Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-10-20
2001-10-16
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120, C435S007100, C435S029000, C436S501000, C706S032000, C711S111000
Reexamination Certificate
active
06303291
ABSTRACT:
TABLE OF CONTENTS
1. FIELD OF THE INVENTION
2. BACKGROUND
3. SUMMARY OF THE INVENTION
4. BRIEF DESCRIPTION OF THE FIGURES
5. DETAILED DESCRIPTION
5.1. INTRODUCTION
5.1.1. DRUG ACTION AND BIOLOGICAL STATE
5.1.2. BIOLOGICAL PATHWAYS
5.2. OVERVIEW OF THE METHODS OF THE INVENTION
5.3. ANALYTIC EMBODIMENTS
5.3.1. DRUG RESPONSE REPRESENTATION
5.3.2. INFLECTION DRUG CONCENTRATION
5.3.3. STATISTICAL ANALYSIS
5.3.4. IMPLEMENTATION SYSTEMS AND METHODS
5.4. MEASUREMENT METHODS
5.4.1. MEASUREMENT OF DRUG RESPONSE DATA
5.4.2. TRANSCRIPTIONAL STATE MEASUREMENT
5.4.2.1. MICROARRAYS GENERALLY
5.4.2.2. PREPARING PROBES FOR MICROARRAYS
5.4.2.3. ATTACHING PROBES TO THE SOLID SURFACE
5.4.2.4. TARGET POLYNUCLEOTIDE MOLECULES
5.4.2.5. HYBRIDIZATION TO MICROARRAYS
5.4.2.6. SIGNAL DETECTION AND DATA ANALYSIS
5.4.2.7. OTHER METHODS OF TRANSCRIPTIONAL STATE MEASUREMENT
5.4.3. MEASUREMENTS OF OTHER ASPECTS OF BIOLOGICAL STATE
5.4.3.1. TRANSLATIONAL STATE MEASUREMENTS
5.4.3.2. ACTIVITY STATE MEASUREMENTS
5.4.3.3. MIXED ASPECTS OF BIOLOGICAL STATE
6. EXAMPLES
6.1. IDENTIFICATION OF DUAL PRIMARY TARGETS
6.2. IDENTIFICATION OF A SINGLE PRIMARY TARGET
6.3. ASSESSING THE CONFIDENCE LEVEL OF BIMODALITY
7. REFERENCES CITED
1. FIELD OF THE INVENTION
The field of this invention relates to methods and systems for characterizing the action of drugs in cells. In particular, the methods and systems of the invention relate to determining whether a drug, drug candidate, or some other compound of interest is altering multiple primary targets in a cell, as well as application of these methods to drug discovery.
2. BACKGROUND
The identification of the multiple primary targets of a drug or drug candidate is a problem of great importance in the process of drug discovery. In particular, one of the major difficulties in drug discovery is the identification of compounds that have selective actions on specific primary targets.
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 biological 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. A critical limitation of this approach is that, often, the mechanisms of action, such as the molecular target(s) and cellular pathway(s) affected by the compound, are unknown, and cannot be determined by the screen. Further, the addition may provide little information about the specificity, either in terms of target or pathways, of the drug's effect. 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 of 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.
The use of high-throughput screens is a powerful methodology for identifying drug candidates, however, it has limitations. In particular, the assay provides little or no information about the effects of a compound at the cellular or organismal level. In order to develop lead compounds into successful drugs, it is necessary not only to find compounds which are able to bind well to the primary target which is being screened, but also to insure that the compounds are not simultaneously interacting with other targets within the cell. These effects must be tested by using the drug in a series of cell biologic and whole animal studies to determine toxicity of 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 et al., 1997, “Molecular Targets for Drug Development,” in DeVita et al., Cancer: Principles & Practice of Oncology, 5th Ed., Lippincott-Raven Publishers, Philadelphia, Pa.).
Several gene expression assays are now becoming practicable for quantitating the drug effect on a large fraction of the genes and proteins in a cell culture (see, e.g., Schena et al., 1995
, Science
270:467-470; Lockhart et al., 1996
, Nature Biotechnology
14:1675-1680; Blanchard et al., 1996
, Nature Biotechnology
14:1649; Ashby et al., U.S. Pat. No. 5,569,588, issued Oct. 29, 1996). Raw data from these gene expression assays 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 or a few genes 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. These data in themselves do not inform an investigator about the pathways affected or primary targets of a drug. They do not indicate which effects result from affects on one particular primary target (e.g., the target screened in a high-throughput assay) versus which effects are the result of other primary targets of the drug.
Knowledge of all the primary targets is necessary in understanding efficacy, side-effects, toxicities, possible failures of efficacy, activation of metabolic responses, etc. Further, the identification of all primary targets of a drug can lead to discovery of alternate primary targets suitable to achieve the original therapeutic response. However, without effective methods of analysis, one is left to ad hoc further experimentation to interpret such gene expression results in terms of biological pathways and mechanisms. Systematic procedures for guiding the interpretation of such data and or such experimentation are needed.
Thus, there is a need for improved (e.g., faster and less expensive) systems and methods to identify multiple primary targets in a cell, based on effective interpretation of such data as gene expression data.
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
3. SUMMARY OF THE INVENTION
The present invention relates to methods and systems for testing and confirming the number of primary targets through which a drug or other compound of interest acts on a cell. The invention also relates to methods and systems for identifying the proteins and genes which are affected by each primary target of a drug or other compound of interest. Further, the invention also relates to methods for drug development based on the methods for testing and confirming the number of primary targets through which a drug or drug candidate acts on a cell.
The principles of the methods of the invention involve analyzing measurements of cellular constituents, such as RNA or protein abundances or activities, in response to varying strengths of drug exposure. The responses are analyzed to determine, for each individual measured cellular constituent, a drug concentration at which the individual cellular constituent is said to be activated (i.e., increased in expression or activity), or de-activated (i.e., decreased in expression or activity). The distribution of the so determined drug concentrations is then analyzed to identify clusters or sets of cellular constituents that are activated at a specific drug concentration. Because a drug will generally have different potencies against different primary targets, the identification of such “expression sets” of cellular constituents is the key element that identifies the existence of primary targets of a drug or compound of interest.
The invention is b
Friend Stephen H.
Stoughton Roland
Forman B J
Jones W. Gary
Pennie & Edmonds LLP
Rosetta Inpharmatics, Inc.
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