METHODS FOR IDENTIFYING GENETIC DETERMINANTS ASSOCIATED WITH MODULATION OF TEST COMPOUND ACTIVITY

Abstract: Disclosed are methods for identifying genetic determinants that modulate the activity of test compounds which affect detectable processes in eukaryotic cells. The process comprises exposing a first cell, whether prokaryotic or eukaryotic, to a test compound to determine if the test compound modulates the detectable process in the cell. If so, the compound is then exposed to a second pool of cells (preferably of the same type as the first pool of cells) which overexpress one or more heterologous polypeptides under conditions which, in the absence of the compound, allow the detectable process to occur. If overexpression of the heterologous polypeptide alters modulation of the detectable process, such modulation allows the detectable process to be correlated with the test compound or with a particular compound and the overexpressed heterologous polypeptide, and the gene encoding the same. Also described are methods for correlating a gene with a detectable cellular process, wherein a first pool of cells, whether prokaryotic or eukaryotic, is exposed to a test compound, determining if the test compound modulates a detectable process in the cells, and if so, exposing the compound to a second pool of cells (preferably of the same type as the first pool of cells) which overexpress a heterologous polypeptide. If overexpression of the heterologous polypeptide alters modulation of the detectable process in the cells by the compound, the heterologous polypeptide responsible for such modulation, and preferably the nucleic acid molecule encoding the same, are characterized and correlated with the detectable process.

Description: METHODS FOR IDENTIFYING GENETIC DETERMINANTS ASSOCIATED WITH MODULATION OF TEST COMPOUND ACTIVITY Technical Field Of The Invention This invention relates to methods for identifying particular modes of action for test compounds which modulate particular detectable cellular processes. Background Of The Invention Methods for identifying compounds which modulate specific cellular pro- cesses, and characterize the gene products which interact with such compounds, are critical for discovering new chemical entities which may be used to develop future therapeutic compounds. The development of new therapeutic compounds can proceed by a variety of methods, but generally falls into one of two approaches. In the traditional approach, screening methods are used to identify compounds that affect a particular tissue or model, without concern for the specific target. The second approach involves the identification of new therapeutic targets, for example, a particular cell type or a receptor on a cell surface or present in the cytoplasm, and screening compounds to identify those which interact with the identified targets. Large collections of compounds, whether synthetically derived or isolated from natural sources, have traditionally served as raw material for screening assays. With more recent technologies such as combinatorial chemistry and phage display, it is relatively straightforward to generate large compound libraries for testing, typically containing from about 10,000 to 100,000 or more related or random compounds for use in high throughput screening protocols. High throughput screening techniques have been enabled by automation of traditional screening methods, although screening large numbers of compounds against one or more specific targets can be a labor and capital intensive endeavor even when implemented in a high throughput fashion. Moreover, these methods rely on specific targets. Summary of the Invention One object of the invention is to provide a method for determining the genetic determinants that can reverse the pharmacological effect of a test compound. It is an object of this invention to provide methods which enable the testing of compounds having activities against a cell type displaying or having a detectable process of interest, and identifying a subset of such test compounds which affect or modulate the detectable process exhibited by the cell. The activities can be previously unidentified. After identification of a subset of test compounds active in modulating the detectable process being studied, those compounds, or a subset thereof, are again screened against cells exhibiting the desired detectable process, although in this second screen the cells have been manipulated to overexpress one or more heterologous polypeptides. Those cells which overexpress one or more heterologous polypeptides which reverse the effect of the test compound on modulating the detectable process in the cell of interest are then characterized, for example by sequencing the nucleic acid that encodes the overexpressed gene product. In this way, the mode of action for a test compound can be identified and cellular processes and constituents responsible therefor can be correlated with a detectable cellular process without requiring a specific target. In addition, the invention dramatically reduces the effort involved in screening by providing simultaneous screening for compounds active on many different targets in a single assay. Thus, the instant methods are particularly suited to efficient screening of multiple cellular targets against large numbers