone, either when incubated with the activator, or when introduced before the
activator.
In other embodiments of the invention, the present system is exploited to
identify
ligands that are antagonists at negatively coupled GPCRs. This can be
achieved, in
accordance with embodiments of the invention, by first incubating cells either
first
with antagonist and then with agonist, or with a combination of antagonist and
agonist, and then treating the cell with an effector protein activator, such
as forskolin,
to stimulate ion flux into the cell. Such reference agonists at negatively
coupled
GPCRs can be identified as just described. The ligand candidate can then be
identified as an antagonist by its ability to modulate the known effect of the
effector
protein activator on ion flow; particularly, an antagonist will counter the
agonist-
mediated decrease in ion flux stimulated by the effector protein activator.
It will be appreciated that the present system can most conveniently be
applied to
identify ligands for GPCRs that are positively coupled, for the reason that
such
ligands can be identified directly by the effect they have on opening
(agonists) or
inhibiting agonist-mediated activation of (antagonists) the CNG channel. To
exploit
this convenience, there is further provided in embodiments of the present
invention, a
system in which GPCRs that are not positively coupled are rendered positively
coupled using recombinant DNA technology. The GPCRs of this type are referred
to
herein as chimeric GPCRs. These chimeric GPCRs have the structural features of
non-positively coupled GPCRs, and the ligand binding properties thereof, but
feature
a third transmembrane loop that has been altered to reproduce a third
transmembrane
loop of a Gs-type GPCR. This has the effect of coupling the otherwise
negatively
coupled GPCR to a Gs-type second messenger system, thereby permitting ligand
binding at the chimeric GPCR to be determined with the convenience available
for Gs
protein coupled receptor ligands.
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CA 02255548 1998-11-14
In other embodiments of the invention, the positive coupling assay format can
be
retained for GPCRs that are not positively coupled, i.e., for those GPCRs
coupled to
Gi, Gq, or G11, by genetically altering the cell to produce a chimeric G-
protein in the
manner described in W098/16557, the disclosure of which is incorporated herein
by
reference. Briefly, the properties of positive coupling intrinsic to Gs
proteins is
conferred by all but about the first 5-30 N-terminal amino acids of the G
protein.
This N terminal region of the G-protein constitutes the GPCR binding domain,
which
can be replaced in the Gs protein by an N terminal domain capable of binding
to the
non-positively coupled GPCR target selected for screening. The result is a G
protein
that will bind a negatively coupled GPCR, but will transduce the signal as
would a
positively coupled GPCR and thereby allow stimulation at that negatively
coupled
receptor to be reported more conveniently by the influx of ion at the CNG
channel.
In still other embodiments of the invention, the present system can be
exploited using
cells that produce a G protein known as the promiscuous G protein, or G 16.
This G
protein is capable of mediating an upregulation of cyclic nucleotide, and
hence an
opening of the CNG channel, regardless of whether the GPCR target is
ordinarily
coupled positively to the Gs protein, or negatively to the Gi protein. The
cloning and
incorporation of the promiscuous G protein into cells is described in
W097/48820,
the contents of which are incorporated herein by reference.
Thus, GPCRs can be classified as being positively or negatively coupled to the
effector protein. Agonist binding to a positively coupled GPCR will result in
an
increase in the intracellular level of the secondary messenger produced by
effector
protein, whereas agonist binding to negatively coupled GPCR will result in a
decrease
in the level of a secondary messenger. Examples of effector proteins include
adenyl
cyclase, guanyl cyclase and phospholipase C.
To identify target receptor ligands in accordance with the invention,
approaches
established in related arts can readily be applied. In particular,
accumulation of
cytosolic canon can be revealed using commercially available dyes that bind
the target
cation to yield a detectable result, for example either an observable change,
for instance
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CA 02255548 1998-11-14
in colour, or an altered wavelength detectable by spectrophotometry, such as
fluorescence detectable by a fluorimeter. In a particularly preferred
embodiment of the
invention, the ligand binding event is revealed by detection of the calcium
ion, and dyes
that fluoresce upon chelating calcium are exploited to report that detection.
To prepare
cells for screening in this fashion, cells are pre-loaded with the dye in an
established
manner, so that colour formation results following subsequent binding, in the
screen, of
a target receptor ligand. Suitable calcium detection dyes include Fura-2, Fluo-
3, Fura-
Red, Bapta-AM, Quin-2, Calcium Green, and the protein, apoaequorin. Detection
of
such dyes can be achieved using a fluorescence detector, such as a Fluoroskan.
As an alternative to measuring ion flux, CNG channel activity can be
determined
indirectly by measuring consequences of ion flow, such as by measuring release
of
acetylcholine from intracellular stores.
When, in accordance with preferred embodiments, the applied screening protocol
relies on the formation of a cyclic nucleotide such as cAMP, it is desirable
to load
cells with a cyclic nucleotide phosphodiesterase inhibitor before incubations
are
commenced. Such agents prevent the formed cyclic nucleotide from being
recycled
to precursor products in the cytosol, and can therefore sustain the calcium
influx
response under detection. Examples of such inhibitors include 3-isobutyl-1-
methylxanthine (IBMX) and 4-[3-(cyclopentyloxy)-4-methoxyphyenyl]-2-
pyrrolidinone sold as Rolipram, which prevent reversion of cAMP, and the
calcium/calmodulin-dependent phosphodiesterase inhibitor, vinpocetin.
For use in the present system, the present invention further provides cells
that are
genetically adapted so that activity at a receptor target is coupled to
activity at an ion
channel, so that altered ion channel activity reports a ligand binding event
at the
receptor. The cells of the present invention are therefore characterized by
the
production of the receptor target, the ion channel and a second messenger
system that
transduces a binding event at the receptor into a detectable activity at the
channel.
