U.S. patent application number 12/790013 was filed with the patent office on 2010-09-30 for high throughput screening assay for the trpm5 ion channel.
This patent application is currently assigned to Redpoint Bio Corporation. Invention is credited to Robert W. BRYANT, M.N. Tulu Buber, S. Paul Lee, R. Kyle Palmer, Qifeng Yang.
Application Number | 20100248988 12/790013 |
Document ID | / |
Family ID | 38023838 |
Filed Date | 2010-09-30 |
United States Patent
Application |
20100248988 |
Kind Code |
A1 |
BRYANT; Robert W. ; et
al. |
September 30, 2010 |
High Throughput Screening Assay for the TRPM5 Ion Channel
Abstract
There exists a need in the art for high throughput screening
assays that can identify compounds that specifically modulate the
activity of fast-acting ion channels, such as TRPM5. Current
methods suffer from a lack of sensitivity, low throughput, and are
labor intensive. The claimed methods provide fluorescent assays
with an optical readout that gives rapid readout of the results,
has a high signal to noise background ratio, are easy to use, can
be modified for automation and miniaturization, and provide
verification that a compound specifically modulates TRPM5.
Inventors: |
BRYANT; Robert W.;
(Princeton, NJ) ; Lee; S. Paul; (Yardley, PA)
; Palmer; R. Kyle; (Cranbury, NJ) ; Yang;
Qifeng; (Ewing, NJ) ; Buber; M.N. Tulu;
(Newtown, PA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Redpoint Bio Corporation
Ewing
NJ
|
Family ID: |
38023838 |
Appl. No.: |
12/790013 |
Filed: |
May 28, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11592180 |
Nov 3, 2006 |
7749730 |
|
|
12790013 |
|
|
|
|
60732636 |
Nov 3, 2005 |
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Current U.S.
Class: |
506/10 ;
536/23.1 |
Current CPC
Class: |
G01N 33/502 20130101;
G01N 33/6872 20130101; G01N 2500/00 20130101 |
Class at
Publication: |
506/10 ;
536/23.1 |
International
Class: |
C40B 30/06 20060101
C40B030/06; C07H 21/04 20060101 C07H021/04 |
Claims
1-14. (canceled)
15. A high throughput screening assay for determining whether a
test compound is a TRPM5 ion channel-specific modulator comprising:
(a) contacting a cell that expresses TRPM5 and has been preloaded
with a membrane potential fluorescent dye, with a test compound in
the presence of potassium chloride; (b) using an optical detector,
measuring the fluorescent intensity of said cell in the presence of
said potential modulating compound; (c) comparing the measured
fluorescent intensity determined in step (b) to the fluorescent
intensity of a different cell that expresses TRPM5 and has been
preloaded with a membrane potential fluorescent dye, in the
presence of potassium chloride and the absence of the test
compound; and (d) evaluating whether the test compound may be a
TRPM5-specific modulator by determining if the ratio of the
fluorescent intensity with potassium chloride and the test compound
to the intensity with potassium chloride in the absence of the test
compound is less than or greater than 1.
16. The assay of claim 15, further comprising selecting a test
compound that enhances TRPM5 activity.
17. The assay of claim 15, further comprising selecting a test
compound that inhibits TRPM5 activity.
18. The assay of claim 15, wherein said cells are located in a
multi-well vessel.
19. The assay of claim 18, wherein said multi-well vessel comprises
up to 96 wells.
20. The assay of claim 18, wherein said multi-well vessel comprises
greater than 96 wells.
21. The assay of claim 18, wherein said multi-well vessel comprises
384 wells.
22. The assay of claim 18, wherein said multi-well vessel comprises
1536 wells.
23-26. (canceled)
27. The assay of claim 15, wherein said membrane potential
fluorescent dye is a Fluorescent Imaging Plate Reader Membrane
Potential (FMP) dye.
28. The assay of claim 15, wherein said optical detector is
selected from the group consisting of: Fluorescent Imaging Plate
Reader (FLIPR.RTM.), FLEXStation, Voltage/Ion Probe Reader (VIPR),
fluorescent microscope and charge-coupled device (CCD) camera, and
Fathway HT.
29. The assay of claim 28, wherein said optical detector is a
FLIPR.RTM..
30-63. (canceled)
64. The assay of claim 15, wherein said cell of step (a) is a cell
selected from the group consisting of: CHO, COS-7, HeLa, HEK 293,
PC-12, and BAF.
65. The assay of claim 64, wherein said cell is a CHO cell.
66. The assay of claim 64, wherein said cell is a HEK 293 cell.
67. The assay of claim 15, wherein the amount of said test compound
that contacts the cell in step (a) is between 0.1 and 1000
.mu.M.
68. The assay of claim 15, wherein the test compound is a small
organic compound having a molecular weight of more than 100 and
less than about 10,000 Daltons.
69. The assay of claim 15, wherein the test compound is selected
from the group consisting of: synthetic organic compounds, chemical
compounds, naturally occurring products, polypeptides, peptides,
and nucleic acids.
70. The assay of claim 15, wherein said membrane potential
fluorescent dye is a voltage-sensitive dye selected from the group
consisting of: modified bisoxonol dyes, sodium dyes, potassium
dyes, and thorium dyes.
71. The assay of claim 15, wherein said membrane potential
fluorescent dye is a dual wavelength FRET-based dye selected from
the group consisting of: DiSBAC2, DiSBAC3, and CC-2-DMPE.
72. The assay of claim 15, wherein said optical detector is a
FLIPR-Tetra.TM..
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Non-Prov. Appl. No.
11/592,180, filed Nov. 3, 2006, which claims the benefit of U.S.
Prov. Appl. No. 60/732,636, filed Nov. 3, 2005, which is herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention is related to a high throughput
screening method for compounds that impact taste. More
specifically, the present invention relates to a screening method
useful in the identification of compounds that affect taste
sensation by modulating the activity of the ion channel TRPM5. The
screening method, using fluorescent membrane potential dyes, allows
for the rapid screening of thousands of compounds by providing a
visual fluorescent readout that can be easily automated.
[0004] 2. Background
[0005] Taste perception not only plays a critical role in the
nutritional status of human beings, but is also essential for the
survival of both lower and higher animals (Margolskee, R. F. J.
Biol. Chem. 277:1-4 (2002); Avenet, P. and Lindemann, B. J.
Membrane Biol. 112:1-8 (1989)). Taste perception is carried out by
taste receptor cells (TRCs). TRCs perceive the multitude of
compounds that are associated with a given taste, and convert that
perception to a signal deciphered by the brain, resulting in sweet,
bitter, sour, salty, or umami (savory) taste.
[0006] TRCs are polarized epithelial cells, meaning they have
specialized apical and basolateral membranes. Taste buds contain
60-100 TRCs, each having a tiny portion of its membrane exposed on
the mucosal surface of the tongue (Kinnamon, S. C. TINS 11:491-496
(1988)). Sensory transduction is initiated by sapid molecules, or
"tastants," that interact with microvillar processes on the apical
membrane of TRCs. The tastants bind specific membrane receptors,
leading to a voltage change across the cell membrane; in turn this
depolarizes, or changes the electric potential of the cell, causing
transmitter release and excitation of primary gustatory nerve
fibers.
[0007] Ion channels are transmembrane proteins that form pores in a
membrane and allow ions to pass from one side to the other
(reviewed in B. Hille (Ed), 1992, Ionic Channels of Excitable
Membranes 2nd ed., Sinauer, Sunderland, Mass.). Although certain
ion channels are open under all physiological membrane conditions
(so-called leaky channels), many channels have "gates" that open in
response to a specific stimulus. As examples, voltage-gated
channels respond to a change in the electric potential across the
membrane, mechanically-gated channels respond to mechanical
stimulation of the membrane, and ligand-gated channels respond to
the binding of specific molecules. Various ligand-gated channels
can open in response to extracellular factors, such as a
neurotransmitters (transmitter-gated channels), or intracellular
factors, such as ions (ion-gated channels), or nucleotides
(nucleotide-gated channels). Still other ion channels are modulated
by interactions with proteins, such as G-proteins (G-protein
coupled receptors or GPCRs).
[0008] Most ion channel proteins mediate the permeation of one
predominant ionic species. For example, sodium (Na.sup.+),
potassium (K.sup.+), chloride (Cl.sup.-), and calcium (Ca.sup.2+)
channels have been identified.
