Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-07-03
2004-06-01
Fredman, Jeffrey (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S007200, C435S029000, C435S252300, C435S254200, C435S455000
Reexamination Certificate
active
06743583
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for high throughput screening of candidate drug compounds, which finds particular use in the rapid and highly sensitive determination of drug bioactivity and drug target identification.
INTRODUCTION
Traditionally the pharmaceutical industry has relied on two principal methods for drug discovery: 1) in vitro, cell-free biochemical assays; and 2) cell-based assays. In in vitro, cell-free biochemical assays, a massive library of compounds is screened against a given target. Biochemical assays identify compounds of interest by detecting the ability of the compound to alter activity of the target (e.g., by decreasing or increasing an enzymatic activity). The rapidity and efficiency of such screening methods have improved with the advent of automated techniques and advances in computer technology, thus facilitating discovery of important drugs (Palmer, 1996
Nature Biotech.
14:513-515).
However, the effectiveness of this high throughput approach to drug screening depends on the ability to design bioassays to test the activity of the target in the screen. The choice of target is then limited, in part, by the efficacy of designing a suitable bioassay amenable to automation. This also requires significant a priori knowledge of the target. In addition, initial target selection is biased since investigators are often forced to select possible targets based only upon a combination of hearsay and empirical experience. Once a compound having a desired activity has been discovered using a biochemical in vitro cell-free assay, several caveats remain including whether the compound will interact with the target in vivo as it did in the cell-free in vitro assay, whether the compound will enter the cell to reach the target, whether it will be stable in vivo, and whether the compound will specifically affect the desired target without affecting non-target gene products, either specifically or nonspecifically. In addition, this screening method makes it difficult to study natural broths or drug mixtures where drug concentrations may be too low to detect any alteration in target activity.
In a second drug discovery method, the compounds are screened for a desired effect in a cell-based assay. Unlike in vitro biochemical assays, cell-based assays are based upon the ability of a compound(s) to affect some function or aspect of an entire organism and will identify compounds that have biologically significant effects. Conventional cell-based assays, however, also have limitations. For example, suitable in vivo assays must be designed, limiting the choice of targets. Cell-based assays can result in identification of compounds that non-specifically affect the cells. For example, investigators can use cell-based assays to identify compounds that generally affect cell growth, but since growth inhibitors may affect any of a variety of cell structures or enzymes, the investigators cannot immediately and directly identify the specific target of the inhibitory compound. In these cases, the drug is either used without knowledge of the target, or more likely, is found to be of limited use due to nonspecific cytotoxic effects.
A direct approach for identification of drug targets is based on the notion that dramatically increasing dosage of the target gene (e.g., through the use of multicopy plasmids), and thus overexpressing the target gene product, will confer resistance to certain drugs. Thus, target gene products can be identified by constructing a library of cells, each cell carrying a multiple copy plasmid expressing a different candidate target gene, and growing the library of cells in the presence of drug to select for those recombinant cells that, by virtue of increased dosage of the target gene, exhibit increased resistance to the drug. Evidence that overexpression of a gene can alter its sensitivity to a drug has been demonstrated (see, e.g. Barnes et al. (1984)
Mol Cell. Biol.
4:2381-88; Rine et al. (1983)
Proc. Natl. Acad. Sci. USA
80:6750-6754; Rine (1991)
Meth. Enzymol.
194:239-251).
Although gene overexpression using high copy number plasmids is a powerful technique for identifying gene products of interest, this approach has certain limitations. Gene product overexpression can itself be lethal to a host cell or can significantly alter the cell's usual biological pathways and processes (Liu, H. et al. (1992)
Genetics
132:665-673). Moreover, the copy number of the plasmid containing the gene of interest is not easily controlled or predictable (Rine, J. (1991), supra). Furthermore, growth under selective conditions, especially for long periods of time (e.g., days to weeks), encourages selection of mutant cells that may be altered in expression of gene products other than the gene product carried on the plasmid (e.g. second site suppressors). Thus, the target gene identified using this selection process does not always identify the true target of the drug. Finally, such gene overexpression assays can be time-consuming, as the method requires additional screening of drug-resistant clones to identify targets.
Another approach, described by Giaever, G. et al. (1999)
Nature Genet.
21(3):278-283, takes advantage of the observation that the copy number of a gene encoding a drug target directly and sensitively determines the host cell's sensitivity to the drug to the degree that altering the target gene copy number by only one copy (e.g., from two copies to one copy) elicited a detectable phenotypic change (e.g., a change in growth rate or fitness of the strain) in the presence of the drug. Thus, drugs and their target gene products can be identified by, for example, identification of heterozygous deletion strains that exhibit a slower growth rate in the presence of drug relative to wildtype. Furthermore, this genomic approach is parallel and quantitative: all strains, and therefore all targets, are simultaneously measured for drug sensitivities. While this approach is powerful, it requires use of currently costly DNA microarrays as well as upfront determination of drug activities, which, though straightforward, can be time consuming.
Ideally, one would like a highly sensitive method for high-throughput screening of thousands of candidate agents simultaneously, either separately, or in cocktails, which would allow both the determination of whether a candidate agent is active as a drug, and what the target of the drug might be. In short, there is a need in the field for a simple assay that provides information about the drug activity of candidate compounds using a rapid, sensitive and inexpensive global indicator of such activity that can be easily detected. In addition, once this drug activity is determined, it can be used to determination the drug target of the compound. The present invention addresses this problem.
LITERATURE
Targeted selection of recombinant yeast clones encoding drug resistance by dramatically increasing gene dosage is described in Rine, J. et al. (1983)
Proc. Natl. Acad. Sci. USA
80:6750-6754.; Rine, J. (1991)
Methods Enzymol.
194:239-251.
Genomic profiling of drug sensitivities via induced haploinsufficiency is described by Giaever, G. et al. (1999)
Nature Genet.
21(3):278-283.
Analysis of yeast deletion mutants using a molecular bar-coding strategy is described in Shoemaker, D. D. et al. (1996)
Nature Genet.
14:450-456.
Drug target validation and identification of “off-target” secondary drug effects using DNA microarrays is described by Marton, M. J. et al. (1998)
Nature Med.
4:1293-1301.
Functional Classification of the
S. cerevisiae
Genome by Gene Deletion and Parallel Analysis in Winzeler, E. et al. (1999)
Science
285:901-906, see also the Stanford University yeast deletion project worldwide website at stanford.edu/group/yeast_deletion
—
3.html.
The complete sequence of the genome of
S. cerevisiae
is available from the Stanford University Saccharomyces genome worldwide website at stanford.edu/Saccharomyces, and is discussed in Goffeau, A et al. (1996)
Science
274:563-567.
SUMMARY OF THE
Davis Ronald W.
Giaever Guri N.
Bozicevic Field & Francis LLP
Francis Carol L.
Strzelecka Teresa
The Board of Trustees of the Leland Stanford Junior University
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