CA 02255548 1998-11-14
As used herein the term "genetically adapted" is used with reference to a cell
which
has been modified by the intervention of man such that the expression of one
or more
endogenous genes of a host cell has been altered to establish a pattern suited
to
assessing ion flow, or an intracellular event influenced by ion flow, on
ligand binding
to a receptor target. This can be achieved, for instance, simply by activating
expression of one or more of an endogenous ion channel or a G-protein coupled
receptor, by intervention at the genomic level, as disclosed in WO 9412650
which is
incorporated herein by reference. Briefly, homologous recombination or
targeting
can be used to replace or disable the regulatory region normally associated
with the
selected gene, which results in a pattern of regulation different from that of
the parent
cell. Alternatively, the level of expression of one or more of a receptor
target, an ion
channel protein or a second messenger system element, such as a G protein, can
be
increased by transiently or stably transfecting a cell with heterologous
nucleic acid
expressing these proteins.
As used herein, "heterologous DNA" includes DNA that does not occur naturally
as
part of the genome in which it is present, and DNA found in a location or
locations in
the genome that differs from that in which it occurs in nature. Heterologous
DNA is
not endogenous to the cell into which it is introduced, but has been obtained
from
another cell or synthesized de novo, and introduced exogenously. Generally,
although not necessarily, such DNA encodes proteins that are not normally
produced
by the recipient cell in which that DNA is expressed.
In a preferred aspect, the present invention thus further provides a cell, or
culture
thereof, that has been genetically adapted to produce (i) a G-protein coupled
receptor
target, (ii) a cyclic nucleotide gated ion channel protein, and (iii) a second
messenger
system through which ion channel activity is modulated in response to ligand
interaction with the G-protein coupled receptor target.
In embodiments of the invention, the GPCR is a Gs-coupled GPCR or a GPCR that
is
other than a Gs-coupled GPCR but has been converted to a Gs-coupled GPCR by
16
CA 02255548 1998-11-14
engineering of its G-protein binding site to a Gs-coupled GPCR; the CNG
channel
protein is an olfactory CNG protein, and the second messenger system
incorporates a
Gs protein or a promiscuous G protein. In still more specific embodiments, the
cell
expresses at least one of such proteins from heterologous DNA coding therefor.
Suitably, at least two of such proteins are expressed from heterologous DNA;
for
instance the GPCR and the CNG channel. Alternatively, the cell can produce all
three such proteins from heterologous DNA.
To provide such cells, in accordance with one embodiment of the invention, a
host
cell that naturally (endogenously) expresses a CNG channel, such as an
olfactory or
retinal host cell, is pre-screened to establish if it expresses a GPCR target.
If it is
established that the CNG channel-expressing cell naturally produces the GPCR
target,
and the endogenous second messenger system is functional, such a cell can be
used to
screen for receptor ligands using the method of the invention. The CNG channel-
producing cell can be assessed for expression of a GPCR target using methods
common in the art, for example, using a competition based assay using labelled
and
non-labelled GPCR reference ligands. If the CNG channel-producing host cell
does
not produce a GPCR target, heterologous DNA coding for the GPCR target can be
introduced therein using standard techniques of molecular biology.
Alternatively, a cell that naturally expresses the GPCR target can be
transformed with
heterologous DNA encoding a CNG channel, for use in the present system.
In specific embodiments of the invention, a host cell is transformed with
heterologous
DNA coding for each of the CNG channel and the GPCR target.
Host cells useful in the present system include the various eukaryotic cells
such as
yeast, Aspergillus, insect, Xenopus, avian and particularly mammalian cells.
Suitable
cells include Chinese hamster ovary (CHO) cells for example of K1 lineage
(ATCC
CCL 61) including the Pros variant (ATCC CRL 1281); the fibroblast-like cells
derived
from SV40-transformed African Green monkey kidney of the CV-1 lineage (ATCC
17
CA 02255548 1998-11-14
CCL 70), marine L-cells, marine 3T3 cells (ATCC CRL 1658), marine C127 cells,
human embryonic kidney cells of the 293 lineage (ATCC CRL 1573), human
carcinoma
cells including those of the HeLa lineage (ATCC CCL 2), and neuroblastoma
cells of
the lines IMR-32 (ATCC CCL 127), SK-N-MC (ATCC HTB 10) and SK-N-SH (ATCC
HTB 11). In specific embodiments, the host cell is a HEK 293 cell.
A variety of gene expression systems have been adapted for use with these
hosts and are
now commercially available, and any one of these systems can be selected to
drive
expression of the receptor-encoding DNA. Expression vectors may be selected to
provide transformed cell lines that express the receptor-encoding DNA either
transiently
or, more desirably, in a stable manner. For transient expression, host cells
are typically
transformed with an expression vector harbouring an origin of replication
functional in a
mammalian cell. For stable expression, such replication origins are
unnecessary, but the
vectors will typically harbour a gene coding for a product that confers on the
transformants a survival advantage, to enable their selection such as a gene
coding for
neomycin resistance in which case the transformants are plated in medium
supplemented
with neomycin.
These systems, available typically in the form of plasmid vectors, incorporate
expression
cassettes the functional components of which include DNA constituting
expression
controlling sequences, which are host-recognized and enable expression of the
receptor-
encoding DNA when linked 5' thereof. The systems further incorporate DNA
sequences that terminate expression when linked 3' of the receptor-encoding
region.
Thus, for expression in the selected mammalian cell host, there is generated a
recombinant DNA expression construct in which the receptor-encoding DNA is
linked
with expression controlling DNA sequences recognized by the host, and which
include a
region 5' of the receptor-encoding DNA to drive expression, and a 3' region to
terminate expression.
Included among the various recombinant DNA expression systems that can be used
to
achieve mammalian cell expression of the receptor-encoding DNA are those that
exploit
18
CA 02255548 1998-11-14
promoters of viruses that infect mammalian cells, such as the promoter from
the
cytomegalovirus (CMV), the Rous sarcoma virus (RSV), simian virus (SV40),
murine
mammary tumour virus (MMTV) and others. Also useful to drive expression are
promoters such as the LTR of retroviruses, insect cell promoters such as those
regulated
by temperature, and isolated from Drosophila, as well as mammalian gene
promoters
such as those regulated by heavy metals i.e. the metalothionein gene promoter,
and
other steroid-inducible promoters.