[0009] One recently discovered ion channel, TRPM5, has been shown
to be essential for taste transduction. Perez et al., Nature
Neuroscience 5:1169-1176 (2002); Zhang et al., Cell 112:293-301
(2003). TRPM5 is a member of the transient receptor potential (TRP)
family of ion channels. TRPM5 forms a channel through the membrane
of the taste receptor cell, and is believed to be activated by
stimulation of a receptor pathway coupled to phospholipase C and by
IP.sub.3-mediated Ca.sup.2+ release. The opening of this channel is
dependent on a rise in Ca.sup.2+ levels. Hofmann et al., Current
Biol. 13:1153-1158 (2003). The activation of this channel leads to
depolarization of the TRC, which in turn leads to transmitter
release and excitation of primary gustatory nerve fibers.
[0010] Because TRPM5 is a necessary part of the taste-perception
machinery, its inhibition prevents an animal from sensing
particular tastes. Although taste perception is a vital function,
the inhibition, or masking, of undesirable tastes is beneficial
under certain circumstances. For example, many active
pharmaceutical ingredients of medicines produce undesirable tastes,
such as a bitter taste. Inhibition of the bitter taste produced by
the medicine may lead to improved acceptance by the patient. In
other circumstances, enhancement of taste may be desirable as in
the case of developing improved artificial sweeteners or in
treatment of taste losses in groups such as the elderly. Mojet et
al., Chem Senses 26:845-60 (2001).
[0011] TRPM5 displays voltage modulation and rapid
activation/deactivation ("opening and closing") kinetics upon
receptor stimulation (Hofmann et al. 2003) which allows for the
passage of monovalent cations, such as sodium and potassium. A
closely related protein, TRPM4b, also shows Ca.sup.2+ dependent
voltage modulation, but opens and closes much slower than TRPM5.
Thus, TRPM5 is the first example of a voltage-modulated,
Ca.sup.2+-activated, monovalent cation channel that has rapid
activation/deactivation kinetics (Hofmann et al. 2003).
[0012] Ion channel activation or inhibition may be determined by
measuring changes in cell membrane potential when cells are exposed
to certain stimuli. This is an indirect method of evaluating ion
channel modulation, as cell membrane potential may be affected by
multiple channels.
[0013] One method for testing ion channel activity is to measure
changes in cell membrane potential using the patch-clamp technique.
(Hamill et al., Nature 294:462-4 (1981)). In this technique, a cell
is attached to an electrode containing a micropipette tip which
directly measures the electrical conditions of the cell. This
allows detailed biophysical characterization of changes in membrane
potential in response to various stimuli. Thus, the patch-clamp
technique can be used as a screening tool to identify compounds
that modulate activity of ion channels. However, this technique is
difficult to master and requires significant expertise to generate
consistent, reliable data. Moreover, this technique is time
consuming and would allow fewer than two or three compounds per day
to be screened for activity.
[0014] Ideally, methods of screening test compounds are high
throughput (i.e., allow for many compounds to be screened quickly),
automated, easy to use, sensitive, and selective. Screening assays
should also provide a high signal to background noise ratio.
(Baxter et al., J. Biomol. Screen. 7:79-85 (2002)). Background
noise is the minimal stimulation that a compound produces
regardless of its effect on the ion channel. The high ratio makes
visualization of positive or negative modulators simpler because
the smallest response will be seen over the background
measurements. This leads to a clear identification of modulating
compounds.
[0015] A potential high throughput method for determining ion
channel modulation utilizes fluorescent dyes that produce a
fluorescent signal when the cell membrane potential changes.
Increases in fluorescence occur, because upon a change in the
membrane potential, the fluorescent dyes "flip" their orientation
in the cell membrane bilayer from an intracellular to extracellular
location. This flip causes an increase in fluorescence that is
easily detected and quantified usually using an optical reader.
Optical readouts of ion channel function are favorable for high
throughput screening because they are potentially sensitive,
versatile, and amenable to miniaturization and automation. Present
day optical readers detect fluorescence from multiple samples in a
short time and can be automated. Fluorescence readouts are used
widely both to monitor intracellular ion concentrations and to
measure membrane potentials.
[0016] In an attempt to overcome some of the shortcomings of
traditional fluorescent dyes, modified bisoxonol fluorescent dyes
such as the FLIPR.RTM. Membrane. Potential dyes (FMP) from
Molecular Devices were developed. FMP dyes have been effective in
correlating fluorescence with membrane potential determined
directly by patch-clamp recording for "slow" ion channels (Baxter
et al., J. Biomol. Screen. 7:79-85 (2002); Behrendt et al., British
J. Pharmacol. 141:737-745 (2004); and Whiteaker et al., J. Biomol.
Screen. 6:305-312 (2001).
[0017] A major challenge in designing a high throughput screening
(HTS) method for compounds that modulate a specific ion channel is
that methods of determining channel activation are indirect. To
identify compounds that affect taste through modulation of TRPM5
activity, there must be a demonstration that the effect of the
compounds on taste is specific to TRPM5 and not also to one or more
of the multitude of other channels and receptors located on the
cell surface. Additionally, since TRPM5 activation is calcium
dependent, specificity of the TRPM5/test compound interaction must
be confirmed by excluding those compounds that also modulate
GPCR-agonist calcium flux.
[0018] Therefore, there exists a need in the art for HTS assays
that can distinguish compounds that modulate taste by specifically
acting on TRPM5, from compounds that may act by other mechanisms
and that may not affect taste perception. The claimed invention
provides HTS methods that give rapid and specific results, have a
high signal to background ratio, and are easy to use.
BRIEF SUMMARY OF THE INVENTION
[0019] A new high throughput screening assay has been discovered
that allows for the rapid screening of compounds that modulate
TRPM5 ion channel activity. The method of the invention is more
selective than methods that rely only on evaluation of a change in
membrane potential. The invention will allow a practitioner to
distinguish agents that are nonspecific modulators of ion channels
from agents that act via modulation of TRPM5. Moreover, the method
will allow thousands of compounds that potentially modulate this
fast ion channel, and affect taste, to be screened quickly and
reliably.
[0020] An embodiment of the present invention is a high throughput
screening assay for screening potential enhancers of the TRPM5 ion
channel comprising contacting a cell expressing TRPM5 with a
suboptimal concentration of an agent that increases intracellular
calcium concentration, wherein the cell has been preloaded with a
membrane potential fluorescent dye; contacting said cell with a
potential enhancing compound; using an optical detector, measuring
the fluorescent intensity of said cell in the presence of said
potential enhancing compound; and comparing the measured
fluorescent intensity to the fluorescent intensity of a different
cell expressing TRPM5 in the presence of an optimal concentration
of an agent that increases intracellular calcium concentration.
[0021] An additional embodiment of the invention is a high
throughput screening assay for determining whether a test compound
is a TRPM5 ion channel-specific modulator comprising contacting a
cell that expresses TRPM5 and has been preloaded with a membrane
potential fluorescent dye, with a test compound in the presence of
potassium chloride; using an optical detector, measuring the
fluorescent intensity of said cell in the presence of said
potential modulating compound; comparing the measured fluorescent
intensity determined above to the fluorescent intensity of a
different cell that expresses TRPM5 and has been preloaded with a
membrane potential dye in the presence of potassium chloride and
the absence of the test compound; and evaluating whether the test
compound may be a TRPM5-specific modulator by determining if the
ratio of the fluorescent intensity with KCl and the test compound
to the intensity with KCl in the absence of the test compound is
less than or greater than 1.
[0022] An additional embodiment of the invention is a high
throughput screening assay for determining whether a test compound
is a TRPM5 ion channel-specific modulator comprising contacting a
cell that expresses TRPM5 and has been preloaded with an
intracellular calcium dye, with a test compound and a suboptimal
concentration of a calcium modulating agent that increases
intracellular calcium concentration; using an optical detector,
measuring the fluorescent intensity of said cell in the presence of
said calcium modulating compound; comparing the measured
fluorescent intensity determined above to the fluorescent intensity
of a different cell that expresses TRPM5 and has been preloaded
with an intracellular calcium dye, in the presence of a suboptimal
concentration of a calcium modulating agent and the absence of the
test compound; and evaluating whether the test compound may be a
TRPM5-specific modulator by determining if the ratio of the
fluorescent intensity with a suboptimal concentration of a calcium
modulating agent and the test compound, to the intensity with a
suboptimal concentration of a calcium modulating agent in the
absence of the test compound is less than or greater than 1.
