Exploiting genomics in the search for new drugs

Abstract

The cellular effects of potentially therapeutic compounds are characterized in mammalian cells and yeast. In the latter case the effects can be characterized on a genome-wide scale by monitoring changes in messenger RNA levels in treated cells with high-density oligonucleotide probe arrays.

We claim:  
     
         1 . A set of at least two probes, wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is down-regulated in response to both compound 52 and flavopiridol.  
     
     
         2 . A set of at least two probes, wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is up-regulated in response to both compound 52 and flavopiridol.  
     
     
         3 . A set of at least two probes, wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is down-regulated in response to compound 52 but not to flavopiridol.  
     
     
         4 . A set of at least two probes, wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is up-regulated in response to compound 52 but not to flavopiridol.  
     
     
         5 . A set of at least two probes, wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is down-regulated in response to flavopiridol but not to compound 52.  
     
     
         6 . A set of at least two probes, wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is up-regulated in response to flavopiridol but not to compound 52.  
     
     
         7 . The set of  claim 1  wherein-the gene is also down-regulated in cdc28-4 mutants.  
     
     
         8 . The set of  claim 2  wherein the gene is also up-regulated in cdc28-4 mutants.  
     
     
         9 . The set of  claim 1  wherein the gene is up-regulated in cdc28-4 mutants.  
     
     
         10 . The set of  claim 2  wherein the gene is down-regulated in cdc28-4 mutants.  
     
     
         11 . A set of at least two probes, wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is down-regulated in cdc28-4 mutants.  
     
     
         12 . A set of at least two probes, wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is down-regulated or up-regulated in response to both compound 52 and flavopiridol.  
     
     
         13 . A set of at least two probes, wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is up-regulated or down-regulated in cdc28-4 mutants.  
     
     
         14 . The set of  claim 13  wherein the up-regulation or down-regulation is at least two fold as compared to wild-type.  
     
     
         15 . A set of at least two probes, wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is up-regulated in cdc28-4 mutants.  
     
     
         16 . The set of  claim 13  wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is not up-regulated or down-regulated in response to compound 52 or flavopiridol.  
     
     
         17 . The set of  claim 13  wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is not up-regulated or down-regulated in response to compound 52.  
     
     
         18 . The set of  claim 13  wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is not up-regulated or down-regulated in response to flavopiridol.  
     
     
         19 . The set of  claim 13  wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is up-regulated or down-regulated in response to compound 52.  
     
     
         20 . The set of  claim 13  wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is up-regulated or down-regulated in response to flavopiridol.  
     
     
         21 . The set of  claim 13  wherein each of said probes comprises a segment of the nucleotide sequence of a gene which is up-regulated or down-regulated in response to both flavopiridol and compound 52.  
     
     
         22 . The set of any of the preceding claims wherein the genes are yeast genes.  
     
     
         23 . The set of  claim 22  wherein the yeast is  Saccharomyces cerevisiae.    
     
     
         24 . The set of any of the preceding claims wherein the genes are human genes.  
     
     
         25 . The set of any of the preceding claims wherein the probes are immobilized on a solid support.  
     
     
         26 . The set of any of the preceding claims wherein the probes are immobilized on an array.  
     
     
         27 . The set of any of the preceding claims wherein up-regulation or down-regulation is determined by a difference of at least three-fold from a control.  
     
     
         28 . The set of any of the preceding claims which comprises at least 3 probes.  
     
     
         29 . The set of any of the preceding claims which comprises at least 5 probes.  
     
     
         30 . The set of any of the preceding claims which comprises at least 7 probes.  
     
     
         31 . The set of any of the preceding claims which comprises at least 9 probes.  
     
     
         32 . The set of any of the preceding claims which comprises at least 11 probes.  
     
     
         33 . The set of any of the preceding claims which comprises at least 20 probes.  
     
     
         34 . The set of any of the preceding claims which comprises at least 30 probes.  
     
     
         35 . The set of any of claims  1 - 31  which consists of less than 10 probes.  
     
     
         36 . The set of any of claims  1 - 32  which comprises less than 20 probes.  
     
     
         37 . The set of any of claims  1 - 33  which consists of less than 30 probes.  
     
     
         38 . The set of any of the preceding claims which consists of less than 100 probes.  
     
     
         39 . The set of any of the preceding claims which consists of less than 1000 probes.  
     
     
         40 . The set of any of the preceding claims which comprises less than 10000 probes.  
     
     
         41 . The set of  claim 25  wherein at least 10% of probes on the solid support comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         42 . The set of  claim 25  wherein at least 20% of probes on the solid support comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         43 . The set of  claim 25  wherein at least 40% of probes on the solid support comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         44 . The set of  claim 25  wherein at least 60% of probes on the solid support comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         45 . The set of  claim 25  wherein at least 80% of probes on the solid support comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         46 . The set of  claim 25  wherein at least 90% of probes on the solid support comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         47 . The set of  claim 26  wherein at least 10% of probes on the array comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         48 . The set of  claim 26  wherein at least 20% of probes on the array comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         49 . The set of  claim 26  wherein at least 40% of probes on the array comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         50 . The set of  claim 26  wherein at least 60% of probes on the array comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         51 . The set of  claim 26  wherein at least 80% of probes on the array comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         52 . The set of  claim 26  wherein at least 90% of probes on the array comprise segments of genes whose regulation is affected by compound 52, flavopiridol, or cdc28-4.  
     