Through the use of the present system, any of the commercially available
chemical
libraries, including small molecules and peptides, may be usefully screened.
In order
to facilitate screening of a large chemical compound bank, the method of the
invention may be automated, thereby allowing high-throughput screening of
compounds, for example, according to the method disclosed in U.S. patent
5,589,351, which is incorporated herein by reference. Briefly, the method
entails the
use of mufti-well plates containing cells according to the invention which are
loaded
with a dye which fluoresces when a metal, e.g., Ca2+, ion is bound. A robotic
device
that can detect fluorescence, such as a Flouroskan, can be used to rapidly
process
such plates. Using this approach, chemical banks comprising hundreds of
thousands
of compounds can be rapidly screened for receptor agonists.
In accordance with another aspect of the invention, the present system is
exploited in
a "multiplexed" fashion, which accelerates the rate at which a chemical
library can
be screened for receptor ligands. This is achieved by exposing the ligand
candidate
simultaneously to a mixed culture of channel-producing cells, in which a first
cell line
produces a first receptor target and a second cell line produces a different,
second
receptor target. So that ion influx can be detected in a way that
discriminates
between cell types, reporter systems that are unique to the cell type are
used. In an
embodiment of the invention, this discrimination is achieved by loading the
different
cell types with different ion chelating dyes that serve as " signature" dyes
for a
particular cell type. For dyes that chelate calcium, for instance, specific
embodiments of the invention include the use of dyes having a fluorescent
signal that
19
CA 02255548 1998-11-14
increases with influx of calcium, such as Fluo-3, and dyes that have a
fluorescent
signal that decreases with influx of calcium, such as Fura Red. Detection of
fluorescence specific to cells loaded with a particular dye can then be
achieved using
filters specific for the wavelength emitted by the given dye.
As in the method described above for single-type cell assays, the multiplexed
assays
are capable of detecting ligand binding at GPCRs that are either positively
coupled or
negatively coupled, and can be used to distinguish between agonists and
antagonists at
a particular GPCR species. Specific embodiments which demonstrate this ability
are
provided in the examples herein.
For use in the multiplexed format of the present system, there is provided, in
another
aspect of the invention, a mixed culture of cells in which each cell is
adapted
genetically to produce (i) a G-protein coupled receptor target, (ii) a cyclic
nucleotide
gated ion channel protein, and (iii) a second messenger system through which
ion
flow through cyclic nucleotide gated channel is modulated in response to
ligand
interaction with the G-protein coupled receptor target; wherein the mixed
culture of
cells includes a first cell type that produces a first type of G-protein
coupled receptor,
and a second cell type that produces a second type of G-protein coupled
receptor
different from said first type of G-protein coupled receptor.
It can be convenient to format the assay by culturing one of the cell lines in
the mixed
culture as an adherent culture, and the other as a suspension culture. In the
alternative, both cultures can be suspension cultures.
With the multiplexed assay format, it will be appreciated that the binding
affinity and
functional properties of a chemical compound for two or more receptors can
simultaneously be determined from a single incubation. This will be helpful
not only
to identify ligands for a particular receptor target, but also to profile
quickly the other
receptor binding properties, and liabilities, of the screened compound. It is
also
within the scope of the present invention to screen two or more chemical
compounds
CA 02255548 1998-11-14
simultaneously, i.e., in a single incubation, with two or more receptor types
to yield
even more information from the present system. Once identified, the target
receptor
ligand can then of course be selected for subsequent drug development.
In another aspect of the invention, the cells are utilized in a modified
screening
protocol, referred to as a "tandem" screening protocol, which incorporates a
step that
utilizes a reference agonist for the target receptor produced by the cells.
The
reference agonist, and the reference response that it generates against the
target
receptor, are utilized to derive additional information from the screening. To
do
this, the screen is run as described above, in which a reaction mixture is
formed by
incubating the cell having the target receptor with a ligand candidate and
then taking a
a reading. In the tandem protocol, a reference agonist is then added to the
reaction
mixture, and the effect of the reference agonist on the original signal is
evaluated. In
the case where the candidate signal is an agonist, no significant difference
in signal
will result from addition of the reference agonist. If the candidate ligand is
an
antagonist, addition of the reference agonist will result in a signal that was
not
originally detected. Similarly, if the candidate ligand is a receptor
modulator, no
signal will be detected initially, but addition of the reference agonist will
result in a
detectable signal that will be greater than normally elicited by the reference
agonist
alone. The tandem protocol, utilizing reference ligands for a particular
receptor, thus
greatly expands the information available from a given incubation.
Published examples of these receptors are provided below, together with their
suitable,
corresponding "reference agonists" [in square brackets] which in many cases
are
simply the endogenous receptor ligand and in other cases are more potent,
synthetic
analogs of the endogenous ligand:
For GPCR receptor targets, reference agonists include natural ligands for
those
receptors, as well as synthetic compounds known to have ligand properties.