[0023] Another embodiment of the claimed invention is a high
throughput screening assay for screening potential enhancers of the
TRPM5 ion channel comprising contacting a cell expressing both
wildtype TRPM5 and a nonfunctional TRPM5 and has been preloaded
with a membrane potential fluorescent dye, with a potential
enhancer in the presence of an agent that increases the calcium
concentration in said cell; using an optical detector, measuring
the fluorescent intensity of said cell in the presence of said
potential enhancer; and comparing the measured fluorescent
intensity determined above to the fluorescent intensity of a cell
that expresses wildtype TRPM5 and that has been preloaded with a
membrane potential dye, in the presence of the potential enhancing
compound to determine the extent of TRPM5 enhancement.
[0024] In some embodiments, the nonfunctional TRPM5 contains a
deletion of the first 1000 base pairs of the TRPM5 gene. In another
embodiment, the nonfunctional TRPM5 contains a deletion of the
first 2000 base pairs of the TRPM5 gene.
[0025] In some embodiments, the claimed method further comprises
selecting a compound that enhances TRPM5 activity. In other
embodiments, the claimed method further comprises selecting a
compound that inhibits TRPM5 activity.
[0026] In additional embodiments, the claimed method is directed to
screening cells that are located in a multi-well vessel. The
multi-well vessels of the claimed invention may contain up to and a
number equaling 96 wells. In another embodiment, the multi-well
vessel comprises greater than 96 wells. In another embodiment, the
multi-well vessel comprises 384 wells. In yet another embodiment,
the multi-well vessel comprises 1536 wells.
[0027] In some embodiments of the claimed invention, agents that
increase calcium concentration are selected from the group
consisting of thrombin, adenosine triphosphate (ATP), carbachol,
and agonists of endogenous G protein coupled receptors (GPCRs). In
one embodiment of the invention, the agent that increases calcium
concentration is a calcium ionophore, e.g. A23187, calcimycin or
ionomycin.
[0028] In some embodiments of the claimed invention, the membrane
potential fluorescent dye is a FMP dye.
[0029] In additional embodiments of the claimed invention, the
optical detector is selected from the group consisting of:
Fluorescent Imaging Plate Reader (FLIPR.RTM.), FLEXStation,
Voltage/Ion Probe Reader (VIPR), fluorescent microscope and
charge-coupled device (CCD) camera, and Pathway HT. In one
embodiment of the invention, the optical detector is a
FLIPR.RTM..
[0030] Further embodiments, features, and advantages of the present
inventions, as well as the structure and operation of the various
embodiments of the present invention, are described in detail below
with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0031] The accompanying drawings, which are incorporated herein and
form a part of the specification, illustrate one or more
embodiments of the present invention and, together with the
description, further serve to explain the principles of the
invention and to enable a person skilled in the pertinent art to
make and use the invention.
[0032] FIG. 1 shows a demonstration of TRPM5-dependent fluorescent
signaling in Chinese Hamster Ovary (CHO) cells. CHO cells
transfected with both the human TRPM5 ion channel and with the
muscarinic 1 (M1) G protein coupled receptor (GPCR) were loaded
with membrane potential dye and stimulated with carbachol, an M1
agonist. This GPCR activation triggers an increase in intracellular
calcium ions in the cell, which in turn opens the TRPM5 ion channel
letting primarily sodium ions into the cell. This depolarization
increases the fluorescent signal of the dye which is measured on
the Fluorescent Imaging Plate Reader (FLIPR.RTM.). Note that in an
assay analyzing the effect of compounds on TRPM5, the compound
would be added prior to activation of TRPM5.
[0033] FIG. 2 shows TRPM5-GFP expression in transiently-transfected
HEK 293 cells by fluorescence microscopy.
[0034] FIG. 3 shows TRPM5 ion channel responses in transiently
transfected HEK 293 cells. FIGS. 3A-C show TRPM5 responses in
transfected cells in response to three GPCR agonists: thrombin
(FIG. 3A), carbachol (FIG. 3B) and adenosine triphosphate (ATP)
(FIG. 3C) measured using a FLEXstation.
[0035] FIG. 4 shows High and Low controls for the TRPM5 high
throughput screening assay using a FLIPR-Tetra.TM.. The assay has a
high signal to noise (High Control vs Low Controls) with a Z' value
of 0.76. A value of Z'>0.5 indicates a robust assay for high
throughput screening. (Zhang, J. H. et al. J. Biomol. Screen.
4:67-73 (1999)).
(Z'=1-((3*SD.sub.HC+3*SD.sub.LC)/(AVG.sub.HC-AVG.sub.LC)).
[0036] FIGS. 5A-5C show stimulation of cells stably expressing
TRPM5 using ATP (FIG. 5A), carbachol (FIG. 5B) or thrombin (FIG.
5C) measured using a FLIPR.RTM..
[0037] FIG. 6 shows results from a TRPM5 high throughput screen on
greater than 85,000 compounds. The data is presented as frequency
distribution of percent inhibition of control responses. Each
compound was tested at a concentration of 10 .mu.M.
[0038] FIG. 7 shows a schematic representation of the TRPM5
specificity filter using Ca++ response and KCl counterscreen
assays.
[0039] FIGS. 8A-8B show that KCl counterscreen (FIG. 8A) and Ca++
flux (FIG. 8B) filters identify non-selective inhibitory
compounds.
[0040] FIGS. 9A-9C show the usefulness of the KCl counterscreen in
the TRPM5 assay to identify TRPM5-specific inhibitors. FIG. 9A
demonstrates the identification of a TRPM5-specific inhibitor
measured using a FLIPR.RTM.. FIG. 9B shows a dose responsive
inhibition of TRPM5 by a compound without inhibiting KCl
depolarization or inhibition of calcium flux activation. FIG. 9C
shows two examples of non-specific inhibition of TRPM5.
[0041] FIG. 10 shows the ability of the KCl counterscreen in the
TRPM5 assay to identify TRPM5-specific enhancer compounds.
[0042] FIG. 11 shows the dose responsive stimulation of TRPM5
activity using a TRPM5-specific enhancer (compound 4).
[0043] FIG. 12 shows that compound 5 (30 .mu.M) produces a very
strong enhancement (17 fold at EC.sub.10) of TRPM5 particularly at
suboptimal concentrations of ATP.
[0044] FIGS. 13A-13B shows the effect of a TRPM5 deletion mutant on
the ability of the calcium ionophore A23187 (FIG. 13A) or carbachol
(FIG. 13B) to cause TRPM5-mediated stimulation.
DETAILED DESCRIPTION OF THE INVENTION
Overview
[0045] The invention is a high throughput screening assay for
compounds that modulate the activity of TRPM5. Since regulators of
TRPM5 are likely to affect taste sensation, the invention,
therefore, provides the first high throughput screening method
useful for the identification of tastants that may specifically
modulate TRPM5. This method is more selective than other screens
for compounds that may impact taste because this method employs
counterscreening, the use of suboptimal dosing, and dominant
negative mutants of TRPM5.
[0046] High throughput refers to processing many compounds in a
short time period. For example, using the invention, greater than
1000 test compounds may be screened for the ability to modulate
TRPM5 activity in one hour. This assay is performed using a cell
that expresses TRPM5. As used in the specification and claims, the
singular form "a", "an" and "the" include plural references unless
the context clearly dictates otherwise. For example, the term "an
ion channel" includes a plurality of ion channels. The term "a
cell" includes a plurality of cells.
[0047] The cell is exposed to a test compound and the ability of
that compound to stimulate opening or to block opening of the
channel is measured. The effect of the test compound is determined
by measuring the change in the cell membrane potential after the
cell is exposed to the compound. A fluorescent dye that responds to
changes in cell membrane potential is used for detection. A means
of evaluating specificity of the ability of the compound to
modulate the channel is performed in parallel with the above
described method. These parallel methods include the use of a
potassium chloride counterscreen, the use of suboptimal doses of
compounds known to stimulate the channel, and the use of a
dominant-negative TRPM5 channel that is biologically inactive.
