Double and triple readout assay systems

Details Double and triple readout assay systems Double and triple readout assay systems

2001-10-25

2003-07-22

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving viable micro-organism

C435S325000, C435S320100, C435S366000, C435S006120, C435S007210, C435S008000, C536S024100

Reexamination Certificate

active

06596506

ABSTRACT:

BACKGROUND OF THE INVENTION
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.
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.
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.
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.
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.
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
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).
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.
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.
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.
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.
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