U.S. patent application number 10/624618 was filed with the patent office on 2004-06-24 for double and triple readout assay systems.
Invention is credited to Kim, Tae Kook.
Application Number | 20040121365 10/624618 |
Document ID | / |
Family ID | 22919727 |
Filed Date | 2004-06-24 |
United States Patent
Application |
20040121365 |
Kind Code |
A1 |
Kim, Tae Kook |
June 24, 2004 |
Double and triple readout assay systems
Abstract
The present invention provides assays for identifying compounds
that affect the transcriptional activity of a protein of interest
or affect the stability of the protein of interest. The triple
readout assay system which can be used to identify compounds that
affect the transcriptional activity of a protein of interest uses
three cell lines to control for non-specific effects such as
sequences flanking the inserted gene and cytotoxicity. The double
readout assay system assesses protein stability and uses a fusion
protein of a reporter and the protein of interest. These assay
systems may be particularly useful in identifying compounds that
affect transcription factors and tumor suppressors. In a particular
embodiment, the tumor suppressor p53 is the target protein being
studied.
Inventors: |
Kim, Tae Kook; (Newton,
MA) |
Correspondence
Address: |
Choate, Hall & Stewart
Exchange Place
53 State Street
Boston
MA
02109
US
|
Family ID: |
22919727 |
Appl. No.: |
10/624618 |
Filed: |
July 22, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10624618 |
Jul 22, 2003 |
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09999504 |
Oct 25, 2001 |
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6596506 |
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60243689 |
Oct 27, 2000 |
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Current U.S.
Class: |
435/6.14 ;
435/7.2 |
Current CPC
Class: |
G01N 33/5011 20130101;
C12Q 1/6897 20130101 |
Class at
Publication: |
435/006 ;
435/007.2 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567 |
Claims
What is claimed is:
1. A method of identifying compounds that affect p53 stability, the
method comprising the steps of: providing a cell line transfected
with a construct expressing p53-fused to a reporter protein and a
control reporter protein; contacting a test compound with cells
from the cell line; contacting cells with a DNA damaging agent; and
comparing levels of the p53 fusion protein and the control reporter
protein.
2. A method of identifying compounds that affect p53 stability, the
method comprising the steps of: providing a cell line transfected
with a construct expressing p53-fused to a reporter protein and a
control reporter protein, and transfected to express E6; contacting
a test compound with cells from the cell line; contacting cells
with a DNA damaging agent; and comparing levels of the p53 fusion
protein and the control reporter protein.
3. The method of claim 1 wherein the cell line is derived from a
cell line known to contain active p53 turnover pathways.
4. The method of claim 1 wherein the cell line is derived from a
cell line that has a relatively low steady state level of p53
protein and shows a significant accumulation of p53 protein
following treatment with a DNA damaging agent.
5. The method of claim 1 wherein the DNA damaging agent is a
chemotherapeutic agent.
6. The method of claim 1 wherein the DNA damaging agent is
adriamycin.
7. The method of claim 1 wherein the cell line is an animal cell
line.
8. The method of claim 1 wherein the cell line is a mammalian cell
line.
9. The method of claim 1 wherein the cell line is a human cell
line.
10. The method of claim 1 wherein the cell line is an RKO cell
line.
11. The method of claim 1 wherein the cell line is an MCF7 cell
line.
12. The method of claim 1 wherein the p53 fusion protein is
detectable by an antibody.
13. The method of claim 1 wherein the control reporter protein is
detectable by an antibody.
14. The method of claim 1 wherein the p53 fusion protein is
detectable by a change in absorbance, change in fluorescence, or
radio-immuno assay.
15. The method of claim 1 wherein the control reporter protein is
detectable by a change in absorbance, change in fluorescence, or a
radio-immuno assay
16. The method of claim 1 wherein the p53 fusion protein and the
control reporter protein are translated from a single mRNA
transcript.
17. The method of claim 16 wherein an internal ribosome entry site
is inserted between a gene encoding the p53 fusion protein and a
gene encoding the control reporter protein.
18. The method of claim 1 wherein a gene encoding the p53 fusion
protein and a gene encoding the control reporter protein are
controlled by a same promoter sequence and regulatory
sequences.
19. The method of claim 1 wherein the reporter protein or the
control reporter protein is selected from the group consisting of
firefly luciferase, secreted alkaline phosphatase, enhanced green
fluorescent protein, horseradish peroxidase, beta-galactosidase,
and renilla luciferase.
20. The method of claim 1 wherein the reporter protein or the
control reporter protein is selected from the group consisting of
firefly luciferase and renilla luciferase.
21. The method of claim 1 wherein the p53 fusion protein and the
control reporter protein are transcribed at substantially the same
level.
22. The method of claim 1 wherein the construct comprises a gene
encoding a p53/FL (firefly luciferase) fusion protein, and a gene
encoding RL (renilla luciferase) protein.
23. The method of claim 22 wherein the construct further comprises
a CMV promoter controlling the expression of the gene encoding the
p53/FL fusion protein and the gene encoding the RL protein, and an
internal ribosome entry site.
24. A kit useful in identifying compounds that affect p53 stability
comprising: a cell line with a construct expressing p52-fused to a
reporter protein and a control reporter protein.
25. A method of identifying compounds that affect stability of a
protein of interest, the method comprising steps of: providing a
cell line transfected with a construct expressing a first reporter
protein fused to the protein of interest, and a second control
reporter protein; contacting a test compound with cells from the
cell line; contacting the cells with a DNA damaging agent; and
comparing levels of the first reporter protein and the second
reporter protein.
26. A method of identifying compounds that affect stability of a
protein of interest, the method comprising steps of: providing a
cell line transfected with a construct expressing a first reporter
protein fused to the protein of interest, and a second control
reporter protein; contacting a test compound with cells from the
cell line; contacting cells with a DNA damaging agent; and
comparing levels of the first reporter protein and the second
reporter protein.