Examples
of reference agonists include: for cannabinoid receptors, CBS [methanandamide]
and
CB2 [1-propyl-2-methyl-3-(1-naphthoyl)indole; for cholecystokinin and gastrin
receptors, CCKA [Boc-Trp-Lys(O-Me-Phe-NH)-Asp-(NMe)Phe-NH2] and CCKB
21
CA 02255548 1998-11-14
[gastrin]; for dopamine receptors, D1 [dihydrexedine], D2 [bromocriptine] and
D3 [7-OH-DPAT]; for serotonin receptors, 5-HT1A [8-OH-DPAT]; 5-HT1B and 5-
HT1D [sumatriptan]; 5-HT2A and 5-HT2B and 5-HT2C [a-Me-5-HT]; 5-HT3 [2-Me-
5-HT]; for neuropeptide Y receptors, Y1 [Leu3l,Pro34-NPY], Y2 [NPY(13-36)], Y4
[pancreatic polypeptide], and YS [PYY(3-36)]; for opioid receptors, mu
[sufentanil],
delta [DAla2-deltorphin I], and ORL1 [nociceptin]; for tachykinin receptors,
NK1
[substance P methyl ester]; for adrenoreceptors, alpha2A [oxymetazoline],
betal
[noradrenaline], beta2 [procaterol]; for GABA receptors, GABA-A [isoguvacine
and
flunitrazepam], and GABA-B [baclofen]
When used in the method of the present invention, the reference agonists are
most
suitably used in the "cold" form, there being no need for radiolabeling given
the
functional nature of the assay in which they are used. Desirably, the
reference agonist
is utilized in a concentration that is in the range from its EC50 to its EC90
concentrations at the target receptor. Concentrations in this range can
prevent the
reference agonist from saturating the receptor, and will thus allow modulation
of its
agonist activity by the ligand candidate to be revealed during the screening
protocol.
The above are representative GPCR targets, many more of which are published in
the
literature and applicable targets for the present method.
The examples below are provided to illustrate the present system. These
examples
are provided by way of illustration and are not included for the purpose of
limiting
the invention.
EXAMPLE 1 - Construction of a CNG channel-producing cell
A) Construction of a full-length rat CNG a clone using PCR.
The rat CNG a gene was cloned into a mammalian expression vector as follows.
Four synthetic oligonucleotides were made as follows:
22
CA 02255548 1998-11-14
P1 5'- GGCATTCGGATCCAAGCCACCATGATGACCGAAAAATCCAATGGTG-3';
P2 5'-CCAAGGCTCT AGAGTCACTTATGGTTATTCAGCAGCAGTTGG-3';
P3 5'- GAGTTCTTTGACCGCACTGAGACA-3' and
P4 5'- GCATTCCAGTGGATGATGACCAAG-3'.
The two oligonucleotides Pl and P2 incorporated the start codon and stop codon
for the
rat CNGa cDNA, (Dhallan et al. (1990), Nature, 374:184-187) respectively. The
two
oligonucleotides P3 and P4 corresponded to nucleotide sequences within the rat
olfactory CNGa open reading frame. The four primers were used to amplify a
full-
length cDNA encoding the rat CNGa channel using standard RT-PCR procedure.
Briefly, reverse transcription (RT) reactions were performed as follows: 25
pmoles of
random primers for the 5' end, and 25 pmoles of P2 were incubated, in a
solution
containing 10 mM KCI, 50 mM Tris-HCl pH 8.3, 3.0 mM MgCl2 , 0.5 mM of each
deoxyribonucleoside triphosphate, 10 mM DTT, 200 units Superscript II Revserse
Transcriptase (BRL) and 5 pg rat total hippocampus RNA in a total volume of
20,1.
The conditions for the RT reaction were 1 hour at 42°C and 95°C
for 5 minutes.
Aliquots (2ul) of the reverse transcribed RNA samples were used in PCR
reactions
using the Vent Polymerase (New England Biolabs) according to the manufacturers
recommended procedure. Initially two PCR reactions were performed, one
reaction
using oligonucleotide primers P1 and P4 and another reaction using P2 and P3.
These
primers sets amplify two overlapping cDNA fragments which together encode a
full-
length CNGa cDNA. PCR reaction was carried out as follows: 1 minute at
94°C
followed by 30 cycles at 94°C for 30 seconds, 55°C for 30
seconds and 72°C for 2
minutes, and finally 72 C for 2 minutes. An aliquot of the PCR reaction was
electrophoresed on a 1 % agarose gel, appropriate bands excised and purified
using
QIAquick gel extraction kit (Qiagen). To construct a full-length CNGa cDNA the
two
overlapping PCR products were combined and used as template in a PCR reaction
using oligonucleotide primers P1 and P2. The PCR reaction was done as follows:
1
minute at 94°C followed by 30 cycles at 94°C for 30 seconds,
55°C for 1 minute and
72°C for 3 minutes, and finally 72°C for 2 minutes. An aliquot
of the PCR reaction
was electrophoresed on a 1 % agarose gel and the expected 2 kilobase band was
excised
and purified using QIAquick gel extraction kit (Qiagen). The full-length CNGa
cDNA
23
CA 02255548 1998-11-14
was initially cloned into the vector pCR-Blunt (Invitrogen). Subsequently, the
CNGa
cDNA was excised from pCR-Blunt using restriction enzymes BamHI and XbaI and
cloned into the mammalian expression vector pcDNA3.1/Zeo (Invitrogen). The
nucleotide sequences of the CNGa clone was determined using an ABI 377
automated
sequencer. The sequence agreed with that previously published by Dhallan et
al.,
supra, and Genbank Acc. No. X55519.
B) Transient transfection in HEK 293 cells.
For transient transfections, cells were plated at a 5 x 105 cells per 100 mm
plate. The
following day at 60-70 % confluence, the cells were transfected with 2.5 ug
CNGa,
cDNA cloned into pcDNA 3.11 Zeo as above, using the standard lipofectamine
transfection protocol (Gibco BRL, 18324-012). On the second day, the cells
were
plated, in triplicate on 96 well poly-D-Lysine coated plates. On day three,
the cells
were assayed for calcium uptake using a Fluoroskan.
C) Detection of Ca2+ flux in CNG channel-producing cells
A HEK 293 cell line stably expressing the CNGa channel was plated in
triplicates on
poly-D-Lysine coated plates at approximately 1 x105 per well 2 days prior to
day of
assay. On the day of the assay, plates were washed lx with 200 ~.I Ca2+ and
Mg2+ free
buffer (145 mM NaCI, 5 mM KCI, 10 mM glucose, 10 mM HEPES pH 7.2; 300
mOsm) per well. The buffer was removed; cells were pre-loaded with dye by
adding 50
pl per well of Fluo-3-AM, and the plates were incubated for 1 hour at RT in
the dark.