of test compounds. One aspect of the present invention concerns methods of identifying modes of action for test compounds which modulate a detectable process in a cell. Such methods comprise exposing a first pool of cells, e.g., prokaryotic or eukaryotic cells, to one or more test compounds under conditions which, in the absence of the test compound, allow a detectable process to occur the cells. It is then determined if exposure to the test compound modulates the detectable process in the cells or subset of the cells, and if so identifying those compounds as active compounds. Active compounds are then exposed to a second pool of cells which overexpress one or more heterologous polypeptides under conditions which, in the absence of the active compound, allow the detectable process to occur. It is then determined if overexpression of the heterologous polypeptide(s) alters modulation of the detectable process mediated by the active compound in the cells. By performing such methods, one is able to identify the mode of action for a test compound which modulates a detectable eukaryotic cell process. In certain embodiments, the cells used in the claimed methods are eukaryotic cells, such as mammalian cells, including canine, feline, ovine, porcine, equine, bovine cells, and human cells. One may use human cells that are disease-specific and correlate with a particular human disease. In some embodiments of the invention, the eukaryotic cells employed to identify which test compounds are active compounds (first population) are the same types of cells which overexpress one or more heterologous polypeptides (second population). In other embodiments, prokaryotic cells are employed, such as, for example, bacterial cells, particularly those which are pathogenic to humans or livestock. Other embodiments of the invention concern high throughput screening methods employing more than one aliquot of the first pool of cells, wherein each aliquot is exposed to a different test compound. Other embodiments of high throughput screening also employ aliquots of the second pool of cells wherein each aliquot is exposed to a different compound identified as an active compound in the initial pre-screening against the first pool or aliquot of a first pool of cells. In one embodiment of the invention, different samples of the second pool of cells overexpress one or more different heterologous polypeptides than are overexpressed in the other samples. In certain embodiments, high throughput screening methods according to the invention are conducted in one or more 96-well microtiter plates, although many other formats are also suitable for high throughput screening, and largely depend on the automated equipment being employed. In certain embodiments, the disclosed methods are used to identify compounds which modulate a detectable cellular process in a negative way, for example by inhibiting (partially or completely) the detectable process in the cells being screened. Preferred detectable processes are those which involve measurable physiological cellular processes, for example, cell growth, cell growth rate, cell migration, nucleic acid replication, nucleic acid synthesis, protein synthesis, protein secretion, cell adhesion, phagocytosis, contact inhibition, and cell death, for example programmed cell death or apoptosis. Other measurable physiological processes include those involving intra- or extra-cellular localization of a cellular component, or expression of -3- a reporter gene. In other embodiments of the invention, modulation involves an increase in the detectable process exhibited by the cells. In certain embodiments of the invention, overexpression of one or more heterologous polypeptides is mediated by a recombinant nucleic acid molecule introduced into the cells. Preferably, the introduced recombinant nucleic acid encodes one or more genes (or functional portions thereof) under the control of a promoter or other cis-acting element required for transcription in the cells. However, promoters or other transcription activation sequences are not essential, particularly when the recombinant nucleic acid being introduced is to be inserted by homologous recombination into a region of the cell s genome functionally adjacent to a tran- scriptional activation sequence sufficient to enable overexpression of one or more genes adjacent thereto. In other embodiments, overexpression may be achieved by upregulating expression of one or more endogenous genes. Upregulation in such embodiments may be achieved by addition or removal of one or more chemicals or other compounds to or from the growth medium. Alternatively, genetic modifications or mutations may result in such upregulation, for instance as may occur upon infection of the cells by certain viruses. Recombinant nucleic acids according to the invention can be incorporated in gene delivery vehicles, for example, viral vectors, liposomes, or nucleic acid/condensing agent compositions, although delivery of "naked" nucleic acids can also be employed. Exemplary viral vectors include retroviral vectors, adeno- associated viral vectors, and alphaviral vectors, particularly viral vectors which are replication defective. Replication defective recombinant retroviral vectors which comprise one or more heterologous genes whose expression is under