Description
RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn. 120 to and is a divisional of U.S. patent application, U.S.
Ser. No. 09/999,504, filed Oct. 25, 2001, which claims priority to
provisional patent application, U.S. Ser. No. 60/243,689, filed
Oct. 27, 2000. The entire contents of each of these applications is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Pharmaceutical screens and biological assays have been used
for decades in the pharmaceutical and biotech industries to
identify lead compounds in the search for new pharmaceutical
agents. In the last decade, the chemist's ability to synthesize
large numbers of chemical compounds in a short amount of time
through techniques such as combinatorial chemistry has greatly
increased (for a recent review of the area of combinatorial
chemistry, please see Geysen et al. Molec. Immunol. 23:709-715,
1986; Houghton et al. Nature 354:84-86, 1991; Frank Tetrahedron
48:9217-9232, 1992; Bunin et al. Proc. Natl. Acad. Sci. USA
91:4708-4712, 1994; Thompson et al. Chem. Rev. 96:555-600, 1996;
Keating et al. Chem. Rev. 97:449-472, 1997; Gennari et al. Liebigs
Ann./Recueil 637-647, 1997; Reddington et al. Science
280:1735-1737, 1998; each of which is incorporated herein by
reference), and it has expanded beyond the capacity of traditional
screening methods. Often, thousands to millions of compounds need
to be screened to identify those having a desired pharmaceutical
property (e.g., anti-neoplastic activity, immunosuppressive
activity, etc.). Many of the currently available screens are
biochemical assay systems in which a compound is added to a
purified or partially purified cell extract to see if it possesses
the desired activity. In contrast to the biochemical assay systems,
currently available cell-based assay systems identify bioactive
molecules that are cell-permeable and work within physiological
environments. However, one of the major drawbacks to cell-based
assay screens is the high false-positive rate resulting from
non-specific effects of the compound within the cell (for examples,
please see Sarver et al. AIDS Res. Hum. Retroviruses 8:659-666,
1992; Witvrouw et al. Antimicrob. Agents Chemother. 36:2628-2633,
1992; each of which is incorporated herein by reference). Given the
fundamental importance of gene regulation in many disease states,
one typical cell-based assay measures the activity of a reporter
gene under the control of a specific reporter. However, inhibition
of reporter gene expression does not necessarily indicate a
specific interference with promoter activity but could reflect a
non-specific inhibition of cellular functions, for example due to
cytotoxicity.
[0003] One particularly important protein in the study of cancer is
the nuclear phosphoprotein, p53. p53 is thought to be mutated in
over 50% of human cancers. Mutations in the p53 gene have been
found in tumors of colon, lung, breast, ovary, bladder, and several
other organs. When mutant forms of the p53 gene are introduced into
primary fibroblasts, these cells become immortalized. The wild type
p53 gene has been shown to suppress the growth of transformed human
cells, but oncogenic forms of p53 lose this suppressor function.
Therefore, the p53 gene has been termed a "tumor suppressor" gene.
Given the role of p53 in tumorigenesis, it has become an important
potential target in the search for new anti-neoplastic agents.
[0004] The wild type p53 may be interfered with functionally. For
example, a transforming viral infection of the cell can interfere
with the p53 protein product. For instance, certain strains of
human papillomavirus (HPV) are transforming and are known to
interfere with the level of p53 protein in the infected cell
because the virus produces a protein, E6, which promotes
degradation of the p53 protein.
[0005] There is also an interest in p53 because p53 protein is
capable of inducing apoptosis in certain cells. In apoptosis, or
"programmed cell death", a series of lethal events for the cell
appear to be generated directly as a result of transcription of
cellular DNA. For example, lymphocytes exposed to glucocorticoids
die by apoptosis. Involution of hormone sensitive tissue such as
breast and prostate that occurs when the trophic hormone is removed
occurs via apoptosis.
[0006] In particular, recent studies have indicated that the
introduction of wild type (non-mutated) p53 into transformed cell
lines that carry a mutant form of p53 induces the cells to undergo
apoptosis with disintegration of nuclear DNA. It is believed that
p53 may suppress tumor development by inducing apoptosis, thus
modulating cell growth.
[0007] Given the importance of p53 in a variety of physiological
and disease states, there is a need for cell-based assays with low
backgrounds that could be used in screening compounds to identify
inhibitors and activators of p53. Moreover, both the rapid increase
of new drug targets through genomics research and the availability
of vast libraries of chemical compounds create an enormous demand
for new technologies which would improve the screening process.
SUMMARY OF THE INVENTION
[0008] The present invention provides assay systems for screening
chemical compounds to identify activators and inhibitors of
proteins of interest (e.g., transcription factors, enzymes, and
tumor suppressors).
[0009] In one aspect, the invention provides a cell-based triple
readout assay system for identifying compounds that affect
transcriptional activity. Three separate cell lines, each
containing a different engineered construct, are used. Two have
been transfected with a construct comprising a reporter gene and a
modulatable transcriptional regulatory sequence known to bind a
selected transcription factor. Each of the two cell lines has a
different reporter gene, and the construct is integrated into the
genome in a different location to control for the effect of
flanking sequences on the transcription of the reporter gene. A
third cell line has been transfected with a construct comprising a
third reporter gene operably linked to a constitutive promoter. The
third cell line is used to assess general cytotoxicity of the test
compound. At least one cell derived from each of the three cell
lines is contacted with the test compound, and the levels of the
reporter genes are assayed and used to determine the specificity of
the test compound on the transcription factor or transcription
factor pathway. In a particularly preferred embodiment, the
transcription factor of interest is p53.