After 1 hour the dye was removed, 200 ~,l of Ca2+ and Mg2+ free buffer was
added per
well and the plates were incubated for a further 30 min at RT in the dark.
Subsequently,
the Ca2+ and Mg2+ free buffer was removed and replaced with 45 ~,1 of Mg2+-
free buffer
containing calcium ion (142 mM NaCI, 5 mM KCI, 2 mM CaCl2, 10 mM glucose, 10
mM HEPES pH 7.2; osmolality = 300 mOsm). A reading was taken using a
Fluoroskan (T=0 time). Compounds studied were made up in a concentrated stock
24
CA 02255548 1998-11-14
using the appropriate solvent, (for example, DMSO for forskolin, dH20 for 8-Br-
cGMP, 8-Br-cAMP and diluted to 4X final concentration and dissolved in 15 p.l
Mg2+
-free buffer), added to the wells and further readings were taken at T=1
minute, T=5
minute etc. using the Fluoroskan.
Results of the study with transient cells are shown in Figure 1 and illustrate
clearly that
calcium ion flux is detected in CNGa-transformed cells (filled shapes) when
treated with
either a cyclic nucleotide (8Br-cGMP) or an adenylate cyclase activator
(forskolin), but
not in mock transfected, similarly treated cells. Figure 2 illustrates results
with cells
stably producing the CNG channel, at increasing concentrations of 8-Br-cGMP
(panel
A), 8-Br-cAMP (panel B) and forskolin (panel C).
EXAMPLE 2 - Incorporation of GPCR into a CNG channel-producing cell
A) Integration of the human SHT6 gene, as GPCR
HEK 293 stable cell line expressing the rat olfactory CNGa channel was
produced
from transiently transfected cells in the manner described in example 1, with
the
following additional steps. On day 3, cells were plated 1:10 and 1:20 in 150
mm
plates in DMEM + 10 % FBS + 400 p,l /ml Zeocin (Invitrogen). Zeocin selection
was maintained for approximately 17 days at which point individual colonies
are
picked and expanded. When sufficient quantities of cells are grown for each
colony,
cells were plated on 96 well poly-D-Lysine coated plates in triplicates.
Approximately 48 hours later the cells were assayed for calcium uptake using a
Flouroskan. Thereafter, in preparation for transfection with a cDNA encoding
the
5HT6 receptor, the cells were plated out a density of approximately 1.8 x106
cell on a
100 mm plate in MEM with 10 % FBS, 1 % glutamine and 400 ug/ml Zeocin
(Invitrogen) and left to attach for 24-48 hours in a 37°C COZ
incubator.
Subsequently, 2 ~,g of a lipofectamine SHT6/cDNA complex was prepared as
follows. The SHT6 receptor gene was cloned using standard PCR, from fetal
brain
cDNA library. The sequence agreed with the published nucleotide sequence
(Genbank L41147 and J. Neurochem. (1996), 66(1): 47-56). The gene was inserted
GJ
CA 02255548 1998-11-14
into the EcoRl/Xbal multiple cloning sites of pcDNA3 (Invitrogen). 2~,g of
pcDNA/SHT6 were diluted in 240,1 serum-free OPTI-MEM. 22,1 lipofectamine was
prepared in 240,1 OPTI-MEM. The 2 solutions mixed gently and incubate at room
temperature for 30-45 minutes. Prior to addition of the DNA/lipofectamine
complex
to a 100 mm plate, cells were washed once with serum-free MEM and 5 ml
serum/antibiotic-free MEM was added.
When cell attachment had taken place 4801 of serum-free OPTI-MEM containing
the
2~,g of 5-HT6 cDNA and 22,1 of lipofectamine was added, dropwise to the plate.
The plate was then gently swirled and left to incubate for 5 hours at
37°C in the COZ
incubator. After 5 hours, the media was removed and fresh MEM with 10 % FBS,
1 % glutamine and 400,ug/ml Zeocin was added. The cells were allowed to
recover
overnight at 37°C.
Cells were then trypsinized after washing with 1XPBS and split into 150 mm
plates at
various dilutions (to ensure well isolated colonies). Cells were allowed to
attach to
the plates for 24 hours. To select for cells containing both the olfactory CNG
channel
and the SHT-6 receptor, the following day the media was changed to MEM
containing 10% FBS, 1 % glutamine, 800-1000wg/ml 6418 (GIBCO/BRL) and
400~,g/ml Zeocin. Media was thereafter changed every 48-96 hours, depending on
cell death. After a sufficient selection time (approximately 2 weeks),
distinct colonies
containing both the CNG channel and the SHT6 receptor appeared on the plate.
Stable cell lines expressing CNG channel and SHT6 receptor cDNA were cloned
using standard procedures. Briefly, sterile cloning rings were placed over
colonies
and 50,1 1X Trypsin was added to the side of each ring to trypsinize cells for
approximately 1-2 minutes. 50,1 growth antibiotic-free media was added and
pipetted
several times. Cells were transferred to 24-well dishes containing 0.5 ml
media. The
following day the media was removed and MEM media containing 10 % FBS, 1
glutamine, 400~cg/ml Zeocin and 800-1000~,g/ml 6418 added.
26
CA 02255548 1998-11-14
B) Integration of the human dopamine Dl receptor, as GPCR
Integration the human D 1 receptor into the HEK 293 stable cell line
expressing the rat
oflactory CNGa channel was achieved in the manner described above for the 5HT6
producing cells. The D1 receptor gene was cloned using standard PCR and
hybridization screening procedures applied to a human genomic library. The
sequence agreed with the published nucleotide sequence (Genbank accession
number
X55758 and Nature, 1990,347(6288):80-83). The gene was inserted into the
EcoRI/XbaI multiple cloning sites of pcDNA3. Three ~,g of pcDNA3/D1 were
diluted in 240~L of serum-free OPTI-MEM. Transfection and selection were then
performed as described above for the 5HT6/CNGa stable cell line.
