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WO2003042411A2 - Use of mutations of mec-1 and its related genes in the identification of compounds for treatment of disease - Google Patents

Use of mutations of mec-1 and its related genes in the identification of compounds for treatment of disease Download PDF

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Publication number
WO2003042411A2
WO2003042411A2 PCT/US2001/029631 US0129631W WO03042411A2 WO 2003042411 A2 WO2003042411 A2 WO 2003042411A2 US 0129631 W US0129631 W US 0129631W WO 03042411 A2 WO03042411 A2 WO 03042411A2
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cell
gene
mecl
drug
yeast
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WO2003042411A3 (en
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Fred Hutchinson Cancer Center
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Priority to EP20010274131 priority patent/EP1366180A2/en
Priority to JP2003544225A priority patent/JP2005509425A/en
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Definitions

  • yeast Saccharomyces cerevisiae strain S288C was determined through an international collaboration of more than 100 laboratories on April
  • yeast genome of about 6000 genes are available publicly and can be found for example on public websites such as for example: http://www.ncbi.nlm.nih.gov/Yeast; http://genome- www.stanford.edu/Saccharomyces/. Other databases exist as well and these databases and links therein to other websites are equally suitable for the purposes of this invention.
  • the examples include but are not limited to Yeast GenBank (A collection of all GenBank sequences that are derived from Saccharomyces cerevisiae); Yeast Swiss-Prot (The collection of Swiss-Prot protein sequences that are derived from Saccharomyces cerevisiae); YPD (The Yeast Protein Database maintained by Proteome, Inc.), and periodic updates thereof the content of which is incorporated herein by way of reference.
  • GenBank A collection of all GenBank sequences that are derived from Saccharomyces cerevisiae
  • Yeast Swiss-Prot The collection of Swiss-Prot protein sequences that are derived from Saccharomyces cerevisiae
  • YPD The Yeast Protein Database maintained by Proteome, Inc.
  • Yeast is often selected as a model due to the ease of manipulation and possibility of screening a large number of candidates in a relatively short period of time. Yeast have the highest rate of recombination and gene conversion among organisms tested, which is several orders of magnitude higher than in mammals.
  • the primary defect would be a mutation in a gene conserved from yeast to humans that is frequently deregulated in tumors (e.g., overexpression of cyclin).
  • Gene products with mutations that specifically kill cells with the primary defect would constitute putative "secondary drug targets" (that is, secondary to the primary defect) whose modulation in tumors may yield great therapeutic advantage.
  • synthetic lethality can also result when there are two mutations that have an additive negative effect on a single essential biological pathway, or when the mutations inactivate two different but functionally overlapping pathways.
  • DNA damage checkpoint is identified, a signal transduction system that detects DNA damage and coordinates repair, replication and cell cycle progression.
  • MEC1 is a homolog of the human ATR gene (Cliby et al, EMBO J. 17:159-169, 1998).
  • ATR is a phosphatidylinositol kinase-related (PIK-related) protein homologous to ATM, which is mutated in ataxia-telangiectasia patients. 3. SUMMARY OF THE INVENTION
  • mbpl-1 It, slmld/YOR080w, rnrl-77, rnr2-72, rnr4-106, cdc21-l, pril-62, poll-101, 141/154, 87, 240, and others.
  • the recombinant eukaryotic cell of the invention can have the primary gene impacted or altered to produce a loss of function.
  • the recombinant cells of the invention can, among other cells, be yeast cells or yeast mutants and human cells or human neoplastic cells.
  • One embodiment of the invention is the synthetic lethal mutant resulting from a point mutation in the mecl gene, which impacts the kinase domain of MEC 1.
  • Another embodiment of the invention is the synthetic lethality resulting from mutations in mecl in combination with deficiencies in lagging strand DNA synthesis.
  • Mass screening and genetic analysis compounds or drug targets are identified that could specifically kill target cells, such as tumor cells.
  • examples of compounds that simulate the activity of found lethal mutants are provided.
  • therapeutic agents are contemplated, which are developed from the identification of essential genes of eukaryotic organisms.
  • Such an identified gene or product thereof serves as a novel target for therapeutics based on a mechanism which is distinct or similar to the mechanisms of existing drugs.
  • Such a compound inhibits the function of a gene product identified by methods disclosed herein, for example, by producing a phenotype or morphology similar to that found in the original mutant strain.
  • the present invention provides for a method of identifying a drug that inhibits the growth or replication of a cell having a mutated MEC 1 gene or an analog or homolog thereof, by contacting, a cell having a mutated MEC1 gene or an analog or homolog thereof with the drug; and determining whether the drug modulates the activity of a wildtype secondary gene which is synthetically lethal when it is mutated and is present in combination with mutated MEC1 gene or an analog or homolog thereof.