[0010] In another aspect, the invention provides a cell-based
double readout assay system for identifying compounds that affect
protein stability/levels in cells. A fusion protein is created
between a protein of interest and a first reporter protein. The
fusion protein and a second reporter protein are expressed in a
cell line to which the test compound is added. The second reporter
protein is used to control for non-specific effects such as
cytotoxicity. The levels of the two proteins (i.e., the fusion
protein and the second reporter protein) are measured to assess the
specificity of the test compound on the protein of interest. In a
particularly preferred embodiment, the two proteins are translated
from the same mRNA transcript of an engineered DNA construct.
[0011] In a particularly preferred embodiment of this aspect of the
present invention, the protein of interest is p53. One critical
point of regulation of p53 occurs at the protein level. Tumor
mutations that affect its conformation typically increase its
half-life, in part by inhibiting its degradation by the
ubiquitin-proteasome pathway. Consistent with its critical role in
tumor suppression, many oncoproteins including human papillomavirus
E6 oncoprotein target the p53 protein and alter its stability.
[0012] In yet another aspect, the invention provides chemical
inhibitors and activators of p53. Such inhibitors and activators
may preferably be identified and/or characterized using one or both
of the inventive triple and double readout assay systems. In
certain clinical situations, it is desirable to suppress the
cellular effects of p53. For example, p53-dependent apoptosis is
thought to contribute to the toxic side effects of anti-cancer
treatment with chemotherapy. In certain preferred embodiments of
the invention, the p53 inhibitors or activators are provided in the
context of a pharmaceutical composition. In a preferred embodiment,
the inhibitors and activators are small molecules.
[0013] In another aspect, the invention provides kits for
performing the double and triple readout assays. Preferably, an
inventive kit contains all the reagents needed to assay a test
compound for its effect on transcription and/or protein
stability/levels. In a particularly preferred embodiment, a kit to
be used in performing the triple readout assay contains the three
cell lines described above. A kit for performing the double readout
assay preferably contains a cell line stably transfected with a
fusion protein and a second reporter protein. In another preferred
embodiment, an inventive kit comprises DNA constructs to be used in
transfecting a cell line. Preferred inventive kits may also contain
additional reagents such as media for growing the cells, enzyme
substrates (e.g., the substrate of luciferase), DNA damaging
compounds (e.g., adriamycin), human papillomavirus E6 oncoprotein,
growth factors, etc.
Definitions
[0014] Unless indicated otherwise, the terms defined below have the
following meanings:
[0015] "Compound": The term "compound" or "chemical compound" as
used herein can include organometallic compounds, polynucleotides,
oligonucleotides, peptides, proteins, organic compounds, metals,
transitional metal complexes, and small molecules. In a
particularly preferred embodiment, the term compound refers to
small molecules (e.g., preferably, non-peptidic and non-oligomeric)
and excludes peptides, polynucleotides, transition metal complexes,
metals, and organometallic compounds.
[0016] "Constitutive promoter": The term constitutive promoter
refers to a promoter that is always "on". In other words, genes
operably linked to a constitutive promoter are always being
transcribed to produce mRNA.
[0017] "Construct": The term construct refers to any polynucleotide
that has been manipulated by the hand of man. Specifically, the
construct is isolated from other sequences that are found in the
natural state. The construct may be produced by recombinant known
in the art such as the polymerase chain reaction. Preferably, the
polynucleotide contains various elements that are operably linked,
and the construct is introduced into a cell. For example, the
construct may contain a promoter operably linked to a coding
sequence, and the construct may be introduced into a cell to cause
the cell to produce the encoded protein. In a preferred embodiment,
the construct has been created or engineered by the hand of man and
does not occur naturally.
[0018] "Fusion protein": The term "fusion protein" refers to a
protein comprising two or more polypeptides that, although
typically unjoined in their native state, are joined by their
respective amino and carboxyl termini through a peptide linkage to
form a single continuous polypeptide. The two or more polypeptide
components can be either directly joined or indirectly joined
through a peptide linker/spacer. The fusion protein may be
translated by a ribosome from mRNA as a single polypeptide, or the
polypeptides may be joined using synthetic or enzymatic
chemistry.
[0019] "Modulatable transcriptional regulatory sequence": The term
"modulatable transcriptional regulatory sequence" refers to a DNA
sequence capable of regulating the initiation of transcription from
the promoter of the reporter gene by the binding of a protein to
the sequence. The protein preferably binds a regulatory sequence of
the construct in which the promoter, modulatable transcriptional
regulatory sequence, and reporter gene are operably linked, and
thereby the protein either up-regulates or down-regulates the
transcription from the promoter.
[0020] "Operably linked": The term operably linked refers to two
segments of polynucleotide sequence that can affect each other. In
a particularly preferred embodiment, one of the two segments is a
sequence that binds a protein (e.g., polymerase, enhancer, and
transcription factor), and the binding of the protein to the
sequence leads to the transcription of a gene sequence located in
the second segment. In another particularly preferred embodiment,
the binding of a molecule (e.g., nucleic acid, small molecule,
protein, and peptide) to one segment may inhibit or enhance the
binding of another molecule (e.g., nucleic acid, small molecule,
protein, and peptide) to the second segment. Preferably, two
operably linked segments are covalently linked, but any type of
association sufficient to achieve the desired results is considered
to be operably linked in the context of the present invention.
[0021] "p53": The term "p53" as used in the present invention
refers to both the gene and protein form of p53 or any homolog of
p53 or member of the family of p53 genes. The homolog should be at
least 50% homologous to the mouse p53 DNA or protein sequence;
preferably, at least 60% homologous, and most preferably, greater
than 75% homologous. A homolog of p53 may also be identified by its
activity such as its ability to suppress the growth of transformed
cells. In another preferred embodiment, the homolog of p53 is
identified by its location in the genome (e.g., location on the
chromosome). In yet another preferred embodiment, the homolog of
p53 is able to hybridize to the p53 gene under standard
hybridization conditions. p53 may also refer to a fragment of a p53
gene. In certain preferred embodiments, p63, p73, and homologs
thereof are considered to be p53 family members. In other preferred
embodiments, homologs are at least 50% homologous within the
central sequence-specific DNA binding domain, the N-terminal
transactivation domains, and/or the C-terminal oligomerization
domain, more preferably greater than 60% homologous.