     
         53 . A method of comparing the specificity of drugs, comprising: 
 contacting a first drug with a first population of cells and a second drug with a second population of said cells;    preparing a transcription indicator from each of the first and the second populations of cells, wherein a transcription indicator is selected from the group consisting of cellular RNA, cellular mRNA, cRNA and cDNA;    preparing a transcription indicator from a third population of said cells which is not contacted with a drug;    hybridizing the transcription indicators to oligonucleotide arrays to form a pattern of hybridization for each of said populations of cells;    comparing each of the first and the second populations' patterns of hybridization to the third population's pattern of hybridization to identify changes induced by the first and the second drugs;    comparing changes induced by the first and second drugs, wherein a drug which effects more changes is less specific than a drug which effects fewer changes.    
     
     
         54 . The method of  claim 53  wherein the first drug is flavopiridol.  
     
     
         55 . The method of  claim 53  wherein the first drug is compound 52.  
     
     
         56 . The method of  claim 53  wherein the cells are yeast cells.  
     
     
         57 . The method of  claim 53  wherein the first and second drugs affect a common target protein.  
     
     
         58 . The method of  claim 53  wherein the cells are mammalian cells.  
     
     
         59 . The method of  claim 53  wherein the array comprises at least 1000 oligonucleotides of distinct sequence.  
     
     
         60 . The method of  claim 53  wherein the array comprises at least 6000 oligonucleotides of distinct sequence.  
     
     
         61 . The method of  claim 53  wherein the first drug has a known beneficial effect and the second drug is identified as useful if it induces a similar pattern of changes.  
     
     
         62 . The method of  claim 53  wherein the drug is a kinase inhibitor.  
     
     
         63 . A method of comparing the effects of a drug to the effects of a mutation, comprising: 
 contacting a drug with a first population of cells;    preparing a transcription indicator from the first population of cells, wherein a transcription indicator is selected from the group consisting of cellular RNA, cellular mRNA, cRNA and cDNA;    preparing a transcription indicator from a second population of cells which population is not contacted with a drug, wherein the second population of cells carry a mutation in a gene of interest relative to the first population of cells;    preparing a transcription indicator from a third population of cells which is not contacted with a drug and which does not carry the mutation;    hybridizing the transcription indicators to oligonucleotide arrays; to form a pattern of hybridization for each of said populations of cells;    comparing each of the first and the second populations' patterns of hybridization to the third population's pattern of hybridization to identify changes caused by the drug and the mutation;    comparing the changes caused by the drug to those caused by the mutation; wherein a drug and a mutation which affect hybridization to one or more common oligonucleotides identifies the gene of interest as a candidate target of the drug; wherein a drug which affects hybridization to both common oligonucleotides and unique oligonucleotides identifies the drug as affecting targets other than the gene.    
     
     
         64 . The method of  claim 63  wherein the drug is flavopiridol.  
     
     
         65 . The method of  claim 63  wherein the drug is compound 52.  
     
     
         66 . The method of  claim 63  wherein the mutation is in a kinase.  
     
     
         67 . The method of  claim 63  wherein the mutaion is in the CDC28 gene.  
     
     
         68 . The method of  claim 63  wherein the cells are yeast cells.  
     
     
         69 . The method of  claim 63  wherein the cells are mammalian cells.  
     
     
         70 . The method of  claim 63  wherein the array comprises at least 1000 oligonucleotides of distinct sequence.  
     
     
         71 . The method of  claim 63  wherein the array comprises at least 6000 oligonucleotides of distinct sequence.  
     
     
         72 . A method of comparing the specificity of drugs, comprising: 
 comparing changes in expression induced by a first drug to those induced by a second drug, wherein a drug which effects more changes is less specific than a drug which effects fewer changes, wherein the changes are determined the process of: 
 contacting the first drug with a first population of cells and the second drug with a second population of said cells;  
 preparing a transcription indicator from each of the first and the second populations of cells, wherein a transcription indicator is selected from the group consisting of cellular RNA, cellular mRNA, cRNA and cDNA;  
 preparing a transcription indicator from a third population of said cells which is not contacted with a drug;  
 hybridizing the transcription indicators to oligonucleotide arrays to form a pattern of hybridization for each of said populations of cells; and  
 comparing a first and a second populations' patterns of hybridization to a third population's pattern of hybridization to identify changes induced by the first and the second drugs.  
   
     
     
         73 . The method of  claim 72  wherein the drug is flavopiridol  
     
     
         74 . The method of  claim 72  wherein the drug is compound 52.  
     
     
         75 . A method of comparing the effects of a drug to the effects of a mutation, comprising: 
 comparing changes in expression caused by a drug to those caused by a mutation, wherein the changes in expression are determined by the process of: 
 contacting the drug with a first population of cells;  
 preparing a transcription indicator from the first population of cells, wherein a transcription indicator is selected from the group consisting of cellular RNA, cellular mRNA, cRNA and cDNA;  
 preparing a transcription indicator from a second population of cells which population is not contacted with a drug, wherein the second population of cells carry the mutation in a gene of interest relative to the first population of cells;  
 preparing a transcription indicator from a third population of cells which is not contacted with a drug and which does not carry the mutation;  
 hybridizing the transcription indicators to oligonucleotide arrays to form a pattern of hybridization for each of said populations of cells;  
 comparing each of the first and the second populations' patterns of hybridization to the third population's pattern of hybridization to identify changes caused by the drug and the mutation;  
   wherein a drug and a mutation which affect hybridization to one or more common oligonucleotides identifies the gene of interest as a candidate target of the drug; wherein a drug which affects hybridization to both common oligonucleotides and unique oligonucleotides identifies the drug as affecting targets other than the gene.    
     
     
         76 . The method of  claim 75  wherein the drug is flavopiridol.  
     
     
         77 . The method of  claim 75  wherein the drug is compound 52.  
     
     
         78 . The method of  claim 75  wherein the mutation is in a kinase.  
     
     
         79 . The method of  claim 75  wherein the mutaion is in the CDC28 gene.