  • the primary gene defect is preferably one found in or associated with a tumor cell or cell affected with cell cycle deregulation.
  • the primary gene defect in the cell provided by the instant method is analogous or homologous to a defect found in or associated with a mammalian or human tumor cell or chromosomally aberrant cell.
  • homologous is meant a direct relationship among a "family" of genes in which certain sequences or domains are strongly conserved among the members of the family.
  • yeast MEC1 gene is homologous to mammalian genes encoding AT-related kinase (Cliby et al., ibid., 1998).
  • “analogous” genes may serve similar or “analogous” functions, but they are not directly related (i.e., sequences are not conserved among analogous genes).
  • the primary gene defect may result in the alteration, loss, or inhibition of a function, for example, a cellular function.
  • the primary gene defect may also result in the enhancement or gain of a function
  • the functions affected can vary widely.
  • the affected functions may include, but are not limited to, the suppression of tumor growth, DNA damage checkpoint, DNA mismatch repair, nucleotide excision repair, O 6 -methylguanine reversal, double-strand break repair, DNA helicase function, signaling, cell cycle control, or apoptosis.
  • defects may be effectively modeled by primary gene defects in other organisms such as Drosophila.
  • втори ⁇ ии By the methods of the present invention, it has been found that certain secondary site mutations can be effected, which may turn out to be lethal to the cell harboring the primary gene defect.
  • Such secondary site mutations may be effected, for example, within a gene selected from the group cdc8-l thymidilate ldnase activity: deoxythymidine monophosphate kinase; rad27, endo and 5' exonuclease; mbpl-1 It, transcription factor slmd/(YOR080w); rnrl-77, ribonucleotide reductase large subunit; rnr2 ⁇ 72, ribonucleoptide reductase small subunit; rnr4-106, ribonucleotide reductase small subunit; cdc21-l, thymidylate synthase; pril-62, DNA primase; POL
  • Double mutants that involve MEC1 and a secondary gene and/or ORF are also contemplated within the scope of this invention.
  • the double mutation of yeast is effected within a gene having a mammalian analog or homolog so that mammalian models can be built which will rely on discoveries made in yeast. These may include but are not limited to yeast CDC8, RAD27, MBP1, SLM1, R R1, RNR2, RNR4, CDC21, PRIl, POL1 among many others.
  • the homologous (by at least 40%) or analogous mammalian gene is selected from this group.
  • a further object of the invention includes the identification of drug or drug candidates.
  • the secondary drug target can be used to screen for a drug or drug candidate that can potentially interact with the secondary drug target, for example to disable its physiological activity.
  • the present invention may provide a drug or drug candidate that interacts with, binds to, or inhibits a particular gene product. Such gene products may include, but are not limited to examples listed supra. It is desirable that the drug or drug candidate exhibit the capacity to inhibit or arrest the growth of a human tumor or benign neoplastic growth.
  • the preferred pharmaceutical composition of the drug comprises oligonucleotide, gene product, homologs or analogs of oligonucleotide or gene product, a small molecule, or a peptide mimetic.
  • an object of this invention is to provide means of controlling unwanted proliferation or differentiation of eukaryotic cells.
  • the term "unwanted proliferation” refers to proliferation of cells which is undesired, be it due to transformation of the cells, e.g., neoplastic or hyperplastic, for purposes of wound healing for example keloid, treatment of restenosis, infection by eukaryotic pathogens and other unwanted smooth muscle proliferation, cosmetic applications, retinopathy resulting from diabetes, etc.
  • unwanted differentiation refers to an undesirable change in the differentiation of a cell, such as where a differentiated cell reverts to an earlier state and recovers or retains an ability to proliferate un-naturally or in an "unwanted” manner.
  • this invention also provides a means for treating various mycotic or yeast pathogens selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus or combination thereof.
  • the object of this invention is to provide a means of controlling unwanted proliferation or differentiation of eukaryotic cells.
  • the term "unwanted proliferation” refers to proliferation of cells which is undesired, be it due to transformation of the cells, e.g., neoplastic or hyperplastic, for purposes of wound healing, treating of restenosis and other unwanted smooth muscle proliferation, cosmetic applications, etc.
  • the term “unwanted differentiation” refers to an undesirable change in the differentiation of a cell, such as senescence.
  • the present invention also embodies diagnostic and prognostic assays, which assess the phenotype and aggressiveness of a disorder by detecting the expression of a cyclin protein or expression of regulating genes or gene products thereof.
  • a further object of this invention includes means of using of gene therapy to induce synthetic lethality in a cell containing at least one defect in a primary gene such as
  • methods of inducing synthetic lethality include introducing a polynucleotide into a cell containing at least one defect in a primary gene such as MEC1 or a homolog or analog thereof wherein the action or expression of the polynucleotide results in the perturbation of a secondary gene that results in synthetic lethality of the cell.