[0022] "p53 binding element": The term "p53 binding element" refers
to a sequence of a polynucleotide that binds the p53 protein. In a
preferred embodiment, the p53 binding element is the p53 binding
element found in the p21, bax, or 14-3-3 gene. The p53 binding
element may comprise multiple binding elements (i.e., be
multimerized).
[0023] "Polynucleotide" or "oligonucleotide": Polynucleotide or
oligonucleotide refers to a polymer of nucleotides. Preferably, the
polynucleotide comprises at least three nucleotides, more
preferably it comprises at least 10 nucleotides, and most
preferably it comprises at least 100 nucleotides. The polymer may
include natural nucleosides (i.e., adenosine, thymidine, guanosine,
cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine,
and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine,
2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,
5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,
C5-bromouridine, C5-fluorouridine, C5-iodouridine,
C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,
7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,
O(6)-methylguanine, and 2-thiocytidine), chemically modified bases,
biologically modified bases (e.g., methylated bases), intercalated
bases, modified sugars (e.g., 2'-fluororibose, ribose,
2'-deoxyribose, arabinose, and hexose), or modified phosphate
groups (e.g., phosphorothioates and 5'-N-phosphoramidite
linkages).
[0024] "Protein": According to the present invention, a "protein"
comprises a polymer of amino acid residues linked together by
peptide bonds. The term, as used herein, refers to proteins,
polypeptides, and peptides of any size, structure, or function.
Typically, a protein will be at least three amino acids long.
Peptide may refer to an individual peptide or a collection of
peptides. Inventive peptides preferably contain only natural amino
acids, although non-natural amino acids (i.e., compounds that do
not occur in nature but that can be incorporated into a polypeptide
chain; see, for example, http://www.cco.caltech.edu/.about.da-
dgrp/Unnatstruct.gif, which displays structures of non-natural
amino acids that have been successfully incorporated into
functional ion channels) and/or amino acid analogs as are known in
the art may alternatively be employed. Also, one or more of the
amino acids in an inventive peptide may be modified, for example,
by the addition of a chemical entity such as a carbohydrate group,
a phosphate group, a hydroxyl group, a farnesyl group, an
isofarnesyl group, a fatty acid group, a linker for conjugation,
functionalization, or other modification, etc. A protein may also
be a single molecule or may be a multi-molecular complex. A protein
may be a fragment of a naturally occurring protein or peptide. A
protein may be naturally occurring, recombinant, or synthetic, or
any combination of these.
[0025] "Reporter gene": As used herein, the term "reporter gene"
refers to a gene whose transcript or any other gene product (e.g.,
protein) is detectable. Preferably, the gene product is also
quantifiable. More preferably, the gene product is detectable using
a standard assay. Most preferably, the gene product is detectable
using a standard assay for which the reagents used in the assay are
available in a kit. In certain preferred embodiments, the reporter
gene encodes a fluorescent protein or an enzyme whose activity is
detectable and preferably quantifiable.
[0026] "Small Molecule": As used herein, the term "small molecule"
refers to a non-peptidic, non-oligomeric organic compound either
synthesized in the laboratory or found in nature. Small molecules,
as used herein, can refer to compounds that are "natural
product-like", however, the term "small molecule" is not limited to
"natural product-like" compounds. Rather, a small molecule is
typically characterized in that it contains several carbon-carbon
bonds, and has a molecular weight of less than 1500, although this
characterization is not intended to be limiting for the purposes of
the present invention. Examples of "small molecules" that occur in
nature include, but are not limited to, taxol, dynemicin, and
rapamycin. Examples of "small molecules"that are synthesized in the
laboratory include, but are not limited to, compounds described in
Tan et al., ("Stereoselective Synthesis of over Two Million
Compounds Having Structural Features Both Reminiscent of Natural
Products and Compatible with Miniaturized Cell-Based Assays" J. Am.
Chem. Soc. 120:8565, 1998) and pending application Ser. No.
08/951,930 "Synthesis of Combinatorial Libraries of Compounds
Reminiscent of Natural Products", the entire contents of which are
incorporated herein by reference.
DESCRIPTION OF THE DRAWING
[0027] FIG. 1 shows the transcriptional activation of p53 binding
sites. (A) Six copies of p53 binding sites from p21, bax, and
14-3-3 gene, and (B) multiple copies (3, 6, and 12) of the p53 site
from 14-3-3 gene were cloned upstream of FL (firefly luciferase)
reporter gene. RKO cells were transfected with these plasmids and
reporter activities were analyzed after treatment with adriamycin.
The results are presented as fold induction, compared to the
luciferase activities from cells untreated with adriamycin.
[0028] FIG. 2 shows a reporter plasmid used for p53 transcriptional
activity. Twelve copies of the p53 site from 14-3-3 gene were
cloned upstream of FL (firefly luciferase) or SEAP (secreted
alkaline phosphatase) reporter gene. This reporter plasmid was used
to monitor the transcriptional activity of p53 in cells.
[0029] FIG. 3 shows transcriptional activation of p53 in a selected
stable cell line by DNA damaging agents. (A) FL (firefly
luciferase) and (B) SEAP (secreted alkaline phosphatase) reporter
activities were determined in the selected clone of stably
transfected RKO cells before and after treatment with various DNA
damaging agents (adriamycin, UV radiation, mitomycin C, and
etopside). The results are presented as fold induction, compared to
the reporter activities from untreated cells.
