Note: Descriptions are shown in the official language in which they were submitted.
CA 02287427 1999-10-19
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"HOMOGENEOUS FLUORESCENCE ASSAY FOR MEASURING THE EFFECT OF
COMPOUNDS ON GENE EXPRESSION"
FIELD OF THE INVENTION
The present invention relates to an improved fluorescence reporter assay
system and its use in screening compounds and combinatorial libraries.
BACKGROUND OF THE INVENTION
Screening of compounds for pharmacological activity is dependent on a
means of measuring the effect of the compounds on a pharmacologically relevant
process, such as activation or inhibition of specific gene expression.
Currently,
this may be done by engineering a DNA construct containing the promoter region
of the gene of interest coupled to a readily detectable 'reporter' gene, such
as an
enzyme or fluorescent protein. The construct is transfected into a recipient
cell
and the effect of compounds on expression of the reporter gene is measured by
assaying the level of enzyme or fluorescent protein produced by the cell.
Examples of widely used reporter genes include chloramphenicol
acetyltransferase (CAT), firefly luciferase, beta galactosidase (beta-gal),
secreted
alkaline phosphatase or green fluorescent protein {GFP). Recently, a system
using bacterial beta lactamase was described, in which a cell permeant
fluorescent
substrate is cleaved by the enzyme, thereby inducing a shift in the
fluorescence
emission wavelength (Patent application no. W09630540-AL; Zlokarnik G et al,
Science 279, 84-88, 1998).
The existing methods have proved to be cumbersome and not readily
adaptable to high throughput screening or for screening combinatorial
libraries.
The CAT, beta-gal and luciferase all require cell lysis which precludes their
use as
selectable markers, making selection of stably transfected cell lines a time
consuming process dependent on co-expressed antibiotic resistance markers.
Additionally, CAT assays require either chromatographic detection of reaction
product or indirect antibody detection which is inconvenient for high
throughput
screening, while luciferase has a limited duration of signal generation, which
in
turn limits the time for signal measurement. GFP has sensitivity limitations
due
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to the presence of only one fluorophore per GFP molecule and therefore
requires
large numbers of cells. The GFP molecule is also extremely stable, making it
unsuitable for monitoring decreases in gene expression. GFP is also unsuitable
for srceening compounds that fluoresce or absorb strongly within its
absorption or
emission wavelength. The latter problem also applies to beta lactamase
readouts,
while secreted alkaline phosphatase suffers from high backgrounds due to
endogenous alkaline phosphatase activity. Multiplexing (allowing for the
measurement of expression of more than one gene per cell) is difficult with
all of
the above readouts. Thus, there is a need in the art for an improved process
for
high throughput screening of file compounds and for screening combinatorial
libraries for agents that modify expression of specific target genes.
Many of the disadvantages of the known methods as well as many of the
needs not met by them are addressed by the present invention which, as
described
more fully hereinafter, provides numerous advantages over the known methods.
SUMMARY OF THE INVENTION
This invention relates to a fluorescence reporter assay system.
This invention also relates to a homogeneous fluorescence reporter assay
system.
This invention also relates to a time resolved fluorescence reporter assay
system.
This invention also relates to a reporter assay system that is compatible
with miniaturized assay formats.
This invention also relates to a method of conducting high throughput
screening using the presently invented fluorescence reporter assay system.
This invention also relates to a method of screening combinatorial libraries
using the presently invented fluorescence reporter assay system.
This invention also relates to compounds identified by the presently
invented fluorescence reporter assay system and presently invented methods.
DETAILED DESCRIPTION OF THE INVENTION
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This invention relates to an improved fluorescence reporter screening
system that allows for gene expression in intact cells, has no washing steps
that
might impede use of the assay in nanowell format, has no endogenous activity
in
untransfected cells, allows measurement of expression of more than one gene
per
cell (multiplexing), has a high signal to noise ratio and a long readout
duration.
The presently invented fluorescence reporter assay system uses an
extracellular marker capable of generating an optical readout. It can be
detected
and quantitated without lysing the cells, is unaffected by compound
fluorescence,
allows multiplexing, requires no washing steps, has no endogenous background
in
mammalian cells and has a low background and long duration of appearance.
The presently invented fluorescence reporter assay system comprises a
nucleotide sequence encoding a promoter/enhancer region, an extracellular
protein
(defined herein as either a cell surface or secreted protein) containing
sequences
coding for one or more binding partners or other molecules capable of
generating
an optical signal; a recipient cell; accessory molecules necessary to produce
and/or enhance the optical signal and an optical detection apparatus.
