Methods for drug target screening

Details Methods for drug target screening Methods for drug target screening

2000-11-10

2002-10-08

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S006120, C435S007100

Reexamination Certificate

active

06461807

ABSTRACT:

TABLE OF CONTENTS
1 FIELD OF THE INVENTION . . .
2 BACKGROUND . . .
3 SUMMARY OF THE INVENTION . . .
4 BRIEF DESCRIPTION OF THE DRAWINGS . . .
5 DETAILED DESCRIPTION . . .
5.1 INTRODUCTION . . .
5.2 METHODS FOR DRUG TARGET SCREENING . . .
5.2.1 ALTERNATIVE EMBODIMENTS . . .
5.2.2 APPLICATIONS TO DRUG DISCOVERY . . .
5.3 TRANSCRIPTIONAL STATE EMBODIMENTS . . .
5.3.1 TRANSCRIPT ARRAYS . . .
5.3.2 OTHER METHODS . . .
5.4 MEASUREMENT OF ALTERNATIVE ASPECTS OF BIOLOGICAL STATE . . .
5.5 CELLULAR MODIFICATION METHODS . . .
5.5.1 GENETIC MODIFICATION . . .
5.5.2 OTHER METHODS . . .
5.6 IDENTIFICATION OF GENETIC DRUG TARGETS . . .
6 EXAMPLES . . .
6.1 SYNTHESIS OF LABELED cDNA . . .
6.2 PRODUCTION OF YEAST GENOME MICROARRAYS . . .
6.3 MAKING YEAST DELETION MUTANTS . . .
6.4 PREPARING TRANSCRIPT ARRAY COMPENDIUM . . .
6.5 IDENTIFICATION OF GENETIC TARGET OF A DRUG
6.6 IDENTIFICATION OF CALCINEURIN AS A FK506 TARGET . . .
6.6.1 CYCLOSPORIN AND FK506 . . .
6.6.2 PRODUCTION OF TRANSCRIPT ARRAYS . . .
6.6.3 TARGETS OF CYCLOSPORIN AND FK506 . . .
6.6.4 TARGETS OF CYCLOSPORIN AND FK506 . . .
7 REFERENCES CITED . . .
1 FIELD OF THE INVENTION
The field of this invention relates to methods for characterizing the action of drugs in cells, in particular for finding direct targets of drugs, as well as application of these methods to drug discovery.
2 BACKGROUND
Drug discovery, a process by which bioactive compounds are identified and preliminarily characterized, is a critical step in the development of treatments for human diseases. Two approaches presently dominate the search for new drugs. The first begins with a screen for compounds that have a desired effect on a cell (e.g., induction of apoptosis), or organism (e.g., inhibition of angiogenesis) as measured in a specific assay. Compounds with the desired activity may then be modified to increase potency, stability, or other properties, and the modified compounds retested in the assay. Thus, a compound that acts as an inhibitor of angiogenesis when tested in a mouse tumor model may be identified, and structurally related compounds synthesized and tested in the same assay. One limitation of this approach is that, often, the mechanism of action and molecular target(s) affected by the compound are unknown, and cannot be determined by the screen. In addition, the assay may provide little information about the specificity of the drug's effect. Finally, the number of compounds that can be screened by assaying biological effects on cells or animals is limited.
In contrast, the second approach to drug screening involves testing numerous compounds for a specific effect on a known molecular target, typically a cloned gene sequence or an isolated enzyme or protein. For example, high-throughput assays can be developed in which numerous compounds can be tested for the ability to change the level of transcription from a specific promoter or the binding of identified proteins. Although the use of high-throughput screens is an extremely powerful methodology for identifying drug candidates, it has limitations. A major drawback is that the assay provides little or no information about the effects of a compound at the cellular or organismal level. These effects must be tested by using the drug in a series of cell biologic and whole animal studies to determine toxicity or side effects in vivo. In fact, analysis of the specificity and toxicity studies of candidate drugs can consume a significant fraction of the drug development process (see, e.g., Oliff, A and S. H. Friend, “Molecular Targets for Drug Development,” in DeVita et al.
Cancer: Principles
&
Practice of Oncoloqy
5th Ed. 1997 Lippincott-Raven Publishers, Philadelphia).
Further, raw data from gene expression analysis are often difficult to coherently interpret. Such measurement technologies typically return numerous genes with altered expression in response to a drug, typically 50-100, possibly up to 1,000 or as few as 10. In the typical case, without more analysis, it is not possible to discern cause and effect from such data alone. The fact that one gene among many has an altered expression in a pair of related biological states yields little or no insight into what caused this change and what the effects of this change are. One is left to ad hoc further experimentation to interpret such gene expression results in terms of biological mechanism. Systematic procedures for guiding the interpretation of such data and such further experimentation, at least in the case of drug target screening, are needed.
Thus, there is a need for improved (e.g., faster and less expensive) methods for characterizing activities and targets of drugs based on effective interpretation of expression data. The present invention provides methods for rapidly characterizing the specificity of candidate drugs and identifying their molecular targets.
3 SUMMARY OF THE INVENTION
The present invention provides methods for identifying targets of a drug in a cell by comparing (i) the effects of the drug on a wild-type cell, (ii) the effects on a wild-type cell of modifications to a putative target of the drug, and (iii) the effects of the drug on a wild-type cell which has had the putative target modified. In various embodiments, the effects on the cell can be determined by measuring gene expression, protein abundances, protein activities, or a combination of such measurements. In various embodiments, modifications to a putative target in the cell can be made by modifications to the genes encoding the target, modification to abundances of RNAs encoding the target, modifications to abundances of target proteins, or modifications to activities of the target proteins. The present invention also provides methods for drug development based on the methods for identifying drug targets.
Accordingly, in a first embodiment, this invention provides a method of determining that a specific cellular constituent present in a cell type is a target of a drug, said method comprising: (a) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that is exposed to said drug in comparison to a cell of said cell type that is not exposed to said drug; (b) identifying cellular constituents as perturbed or as not perturbed in a cell of said cell type that both is exposed to said drug and also has said specific cellular constituent modified in comparison to a cell of said cell type that has said specific cellular constituent modified and is not exposed to said drug; (c) identifying cellular constituents that drop out by a method comprising determining each of said cellular constituents that is both identified in step (a) as perturbed and that is also identified in step (b) as either differently perturbed or not perturbed; and (d) ascertaining if each said cellular constituent identified in step (c) to drop out is also identified as perturbed in a cell of said cell type that has said specific cellular constituent modified in comparison to a cell of said cell type that does not have said specific cellular constituent modified, whereby said specific cellular constituent is determined as a target of said drug.
In one aspect of the first embodiment, this invention further provides that said ascertaining step further comprises ascertaining if each said cellular constituent that is identified-in step (c) to drop out and is identified as perturbed in said ascertaining step is also identified as similarly perturbed in step (a). In a second aspect of the first embodiment, this invention further provides that step (c) further comprises excluding said specific cellular constituent from said cellular constituents identified to drop out, and wherein step (d) further comprises excluding said specific cellular constituent from said cellular constituents identified as perturbed.
In a second embodiment, this invention provides a method of determining that a specific gene (or genes) or a product of a specific gene (or products of specific genes) present in a cell type is a target of a drug, said method comprising: (a) identifying genes wh

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