[0030] FIG. 4 shows triple readout for p53 transcriptional
activity. An equal number of each of the three different RKO
reporter cell lines (containing FL and SEAP under the control of
p53, an RL under the control of the CMV promoter) was cultured in
each well of 384-well plates. Chemicals were transferred into the
wells using pin arrays and preincubated with cells before addition
or adriamycin (0.5-1.0 .mu.M). After .about.24 hours, SEAP activity
was determined from culture media using the SEAP Assay System
(Promega). FL and RL activities were determined from cell lysates
using the Dual Luciferase Assay System (Promega). In the screens,
we identified chemicals that specifically affected p53-dependent FL
and SEAP expression, without affecting CMV promoter-driven RL
expression.
[0031] FIG. 5 shows stabilization of p53 protein by DNA damaging
agents and destabilization of p53 protein by E6 oncoprotein. (A)
p53-FL (firefly luciferase) and RL (renilla luciferase) reporter
activities were determined in the selected MCF7 clone before and
after treatment with adriamycin. The results are presented as fold
induction (p53-FL/RL), compared to the luciferase activities from
cells untreated with adriamycin. (B) p53-FL and RL reporter
activities were determined in the selected MCF7 clone in the
absence or presence of expression of E6 oncoprotein. The results
are presented as fold induction (p53-FL/RL), compared to the
luciferase activities from untreated cells.
[0032] FIG. 6 shows a reporter plasmid used for p53 protein
stability and levels. The p53 gene was fused with FL (firefly
luciferase) reporter gene. p53-FL fusion protein was expressed
together with RL (renilla luciferase) reporter protein through IRES
(internal ribosome entry site) under the control of CMV promoter.
This reporter plasmid was used to monitor the stability of p53
protein in cells.
[0033] FIG. 7 shows the double readout assay for p53 protein
stability. A 384-well plate was used to culture MCF7 cells
expressing p53-FL and RL proteins, both under control of the CMV
promoter. Chemicals were transferred into 384-well plates using pin
arrays and preincubated with cells before addition of adriamycin (1
.mu.M). After .about.24 hours, p53-FL and RL activities were
determined from cell lysates using the Dual Luciferase Assay System
(Promega). In the screens, chemical compounds were identified that
specifically affect the stability/levels of p53FL protein, without
affecting those of the RL protein.
[0034] FIG. 8 shows specific chemicals identified from multiple
readouts. (A) The triple and double readout systems were
effectively used to screen specific chemicals in a diverse
collection of 16,320 synthetic chemicals (Chembridge Corporation),
which affect the transcriptional activity and protein stability of
p53, respectively. (B) Shown in an example of the selected
chemicals which affected p53-dependent FL and SEAP expression,
without affecting CMV promoter-driven RL expression.
[0035] FIG. 9 shows double and triple readouts for signaling
pathways as a drug-discovery platform. Using multiple readouts,
these assays can be used to effectively screen for small molecules
specific to p53. Some chemicals may affect transcriptional activity
of p53 by up-regulating or down-regulating p53 protein levels. The
other chemicals, those whose activity was detected in the triple
readout, but not the double readout, presumably modulate
p53transcriptional activity without affecting p53 protein
stability. Thus, these readouts enabled us to select and categorize
chemicals with potent and specific effects on p53 protein stability
and/or transcriptional activity. These assay systems lay out the
basis of systematic screening for specific chemical ligands in many
other signaling pathways.
DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
[0036] The present invention provides systems for identifying
inhibitors and activators of genes and gene products using
cell-based assays with built-in controls to minimize the number of
non-specific hits (i.e., background). The inventive assay systems
eliminate the need for multiple screenings, which can be costly and
time-consuming. The inventive assays determine the effect of a test
compound on transcription and levels/stability of the protein of
interest.
Triple Readout Assay System
[0037] The present invention provides a cell-based triple readout
assay system that involves the measurement of the activity or level
of a reporter gene product under the control of a specific
promoter. Past efforts to assay reporter gene activity were
complicated by problems including non-specific inhibition of
reporter gene expression, which could be due to non-specific
inhibition of cellular functions by cytotoxicity. Before the
present invention, in order to distinguish between "true"
inhibition and other non-specific effects, a number of
time-consuming and costly experiments would need to be performed.
Also, another problem associated with many available reporter gene
assays is that upon integration of the reporter gene into the
genome the transcription of the reporter gene could be influenced
by DNA sequences around which the reporter gene was stably
inserted.
[0038] The inventive triple readout assay overcomes these
limitations and therefore reduces or eliminates the need for
multiple screenings. The inventive cell-based triple readout assay
involves three separate cell lines. Two of the cell lines express a
reporter gene under the control of the same modulatable
transcriptional regulatory sequence. The third cell line expresses
a reporter gene under the control of a constitutive promoter. The
influence of the DNA sequences adjacent to where the reporter gene
was stably inserted is controlled for by the first two cell lines.
General effects of cytotoxicity or inhibition of gene expression
are eliminated using the third cell line that uses a constitutive
promoter.
[0039] In a particularly preferred embodiment, the transcription
factor being studied is p53. In unstressed cells, p53 is present at
low levels and exists in a latent, inactive form. Levels and/or
activity of p53 increase in response to a variety of stimuli
including genotoxic stress. Active p53 protein accumulates in the
nucleus, binding to its responsive DNA elements and inducing the
transcription of its target genes. Under inappropriate growth
conditions, p53 can integrate a variety of signals to the specific
gene regulation for protection of cells against neoplastic
transformation. Consistent with its critical role in tumor
suppression, the majority of the p53 mutations associated with
human tumors, in addition to many oncoproteins, decrease p53's
sequence-specific DNA binding and transcriptional activity. Thus,
small molecules that modulate transcriptional activity of p53 are
important for therapeutic purposes.