Preferably,
the extracellular protein is xenogeneic (defined herein as a protein that is
not
naturally produced by the recipient cell type). The nucleic acid construct is
prepared such that expression of the cell surface or secreted protein is
driven by
the promoter/enhancer region, the latter being derived from a
pharmacologically
relevant protein target. An example of such a target is the pro-alpha 1 chain
of
Type 1 collagen which is specifically expressed in osteoblasts and is linked
to
formation of new bone matrix (Identification of a minimal sequence of the
mouse
pro-alpha 1 (I) collagen promoter that confers high-level osteoblast
expression in
transgenic mice and that binds a protein selectively present in osteoblasts.
Rossert-JA; Chen-SS; Eberspaecher-H; Smith-CN; de-Crombrugghe-B Proc-
Natl-Acad-Sci-USA. 1996; 93(3): 1027-31 ). The extracellular protein is
constructed with the optical signal-generating sequences inserted in frame
within
or appended to the coding sequence and carries additional sequences specifying
membrane targeting or secretion (Duffaud GD et al, in: Current Topics in
Membranes and Transport, Chapter 2 Vol. 24, Academic Press NY, 1985). The
optical signal may be generated directly, for example, by inserting sequences
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encoding for firefly luciferase. Preferably, the optical signal may be
generated
indirectly, for example, by providing binding sites for exogenous binding
partners
that contain optically active constituents such as fluorescent dyes or enzymes
capable of generating optically detectable products. In either case, the use
of
polypeptide sequences that are not naturally present on or produced by the
recipient cell (xenogeneic sequences) is preferred to minimize background. An
example of a suitable extracellular protein with an indirect optical readout
is the
epidermal growth factor (EGF) receptor, with two or more defined epitope tags
inserted into the extracellular domain. A defined epitope tag (DET) is
preferably
a short non-mammalian polypeptide sequence that is recognized by a specific
monoclonal antibody, such as the 11 amino acid epitope from Human
Immunodeficiency Virus Type 1 (HIV-I) envelope protein gp120 (or gp160) that
is recognized by the monoclonal antibody 178.1 (see, e.g., Thiriart et al., J.
Immunol., 143: I832-1836 (1989)), which was prepared by immunization of mice
with a yeast-expressed HIV-1 gp 160 molecule from strain BH10 (Ratner et al.,
Nature, 313: 277-284 (1985)). Another example of a DET is the commercially
available FLAG tagging system from Eastman Kodak, Rochester NY. In this
example, each molecule of the EGF receptor carries tandem copies of each of
two
DETs and the copies may be repeated within the molecule to afford additional
binding sites. Alternatively, the DET sequences may be inserted into a
secreted
protein such as human growth hormone. It will be appreciated that there are
many
variations for implementation of such an extracellular protein. All such
variations
are included within the scope of this invention.
Thus, the minimal essential features of the construct are a
promoter/enhancer region, a membrane or secretion-targeting signal
sequence(s),
a transmembrane or membrane insertion region for cell surface proteins and a
polypeptide that is directly or indirectly capable of generating an optically
readable signal.
An additional, preferred, component of this invention is the provision of
accessory molecules for generation and or enhancement of the optical signal.
Such molecules include but are not limited to antibodies labeled with
fluorescent
dyes, substrates for light-generating enzymes or color-generating reagents for
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enzymes. In the preferred example, two exogenous binding partners are
provided,
each of which recognizes one of the DET sequences that was inserted into the
extracellular domain of the extracellular protein. Alternatively, one or both
exogenous binding partners may recognize spatially discrete regions of a
naturally
occurring extracellular protein used in the above promoter/enhancer construct
and
which is not present on or produced by the chosen recipient cell, such that
when
bound, the two exogenous binding partners are separated by a distance
comparable to or less than the energy transfer radius for the chosen
fluorophores.
Examples of such exogenous binding partners are monoclonal antibodies to
DETs. These binding partners are each labelled, preferably with fluorescent
dyes
capable of resonance energy transfer. Preferably, one of the dyes is capable
of
time resolved fluorescence (luminescence). Examples of such dyes are terbium
chelate/cryptates (Selvin P R and Hearst (J E. Proc. Natl. Acad. Sci. 91:
10024-
10028, 1994) and tetramethyl rhodamine (Molecular Probes, Eugene, OR).
Methods for labelling antibodies with fluorescent dyes are well known in the
art.
An additionally preferred component of this invention is a recipient cell.
This cell may be of eukaryotic or prokaryotic origin and in the former case
may
be somatic or germline. The preferred cell type is mammalian, preferably a
human cell line that can be maintained in culture. An example of such a cell
line
is the human embryonic kidney cell line HEK293 which is available from the
American Type Culture Collection. Methods for transfection of mammalian cells
with nucleic acid constructs are well known to one skilled in the art.
Depending
on the nature of the promoter, it may be necessary to stimulate the cells with
a
hormone or other substance to initiate expresion of the extracellular protein.