[0048] While specific configurations and arrangements are
discussed, it should be understood that this is done for
illustrative purposes only. A person skilled in the pertinent art
will recognize that other configurations and arrangements can be
used without departing from the spirit and scope of the present
invention. It will be apparent to a person skilled in the pertinent
art that this invention can also be employed in a variety of other
applications.
Cells
[0049] Cells for use in the method of the invention contain either
a functional or non-functional TRPM5. The practitioner may use
cells in which TRPM5 is endogenous or may introduce TRPM5 into a
cell. If TRPM5 is endogenous to the cell, but the level of
expression is not optimum, the practitioner may increase the level
of expression of TRPM5 in the cell. Where a given cell does not
produce TRPM5 at all, or at sufficient levels, a TRPM5 nucleic acid
may be introduced into a host cell for expression and insertion
into the cell membrane. The introduction, which may be generally
referred to without limitation as "transformation", may employ any
available technique. For eukaryotic cells, suitable techniques may
include calcium phosphate transfection, DEAE-Dextran,
electroporation, liposome-mediated transfection and transduction
using retrovirus or other virus, e.g. vaccinia or, for insect
cells, baculovirus. General aspects of mammalian cell host system
transformations have been described in U.S. Pat. No. 4,399,216. For
various techniques for transforming mammalian cells, see Keown et
al., Meth. Enzym., 185:527-537 (1990) and Mansour et al., Nature
336:348-352 (1988). As is described in detail below, TRPM5 can also
be rendered non-functional. Biologically inactive TRPM5 can be
introduced into cells using any of the above-described techniques.
Cells expressing inactive TRPM5 are useful for confirmation of the
specificity of TRPM5 activation.
[0050] The TRPM5 gene is expressed as a 4.5 kb transcript in a
variety of fetal and adult tissues (Prawitt et al. Hum. Mol. Gen.
9:203-216 (2000)). Human TRPM5 has a putative reading frame
containing 24 exons which encode an 1165 amino acid, membrane
spanning polypeptide. The National Center for Biotechnology
Information (NCBI) database lists several sequences for both the
nucleic acid (NP.sub.--064673, NP.sub.--055370, AAP44477, AAP44476)
and amino acid (NM.sub.--014555, NM.sub.--020277, AY280364,
AY280365) sequences for both the human and mouse forms of TRPM5,
respectively. The inclusion of the above sequences is for the
purpose of illustration of the TRPM5 genetic sequence, however the
invention is not limited to one of the disclosed sequences.
[0051] It is recognized in the art that there can be significant
heterogeneity in a gene sequence depending on the source of the
isolated sequence. The invention contemplates the use of
conservatively modified variants of TRPM5. Conservatively modified
variants applies to both amino acid and nucleic acid sequences.
With respect to particular nucleic acid sequences, conservatively
modified variants refers to those nucleic acids which encode
identical or essentially identical amino acid sequences, or where
the nucleic acid does not encode an amino acid sequence to
essentially identical sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode any given protein.
[0052] For instance, the codons GCA, GCC, GCG and GCU all encode
the amino acid alanine. Thus, at every position where an alanine is
specified by a codon, the codon can be altered to any of the
corresponding codons described without altering the encoded
polypeptide. Such nucleic acid variations are "silent variations,"
which are one species of conservatively modified variations. Every
nucleic acid sequence herein, which encodes a polypeptide, also
describes every possible silent variation of the nucleic acid. One
of skill will recognize that each codon in a nucleic acid (except
AUG, which is ordinarily the only codon for methionine, and TGG,
which is ordinarily the only codon for tryptophan) can be modified
to yield a functionally identical molecule. Accordingly, each
silent variation of a nucleic acid, which encodes a polypeptide, is
implicit in each described sequence.
[0053] Conservative substitution tables providing functionally
similar amino acids are well known in the art. For example, one
exemplary guideline to select conservative substitutions includes
(original residue followed by exemplary substitution): ala/gly or
ser; arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp;
gly/ala or pro; his/asn or gln; ile/leu or val; leu/ile or val;
lys/arg or gln or glu; met/leu or tyr or ile; phe/met or leu or
tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu. An
alternative exemplary guideline uses the following six groups, each
containing amino acids that are conservative substitutions for one
another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic
acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4)
Arginine (R), Lysine (I); 5) Isoleucine (I), Leucine (L),
Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),
Tryptophan (W); (see also, e.g., Creighton, Proteins, W. H. Freeman
and Company (1984); Schultz and Schimer, Principles of Protein
Structure, Springer-Verlag (1979)). One of skill in the art will
appreciate that the above-identified substitutions are not the only
possible conservative substitutions. For example, for some
purposes, one may regard all charged amino acids as conservative
substitutions for each other whether they are positive or negative.
In addition, individual substitutions, deletions or additions that
alter, add or delete a single amino acid or a small percentage of
amino acids in an encoded sequence can also be considered
"conservatively modified variations."
[0054] Dominant negative forms of TRPM5 may also be used in the
high throughput screening assay to identify compounds that
specifically modulate TRPM5. By "dominant negative" herein is meant
a protein comprising at least one variant TRPM5 monomer that
competes for binding to wildtype subunits such that the protein
retains the ability to form an ion channel but it cannot regulate
the flux of monovalent cations. Depending on the composition of the
ion channel, the degree to which monovalent cation flux is
inhibited will vary.
[0055] The variant TRPM5 proteins of the invention comprise
non-conservative modifications (e.g. substitutions). By
"nonconservative" modification herein is meant a modification in
which the wildtype residue and the mutant residue differ
significantly in one or more physical properties, including
hydrophobicity, charge, size, and shape. For example, modifications
from a polar residue to a nonpolar residue or vice-versa,
modifications from positively charged residues to negatively
charged residues or vice versa, and modifications from large
residues to small residues or vice versa are nonconservative
modifications. For example, substitutions may be made which more
significantly affect: the structure of the polypeptide backbone in
the area of the alteration, for example the alpha-helical or
beta-sheet structure; the charge or hydrophobicity of the molecule
at the target site; or the bulk of the side chain. The
substitutions which in general are expected to produce the greatest
changes in the polypeptide's properties are those in which (a) a
hydrophilic residue, e.g. seryl or threonyl, is substituted for (or
by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,
valyl or alanyl; (b) a cysteine or proline is substituted for (or
by) any other residue; (c) a residue having an electropositive side
chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)
an electronegative residue, e.g. glutamyl or aspartyl; or (d) a
residue having a bulky side chain, e.g. phenylalanine, is
substituted for (or by) one not having a side chain, e.g. glycine.
In one embodiment, the variant TRPM5 proteins of the present
invention have at least one nonconservative modification. In one
embodiment, the variant TRPM5 protein results from translation of a
polynucleotide in which the first 1000 base pairs of the TRPM5 gene
have been deleted. In another embodiment, the variant TRPM5 protein
results from translation of a polynucleotide in which the first
2000 base pairs of the TRPM5 gene have been deleted.
[0056] The variant proteins may be generated, for example, by using
a PDA.TM. system previously described in U.S. Pat. Nos. 6,188,965;
6,296,312; 6,403,312; alanine scanning (see U.S. Pat. No.
5,506,107), gene shuffling (WO 01/25277), site saturation
mutagenesis, mean field, sequence homology, polymerase chain
reaction (PCR) or other methods known to those of skill in the art
that guide the selection of point or deletion mutation sites and
types.
[0057] The cells used in methods of the present invention may be
present in, or extracted from, organisms, may be cells or cell
lines transiently or permanently transfected or transformed with
the appropriate proteins or nucleic acids encoding them, or may be
cells or cell lines that express the required TRPM5 from endogenous
(i.e. not artificially introduced) genes.
[0058] Expression of the TRPM5 protein refers to the translation of
the TRPM5 polypeptide from a TRPM5 gene sequence either from an
endogenous gene or from nucleic acid introduced into a cell. The
term "in situ" where used herein includes all these possibilities.
Thus in situ methods may be performed in a suitably responsive cell
line which expresses the TRPM5 (either as a native channel, or from
a nucleic acid introduced into the cell). The cell line may be in
tissue culture or may be, for example, a cell line xenograft in a
non-human animal subject.