[0040] Any cell line may be used in developing the cell lines used
in the inventive triple readout assay. The cells may be animal,
plant, bacterial, or fungal cells. In a preferred embodiment, the
cell line is a mammalian cell line. In a particularly preferred
embodiment, the cell line is a human cell line. The chosen cell
line may satisfy other requirements particular to the protein of
interest and known to one of skill in this art; for example, if one
were interested in regulating p53-dependent transcription, the cell
line should preferably (1) express wild-type p53; (2) show a high
level of induction of p53 after treatment with DNA damaging agents;
and/or (3) have functionally intact p53-mediated growth arrest
pathways. A particularly preferred cell line used in studying p53
is the RKO cell line, a colon cancer cell line. Those of ordinary
skill in this art will readily appreciate the multitude of cell
lines that could be utilized in the assay systems and would be able
to choose a cell line based on multiple factors affecting the gene
or protein being studied in the inventive assay.
[0041] The modulatable transcriptional regulatory sequence is
modulated by the protein of interest or by a protein which is
secondarily or indirectly modulated by the protein of interest. In
some embodiments, the protein of interest directly modulates
transcription by binding to the regulatory sequence directly or
indirectly. In other embodiments, the protein of interest acts
indirectly to modulate transcription. The protein of interest may
act indirectly through a single- or multi-step biological pathway.
The responsiveness of the regulatory sequence may depend on the
presence or absence, or activity level, of the protein of
interest.
[0042] To give but one example, in which p53 is the protein of
interest, the modulatable transcriptional regulatory sequence
comprises at least one p53 binding element. Any p53 binding element
may be operably linked to the reporter gene. In a preferred
embodiment, the p53 binding element shows a high level of induction
of the operably linked reporter gene in response to DNA damage.
Examples of p53 binding elements can be found in the following
three target genes: p21, bax, and 14-3-3. In a particularly
preferred embodiment, the p53 binding element is multimerized. For
example, six to twelve copies of the element may result in nearly
saturated levels of p53 induction. In other embodiments, the p53
may act as a repressor, and down-regulation of the reporter gene is
assayed.
[0043] The reporter gene may encode any gene product (i.e.,
transcript and protein) that is detectable by any known method
(e.g., Northern analysis, Western analysis, radio-immuno assay,
enzymatic assay, change in fluorescence, change in absorbance,
etc.), and preferably the level of the gene product is quantifiable
by any known method (please see, Molecular Cloning: A Laboratory
Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold
Spring Harbor Laboratory Press: 1989); Nucleic Acid Hybridization
(B. D. Hames & S. J. Higgins eds. 1984); the treatise, Methods
in Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods
in Cell and Molecular Biology (Mayer and Walker, eds., Academic
Press, London, 1987); Ausubel et al. Current Protocols in Molecular
Biology (John Wiley & Sons, Inc., New York, 1999);
Transcription and Translation (B. D. Hames & S. J. Higgins eds.
1984); Handbook of Experimental Immunology, Volumes I-IV (D. M.
Weir and C. C. Blackwell, eds., 1986); each of which is
incorporated herein by reference). In a preferred embodiment, the
reporter gene is an enzyme (e.g., kinases, phosphatases,
luciferases, .beta.-galactosidase, reductases, synthases,
horseradish peroxidase, synthetases, etc.). The enzyme itself may
be detectable or the activity of the enzyme may be used to
indirectly measure the level of the enzyme.
[0044] In a particularly preferred embodiment of the present
invention, at least one of the reporter genes of the assay system
encodes a luciferase protein. Luciferases may come from any
organism including the North American firefly, Photinus pyralis,
the sea pansy, Renilla reniformis, and the bacterium, Vibrio
fischeri. A more complete description of luciferases and their use
as transcriptional reporters can be found in De Wet et al.
("Cloning of firefly luciferase cDNA and the expression of active
luciferase in Escherichia coli" Proc. Natl. Acad. Sci. USA 82:7870,
1985; incorporated herein by reference) and Engebrecht et al.
("Measuring gene expression with light" Science 227:1345, 1985;
incorporated herein by reference).
[0045] In another particularly preferred embodiment of the present
invention, at least one of the reporter genes of the assay system
encodes an alkaline phosphatase. The phosphatase gene may be from
any species. A particularly preferred alkaline phosphatase is
secreted alkaline phosphatase (SEAP).
[0046] In another particularly preferred embodiment, the reporter
gene encodes an enhanced green fluorescent protein (EGFP). For more
details in the use of enhanced green fluorescent protein as a
reporter, please see Cinelli et al. (Photochem. Photobiol.
71(6):771-776, 2000; incorporated herein by reference); Kiss-Toth
et al. (J. Immunol. Methods 239(1-2):125-135, 2000; incorporated
herein by reference); and Millet et al. (Toxicol. Sci. 55(1):69-77,
2000; incorporated herein by reference).
Double Readout Assay System
[0047] The level of certain proteins in the cell regulates many
cellular processes. Therefore, the degradation, stability, and
accumulation of proteins are highly controlled in the cell. For
example, one pathway which regulates the degradation of proteins is
the ubiquitin-proteasome pathway. The ability to regulate specific
protein degradation pathways or to affect the stability of specific
proteins would allow for a new approach in treating diseases such
as cancer, autoimmune diseases, and neurological diseases.
[0048] Traditional assays for identifying compounds that effect
protein stability have been hampered by the large number of
non-specific effects. For example, an increase in protein stability
may reflect a non-specific inhibition of cellular functions by the
cytotoxic effect of the compound rather than a specific effect on
the protein's stability.