  • Perturbation of the secondary gene includes but is not limited to the up-regulation, down-regulation, elimination or disruption of the secondary gene.
  • antisense polynucleotides are described generally in, for example, Mol and Van der rul, eds., Antisense Nucleic Acids and Proteins Fundamentals and Applications, New York, NY, 1992, incorporated by reference herein in its entirety.
  • Suitable antisense oligonucleotides are at least 15 nucleotides in length and up to and including the upstream untranslated and associated coding sequences of the secondary gene of choice.
  • Suitable target sequences for antisense polynucleotides include intron-exon junctions (to prevent proper splicing), regions in which DNA/RNA hybrids will prevent transport of mRNA from the nucleus to the cytoplasm, initiation factor binding sites, ribosome binding sites, and sites that interfere with ribosome progression.
  • a particularly preferred target region for antisense polynucleotides is the 5' untranslated region of the secondary gene of choice.
  • Antisense polynucleotides targeted to the secondary gene of choice may also be prepared by inserting a DNA molecule containing the target polynucleotide sequence into a suitable expression vector such that the DNA molecule is inserted downstream of a promoter in a reverse orientation as compared to the gene itself.
  • the expression vector may then be transduced, transformed or transfected into a suitable cell resulting in the expression of antisense polynucleotides.
  • antisense oligonucleotides may be synthesized using standard manual or automated synthesis techniques. Synthesized oligonucleotides may be introduced into suitable cells by a variety of means including electroporation (e.g., as described in Yang et al., Nucl. Acids.
  • antisense oligonucleotide administration method will be evident to one skilled in the art.
  • Stabilizing agents include intercalating agents that are covalently attached to either or both ends of the oligonucleotide.
  • Oligonucleotides may be made resistant to nucleases by, for example, modifications to the phosphodiester backbone by the introduction of phosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates, phosphoramidates or phosphorodithioates. Oligonucleotides may also be made nuclease resistant by the synthesis of the oligonucleotides with alpha-anomers of the deoxyribonucleotides, as generally described in Mol and Van der Krul (ibid.).
  • polynucleotide-based inhibitors of the present invention include the triplex forming oligonucleotides, sequence-specific DNA binding drugs that interfere with target gene transcription.
  • Triplex-forming oligonucleotides are generally described in Maher, Bioessays 14: 807-815, 1992; Gee et al., Gene 149: 109- 114, 1994; Noonberg et al, Gene 149: 123-126, 1994; Song et al, Ann. NY Acad. Sci. 761: 97-108, 1995; Westin et al., Nuc. Acids. Res. 23: 2184-2191, 1995; and Wand and
  • oligonucleotides form triple helical complexes under physiological conditions on double-stranded DNA, selectively inhibiting gene transcription by physically blocking RNA polymerase or transcription factor access to the DNA template. See also, e.g., WO 95/25818; WO 95/20404; WO 94/15616; WO 94/04550; and WO 93/09788, each of which is incorporated herein by reference.
  • the triplex forming oligonucleotides may contain either a nucleotide or non-nucleotide tail to enhance the inhibition of transcription factor binding.
  • the triplex forming oligonucleotides are targeted to the secondary genes: CDC8, RAD27, MBP1, SLMl/YOR080w, RNR1, RNR2, RNR4, CDC21, PRIl, POL1, 141/154, 87, and 240 and their respective analogs and homologs.
  • a suitable sequence will be guided by, for example, the type of inhibitor (i.e., triplex forming oligonucleotide or antisense polynucleotide) and the species to be treated. It may be preferable to choose sequences that are conserved between species to permit use in readily available animal models.
  • the present invention also provides compositions and methods for using ribozyme inhibitors for inhibiting secondary gene expression.
  • the ribozymes can be administered in a variety of ways, including by gene therapy targeted to a desired cell.
  • the ribozyme of the invention may target the RNA transcripts of the gene of interest in a sequence-specific manner.
  • ribozymes may be designed to specifically inhibit the transcription of the secondary gene
  • Each ribozyme molecule is designed to contain a catalytically active segment capable of cleaving secondary gene RNA, and further comprises flanking sequences having a nucleotide sequence complementary to portions of the targeted RNA.
  • flanking sequences to the target sequence do not need to be completely complimentary, however, as the flanking sequences need only be sufficiently complimentary to form a duplex with the target RNA and to allow the catalytically active segment of the ribozyme to cleave at the target sites. Thus, the flanking sequences need only be sufficiently complimentary to pemiit the ribozyme to be hybridizable with the target RNA under physiological conditions.