EXAMPLE 3 Screening for 5HT6 receptor ligands
To prepare for screening of 5HT6 receptor ligand candidates, the HEK293 cell
line
stably expressing both the r CNGa channel and the h5HT6 receptor was plated in
triplicate on poly-D-Lysine coated plates at approximately 3x105cells/mL two
days
prior to assay. On the day of the assay, plates were washed once with 2001 of
CNG
buffer (142 mM NaCI, 5mM KCI, 2 mM CaCl2, 10 mM glucose, lOmM HEPES
pH7.2; osmolality = 300mOsm) per well. The buffer was removed, and 35~M of
Fluo-3-AM dye (Molecular Probes) was added per well in a volume of 50~L. The
plates were incubated for 1 hour at room temperature in the dark. After 1
hour, the
dye was removed and cells were washed twice with 200,1 of CNG buffer.
Subsequently, 200pL of CNG buffer was added to each well and the plates were
incubated for a further 10 minutes at room temperature in the dark.
A) Agonist screening
Calcium influx was measured following 10 minutes of incubation with 15~L of
10~.M
each of the 5HT6 receptor ligands 5-hydroxytryptamine (5-HT), 5-
carboxytryptamine
(5-CT) and 5-methoxytryptamine (5-MeOT). Fluorescence was detected by
27
CA 02255548 1998-11-14
Fluoroskan as noted above, and the results are shown in Panel A of Figure 3.
As
anticipated, incubation with these agonists of the Gs-type SHT6 receptor
resulted in
stimulation of calcium influx, as revealed by the increased fluorescence
relative to
controls. Similar results were obtained when the agonists were assessed
against
CNG-producing cells that transiently produce the SHT6 receptor.
B) Antagonist effect
Panel B of Figure 3 illustrates the response seen with the noted known
antagonists of
the SHT6 receptor, using the same protocol as with the agonists. It will be
noted
that, as expected, no stimution of calcium influx resulted.
EXAMPLE 4 - Screening for D 1 receptor ligands
A) Agonist screening
In the manner described in Example 3A for SHT6 agonist screening, but using
the
CNG-producing D1 receptor cell line as obtained in Example 2B, calcium influx
was
measured following 5 minutes of incubation with each of the D 1 receptor
agonists
(10~.M final concentration, 60uL final volume) noted in Figure 4, panel A. As
the
results show, interaction between the Gs-type D 1 receptor the agonists
clearly
resulted in a significant influx of calcium ion, relative to mock treated
controls.
B) Antagonist effect
Using the CNG-producing D 1 receptor cell line as obtained in Example 2B, the
D 1
receptor antagonists noted in Figure 4 panel B were assessed for their effect
on
calcium influx. Antagonists were added in lSuL volumes, to a final
concentration of
lOuM. As shown, the antagonist properties of these compounds are revealed by
the
absence of detected calcium influx.
C) Antagonist screening
To identify ligand candidates as functional antagonists, the following
protocol was
developed, which studies the ability of an antagonist candidate to inhibit
calcium
28
CA 02255548 1998-11-14
influx mediated by a reference agonist.
Cells were dye-loaded and plated in the manner described in Example 3. Cell-
borne
D 1 receptors were then first saturated with the selected antagonist
incubating the cells
with 50p.L of the selected antagonist at 10~M for five minutes. Incubation
buffer was
then replaced with CNG buffer containing IBMX (0. l l lmg/mL) in a volume of
30uL, in order to maintain the cytosolic levels of cAMP formed upon subsequent
addition of the reference agonist.
An initial t=0 reading was taken on the fluorescence reader (Fluoroskan).
Following
this read, 15~,L of the selected antagonist (IOpM final) were added to the pre-
treated
wells, together with lSuL of dopamine, the reference agonist, at a final
concentration
of lpM. Subsequent reads were taken at T=5 minutes, and T=10 minutes.
As shown in Figure 5, results reveal that in the presence of antagonists
flupentixol
and SCH23390, dopamine response was inhibited (panel B). In panel A cells were
not pretreated with antagonist.
EXAMPLE 5 - Multiplexed Screening
The examples which follow utilize CNG channel-producing cells that incorporate
either no GPCR (for control) or the Gs-coupled 5HT6 or D1 receptors. These
cell
lines were prepared as described in the preceding examples unless otherwise
noted
below.
A) Cell preparation
Dye Loading of HEK293 cells stably expressing rCNG channel
A HEK2,93 cell line stably expressing the r CNGa channel was plated in
triplicate on
poly-D-Lysine coated plates at approximately 3x105cells/mL 2 days prior to day
of
assay. On the day of the assay, plates were washed once with 200p1 of CNG
buffer
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CA 02255548 1998-11-14
(142 mM NaCI, SmM KC1, 2 mM CaCl2, 10 mM glucose, IOmM HEPES pH7.2;
osmolality = 300mOsm) per well. The buffer was removed, and SOpM of Fura Red-
AM dye (Molecular Probes) in a volume of SOwL was added per well, and the
plates
were incubated for 1 hour at room temperature in the dark. After 1 hour, the
dye
S was removed and cells were washed with 200~L of CNG buffer, twice.
Subsequently, 200wL of CNG buffer was added to each well and the plates were
incubated for a further 15 min at room temperature in the dark. Subsequently,
the
CNG buffer was removed and replaced with 45~L of HEK293 cells stably
expressing
both the rCNG channel and the hDl receptor (See below).