[0049] To give but one example of a protein whose level is critical
in a cellular process, p53 is a tumor suppressor protein whose
level has been found to be important in tumorigenesis. Mutations in
p53 that affect its conformation typically increase its half-life,
in part by inhibiting its degradation by the ubiquitin-proteasome
pathway. In unstressed cells, p53 is present at low levels and
exists in a latent, inactive form. If the cell is then stressed
(e.g., genotoxic stress), the levels and/or activity of p53
increase. Active p53 protein then accumulates in the nucleus of the
cell, binds to its responsive DNA elements, and induces the
expression of its target genes. Under inappropriate growth
conditions, p53 can integrate a variety of signals for protection
of the cells against neoplastic transformation. Consistent with
p53's critical role in tumor suppression, many oncoproteins target
p53 and its protein stability, including the human papillomavirus
E6 oncoprotein. Therefore, compounds that could modulate p53
protein stability are important for therapeutic purposes.
[0050] In the inventive double readout assay system, the protein of
interest (e.g., p53) is fused to a first reporter protein, for
example using standard recombinant DNA techniques (Ausubel et al.,
Current Protocols in Molecular Biology (John Wiley & Sons,
Inc., New York, 1999); Sambrook, Fritsch, and Maniatis, eds.,
Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., (Cold Spring
Harbor Laboratory Press, 1989); each of which is incorporated
herein by reference). Both the fusion protein and a control
reporter protein are expressed in the cell. The cell(s) are
contacted with various test compounds, and the levels of the fusion
protein and the control reporter protein are measured. The level of
the fusion protein when compared to the level of the control
reporter protein would allow for the identification of compounds
specifically affecting the stability of the protein of interest due
to their effects on the ratio of fusion protein to control. In a
preferred embodiment, at least a 50% change in the level of the
fusion protein when compared to that of the control reporter
protein is considered to be a hit, more preferably there is at
least a 150% change, and most preferably there is at a 500%
change.
[0051] The reporter proteins may be any protein or peptide as
described above in the triple readout assay system. Preferably, the
presence of the protein or peptide can be detected using standard
techniques (e.g., radioimmunoassay, radio-labeling, immunoassay,
assay for enzymatic activity, absorbance, fluorescence,
luminescence, and Western blot). More preferably, the level of each
of the reporter proteins is easily quantifiable using standard
techniques even at low levels. In a preferred embodiment, the
reporter proteins are related so that their levels are easily
comparable.
[0052] In a particularly preferred embodiment, the reporter
proteins are luciferases as described above in the triple readout
assay system. Preferably, the luciferases are distinguishable from
one another if two luciferases are used as the reporter proteins.
In a particularly preferred embodiment, one reporter protein is
firefly luciferase (FL) from Photinus pyralis, and the other is
Renilla luciferase (RL) from Renilla reniformis. The protein levels
may be determined using the Dual-Luciferase.RTM. Assay System
(Promega) (Dual-Luciferase.RTM. Reporter 1000 Assay System,
Technical Manual No. 046, Promega Corp., Madison, Wis., 1999;
incorporated herein by reference).
[0053] Preferably, the genes encoding the fusion protein and the
control reporter protein are transcribed at substantially the same
level. In a particularly preferred embodiment, the two genes of the
proteins are transcribed together to create one transcript. An
internal ribosome entry site (IRES) may be inserted between the two
genes to ensure translation of both encoded proteins. In another
particularly preferred embodiment, the two genes are under the
control of the same promoter sequences and regulatory elements.
[0054] As with the inventive triple readout assay system, any cell
line in which the proteins are expressed may be employed in the
inventive double readout system. Preferably, in the example of p53,
the cell line contains active p53 turnover pathways, the
steady-state level of p53 protein is relatively low, and the level
of p53 protein significantly increases following stress stimuli
(e.g., treatment with chemotherapeutic DNA damaging agents). In a
particularly preferred embodiment, the cell line is MCF7.
[0055] In another particularly preferred embodiment, the cell line
is transfected with the fusion protein gene and the control
reporter gene, and the resulting cells are tested for stabilization
of p53 protein after treatment with adriamycin. Preferably, the
clones show a 5-fold increase in the level of the fusion protein.
More preferably, the clones show a 10-fold increase; and most
preferably, the clones show a 20-fold increase.
[0056] The cell line may be transfected with other genes that
affect p53 degradation. In a particularly preferred embodiment, the
cell line is transfected with the human papillomavirus oncoprotein
E6. Many other oncoproteins may be used in this embodiment of the
invention. As will be appreciated by one of skill in this art, any
gene that may affect the stability/level of the protein being
investigated may be transfected into the cell line.
Test Compounds
[0057] The compounds to be screened in the inventive double and
triple readout assays may be provided by any means known in the
art. The test compounds may be polynucleotides, peptides, proteins,
small molecules, organic molecules, inorganic molecules,
peptidomimetics, antibodies, or other chemical compounds. The
compounds may be prepared by purification or isolation from a
source (e.g., plant, fungus, animal, bacteria, soil sample, etc.),
or by synthesis. The synthesized compounds may be created by more
conventional one-by-one synthetic methods or by combinatorial
chemistry methods through rapid parallel and/or automated
synthesis. The compounds may be provided in crude or pure forms.
The compounds may be natural products or derivatives of natural
products. In another preferred embodiment, the compounds are
provided from the historical compound files of large pharmaceutical
and chemical companies. Preferably, the compounds are provided as
libraries of chemical compounds.
[0058] The compounds are screened by the methods described above to
identify activators and inhibitors of the protein/gene of interest.
To give but one example, the methods may be used to identify
activators and inhibitors of p53. Chemical inhibitors of p53 are
useful in preventing or minimizing the toxic side effects derived
from p53-dependent apoptosis (e.g., those side effects seen in
individuals undergoing chemotherapy and radiation therapy).
Chemical activators of p53 would be useful in developing new
anti-neoplastic agents. In a particularly preferred embodiment, the
compounds may activate or inhibit the protein/gene of interest by
two-fold, more preferably five-fold, and most preferably
ten-fold.
