Targeted methods of drug screening using co-culture methods

Details Targeted methods of drug screening using co-culture methods Targeted methods of drug screening using co-culture methods

1999-12-03

2003-02-11

Achutamurthy, Ponnathapura (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S004000, C435S006120, C435S007710

Reexamination Certificate

active

06518035

ABSTRACT:

1. FIELD OF THE INVENTION
The present invention relates to methods of screening for molecules, including drugs, that target and inhibit specific proteins or cellular pathways that affect the proliferation, growth or survival of a cell or organism. The methods are based on a co-culture assay, and can be applied to bacteria, yeast,
C. elegans,
and cultured cells, such as mammalian, insect and plant cells.
2. BACKGROUND OF THE INVENTION
2.1. Co-culture Assay
Co-culture experiments have been utilized extensively to identify genes that contribute to the fitness of cells. Giaever et al. (1999, Nat. Genet. 21:278-283) recently showed that from a large pool of cells with distinct genotypes, cells could be identified that had slight differences in fitness when grown in the presence of inhibitors. The genotypes that were responsible for the altered fitness were heterozygous mutations in diploid cells. Thus, this technique was sensitive enough to identify changes in fitness that resulted from the difference between one and two copies of a given gene.
2.2. Identification of Drug Targets
It has been shown that overexpression of a drug's target protein in a cell confers resistance to the cell against the drug. The resistance conferred by overexpression of a target gene has been used as a basis for screening yeast populations transformed with an expression plasmid library for yeast colonies that are resistant to tunicamycin, compactin or ethionine (Rine et al., 1983, Proc. Natl. Acad. Sci. USA 80:6750-6754; Launhardt et al., 1998, Yeast 14:935-942). The ability of a colony to grow after treatment with a drug indicates that the plasmid harbored by the colony directs expression of a protein that confers resistance to the drug, i.e., that the protein is the target of that drug.
2.3. Methods of Drug Discovery
Identification of targets for drug development is a laborious process that has had a low rate of success. Accordingly, there is a need in the art for novel methods for the development of novel drugs and therapies that modulate specific cellular pathways. The present invention provides a method for screening for compounds which specifically inhibit such target pathways. Traditional methods for identifying inhibitors of specific cellular targets typically involve in vitro assays that can directly measure the biochemical activity of an enzyme or the binding of a ligand to a receptor. Alternative methods for identifying inhibitors utilize reporter genes in intact cells that are up- or down-regulated when a specific process has been modulated in the cell by a test compound. While these approaches have been successfully used to identify pharmaceutical lead compounds, they require a considerable amount of lead time and labor to develop prior to screening thousands to hundreds of thousands of chemical compounds or natural products.
Similarly, new antibiotics are desperately needed. The widespread use of antibiotics over the past half century has lead to the emergence of bacterial strains that are resistant to nearly all antibiotics now in use. Thus there is an immediate need to develop fast and efficient methods for producing new antibiotics to combat the increasing number of these antibiotic-resistant strains (Chopra et al., 1997, Antimicrob. Agents Chemother., 37:1563-1571; Cohen, 1992, Science, 257:1050-1055; Kunin, 1993, Ann. Intern. Med., 118:557-561; Neu, 1992, Science, 257:1064-1073; Tenover & Hughes, 1996, JAMA, 275:300-304).
Traditional approaches to antibiotic development have failed to meet these needs. One commonly used approach involves chemical modification of an existing antibiotic to produce a more potent formulation. Another approach involves screening for compounds that target the resistance mechanism of a known antibiotic. Such compounds are then be used in conjunction with the known antibiotic to improve its efficacy. These approaches have been somewhat successful, but are research intensive and such drugs tend to target the same bacterial processes as existing antibiotics, and thus, like the earlier breed of antibiotics, are likely to quickly encounter resistance. A second approach has involved mass screening of compounds for their ability to inhibit bacterial growth. Using microbiological assays, natural products and semisynthetic or synthetic chemicals are screened for their ability to kill or arrest the growth of a target pathogen. At least initially, this approach has the advantage of being simple and relatively inexpensive, and allowing rapid testing of large libraries of compounds. However, the promising lead compounds that emerge from such screens subsequently must be tested for host toxicity. Furthermore, since such screens are result-oriented and blind to mechanism, further studies must be done in order to precisely understand the drug's mechanism of action and to identify its target in the cell.
The genomes of several pathogenic microorganisms, such as
Escherichia coli, Helicobacter pylori,
and
Chlamydia trachomatis
, recently have been sequenced (Blattner et al., 1997, Science 277: 1453; Tomb et al., 1997, Nature, 388: 539-547). The availability of gene sequences encoding all proteins of these bacteria provides an unprecedented opportunity for understanding and manipulating bacterial genomes at the molecular level. A number of genes are known or are suspected to be essential to growth, survival or virulence. Such genes could be ideal targets in screening for novel antibiotics.
The present invention provides screening methods for the identification of drugs or antibiotics that target specific proteins using co-culture methods.
Citation or discussion of a reference herein shall not be construed as an admission that such is prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention provides methods for screening for a molecule that inhibits the expression or activity of a protein encoded by a target gene which affects the fitness of a cell. The methods comprise co-culturing a first cell and a second cell, wherein the first cell has higher expression or activity of the protein encoded by the target gene (“target protein”) than the second cell, and wherein the first cell further comprises and expresses a reporter gene that is substantially not expressed in said second cell and wherein the first cell and second cell are of the same species and cell type, wherein said target protein affects the fitness of the first cell and second cell, wherein the first cell further comprises and expresses a reporter gene that is substantially not expressed in said second cell, and wherein the first cell and second cell are of the same species and cell type; and measuring the activity or amount of protein encoded by the reporter gene, wherein the activity or amount of protein encoded by the reporter gene is indicative of whether the test molecule inhibits the target gene.
In certain specific embodiments, the first and second cells are selected from the group consisting of a bacterial cell, a yeast cell, an insect cell, a mammalian cell and a plant cell.
In an alternative embodiment, the first and second cells can be groups of cells, e.g., individual multicellular organisms of the same species. In a preferred mode of the embodiment, the species is
C. elegans.
In one embodiment, the first cell has wild-type levels of target protein expression or activity and the second cell has reduced levels of target protein expression or activity relative to wild type levels of expression or activity. In an alternative embodiment, the first cell has elevated levels of target protein expression or activity relative to wild type levels of expression or activity and the second cell has wild-type levels of target gene expression or activity. In another alternative embodiment, the first cell has elevated levels of target protein expression or activity relative to wild type levels of expression or activity and the second cell has reduced levels of target protein expression or activity relative to wild type levels of expression or activity.
The reduced level of target

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