Dye Loading of HEK293 cells stably expressing both rCNG channel and h5HT6
receptor:
A HEK293 cell line stably expressing both the r CNGa channel and the h5HT6
receptor was plated in triplicate on poly-D-Lysine coated plates at
approximately
3x105cells/mL two days prior to day of assay. On the day of the assay, plates
were
washed once with 200p.L of CNG buffer (142 mM NaCI, SmM KCI, 2 mM CaCl2, 10
mM glucose, lOmM HEPES pH7.2; osmolality = 300mOsm) per well. The buffer
was removed, SO~M of Fura Red-AM (Molecular Probes) dye in a volume of SO~L
was added per well, and the plates were incubated for 1 hour at room
temperature in
the dark. After 1 hour, the dye was removed and cells were washed twice with
200~L of CNG buffer. Subsequently, 200~L of CNG buffer was added to each well
and the plates were incubated for a further 15 minutes at room temperature in
the
dark. Subsequently, the CNG buffer was removed and replaced with 45~L of
HEK293 cells stably expressing both the rCNG channel and the hD 1 receptor
(See
below).
Dye Loading of HEK293 cells stably expressing both rCNG channel and Dl
receptor
A HEK293 cell line stably expressing both the r CNGa channel and the D 1
receptor
were grown to 90 % confluency in a T75 flask. On the day of the assay, cells
at this
CA 02255548 1998-11-14
confluency, were washed once with PBS and dislodged by trypsinization. Cells
were
resuspended in CNG buffer by gentle trituration. Subsequently, IOp,M of Fluo-3-
AM
dye (Molecular Probes) was added to the cell suspension, and incubated for 1
hour at
room temperature in the dark. After 1 hour, cells were centrifuged at 1000 rpm
for 5
minutes, and the dye removed. Cells were resuspended in CNG buffer and washed
twice by centrifugation at 1000 rpm at room temperature. Following the last
wash,
cells were resuspended to a density of approximately 5x105 cells/mL in CNG
buffer
supplemented with 0.111 mg/mL IBMX (Sigma), and cell clumping was minimized
by gentle trituration.
B) Agonist Screening
1) Multiplexing cells producing CNG alone with cells producing CNG
and Dl receptor
HEK293 cells loaded with Fluo-3-AM which express rCNG and the D 1 receptor
(see
above) were added (45p1) to 96-well plates containing adherent Fura Red-AM
loaded
HEK293 cells stably expressing the rCNG channel (see above). A reading was
taken
using the fluorescence reader (Fluoroskan) as T=0. Compounds studied (see
Figure
6) were dissolved in the appropriate solvent (for example, dH20 for 8Br-cGMP
and
8Br-cAMP, and CNG buffer for Dopamine), diluted to four times final
concentration
in CNG buffer and added in a volume of 15 pl to the appropriate wells. Further
readings were taken at T=1 minute, T=5 minutes, and T=10 minutes, using the
Fluoroskan. Fluoroskan readings were taken using Excitation Filter 485 and
Emission Filter 538 for measurement of fluorescence response in cells loaded
with
Fluo-3-AM, and Excitation Filter 485 and Emission Filter 660 for measurement
of
fluorescence response in cells loaded with Fura Red-AM.
As shown in Figure 6, cells loaded with Fura Red respond to direct CNG channel
activation with a reduction in the mixed culture that are fluorescence signal
(panel A),
whereas cells in the mixed culture that are loaded with Fluo-3 respond to
direct
channel activation with an increase in fluorescence signal (panel B). This
result is
31
CA 02255548 1998-11-14
also reflected in results with D1 receptor ligands. As shown in panel C,
incubation of
D 1 agonists with cells producing only the CNG channel, and loaded only with
Fura
Red, showed no influx of calcium as expected. Also as expected, the D 1
receptor
antagonists, flupentixol and haloperidol, also failed to stimulate calcium
influx, as
revealed by the absence of any decrease in Fura Red fluorescence. On the other
hand, as shown in Figure 6 panel D, Fluo-3-loaded cells producing the D 1
receptor
and the CNG channel elicited a significant calcium influx when incubated with
the D 1
receptor agonists. As expected, no calcium influx, and accordingly no increase
in
Fluo-3 fluorescence, was detected when the antagonists flupentixol and
haloperidol
were incubated.
2) Multiplexing cells stably producing both rCNG channel and 5HT6
receptor with cells stably producing both rCNG channel and Dl receptor
HEK293 cells stably expressing both rCNG and the 5HT6 receptor were
grown as adherent cultures in 96 well Poly-D-Lysine coated plates, and loaded
with the specific calcium indicator Fura Red-AM (50pM). HEK293 cells
stably expressing both rCNG and the D 1 receptor were grown to 90
confluency in T75 flasks and dislodged by trypsinization. Cells were
resuspended in CNG buffer to a density of approximately 5x105 cells/mL, and
loaded in suspension with the calcium specific indicator Fluo-3-AM (IOp,M).
Suspension cells were added together with the adherent cells, and calcium
influx was measured following 10 minutes of incubation with a CNG channel
activator, 5mM 8Br-cGMP, D1 selective agonists: IOpM Dopamine, 10~M
ADTN, lOuM SKF38393, partial agonists, IOpM Apomorphine, 10~M
Pergolide, or antagonists: lOuM Flupentixol, 10~M Haloperidol, and 5HT6
selective agonists: lOuM SHT, lOuM SCT, 10~M (+)Lisuride, or
antagonists: IOpM Clozapine, and lOpM Methiothepin. Calcium influx via
5HT6 receptor activation/inactivation was measured using Excitation Filter
485 and Emmision Filter 660, which are specific for the calcium indicator
Fura Red-AM (A, C). D1 receptor activation/inactivation was measured using
32
CA 02255548 1998-11-14
Excitation Filter 485 and Emmision Filter 538 which are specific for Fluo-3-
AM (B,D).
As shown in Figure 7, 5HT6 specific agonist activity was observed only in the
cell line producing CNG channel and 5HT6 receptor loaded with Fura Red
(panel B); these cells did not respond to D1 specific agonists (panel A). The
cell line producing CNG channel and D 1 receptor loaded with Fluo-3 was
responsive only to D1 specific agonist activity (panel B), and was
unresponsive to 5HT6 specific agonists (panel D). Non-selective agonists
lisuride and apomorphine activated D 1 and 5HT6 receptors in both cell lines
(see panels A, B, C, D).