Kits
[0059] The present invention also provides kits for performing the
double and triple readout assays. Preferably, these kits include
all the reagents necessary to assay a test compound or library of
test compounds. The kits may include cell lines, stably transfected
cell lines as described above, media, growth factors, DNA damaging
agents (e.g., bleomycin, adriamycin), human papillomavirus E6
oncoprotein, DNA constructs, enzyme substrate (e.g., substrate of
the luciferase enzyme being assayed for), etc.
[0060] These and other aspects of the present invention will be
further appreciated upon consideration of the following Examples,
which are intended to illustrate certain particular embodiments of
the invention but are not intended to limit its scope, as defined
by the claims.
EXAMPLES
Example 1
[0061] Triple Readout for p53 Transcriptional Activity
[0062] Three different reporter cell lines (one containing FL under
the control of p53, a second containing SEAP under the control of
p53, and a third containing RL under the control of the CMV
promoter) were established by selection with hygromycin (400
.mu.g/mL) after transfection of RKO cells with the reporter plasmid
(see FIG. 2). In the screen, cells derived from the three cell
lines were mixed, and the activities of the three different
reporter proteins were assayed in a single well after incubation
with chemicals as described below.
[0063] The cells lines were maintained in McCoy media supplemented
with antibiotics and 10% heat-inactivated fetal bovine serum (FBS)
to exclude SEAP activity in the serum. After monomerization of
reporter cells with trypsin, an equal number of cells from each of
the three cells lines was mixed in a flask, and 30 .mu.L of the
resulting cell mixture was dispensed in each well of a 384-well
plate (5.times.10.sup.3 cells/well) using an automatic dispenser
(Multidrop 384, Labsystems). After 12-16 hours, test chemical
compounds were introduced into the wells using pin arrays resulting
in a final concentration of .about.5 .mu.g/mL of the test compound
and pre-incubated with the cells for 2 hours followed by the
addition of adriamycin (0.5-1.0 .mu.M). After 24 hours, activities
of reporter proteins were determined using corresponding assay
systems according to the manufacturer's instructions.
[0064] For the SEAP assay, 5 .mu.L of each of the culture media was
transferred to another 384-well plate and incubated with 55 .mu.L
of the SEAP assay mixture (EscAPe SEAP Chemiluminescent Assay
System, Clontech Laboratories) at room temperature for 20 minutes.
SEAP activity was measured using a luminescent reader (Wallac
VICTOR.sup.2, PerkinElmer Life Sciences).
[0065] The Dual Luciferase Reporter Assay System (Promega) was used
for FL and RL assays. In the original 384-well culture plate, the
remaining media was removed, and the cells were washed once with 40
.mu.L of phosphate buffer-saline (PBS). The washed cells were
incubated with Passive Lysis Buffer (PLB, 5 .mu.L/well) for 20
minutes at room temperature with mild-shaking. Then, the Luciferase
Assay Reagent II (LAR II, 25 .mu.L/well) was added directly to the
cell lysates, and FL activity was determined using a luminescent
reader (Wallac VICTOR.sup.2, PerkinElmer Life Sciences). After
measuring the FL activity, Stop & Glo Reagent (25 .mu.L/well)
was added, and the RL activity was determined. In the screens,
chemical compounds were identified that specifically affected
p53-dependent FL and SEAP expression, without affecting CMV
promoter-driven RL expression.
Example 2
[0066] Double Readout Assay for p53 Protein Stability
[0067] A plasmid expressing both the p53/FL fusion gene and RL gene
under the control of a single CMV promoter with an internal
ribosome entry site (IRES) was created (see FIG. 6). Co-expression
of p53/FL and RL proteins by taking advantage of the internal
ribosome entry site makes it possible to normalize the levels of
p53/FL with those of RL in various assay conditions. The reporter
cell line was established by selecting MCF7 cells tranfected with
the IRES expression plasmid using media containing Geneticin (G418)
(700 .mu.g/mL). In the screen, the activities of the two different
reporter proteins, p53/FL and RL, were measured to determine the
effect of the test chemical compounds.
[0068] Cells derived from the reporter cell line were maintained in
DMEM media supplemented with antibiotics and 10% FBS. After
monomerization of reporter cells with trypsin treatment, an equal
number of cells in 30 .mu.L volume was dispensed in each well of a
384-well plate (5.times.10.sup.3 cells/well) using an automatic
dispenser (Multidrop 384, Labsystems). After 12-16 hours, test
chemical compounds were introduced in the wells using pin arrays at
a final concentration of .about.5 .mu.g/mL of the test compound and
pre-incubated with the cells for 2 hours, followed by the addition
of adriamycin (1.0 .mu.M).
[0069] After 24 hours, activities of p53/FL and RL were determined
in the same wells using the Dual Luciferase Reporter Assay System
(Promega). In the 384-well culture plate, the remaining media was
removed from the cells, and the cells were washed once with 40
.mu.L of PBS. The washed cells were then incubated with Passive
Lysis Buffer (PLB, 5 .mu.L/well) for 20 minutes at room temperature
with mild shaking. After the Luciferase Assay Reagent II (LAR II,
25 .mu.L/well) was added directly to the cell lysates, FL activity
was measured using a luminescence reader (Wallac VICTOR.sup.2,
PerkinElmer Life Sciences). The Stop & Glo Reagent (25
.mu.L/well) was then added to determine the RL activity. In the
screen, test chemical compounds were identified that specifically
affect the stabilization of p53-FL without affecting that of RL.
The observed effects of these chemical compounds were confirmed by
immunoblot analyses of endogenous p53 protein and other endogenous
control proteins such as actin.
Other Embodiments
[0070] Those of ordinary skill in the art will readily appreciate
that the foregoing represents merely certain preferred embodiments
of the invention. Various changes and modifications to the
procedures and compositions described above can be made without
departing from the spirit or scope of the present invention, as set
forth in the following claims.
* * * * *
References