the control of an efficient promoter or other transcription activation sequence are useful in the invention. In certain embodiments, such activation sequences are inducible promoters. Retroviral vectors are particularly useful because they enable stable, long term expression. See U.S. patents 5,716,832, 5,591,624, 5,693,522, 5,716,613, 5,716,826, and 5,662,896, each of which is hereby incorporated by reference, for a description of producing replication defective retroviral vectors and packaging cell lines therefor. As with retroviruses, the host range of other gene delivery vehicle being used can be manipulated to target specific cell types. _4_ Certain embodiments of this aspect of the invention concern overexpression of a library of polypeptides, such as may be encoded by a library of heterologous genes, in the second pool of cells. Such a library can be a cDNA library prepared from messenger RNA isolated from cells of the same species as are represented in the second pool of cells. In a complete embodiment, the cDNA library is fully representative of all genes expressed in the organism in which the cell was derived. Alternatively, the library may be less than fully representative of the organism, as may be obtained by generating a cDNA library from a specific cell type of an organism, e.g., a hepatocyte or nerve cell. Moreover, libraries according to the invention may be further refined by techniques wherein "housekeeping" genes common to most or all cell types of the organism are eliminated by subtractive cross- hybridization. In yet another embodiment, the cDNA library is at least partially comprised of a custom library containing preselected cDNAs pooled for the purpose of conducting the particular assay. Certain embodiments of the invention concern characterization of a heterologous polypeptide, or a gene encoding the heterologous polypeptide, which is found to modulate the activity of an active compound through its overexpression in the second pool of cells. With respect to characterization of polypeptides, various techniques can employed, for example antibody- or other high-affinity receptor-based detection. With respect to nucleic acid characterization, hybridization or sequencing techniques are typically utilized. Sequencing typically comprises determining at least a portion of a nucleotide sequence of the heterologous gene or genes which are responsible for overexpression of the heterologous polypeptides which modulate activity of the active compound. In some circumstances it is desirable to amplify heterologous genes prior to sequencing. To facilitate amplification, the vector in which the heterologous genes are inserted preferably contains a unique amplifiable sequence 5' and 3' to the region where the heterologous gene is inserted into the vector. Amplification primers which have nucleotide sequences substantially complementary to the unique amplifiable sequences can then be used to amplify the heterologous gene insert by an appropriate amplification methodology, e.g., PCR (polymerase chain reaction), transcription- mediated amplification, ligase chain reaction, or strand displacement amplification. In addition, or alternatively, the vector may also contain a "tag" region for amplification. When tags are used, it is preferred that there is a one-to-one -5- correspondence between a particular tag and the heterologous gene of unknown sequence included in the same vector. In this way, a specific tag corresponds to the specific heterologous gene carries in the recombinant nucleic acid. Another aspect of the invention concerns methods of correlating a gene with a detectable cellular process, such methods being based on exposing a first pool of cells to one or more test compounds under conditions which, in the absence of the test compound, allow the detectable process to occur, provided that in the event more than one test compound is to be screened, an aliquot of the first pool of cells is used for each such test compound. Following exposure of a test compound to the first pool of cells or an aliquot thereof, it is determined if one or more of the test compounds modulates a detectable process in such cells, and if so identifying those test compound(s) as an active compound. The active compound or compounds are then exposed to a second pool of cells which overexpress one or more heterologous polypeptides under conditions which, in the absence of an active compound, allow the detectable process to occur. If overexpression of a heterologous polypeptide in the cell alters modulation of the detectable process, the heterologous gene or nucleic acid molecule encoding the heterologous is then sequenced and correlated with the detectable process. Detailed Description Definitions When used in this application, the following terms will have meanings described below, unless otherwise specifically indicated. "Mode of action" for a test compound refers to the cellular process or processes affected by the test compound when administered to a cell. A mode of action is based on the activity of one or more genetic determinants. "Test compound" refers to any molecule, synthetic or naturally occurring, used in the practice of the present invention. Such compounds include, without limitation, nucleic acids, e.g., oligonucleotides, ribozymes, and antisense molecules (including without limitation RNA, DNA, RNA/DNA hybrids, peptide nucleic acids, and polynucleotide analogs having altered backbone and/or bass structures or other chemical modifications); proteins, polypeptides, carbohydrates, lipids, and small molecule drug candidates. "Small molecules" are, for example, naturally occurring -6- compounds (for example, derived from plant extracts, microbial broths, and the like) or synthetic organic or organometallic compounds having molecular weights of less than about 10,000 daltons, preferably less than about 5,000 daltons, and most preferably less than about 1,500 daltons. The term "test conditions" refers to a variation in the environment other than the presence of a test compound, which perturbs the metabolism or activity of a test cell. Examples of test conditions include elevated or depressed temperature, altered concentration of nutrients (other than proteins), adhesion or contact surfaces, gas concentrations, rate of temperature change, presence or absence of other cells, and the like. "Modulate" means increasing or decreasing a particular activity or detectable process of a cell. Modulation of an activity ranges from partial to complete. "Detectable process" refers to a cellular process which is observable or measurable. Representative examples of such processes include measurable physiological processes such as cell growth, changes in cell growth rate, cell migration, nucleic acid synthesis, protein synthesis, protein secretion, cell adhesion, phagocytosis, contact inhibition, apoptosis and cell death. The mechanism or mechanisms used to observe or measure the process depends on the process being detected. For example, detection may depend on the intracellular or extracellular localization of a specific cellular component, such as a protein, lipid, carbohydrate, or nucleic acid. The term "cell" refers to any cell, and includes both prokaryotic and eukaryotic cells. "Prokaryotic cell" refers to any cell lacking a membrane-bound nucleus, for example, bacterial cells. "Eukaryotic cell" refers to any cell which has a membrane-bound nucleus. Eukaryotic cells used in the practice of this invention may be derived from single-celled organisms such as fungi or from multi-cellular organisms such as plants and animals, for example, higher animals such as birds and mammals, e.g., human, bovine, canine, feline, equine, ovine, and porcine animals, and can be normal cells, cell lines, or cells associated with a particular disease state, for example, cancer. The cells used may represent a recognized disease model. "Pool" of cells refers to a population of cells, and includes cell populations comprised of different cells types, e.g., prokaryotic and eukaryotic cells, or different types of eukaryotic cells, e.g., human hepatocytes and lymphocytes. A pool of cells -7- can be derived from the same cell source, e.g., cells of a particular cell line such as HeLa cells. An "aliquot" of such a pool refers to a subset of the pool, e.g., a 100 μL sample of a 10 mL culture of cells. The term "specifically altered" as used herein refers to the alteration of the activity of a heterologous gene, by changing the level of expression of a gene (up or down) found in the first cell type, or the specific activity of its protein product. Specific alteration includes overexpression of the gene product, upregulation and down-regulation of the gene, inhibition of the gene activity and/or transcription, and mutation of the gene that alters the biological activity of its product. The terms "overexpress" and "overexpression" refers to expression of a gene in a cell at a level higher than normally expressed in a cell of that type under the particular growth conditions employed. Thus, if a gene is not expressed in the particular cell type under the growth conditions employed, any expression of that gene would constitute overexpression. Overexpression of a gene can be mediated by introduction of a heterologous gene into the cell, such as by transfection. Alternatively, overexpression can also be achieved by manipulating the cell to upregulate expression of the naturally occurring gene in that cell, for instance by virus-mediated mutagenesis or exposure to a chemical or other compound which directly or indirectly leads to upregulation of expression of the gene. "Specific inhibition" refers to the inhibition of the activity of a specific gene. Instead of overexpressing a gene native to the first cell, one can inhibit the function of that gene (where native to the second cell population) specifically, for example by using antisense polynucleotides or ribozymes in the second cell population. This embodiment is particularly useful for studying the effects of compounds which act as agonists, or by upregulating the activity of a gene. "Gene" refers to a polynucleotide that encodes a polypeptide, i.e., two or more amino acids linked by a peptide bond. Thus, as used herein, "gene" can refer to the entire coding region for a protein, in genomic or cDNA form, or an open reading frame (ORF), or fragments thereof. The term "reporter gene" refers to a polynucleotide that provides a detectable signal following transcription. The signal can be direct or indirect, and can be transcriptional (for example, by providing a unique or characteristic sequence detected by