C) Antagonist Screening
The compound of interest (10~M final concentration) was added to a combined
mixture of two HEK293 cell lines stably expressing rCNG channel and either
h5HT6
receptor or hD 1 receptor (prepared and dye loaded as described above) in a
volume
of 10 pL. Cells were pretreated with this compound for 5 minutes. Immediately
following this pretreatment an initial t=0 reading was taken on the
fluorescence
reader (Fluoroskan). Following this reading, 10 ~,L of known agonist at a
final
concentration of 1mM (serotonin for 5HT6 and dopamine for D1) was added to
each
well. Subsequent reads were taken at T=5 minutes, and T=10 minutes. Excitation
filter 485 and Emission filter 538 were used for measurement of fluorescence
response in cells loaded with Fluo-3-AM. Excitation filter 485 and Emission
filter
660 were used for measurement of fluorescence response in cells loaded with
Fura
Red-AM. Antagonistic properties of these compounds were assessed based on
reversal of either serotonin and/or reversal of dopamine stimulated agonist
activity.
Particularly, HEK293 cells stably expressing both rCNG and the 5HT6 receptor
were
grown as adherent cultures in 96 well Poly-D-Lysine coated plates, and loaded
with
the specific calcium indicator Fura Red-AM (50 M). HEK293 cells stably
expressing both rCNG and the D 1 receptor were grown to 90 % confluency in T75
33
CA 02255548 1998-11-14
flasks and dislodged by trypsinization. Cells were resuspended in CNG buffer
to a
density of approximately 5x105 cells/mL, and loaded in suspension with the
calcium
specific indicator Fluo-3-AM (10~M). Suspension cells were added together with
the
adherent cells. Multiplexed cells were pretreated for 5 minutes with lOmM of
clozapine (SHT6 selective antagonist), flupentixol (D1 selective antagonist)
or
propranolol (non-selective antagonist). Following this pretreatment, 1mM of
the
SHT6 selective agonist, serotonin (A) or the D1 selective agonist, dopamine
(B), was
added to each well. Calcium influx via SHT6 receptor activation/inactivation s
measured using Excitation filter 485 and Emmision filter 660, which are
specific for
the calcium indicator Fura Red-AM (A). D1 receptor activation/inactivation was
measured using Excitation filter 485 and Emmision filter 538 which are
specific for
Fluo-3-AM (B).
Results are presented in Figure 8, as a percentage reversal of agonist
(serotonin (A)
or dopamine (B)) response due to the effect of various antagonists. As
summarized in
panel A, the SHT-stimulated response could be reversed by clozapine, and
partially
reversed by flupentixol. Propranolol had no effect. In panel B, the dopamine-
stimulated response is fully reversed by flupentixol and only slightly
reversed by
propranolol. Clozapine had no effect.
EXAMPLE 6 Tandem Screening Protocol
A HEK293 cell line stably expressing both the rCNGchannel and a Gs-coupled
receptor (eg.Human Dopamine D1) was plated in triplicates on poly-D-Lysine
coated
plates at approximately 3x105cells/mL two days prior to day of assay. On the
day of
the assay, plates were washed once with 200uL of CNG buffer (142 mM NaCI, 5mM
KCI, 2 mM CaCl2, 10 mM glucose, lOmM HEPES pH7.2; osmolality = 300mOsm)
per well. The buffer was removed, and 35uM of Fluo-3-AM dye (Molecular Probes)
was added per well in a volume of SOuL. The plates were incubated for 1 hour
at
room temperature in the dark. After 1 hour, the dye was removed and cells were
washed twice with 200uL of CNG buffer. Subsequently, 200uL of CNG buffer was
added to each well and the plates were incubated for a further 10 minutes at
room
34
CA 02255548 1998-11-14
temperature in the dark.
Following a 10 minute wash with CNG buffer, 30uL of CNG buffer supplemented
with O.l l lmg/mL IBMX was added to each well. An initial t=0 reading was
taken
on the fluorescence reader (Fluoroskan). The compound of interest was added to
cells (lOuM final concentration) in a volume of lSuL. Cells were pretreated
with this
compound for 5 minutes and a t=5 reading was taken. Immediately following this
read, lSuL of known reference agonist (eg. SKF38393 for the human dopamine
receptor D1) at a final concentration of luM was added to each pretreated
well, and
subsequent reads were taken at t=2 minutes, and t=5 minutes.
Ligand candidates can be identified functionally as follows:
Agonist Identification: If the compound of interest is an agonist (e.g.
Dopamine),
an increase in fluorescence will be seen 5 minutes post compound addition (t=5
reading). This increase in fluorescence will not be significantly altered
following
incubation with the known agonist (eg. SKF38393) as shown in Figure 9a and
figure
10a.
Antagonist Identification: If the compound of interest is an antagonist (e.g.
Flupentixol, SCH23390) no significant change in baseline fluorescence will be
detected 5 minutes post compound addition (t=5 reading), and addition of the
known
agonist (eg. SKF38393) will have a blunted or no increase in fluorescence
which
should be related to the potency of the antagonist (figure 9b and figure lOb).
'Inactive Compound' Identification: If the compound of interest is ineffective
or a
(e.g. Propranolol at the human dopamine D1 receptor) no significant change in
baseline fluorescence will be detected 5 minutes post compound addition (t=5
reading), and the addition of the known agonist will cause an expected
increase in
fluorescence (figure 9c and figure lOc).
CA 02255548 1998-11-14
d) Receptor Modulator Identification: If the compound of interest is a
positive
receptor modulator, no significant increase in baseline fluorescence will be
detected
minutes post compound addition, and the addition of the known agonist will
cause
an increase in fluorescence to a greater extent than is normally expected for
agonist
alone (figure 9d).
36