[0059] As used herein, the term "cell membrane" refers to a lipid
bilayer surrounding a biological compartment, and encompasses an
entire cell comprising such a membrane, or a portion of a cell.
[0060] For stable transfection of mammalian cells, depending upon
the expression vector and transfection technique used, only a small
fraction of cells may integrate the foreign DNA into their genome.
In order to identify and select these integrants, a gene that
encodes a selectable marker (e.g., resistance to antibiotics) is
generally introduced into the host cell along with the gene of
interest. Preferred selectable markers include those which confer
resistance to drugs, such as G418, hygromycin and methotrexate. A
nucleic acid encoding a selectable marker can be introduced into a
host cell in the same vector as that encoding TRPM5, or can be
introduced in a separate vector. Cells stably transfected with the
introduced nucleic acid can be identified by drug selection (e.g.,
cells that have incorporated the selectable marker gene will
survive, while the other cells die).
[0061] It should be noted that expression of TRPM5 can also be
controlled by any of a number of inducible promoters known in the
art, such as a tetracycline responsive element, TRE. For example,
TRPM5 can be selectively presented on the cell membrane by
controlled expression using the Tet-on and Tet-off expression
systems provided by Clontech (Gossen, M. and Bujard, H. Proc. Natl.
Acad. Sci. USA 89: 5547-5551 (1992)). In the Tet-on system, gene
expression is activated by the addition of a tetracycline
derivative doxycycline (Dox), whereas in the Tet-off system, gene
expression is turned on by the withdrawal of tetracyline (Tc) or
Dox. Any other inducible mammalian gene expression system may also
be used. Examples include systems using heat shock factors, steroid
hormones, heavy metal ions, phorbol ester and interferons to
conditionally expressing genes in mammalian cells.
[0062] The cell lines used in assays of the invention may be used
to achieve transient expression of TRPM5, or may be stably
transfected with constructs that express a TRPM5 peptide. Means to
generate stably transformed cell lines are well known in the art
and such means may be used here. Examples of cells include, but are
not limited to Chinese Hamster Ovary (CHO) cells, COS-7, HeLa, HEK
293, PC-12, and BAF.
[0063] The level of TRPM5 expression in a cell may be increased by
introducing a TRPM5 nucleic acid into the cells or by causing or
allowing expression from a heterologous nucleic acid encoding
TRPM5. A cell may be used that endogenously expresses TRPM5 without
the introduction of heterologous genes. Such a cell may
endogenously express sufficient levels of TRPM5 for use in the
methods of the invention, or may express only low levels of TRPM5
which require supplementation as described herein.
[0064] The level of TRPM5 expression in a cell may also be
increased by increasing the levels of expression of the endogenous
gene. Endogenous gene activation techniques are known in the art
and include, but are not limited to, the use of viral promoters (WO
93/09222; WO 94/12650 and WO 95/31560) and artificial transcription
factors (Park et al. Nat. Biotech. 21:1208-1214 (2003).
[0065] The level of TRPM5 expression in a cell may be determined by
techniques known in the art, including but not limited to, nucleic
acid hybridization, polymerase chain reaction, RNase protection,
dot blotting, immunocytochemistry and Western blotting.
Alternatively, TRPM5 expression can be measured using a reporter
gene system. Such systems, which include for example red or green
fluorescent protein (see, e.g. Mistili and Spector, Nature
Biotechnology 15:961-964 (1997), allow visualization of the
reporter gene using standard techniques known to those of skill in
the art, for example, fluorescence microscopy. Furthermore, the
ability of TRPM5 to be activated by known positive modulating
compounds, such as thrombin, may be determined following
manipulation of the TRPM5 expressing cells.
[0066] Cells described herein may be cultured in any conventional
nutrient media. The culture conditions, such as media, temperature,
pH and the like, can be selected by the skilled artisan without
undue experimentation. In general, principles, protocols, and
practical techniques for maximizing the productivity of cell
cultures can be found in "Mammalian Cell Biotechnology: a Practical
Approach", M. Butler, ed. JRL Press, (1991) and Sambrook et al,
supra.
Intracellular Calcium Activation
[0067] TRPM5 is a calcium-activated ion channel permeable to
monovalent cations such as sodium. Therefore, in order to observe
channel activity, calcium stores within the cells must first be
activated. There are many methods to activate intracellular calcium
stores and many calcium activating agents are known in the art and
include, but are not limited to thrombin, adenosine triphosphate
(ATP), carbachol, and calcium ionophores (e.g. A23187). While
nanomolar increases in calcium concentration ranges are required
for TRPM5 channel activation, the concentration ranges useful for
the claimed invention are known in the art, e.g., between
10.sup.-10 to 10.sup.-4 M for ATP, however, the precise
concentration may vary depending on a variety of factors including
cell type and time of incubation. The increased calcium
concentration can be confirmed using calcium sensitive dyes, e.g.,
Fluo 3, Fluo 4, or FLIPR calcium. 3 dye and single cell imaging
techniques in conjunction with Fura2.
[0068] As described below, application of suboptimal doses of
calcium activating agents can be used as a secondary screen for
TRPM5 modulating specificity. Test cells are incubated with lower
doses of the calcium activating agents described above, such that a
fluorescent response that is lower than the maximum achievable
response is generated. Generally, the dose is referred to as the
effect concentration or EC.sub.20-30, which relates to the effect
condition where the fluorescent intensity is 20-30% of the maximal
response. As used herein, "EC" refers to effect condition, such
that EC.sub.20 refers to the effect condition where the fluorescent
intensity is 20% of the maximal response is generated. Upon the
addition of a TRPM5-specific activating compound, this low response
will be increased to at, or near, maximal levels of activation.
[0069] Counterscreening techniques are also useful for identifying
TRPM5-specific modulating compounds. The ability to distinguish
compounds specific for TRPM5 inhibition and activation from
compounds that modulate other ion channels, in addition to, or
instead of TRPM5, particularly channels not involved in taste
transduction is vital. As described in greater detail below,
potassium chloride non-specifically activates a number of ion
channels, but not TRPM5. Therefore, KCl activation can be used as a
counterscreen to identify TRPM5-specific modulating compounds.
Fluorescent Dyes
[0070] Voltage sensitive dyes that may be used in the assays and
methods of the invention have been used to address cellular
membrane potentials (Zochowski et al., Biol. Bull. 198:1-21
(2000)). Membrane potential dyes or voltage-sensitive dyes refer to
molecules or combinations of molecules that enter depolarized
cells, bind to intracellular proteins or membranes and exhibit
enhanced fluorescence. These dyes can be used to detect changes in
the activity of an ion channel such as TRPM5, expressed in a cell.
Voltage-sensitive dyes include, but are not limited to, modified
bisoxonol dyes, sodium dyes, potassium dyes and thorium dyes. The
dyes enter cells and bind to intracellular proteins or membranes,
therein exhibiting enhanced fluorescence and red spectral shifts
(Epps et al., Chem. Phys. Lipids 69:137-150 (1994)). Increased
depolarization results in more influx of the anionic dye and thus
an increase in fluorescence.
[0071] The TRPM5 cells of the assay are preloaded with the membrane
potential dyes for 30-240 minutes prior to addition of test
compounds. Preloading refers to the addition of the fluorescent dye
for a period prior to test compound addition during which the dye
enters the cell and binds to intracellular lipophilic moieties.
[0072] In one embodiment, the membrane potential dyes are FMP dyes
available from Molecular Devices (Catalog Nos. R8034, R8123). In
other embodiments, suitable dyes could include dual wavelength
FRET-based dyes such as DiSBAC2, DiSBAC3, and CC-2-DMPE (Invitrogen
Cat. No. K1016). [Chemical Name Pacific Blue.TM.
1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine,
triethylammonium salt]. Cells are typically treated with 1 to 10
.mu.M buffered solutions of the dye for 20 to 60 minutes at
37.degree. C.
[0073] Dyes that measure intracellular calcium levels are also used
to confirm TRPM5 specificity. In one embodiment, the intracellular
calcium dye is the FLIPR Calcium 3 dye available from Molecular
Devices (Part Number: R8091). In other embodiments, suitable dyes
such as Fluo-3, Fluo-4 (Invitrogen (Cat. Numbers F14242 and F14202)
can be used to measure increases in intercellular calcium. Cells
are typically treated with 1 to 10 .mu.M buffered solutions of the
dye for 20 to 60 minutes at 37.degree. C. In some cases it is
necessary to remove the dye solutions from the cells and add fresh
assay buffer before proceeding with the assay.