hybridization) or translational (for example, by providing a distinct -8- surface antigen, a chromophore or fluorescent protein, a chromogenic enzyme, and the like). The term "heterologous polynucleotide" refers to a polynucleotide that is foreign to the host cell, or that is native to the host cell but operatively associated with a promoter other than its native promoter. "Heterologous polynucleotides" further include complementary and antisense sequences capable of inhibiting expression, and mutated sequences affecting the biological activity of the product. For the purposes of this patent, "native" polynucleotides include polynucleotide sequences capable of specifically inhibiting transcription and/or expression of a gene or cDNA found in the first cell type. A "heterologous polypeptide" is a protein or polypeptide product of a heterologous gene. An "inducible expression system" refers to a nucleic acid the expression of which is regulated. Under certain conditions, the gene(s) encoded by such a system may be induced, for instance, by the addition of a chemical or some other change in environmental conditions. A "library" of heterologous genes refers to a collection of two or more such genes, preferably at least about 10 genes, more preferably at least about 20, most preferably about 100 or more. As applied to cDNA, such a library can be prepared from total RNA, or preferably mRNA, from one or more cell types. A "fully representative library" is one containing all genes expressed in a particular cell type, developmental stage, or organism, for example. Preferred cDNA libraries include those which are "subtractive," in which certain mRNAs are removed by a cross hybridization reaction designed to remove genes that do not contribute to the attributes of a specific cell type which distinguish it from other cell types. As a result, such a library will generally lack members common to all or other cell types, for example "housekeeping" enzyme genes. As used herein, "high throughput" refers to screening techniques wherein more than about 10, preferably more than about 100, and more preferably more than about 1,000, compounds are screened in an automated fashion in a single experiment according to the methods described herein. General Methods -9- In the practice of the claimed method, the order of genomics driven drug discovery is reversed. Genomics approaches usually provide large sets of genes (sometimes all genes , when the organism is entirely sequenced) suspected to be involved in a particular cellular process. It also provides tools to investigate these genes and prioritize them with respect to the process of interest. This allows one to focus on a few novel and useful genes around which drug screens can be designed. In the practice of the invention, one first identifies a set of compounds that affect a process of interest. Genomic techniques are then used to identify sets of genes that modulate the effect of these compounds. This concept can be applied to many different situations. The requirements are (1) a relevant cell type, (2) a scorable phenotype relevant to the ultimate therapeutic goal, preferably amenable to high- throughput screening, and (3) a method to introduce gene-specific alterations in either the level of expression of a gene or in the specific activity of the encoded gene product. Thus the method comprises two parts: first, one identifies a set of compounds affecting the process of interest, preferably in the cell of interest, preferably using the most relevant readout for the desired outcome, and then one identifies a set of genes (for example all genes) that modulate the effect of these compounds. One can use the same cell type, and the same phenotype as a readout. Relevant cell types can be prokaryotic or eukaryotic, such as a mammalian cell line, and can be primary cultures, permanent cell cultures, and the like. In cases in which a particular cell type is known to be involved in a disease process under investigation, one can use primary cells of that type, or a model cell can be substituted. The second pool of cells employed in screening can be any type of cell, but is preferably eukaryotic. The scorable phenotype is preferably relevant to the cellular process under investigation, and preferably simple to observe. For example, if the therapy under investigation is cancer metastasis, the phenotype can be anchorage-independent growth. Alternatively, the phenotype can be read by employing a hybridization array, and directly determining the concentration of mRNAs produced in response to the test compounds. Alternatively, the scorable phenotype can be expression of a reporter gene or cell surface antigen, which permits one to label cells with antibodies or other binding ligands, and sort the cells using FACS, panning and the like. -10- The second pool of cells comprises at least one heterologous polynucleotide found in the first cell (and thus a possible target of the test compound), where expression of the heterologous polynucleotide is specifically altered in comparison to the first cell. The heterologous polynucleotide can be provided on a plasmid or other non-genomic vector, or can be integrated into the second cell population's genome (for example, by using a retroviral vector, homologous recombination, and the like). The specific