EP1070145A1 - Procedes d'identification de determinants genetiques associes a la modulation de l'activite de composes de test - Google Patents

Procedes d'identification de determinants genetiques associes a la modulation de l'activite de composes de test

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Publication number
EP1070145A1
EP1070145A1 EP99918480A EP99918480A EP1070145A1 EP 1070145 A1 EP1070145 A1 EP 1070145A1 EP 99918480 A EP99918480 A EP 99918480A EP 99918480 A EP99918480 A EP 99918480A EP 1070145 A1 EP1070145 A1 EP 1070145A1
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European Patent Office
Prior art keywords
cells
pool
cell
gene
detectable
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EP99918480A
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German (de)
English (en)
Inventor
Georges Natsoulis
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Iconix Pharmaceuticals Inc
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Iconix Pharmaceuticals Inc
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Publication of EP1070145A1 publication Critical patent/EP1070145A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1048—SELEX
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • This invention relates to methods for identifying particular modes of action for test compounds which modulate particular detectable cellular processes.
  • 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.
  • new therapeutic targets for example, a particular cell type or a receptor on a cell surface or present in the cytoplasm
  • 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.
  • 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.
  • the activities can be previously unidentified.
  • 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.
  • 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.
  • the invention dramatically reduces the effort involved in screening by providing simultaneous screening for compounds active on many different targets in a single assay.
  • 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.
  • a first pool of cells e.g., prokaryotic or eukaryotic cells
  • 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.
  • eukaryotic cells such as mammalian cells, including canine, feline, ovine, porcine, equine, bovine cells, and human cells.
  • human cells that are disease-specific and correlate with a particular human disease.
  • 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).
  • prokaryotic cells are employed, such as, for example, bacterial cells, particularly those which are pathogenic to humans or livestock.
  • inventions 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.
  • different samples of the second pool of cells overexpress one or more different heterologous polypeptides than are overexpressed in the other samples.
  • high throughput screening methods 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.
  • 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
  • modulation involves an increase in the detectable process exhibited by the cells.
  • overexpression of one or more heterologous polypeptides is mediated by a recombinant nucleic acid molecule introduced into the cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • the host range of other gene delivery vehicle being used can be manipulated to target specific cell types.
  • a library of polypeptides such as may be encoded by a library of heterologous genes, in the second pool of cells.
  • 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.
  • the cDNA library is fully representative of all genes expressed in the organism in which the cell was derived.
  • 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.
  • 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.
  • 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.
  • various techniques can employed, for example antibody- or other high-affinity receptor-based detection.
  • 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.
  • 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.
  • the vector may also contain a "tag" region for amplification. When tags are used, it is preferred that there is a one-to-one
  • 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.
  • 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.
  • 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
  • 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.
  • 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 e.g., prokaryotic and eukaryotic cells, or different types of eukaryotic cells, e.g., human hepatocytes and lymphocytes.
  • -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 ⁇ L 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.
  • 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.
  • Gene refers to a polynucleotide that encodes a polypeptide, i.e., two or more amino acids linked by a peptide bond.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the phenotype can be read by employing a hybridization array, and directly determining the concentration of mRNAs produced in response to the test compounds.
  • 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.
  • 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.
  • 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.
  • a strong promoter include those for, without limitation, Tet, GAL, ecdysone, baculovirus, ADH, GAP, CMV, SV40, metallothionein, hybrid promoters, and the like.
  • the heterologous polynucleotide can be integrated into the host cell genome in sufficient proximity to a native promoter.
  • 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.
  • the coding sequence can be provided either on a plasmid or other extra-genomic element, or can be integrated into the host cell genome.
  • 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)
  • heterologous polynucleotide responsible for the activity is identified by any suitable means.
  • the heterologous polynucleotide can be identified by sequencing.
  • 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 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 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.
  • 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 ⁇ g/ml to 0.5 ⁇ g/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.
  • 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.
  • 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
  • the ERG24 gene may be the target of compound CmpdG.
  • 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.
  • Example 2 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.
  • 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.
  • 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.
  • the overexpressed gene can be sequenced directly. In these cases, the fibroblasts can be pooled, and assayed simultaneously.
  • CMV Cytomegalo virus
  • -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.

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Abstract

L'invention concerne des procédés d'identification de déterminants génétiques modulant l'activité de composés de test qui affectent des procédés décelables dans les cellules eucaryotes. Le procédé consiste à exposer une première cellule, procaryote ou eucaryote, à un composé de test pour déterminer si le composé de test module le procédé décelable dans la cellule. Le cas échéant, le composé est exposé à un second groupe de cellules (de préférence du même type des celles du premier groupe ) exprimant en excès un ou plusieurs polypeptides hétérologues dans des conditions qui, en l'absence du composé, permettent au procédé décelable d'avoir lieu. Si l'expression excessive des polypeptides hétérologues modifie la modulation du procédé décelable, cette modulation permet la corrélation du procédé décelable avec le composé de test ou avec un composé particulier et le polypeptide hétérologue exprimé en excès et le gène codant pour celui-ci. Font aussi l'objet de cette invention des procédés de mise en corrélation d'un gène avec un procédé cellulaire décelable, dans lequel un premier groupe de cellules, procaryotes ou eucaryotes, est soumis à un composé de test, dans le but de déterminer si le composé de test module un procédé décelable dans les cellules et, le cas échéant, le composé est soumis à un second groupe de cellules (de préférence du même type de celles du premier groupe ) qui expriment en excès un polypeptide hétérologue. Si l'expression excessive du polypeptide hétérologue modifie la modulation du procédé décelable dans les cellules par le composé, le polypeptide hétérologue impliqué dans cette modulation et, de préférence la molécule d'acide nucléique codant pour celui-ci, sont caractérisés et mis en corrélation avec le procédé décelable.
EP99918480A 1998-04-09 1999-04-09 Procedes d'identification de determinants genetiques associes a la modulation de l'activite de composes de test Withdrawn EP1070145A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US8122498P 1998-04-09 1998-04-09
US81224P 1998-04-09
PCT/US1999/007802 WO1999053098A1 (fr) 1998-04-09 1999-04-09 Procedes d'identification de determinants genetiques associes a la modulation de l'activite de composes de test

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US6653075B2 (en) 1999-12-16 2003-11-25 Iconix Pharmaceuticals, Inc. Random domain mapping
EP1683874A3 (fr) 2000-08-29 2006-10-11 YEDA RESEARCH AND DEVELOPMENT Co. LTD. Méthode pour l'isolement de gènes codant pour des protéines à fonction spécifique et pour le criblage d'agents pharmaceutiques actifs

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