Assay Detection
[0074] Detecting and recording alterations in the spectral
characteristics of the dye in response to changes in membrane
potential may be performed by any means known to those skilled in
the art. As used herein, a "recording" refers to collecting and/or
storing data obtained from processed fluorescent signals, such as
are obtained in fluorescent imaging analysis.
[0075] In some embodiments, the assays of the present invention are
performed on isolated cells using microscopic imaging to detect
changes in spectral (i.e., fluorescent) properties. In other
embodiments, the assay is performed in a multi-well format and
spectral characteristics are determined using a microplate
reader.
[0076] By "well" it is meant generally a bounded area within a
container, which may be either discrete (e.g., to provide for an
isolated sample) or in communication with one or more other bounded
areas (e.g., to provide for fluid communication between one or more
samples in a well). For example, cells grown on a substrate are
normally contained within a well that may also contain culture
medium for living cells. Substrates can comprise any suitable
material, such as plastic, glass, and the like. Plastic is
conventionally used for maintenance and/or growth of cells in
vitro.
[0077] A "multi-well vessel", as noted above, is an example of a
substrate comprising more than one well in an array. Multi-well
vessels useful in the invention can be of any of a variety of
standard formats (e.g., plates having 2, 4, 6, 24, 96, 384, or
1536, etc., wells), but can also be in a non-standard format (e.g.,
plates having 3, 5, 7, etc., wells).
[0078] A suitable configuration for single cell imaging involves
the use of a microscope equipped with a computer system. One
example of such a configuration, ATTO's Attofluor.RTM.
RatioVision.RTM. real-time digital fluorescence analyzer from Carl
Zeiss, is a completely integrated work station for the analysis of
fluorescent probes in living cells and prepared specimens (ATTO,
Rockville, Md.). The system can observe ions either individually or
simultaneously in combinations limited only by the optical
properties of the probes in use. The standard imaging system is
capable of performing multiple dye experiments such as FMP (for
sodium) combined with GFP (for transfection) in the same cells over
the same period of time. Ratio images and graphical data from
multiple dyes are displayed online.
[0079] When the assays of the invention are performed in a
multi-well format, a suitable device for detecting changes in
spectral qualities of the dyes used is a multi-well microplate
reader. Suitable devices are commercially available, for example,
from Molecular Devices (FLEXstation.RTM. microplate reader and
fluid transfer system or FLIPR.RTM. system), from Hamamatsu (FDSS
6000) and the "VIPR" voltage ion probe reader (Aurora, Bioscience
Corp. CA, USA). The FLIPR-Tetra.TM. is a second generation reader
that provides real-time kinetic cell-based assays using up to 1536
simultaneous liquid transfer systems. All of these systems can be
used with commercially available dyes such as FMP, which excites in
the visible wavelength range.
[0080] Using the FLIPR.RTM. system, the change in fluorescent
intensity is monitored over time and is graphically displayed as
shown, for example in FIGS. 9A-9C. The addition of TRPM5 enhancing
compounds causes an increase in fluorescence, while TRPM5 blocking
compounds block this increase.
[0081] Several commercial fluorescence detectors are available that
can inject liquid into a single well or simultaneously into
multiple wells. These include, but are not limited to, the
Molecular Devices FlexStation (eight wells), BMG NovoStar (two
wells) and Aurora VIPR (eight wells). Typically, these instruments
require 12 to 96 minutes to read a 96-well plate in flash
luminescence or fluorescence mode (1 min/well). An alternative
method is to inject the modulator into all sample wells at the same
time and measure the luminescence in the whole plate by imaging
with a charge-coupled device (CCD) camera, similar to the way that
calcium responses are read by calcium-sensitive fluorescent dyes in
the FLIPR.RTM., FLIPR-384 or FLIPR-Tetra.TM. instruments. Other
fluorescence imaging systems with integrated liquid handling are
expected from other commercial suppliers such as the second
generation LEADSEEKER from Amersham, the Perkin Elmer
CellLux-Cellular Fluorescence Workstation and the Hamamatsu
FDSS6000 System. These instruments can generally be configured to
proper excitation and emission settings to read FMP dye
(540.sub.ex.+-.15 nm, 570.sub.em.+-.15 nm) and calcium dye
(490.sub.ex.+-.15 nm, 530.sub.em.+-.15 nm). The excitation/emission
characteristics differ for each dye, therefore, the instruments are
configured to detect the dye chosen for each assay.
Test Compounds
[0082] Test compounds employed in the screening methods of this
invention include for example, without limitation, synthetic
organic compounds, chemical compounds, naturally occurring
products, polypeptides and peptides, nucleic acids, etc.
[0083] Essentially any chemical compound can be used as a potential
modulator or ligand in the assays of the invention. Most often
compounds dissolved in aqueous or organic (especially dimethyl
sulfoxide- or DMSO-based) solutions are used. The assays are
designed to screen large chemical libraries by automating the assay
steps. The compounds are provided from any convenient source to the
cells. The assays are typically run in parallel (e.g., in
microtiter formats on microtiter plates in robotic assays with
different test compounds in different wells on the same plate). It
will be appreciated that there are many suppliers of chemical
compounds, including ChemDiv (San Diego, Calif.), Sigma-Aldrich
(St. Louis, Mo.), Fluka Chemika-Biochemica-Analytika (Buchs
Switzerland) and the like.
[0084] "Modulating" as used herein includes any effect on the
functional activity of TRPM5. This includes blocking or inhibiting
the activity of the channel in the presence of, or in response to,
an appropriate stimulator. Alternatively, modulators may enhance
the activity of the channel. "Enhance" as used herein, includes any
increase in the functional activity of TRPM5.
[0085] In one embodiment, the high throughput screening methods
involve providing a small organic molecule or peptide library
containing a large number of potential TRPM5 modulators. Such
"chemical libraries" are then screened in one or more assays, as
described herein, to identify those library members (particular
chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual products.
[0086] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0087] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication No. WO
93/20242), random bio-oligomers (e.g., PCT Publication No. WO
92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514),
diversomers such as hydantoins, benzodiazepines and dipeptides
(Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)),
vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.
114:6568 (1992)), nonpeptidal peptidomimetics with glucose
scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218
(1992)), analogous organic syntheses of small compound libraries
(Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates
(Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates
(Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid
libraries (see Ausubel, Berger and Sambrook, all supra), peptide
nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083),
antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology,
14:309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see,
e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No.
5,593,853), small organic molecule libraries (see, e.g.,
isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and
metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat.
Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No.
5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the
like).
[0088] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A Applied
Biosystems, Foster City, Calif.; 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J.; Asinex, Moscow, Russia; Tripos, Inc., St. Louis, Mo.;
ChemStar, Ltd, Moscow, Russia; 3D Pharmaceuticals, Exton, Pa.;
Martek Biosciences, Columbia, Md.; etc.).
[0089] Candidate agents, compounds, drugs, and the like encompass
numerous chemical classes, though typically they are organic
molecules, preferably small organic compounds having a molecular
weight of more than 100 and less than about 10,000 daltons,
preferably, less than about 2000 to 5000 daltons. Candidate
compounds may comprise functional groups necessary for structural
interaction with proteins, particularly hydrogen bonding, and
typically include at least an amine, carbonyl, hydroxyl or carboxyl
group, preferably at least two of the functional chemical groups.
The candidate compounds may comprise cyclical carbon or
heterocyclic structures, and/or aromatic or polyaromatic structures
substituted with one or more of the above functional groups.
Candidate compounds are also found among biomolecules including
peptides, saccharides, fatty acids, steroids, purines, pyrimidines,
derivatives, structural analogs or combinations thereof.
[0090] A variety of other reagents may be included in the screening
assay according to the present invention. Such reagents include,
but are not limited to, salts, solvents, neutral proteins, e.g.
albumin, detergents, etc., which may be used to facilitate optimal
protein-protein binding and/or to reduce non-specific or background
interactions. Examples of solvents include, but are not limited to,
dimethyl sulfoxide (DMSO), ethanol and acetone, and are generally
used at a concentration of less than or equal to 1% (v/v) of the
total assay volume. In addition, reagents that otherwise improve
the efficiency of the assay, such as protease inhibitors,
anti-microbial agents, etc. may be used. Further, the mixture of
components in the method may be added in any order that provides
for the requisite binding.