alteration selected will depend on the cellular process under study, and the desired or suspected mode of action of the test compounds. Overexpression of the heterologous polynucleotide is conveniently provided by inserting the coding sequence operatively associated with a strong promoter, which can be either regulated or constitutive, and can be native or heterologous to the host cell. Suitable promoters include those for, without limitation, Tet, GAL, ecdysone, baculovirus, ADH, GAP, CMV, SV40, metallothionein, hybrid promoters, and the like. Alternatively, the heterologous polynucleotide can be integrated into the host cell genome in sufficient proximity to a native promoter. Alternatively, a native gene can be specifically inhibited, for example, by inserting a heterologous polynucleotide that provides an antisense polynucleotide or a ribozyme specific for the gene, using methods known in the art. Again, the coding sequence can be provided either on a plasmid or other extra-genomic element, or can be integrated into the host cell genome. Finally, the heterologous polynucleotide can be mutated, for example by random mutagenesis, point mutation at an active site, truncation, and the like, in such a way that a biological activity of the protein product is altered. For example, one can generate a dominant mutation resulting in a protein that retains the original activity of the native protein but no longer binds the test compound. Expression of the mutant reverses the compound-induced phenotype (See, e.g., G. Barnes et al., Mol Cell Biol (1984) 4(11):2381-88). If desired, a plurality of different mutated sequences can be used, and in fact can be used to simultaneously determine the active portions of the sequence along with identifying the corresponding gene as a target of the test compound. The second pool of cells is screened for response to the test compounds identified as active with the first cell type. Cells in the second pool that are capable of counteracting or reversing the activity of the test compound to a detectable degree) -11- are identified, and the heterologous polynucleotide responsible for the activity is identified by any suitable means. The heterologous polynucleotide can be identified by sequencing. The information obtained is useful for a number of purposes. For example, the genes identified as capable of reversing the activity of a compound are candidate targets of the compound. If the compound is a microbicide, these genes may form part of an organism's resistance mechanism. Further, such information could be used to design combination therapies that affect different genes (and different cellular processes), making it more difficult for resistance to arise. Example 1 (Antifungal drug development) The yeast Saccharomyces cerevisiae was used as a target for antifungal research. The scorable phenotype employed was growth inhibition in the presence of test compounds. The method of introducing gene-specific alteration is to transform high copy libraries selecting for compound resistance. (A) Identification of antifungal compounds. A set of compounds exhibiting antifungal activity was identified by screening a diverse collection of compounds against four fungal species: Saccharomyces cerevisiae, Candida albicans, Aspergillus fumigatus, and Cryptococccus neoformans. Approximately 100,000 compounds in DMSO were screened in a 96-well microtiter format. The screening conditions are summarized in Table 1 : Table 1 : Screening Conditions S. cerevisiae C. albicans C. neoformans A. fumigatus Inoculum (OD Λ , n ) 0.08 0.002 0.2 1:600 [compound] 20 30 20 30 (μg/ml) [DMSO1 2% 3% 2% 3% Incubation 30°C 30°C 30°C 37°C temperature (°C) Incubation time 24 20 46 46 (hrs) The result of the screen in summarized in Table 2: Table 2: Screening results -12- Species Compounds Hits (#) Compounds active screened (#) against >1 species (#) A. fumigatus 93660 1919 1045 C. albicans 94156 1920 1351 C. neoformans 92990 3158 1451 S. cerevisiae 95263 1685 1205 Of the compounds screened, 359 were active against all four species. S. cerevisiae was the only cell type used in the subsequent step of the method because it is most amenable to genetic manipulations. The other species were included at this stage because they are important pathogens and maximize the relevance of the set of compounds identified by this screen. (B) Identification of sets of genes modulating the effect of antifungal compounds. In this experiment, a set of genes capable of reversing the effect of some of the previously identified antifungal compounds (i.e., provide resistance to the drug) was identified. It is known that overexpression-based resistance to an antimicrobial compound can occur through a variety of mechanisms. MIC determination in solid medium: For each compound to be tested, the minimum inhibitory concentration in solid medium was determined. The test was performed in 24 well plates containing 1 ml of YPD agar. The compound was serially diluted from 128 μg/ml to 0.5 μg/ml. Each well was inoculated with 50,000 cfu. The plates were incubated for 24 hours at 26°C. The lowest concentration of compound capable of inhibiting the growth of the inoculum is the MIC. Generation of pools of overexpressor cells: Here, two yeast genomic DNA 2- micron plasmid libraries capable of overexpressing