An additionally preferred component of this invention is a device capable
of detecting and measuring the optical signal. Such a device should produce a
numeric or graphical output that is related to the intensity and wavelength of
the
optical signal. It should be sensitive to the wavelength of the optical signal
and
may contain a source of electromagnetic radiation to assist in the generation
of the
optical signal, for example, an excitation source for fluorophores. It may
also
incorporate a time delay mechanism such that excitation radiation may be
applied
for a discrete time interval and measurement may be initiated at a specific
time
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after termination of the excitation. Examples of such devices are
fluorimeters,
fluorescence correlation spectrometers, luminometers, spectrophotometers, CCD
cameras, photon counters and fluorescence activated cell sorters. Such devices
are readily available from commercial sources.
In utilizing the presently invented fluorescence reporter assay system, the
polynucleotide construct is transfected into the chosen recipient cell. If the
promoter is active in the cellular environment, it drives expression of the
extracellular protein, which leads to display on the cell surface or secretion
of the
protein carrying the tandem binding partners. Cells that express high levels
of a
construct utilizing a cell surface protein are selected by addition of the
labelled
exogenous binding partners, followed by fluorescence activated cell sorting.
Cells
that produce secreted markers are cloned by limiting dilution and selected by
monitoring of the culture media for the optical signal.
To detect the optical signal, accessory molecules are added to the culture
media. For example, monoclonal anti-DET antibodies labeled with fluorescent
dyes that are capable of resonance energy transfer are added. Upon binding to
their respective recognition sites on the extracellular protein, the
fluorescent dyes
are brought within their critical radii for resonance energy transfer and
emission
from the donor or acceptor dye is used to select cells that express high
levels of
the extracellular protein. These cells may be subjected to iterative
selections as
above to generate stable transfectants. The optical signal is detected by
using a
suitable detection device as above.
Once a stably transfected cell population has been obtained, the cells are
used for screening compound collections or combinatorial libraries. Cells are
plated in test wells in groups, preferably of more than 100 cells, to average
out
cell-to cell variations in reporter expression levels. Test compounds are
added
with or without an inducing hormone or other substance as appropriate for the
promoter region of interest. After a suitable interval to allow for optimum
expression and surface display or secretion of the reporter, accessory
reagents are
added to each well. The optical signal is quantitated using a suitable
detection
device.
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For multiplexed assays, cells are co-transfected with two or more
constructs of the same extracellular protein but with different promoter
regions
driving each construct. For each construct, the optical signal-generating
sequences are chosen to give distinct signals. For example, peptide binding
partners are chosen such that they interact with distinct exogenous binding
partners. In this example, cells may be transfected with promoter 1 linked to
DETI-DET2 and with promoter 2 linked to DETI-DET3 or DET3-DET4. The
accessory molecules are each labelled with a fluorophore of distinct
absorption or
emission wavelength. For example, an antibody to DET1 could be labeled with
terbium chelate/cryptate (emission 545 nm), anti-DET2 with TMR (excitation
550 nm emission 575 nm) and anti-DET3 with samarium chelate/cryptate
(emission 655 nm) and DET4 with Cy5 (Amersham, Arlington Heights IL)
(excitation 650nm emission 670nm). Stable transfectants are selected as above
for expression of both constructs. Screening of test compounds is done as
above
but fluorescence emission is monitored both at 575 nm and 670 nm, so that
differential effects of compounds on one or other promoter activity can be
measured.
The advantages of this process compared with existing reporter gene
systems are 1 ) it allows the use of time resolving dyes which are insensitive
to
fluorescence of test compounds or cellular components, 2) it allows
unrestricted
choice of fluorophores for multiplexing and avoidance of quenching by test
compounds, 3) it does not require cell lysis so the selection of stable
transfectants
is simplified, 4) the signal is present for a long duration so that time-
dependent
imaging can be used for detection 5) multiple fluorophores can be conjugated
to
each accessory molecule and multiple binding partner sequences can be added to
the extracellular protein for increased sensitivity 6) no washing steps are
needed
which simplifies high throughput screening 7) the binding partners can be
chosen
from microbial, viral, fungal, insect or artificial sources so that no such
molecules
occur endogenously on mammalian cells 8) signal to noise ratios are enhanced
by
the requirement that two specific binding events must occur in close proximity
to
one another for signal generation, and by the use of time resolving dyes.
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Without further elaboration, it is believed that one skilled in the art can,
using the preceding description, utilize the present invention to its fullest
extent.
While the preferred embodiments of the invention are illustrated by the above,
it
is to be understood that the invention is not limited to the precise
instructions
herein disclosed and that the right to all modifications coming within the
scope of
the following claims is reserved.
_g_