[0091] The compounds identified using the disclosed assay are
potentially useful as ingredients or flavorants in ingestible
compositions, i.e., foods and beverages as wells as orally
administered medicinals. Compounds that modulate taste perception
can be used alone or in combination as flavorants in foods or
beverages. The amount of such compound(s) will be an amount that
yields the desired degree of modulated taste perception of which
starting concentrations may generally be between 0.1 and 1000
.mu.M.
EXAMPLES
Example 1
Imaging-Based High Throughput Screening Assay Using
Transiently-Transfected Cells
[0092] As described in greater detail below, HEK 293 cells,
transiently transfected with a plasmid bearing the human TRPM5
gene, were used to develop the high throughput screening assay.
Indirect measurement of the changes in Na.sup.+ ions within the HEK
293 cells were made using a FMP dye and stimulation of the cells
using calcium activating agents.
Plasmid Construction
[0093] First strand cDNA was synthesized by Thermoscript RT-PCR
System (Invitrogen) from human small intestine poly A+ RNA (BD
Biosciences) and the full length hTRPM5 was amplified by PCR using
GC Melt (BD Biosciences). The product was PCR purified by Pure Link
PCR Purification (Invitrogen) and inserted into a vector using the
TOPO TA Cloning Kit (Invitrogen). After sequencing, 6 mutations
were found and the mutations were corrected using the Quick Change
Multi Site Directed Mutagenesis Kit (Stratagene) in 2 rounds. Three
mutations were corrected in each round. The full length TRPM5 was
excised from the TOPO TA vector using the EcoRI and NotI
restriction enzymes and ligated in the pENTR 3C vector, which had
also been digested with EcoRI and NotI. The insert and vector bands
were gel extracted and purified using the SNAP Gel Purification Kit
(Invitrogen). Finally, LR Recombination Reaction (Invitrogen) was
used to insert the entry clone into destination vectors of interest
(e.g., pT-Rex-DEST 30, pcDNA-DEST 53, pcDNA 3.2/v5-DEST and pcDNA
6.2/V5-DEST).
Transfection
[0094] 1.0.times.10.sup.6 HEK 293 cells (ATCC) were plated in each
well of a 6-well tissue culture dish overnight. The following day,
cells were transfected with 4 .mu.g of a pcDNA3.2 vector containing
TRPM5 cDNA and 8 .mu.l of Lipofectamine 2000 (Invitrogen),
according to the manufacturer's protocol, and incubated overnight.
The following day, transfected cells were trypsinized and seeded
into 96-well black, clear bottom, poly-D-lysine plates (Corning) at
a density of 70,000 cells/well in a 100 .mu.l volume and incubated
in a 37.degree. C./5% CO.sub.2 incubator overnight.
Fluorescence Microscopy
[0095] To confirm that the HEK transfected cells expressed TRPM5,
cells transiently-transfected with 6 .mu.g of plasmid DNA
expressing TRPM5 (as described above) and grown on Lab TekII
Chamber slides, were evaluated. Control, untransfected cells were
grown in parallel with the transfected cells. The fluorescent
emission of the GFP-TRPM5 expressing cells was detected using the
green detection channel (515-530 nm) of a fluorescent
microscope.
Membrane Potential Assay
[0096] Once the expression of TRPM5 was confirmed in the HEK cells,
100 .mu.l of the Blue or Red FMP dye (Molecular Devices) was added
to, each well of plates seeded with the transiently transfected
cells. The plate was then incubated in a 37.degree. C./5% CO.sub.2
incubator for 1 hour. The plate was read in a FLEXStation
microplate reader (Molecular Devices) with an excitation of 530 nm
and an emission of 565 nm. The fluorescence was monitored for 3
minutes upon exposure of the cells to a calcium activating agent
(carbachol, thrombin peptide or ATP).
Results
[0097] The TRPM5 plasmid was readily expressed as demonstrated by
the appearance of bright green HEK 293 cells that were transfected
with the GFP-TRPM5 plasmid (FIG. 2).
[0098] Demonstration of TRPM5 response to stimuli is shown in FIG.
3A-3C. TRPM5 transfected cells were loaded with FMP dye and then
treated with thrombin (FIG. 3A), carbachol (FIG. 3B), or ATP (FIG.
3C) and monitored for an increase in cellular fluorescence in the
FLEXstation. All three agents generated a strong spike in relative
fluorescence within the first 30 seconds of agonist addition. The
response was transient in nature as well, as fluorescence levels
returned to near baseline levels by approximately 1 minute
post-agonist addition. Mock treated cells produced a low response
in both the ATP and carbachol treated cells, however a high degree
of background fluorescence was observed in the thrombin treated
group. The fluorescence of the thrombin treated cells was greater
than 4-fold over background, therefore the background fluorescence
did not interfere with data interpretation.
[0099] The applicability of the screening method of the invention
to a high throughput format is demonstrated in FIG. 4, where
samples in a 384-well plate were evaluated in a 5 minute assay on
the FLIPR-Tetra.TM. (Molecular Devices). TRPM5-Transfected HEK
cells, 15,000/well, were seeded overnight on poly-D-lysine coated
384 well plates in 20 .mu.l media. Membrane potential dye, 20
.mu.l/well, was added and the plates incubated for 1 hour at
37.degree. C. Plates were placed in a FLIPR-Tetra.TM. and
fluorescence readings were taken using appropriate filters. After
10 seconds, 10 .mu.l of either buffer or a deactivating agent (ATP)
were added to the cells (first addition). At 200 seconds a second
addition of 10 .mu.l of ATP was added to all cells. A strong,
reproducible TRP M5 response was seen in those cells that received
buffer (High Control), while those that were initially stimulated
with ATP became deactivated and failed to respond to a second
addition of ATP (Low Control). There was a >5 fold difference in
peak heights (maximum-minimum values over each peak) between the
High and Low controls, demonstrating that the assay is suitable for
high throughput screening. Furthermore a calculation of Z'* gave a
value of 0.76, greater than the HTS acceptable value of 0.5.
(Z'=1-((3*SDhigh control+3*SD low control)/(High Control-Low
Control)).
Example 2
Imaging-Based High Throughput Screening Assay Using
Stably-Transfected Cells
[0100] Stimulation of TRPM5 was also visible in HEK cells
stably-expressing TRPM5. Following confirmation of TRPM5
expression, the ability to regulate TRPM5 activity was analyzed as
described above.
Plasmid Construction and Transfection
[0101] HEK cells stably-expressing TRPM5 were generated using the
pcDNA 3.2 vector containing hTRPM5 using the technique described
above. Stable clones were generated by transfecting
1.0.times.10.sup.6 HEK 293 cells with 4 .mu.g of pcDNA 3.2-TrpM5 in
a 35 mm tissue culture dish. Two days post-transfection, the cells
were trypsinized and diluted 1:10 and 1:100 in growth medium
containing 1 mg/ml Geneticin (Invitrogen) to select for single
clones. Cells were maintained in this medium until single
individual clones could be isolated and expanded. Upon selection of
individual clones, cells were maintained in medium containing 0.25
mg/ml Geneticin to maintain the selective pressure. Individual
clones were then examined with membrane potential dye in the
FLEXstation or FLIPR.RTM. as described above. Those clones with the
largest fluorescent response to ATP and carbachol were then
selected and examined for further analysis. Selected clones with
the highest EC.sub.50 to ATP and carbachol were then expanded and
used for the high throughput screening assays.
Results
[0102] TRPM5 stably expressed in HEK cells was analyzed for its
ability to respond to different concentrations of several GPCR
agonists. The assay was performed on a FLIPR.RTM. using the
excitation 510-545 nm and emission 565-625 nm filter sets. Assay
plates containing stably expressing HEK cells were loaded with
1.times. Membrane Potential Assay Dye Red (Molecular Devices) for
one hour in a 37.degree. C. and 5% CO.sub.2 incubator. The plates
were then removed from the incubator and equilibrated to room
temperature for 15 minutes before reading on the FLIPR.RTM.. The
plates were read on the FLIPR.RTM. for a total of 3 minutes.