random genes were employed. One of these uses the yEP24vector (Carlson and Botstein, Cell (1982) 28:145), the other library is in the pRS203 vector (gift of Philip Hieter). Transformants (200,000 and 275,000 for the two libraries respectively) were generated, which correspond to more than a 50-fold coverage of the genome. The transformants were generated using the Lithium Acetate transformation procedure (Rose et al., Methods in Yeast Genetics, (Cold Spring Harbor Laboratory Press, 1990)). After 3 days of growth at 26°C the transformant colonies were recovered from the plate and frozen in aliquots at -80°C. -13- Selection for resistance: Approximately 1 x 10 7 colony forming units from one of the transformant pools were plated on 60 mm Petri dishes containing 8 ml of YPD agar, including 2x the previously determined MIC concentration of the compound to be tested. Resistant colonies that emerged after 3 days growth on solid YPD agar containing the compound were harvested and pooled. Plasmid DNA was recovered from the yeast cells as described by Rose et al., supra. Amplification of this material was achieved by transformation and subsequent isolation of plasmid DNA in E. coli. Identification of genes responsible for the resistance using DNA microarrays: Approximately 200 ng of plasmid DNA was labeled with the fluorescent nucleotide analog Cy3-dUTP (Amersham Pharmacia PA53022) using a commercially-available nick translation kit (Amersham Pharmacia N5500). The resulting labeled DNA was purified and used to probe a DNA microarray as described by Eisen and Brown (Meth EnzymoL in press). Arrays were prepared as described by Shalon et al., Genome Res.(l 996) 6:639, incorporated herein by reference in full, using PCR products generated using gene PAIRS primers (Research Genetics, Huntsville AL). This micro- array contains over 930 yeast genes including all genes known to be essential for vegetative growth, all ABC transporters, as well as other genes known to be involved in chemical resistance or sensitivity. A positive hybridization signal in this experiment is indicated that the corresponding gene (or a gene linked to it) is responsible for the compound resistance. When a positive signal was identified, the corresponding ORF was cloned, as well as its close genetic neighbors on an expression construct under the control of the yeast ADH1 promoter. Each of these constructs was transformed back into yeast and the resulting transformants were tested for increased resistance to the compound. Results: Using this procedure, one or more resistance genes were identified for at least half the compounds tested (see Table 3). The most commonly identified genes are likely to be involved in the transport of the compound in and out of the cell. This effect can be direct (for example, SNQ2, a multidrug transporter of the ABC family of transporters) or indirect (for example, YAPl and YAP2, two transcription factors known to activate multidrug resistance genes). This is very useful information in the context of antifungal development, because it indicates potential resistance mechanisms against the drug. In some cases, the isolated gene can be the molecular -14- target for the drug. For example, in Table 3 below, the ERG24 gene may be the target of compound CmpdG. In yet other cases, some of the genes isolated by this method can be indicative of the mode of action of the compound, without necessarily being the molecular target of the compound. This is the case with the DDIl and the MAGI gene providing resistance for CmpdC. These genes are induced by DNA damage and the fact that they provide resistance for the compound may indicate that this compound is a DNA damaging agent. Table 3: Active compounds and affected genes. Compound Gene Description CmpdA YAPl Transcription factor involved in drug resistance and oxidative stress response CADI Transcriptional activator involved in multidrug resistance SNQ2 ABC transport protein CmpdB YAPl Transcription factor involved in drug resistance and oxidative stress response CmpdC PRP39 Ul snRNA-associated protein required for commitment of pre-mRNA to splicing pathway MAGI DNA-3-methyladenine glycosidase; excises alkylation- damaged DNA base DDIl DNA damage inducible gene CmpdD SNQ2 ABC transport protein CLB1 Gl cyclin SUI3 Translation initiation factor eIF2β subunit CmpdE YAPl Transcription factor involved in drug resistance and oxidative stress response SNQ2 ABC transport protein CmpdF YAPl Transcription factor involved in drug resistance and oxidative stress response SNQ2 ABC transport protein CmpdG ERG24 C14 sterol reductase (ergosterol biosynthesis) POP3 Involved in processing of rRNA and tRNA precursors RAP1 DNA-binding protein with repressor and activator activities CmpdH PDR1 Transcription factor involved in drug resistance Cmpdl RRP43 Required for 3' processing of ribosomal 5.8S rRNA RNR4 Ribonucleotide reductase -15- Example 2 (Adhesion) A library of 100,000 test compounds is individually screened against an aliquot of the fibroblasts to identify compounds which alter the cells' ability to adhere to a substrate, e.g., a plastic culture dish. I.e., the detectable process being studied in this instance is adhesion to a substrate. The test compounds that modulate the cells adhesion ability are identified and termed "active compounds." Each