Baseline fluorescence was obtained on the FLIPR.RTM. for 10 seconds
followed by addition of each agonist by the FLIPR.RTM. and read for
an additional 2 minutes and 50 seconds. FIG. 5 shows that two
TRPM5-expressing clones were stimulated by varying concentrations
of the GPCR agonists as evidenced by an increase in the relative
fluorescence of TRPM5-expressing cells compared to sham transfected
cells. The values on the graph represent the difference in the
maximum minus the minimum fluorescence upon agonist addition.
Individual clones are represented by clone number, while the pool
of clones represents the sum of all cells that were resistant to
selection. In all cases, clone 1 gave the strongest response to all
3 agonists (ATP, carbachol, and thrombin peptide, FIGS. 5A-5C,
respectively). Clone 5 and pooled clones generated a lower response
in comparison to clone 1. However, both the clone 5 and pool
responses were a minimum 3-fold higher than fluorescence in
non-transfected cells. Sham, non-transfected cells showed little or
no response at any agonist concentration.
Example 3
High Throughput Screening Assay Using Suboptimal Concentrations of
Calcium-Activating Agents
[0103] Specificity of potential activating compounds may be
identified using suboptimal concentrations of agents that increase
intracellular calcium levels. In this type of assay, rather than
using a high concentration of, for example carbachol, a reduced
concentration is added to TRPM5-expressing cells with or without an
additional test compound. Enhancers of TRPM5 activity are those
test compounds that increase the fluorescent intensity in reduced
carbachol treated cells, to the level seen in cells treated to a
high dose.
[0104] A carbachol dose response curve was generated for the TRPM5
expressing cells so that the suboptimal concentration range could
be determined. Cells expressing TRPM5 were incubated with an
EC.sub.20-EC.sub.30 level of carbachol (0.3 to 1 .mu.M) prior to
addition of test compounds. Mock incubated and EC.sub.100 treated
cells were used as controls. Test compounds that increased the
fluorescent intensity of EC.sub.20-EC.sub.30 treated cells to
levels approaching EC.sub.100 treated cells were classified as
activators of TRPM5.
Example 4
KC.sub.1-Counterscreen for TRPM5 Specificity
[0105] The need for enhanced specificity assays for TRPM5
activation is shown in FIG. 6. Greater than 85,000 compounds were
screened using the above-described high throughput screening assays
and the Gaussian distribution of inhibition values was plotted. As
is visible in the figure, most of the compounds were within the -25
to +25 percent range of inhibition of the control response.
Therefore, in order to identify TRPM5 modulating compounds with
greater specificity, compounds that also act on other ion channels
would have to be removed from the analysis.
[0106] KCl activates a number of ion channels, but not TRPM5.
Therefore, KCl can be used as a counterscreen to identify
modulating compounds specific for TRPM5.
[0107] The ideal blocker would block TRPM5 but not other channels.
The TRPM5 assay is conducted as described in Example 3, utilizing a
membrane potential dye. A test compound is added, and the cells are
then stimulated with ATP to trigger the channel, leading to a dye
response. The process is shown schematically in FIG. 7. The KCl
counterscreen is performed as described in Example 3, with
identical cells, pretreated with the same compound, but the
stimulus was 20 mM KCl, not ATP. KCl stimulated and unstimulated
responses are used as controls. An example of a non-selective
inhibitory compound as identified using the KCl counterscreen is
shown in FIG. 8A. Compound F001344, A3 (structure shown below)
inhibits TRPM5, but also the KCl responses (arrows). An additional
specificity assay utilizes a Ca++ flux dye (Calcium 3 Dye, Part No.
R8091) to determine whether or not the compound interferes with
agonist-induced Ca++ flux response. An example of a non-selective
inhibitory compound as identified by the Ca++ flux assay is shown
in FIG. 8B. Compound F0013488, C13 (structure shown below) inhibits
TRPM5, but also activates the Ca++ flux response (arrows).
##STR00001##
[0108] FIG. 9A shows FLIPR traces in a TRPM5 assay for 4
concentrations of a test compound, compound 1 (structure shown
below). Panel 1 shows dose responsive inhibition of the TRPM5
response. Panels 2 and 3 demonstrate that increasing dose of the
compound does not alter KCl or Ca++ responses. The quantitation of
the these results is shown in FIG. 9B. Examples of two additional
test compounds (compounds 2 and 3) are shown in FIG. 9C, which
shows non-specific inhibition TPRM5, where compound 2 also inhibits
the KCl response and compound 3 inhibits the Ca++ response.
##STR00002##
[0109] The KCl counterscreen is also useful for the identification
of selective TRPM5 enhancing compounds. FIG. 10 shows the selective
enhancement of TRPM5. The counterscreen experiments were performed
as described above in the presence of test compound 4. TRPM5
expressing HEK and CHO cells demonstrated a 131% and 135% maximal
stimulation upon addition of test compound 4, respectively.
Addition of increasing amounts of test compound 4 also resulted in
a dose-dependent increase in TRPM5 activity (FIG. 11). Furthermore,
very strong enhancement is seen at suboptimal (EC.sub.10)
concentrations of ATP agonist using compound 5 (FIG. 12).
Example 5
High Throughput Assay Using a Dominant Negative TRPM5
[0110] Deletion mutants were generated to examine whether
specificity for TRPM5 could be achieved using a dominant negative
form of the channel. The N1000 deletion mutant is a form of mTRPM5
in which the first 1000 base pairs of the gene have been deleted
and the N2000 deletion mutant contains a deletion of the first 2000
base pairs of the gene. The first 2000 base pairs of the gene
correspond to the amino-terminal domain of the mTRPM5 ion channel.
Deletion of this region results in a truncated version of the
protein where the entire amino-terminal domain is removed and the
protein begins with the first transmembrane region of the ion
channel. The deletion mutants were constructed by PCR using primers
designed to amplify the gene with the first 1000 base pairs deleted
and the first 2000 base pairs deleted, respectively. The
experiments described below were performed as previously described
in terms of number of cells used, incubation times and dyes.
[0111] Experiments were performed by comparing transfection of
different ratios of the deletion mutants to wildtype mTRPM5 as
compared to the wildtype mTRPM5 with a null vector. The total
amount of transfected DNA was kept constant at 4 .mu.g.
1.times.10.sup.6 HEK 293 cells were plated in 6 well dishes
overnight. Ratios of deletion mutant mTRPM5/wildtype mTRPM5 and
wildtype mTRPM5/pSV3-neo were then transfected into the HEK 293
cells using Lipofectamine 2000 as indicated in Table 1. One day
following transfection, 15,000 cells/well were plated on 384 plates
and maintained in an incubator overnight. The following day the
cells were loaded with membrane potential dye at 37.degree. C. and
response to A23187 and carbachol dose responses were compared.
TABLE-US-00001 TABLE 1 Transfections in HEK 293 Cells Followed by
TRPM5 Assay Experimental Design Wt-mTrpM5 3.8 .mu.g 3 .mu.g 2 .mu.g
1 .mu.g 0.2 .mu.g 4 .mu.g N1000 0.2 .mu.g 1 .mu.g 2 .mu.g 3 .mu.g
3.8 .mu.g 0 .mu.g pSV3-neo 0.2 .mu.g 1 .mu.g 2 .mu.g 3 .mu.g 3.8
.mu.g 0 .mu.g
[0112] As shown in FIGS. 13A-13B, as the concentration of the
deletion mutant increases, the relative fluorescence in response to
A23187 (FIG. 13A) or carbachol (FIG. 13B) decreases. However, the
decrease is absent in the presence of the null vector (pSV3-neo).
In addition, there is no effect on the calcium response to the
ligands, indicating that the decrease in the membrane potential
response cannot be attributed to altering the calcium
concentration.
[0113] While various embodiments of the present invention have been
described above, it should be understood that they have been
presented by way of example only, and not limitation. It will be
apparent to persons skilled in the relevant art that various
changes in form and detail can be made therein without departing
from the spirit and scope of the invention. Thus, the breadth and
scope of the present invention should not be limited by any of the
above-described exemplary embodiments, but should be defined only
in accordance with the following claims and their equivalents. All
publications, patents and patent applications cited herein are
incorporated by reference in their entirety into the
disclosure.
* * * * *