of the active compounds is then individually screened against each of about 15,000 fibroblast clones (representing the fibroblast-specific clones currently available from the I.M.A.G.E. Consortium), each of which overexpresses a different human gene (delivered via a recombinant retroviral vector). Those clones or pool samples which alter the ability of the corresponding active compound to modulate, e.g., inhibit, cell adhesion are then isolated and the heterologous gene(s) therein is characterized. In this case, fibroblasts with inhibited adhesion are eluted from the substrate, and those remaining (having adhesion restored by the overexpressed gene) are examined. If the cells have been cultured and tested individually, the overexpressed gene will already be known. Alternatively, the overexpressing cells can be labeled , for example with a polynucleotide marker unique to each overexpressed gene, and the genes in the adherent cells identified on the basis of their markers. Alternatively, the overexpressed gene can be sequenced directly. In these cases, the fibroblasts can be pooled, and assayed simultaneously. Example 3 (CMV Assay) Cytomegalo virus (CMV) contains a large number of genes, many of which are still uncharacterized. There are a number of CMV-susceptible cell lines, and laboratory strains of CMV available for study. HFF cells are infected with the RC256 strain of CMV (Hippenmeyer and Dilworth, Antiviral Res (1996) 32:35-42), and are then contacted with a large variety of test compounds. Compounds that demonstrate activity against CMV (i.e., which prevent cell death due to CMV infection) are identified and used in the second phase. -16- HFF cells are transfected with each of the 175 CMV genes individually, using retroviral vectors that provide for overexpression of a CMV gene, and are then infected with CMV RC256 in the presence of the selected compounds identified in phase 1. In most cases, the compound will still protect the cell line from the effects of CMV infection. However, the CMV gene overexpressed in some cell lines will be able to overcome the effect of the test compound, resulting in death of that cell line from CMV infection. The effective genes are then identified, and are possible targets of the test compound. -17-

Claims: What is claimed: 1.) A method for identifying genes modulating the effect of a test compound on a cellular process, comprising: a) Contacting a first cell with a plurality of test compounds; b) Identifying a test compound having an activity that alters a detectable property of said first cell; c) Providing a second pool of cells, wherein said cells of said second pool comprise a gene native to said first cell, wherein the activity of the gene is specifically altered with respect to the first cell; d) Contacting said second pool with an active test compound; and e) Identifying any genes in said second pool that counteract the activity of said active test compound. 2.) The method of claim 1, wherein the activity of the gene is altered by overexpression. 3.) The method of claim 1, wherein the activity of the gene is altered by specific inhibition. 4.) The method of claim 1, wherein the activity of the gene is altered by mutation. 5.) The method of claim 1.), wherein said detectable property comprises a detectable cellular process. 6.) The method of claim 5.), wherein said detectable cellular process is associated with a pathological state. 7.) The method of claim 1.), wherein said first pool of cells comprises eukaryotic cells. 8.) The method of claim 7.), wherein said first pool of cells comprise mammalian cells. -18- 9.) The method of claim 7.), wherein said first pool of cells comprise fungal cells. 10.) The method of claim 1.), wherein the cells of said second pool overexpress a plurality of genes native to said first pool. 11.) The method of claim 10.), wherein the cells of said second pool overexpress at least about 100 of the genes native to said first pool. 12.) The method of claim 11.), wherein the cells of said second pool overexpress substantially all of the genes native to said first pool. 13.) The method of claim 1.), wherein the identity of overexpressed genes in said second pool are predetermined. 14.) The method of claim 1, wherein step e) comprises sequencing said overexpressed genes. 15.) The method of claim 1 , wherein the detectable property is selected from the group consisting of cell growth, cell growth rate, cell migration, nucleic acid replication, nucleic acid synthesis, protein synthesis, protein secretion, surface antigen expression, reporter gene expression, cell adhesion, phagocytosis, contact inhibition, apoptosis, and cell death. 16.) The method of claim 15.), wherein the detectable property comprises growth. 17.) The method of claim 15.), wherein the detectable property comprises apoptosis. 18.) The method of claim 15.), wherein the detectable property comprises adhesion. ■19- 19.) The method of claim 15.), wherein said detectable property comprises expression of a reporter gene. 20.) The method of claim 15.), wherein said detectable property comprises expression of a cell surface antigen. -20-

Inventors: NATSOULIS GEORGES, NATSOULIS, GEORGES
Applicants: ICONIX PHARM INC, ICONIX PHARMACEUTICALS INC.
Application year: 1999
Main classification: C12Q1
IPC codes: C12N15, C12Q1