  • ribozyme means an RNA molecule having an enzymatic activity that is able to cleave or splice other separate RNA molecules in a nucleotide base sequence specific manner.
  • RNA molecule which has complementarity in a substrate binding region to a specific RNA target (e.g. CDC8, RAD27, MBP1, SLMl/YOR080w, RNR1, RNR2, RNR4, CDC21, PRIl, POL1, 141/154, 87 or 240 RNA), and also has enzymatic activity that is active to cleave and/or splice RNA in that target, thereby altering the target molecule.
  • a specific RNA target e.g. CDC8, RAD27, MBP1, SLMl/YOR080w, RNR1, RNR2, RNR4, CDC21, PRIl, POL1, 141/154, 87 or 240 RNA
  • the enzymatic RNA molecule is formed in a hammerhead motif, but the ribozyme may also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RNAse P RNA (in association with an RNA guide sequence).
  • hammerhead motifs are described by Rossi et al, AIDS Res. Hum. Retrovir. 8: 183, 1992
  • hairpin motifs are described by Hampel et al., Biochem. 28:4929, 1989 and Hampel et al., Nucl. Acids Res. 18: 299, 1990
  • the hepatitis delta virus motif is exemplified in Perrotta and Been, Biochem.
  • RNAseP motif is described in Guerrier-Takada et al., Cell 35: 849, 1983, and examples of the group I intron motif are described in Cech et al., U.S. Patent 4,987,071, each of the foregoing disclosures being incorporated herein by reference.
  • These specific motifs are not limiting in the present invention and those of skill in the art will recognize that an enzymatic RNA molecule of the invention has a specific substrate binding site which is complementary to one or more of the target RNA regions and that it has nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
  • flanking sequences upstream and downstream of the ribozyme catalytic site may comprise segments of any length that effectively imparts the desired degree of targeting specificity for the ribozyme.
  • a flanking sequence comprises from about 4 to about 24 nucleotides, more preferably from about 6 to about 15 nucleotides, and typically about 9 to 12 nucleotides, and results in base pairing to the substrate sequence immediately upstream and downstream of the RNA sequences which comprise the cleavage site.
  • Polynucleotide inhibitors e.g., triplex forming oligonucleotides, antisense oligonucleotide, ribozyme, etc., or a combination of such inhibitors targeted to different portions of the target DNA or corresponding RNA can be delivered in a wide variety of ways to targeted cells to facilitate inhibition of the gene of interest.
  • the oligonucleotides can be administered as synthetic oligonucleotides or expressed from an expression vector.
  • the oligonucleotide can be administered ex vivo, i.e., contacted with target cells that have been removed from an individual or other cell source, treated and returned, or the oligonucleotide molecule can be administered in vivo.
  • mitogens e.g., serum mitogens (SCF, IL-3, EPO, TPO, etc.
  • POL1 proteins involved in aspects of the cell cycle including, but not limited to aspects of DNA replication, DNA repair, transcription control and nucleotide cycling.
  • POL1 for example is required for mitotic DNA synthesis, premeiotic DNA synthesis, recombination, and full sporulation and its null mutant is inviable.
  • Poll(ts) mutants show blocked cell division at 36 degrees C.
  • CDC8 and its homologs and analogs encode proteins with thymidilate kinase activity.
  • RAD27 and its homologs and analogs encode proteins with endo and 5' exonuclease activity.
  • MBP1 and its homologs and analogs encode proteins that act as transcriptions factors.
  • RNR proteins act as subunits of ribonuclease reductase.
  • CDC21 and its homologs and analogs encode proteins that act as thymidilate synthases.
  • PRIl and its homologs and analogs encode proteins that act as a DNA primase.
  • genes that are synthetic lethal in combination with mutations in the primary gene MEC1 permit the development of screens for agents that can mimic the secondary gene mutation. Such agents are useful therapeutics within the context of this invention for the treatment of diseases associated with aberrant proliferation.
  • the secondary genes of the present invention are used, for example, within assays to identify therapeutic agents for the treatment of proliferative diseases such as cancer wherein the disease is characterized by a mutation in the primary gene, such as mutations in MEC lor an analog or homolog thereof.
  • screening assays may be carried out to identify agents, including candidate drugs, that modulate the activity of the secondary gene or the secondary gene product or an analog or homolog thereof, wherein such modulation results in the inhibition or reduction in activity of secondary gene product, the up-regulation, down-regulation, elimination or disruption of said secondary gene.
  • agents including candidate drugs, that modulate the activity of the secondary gene or the secondary gene product or an analog or homolog thereof, wherein such modulation results in the inhibition or reduction in activity of secondary gene product, the up-regulation, down-regulation, elimination or disruption of said secondary gene.
  • Assays in this context may use whole cells expressing the target gene, cell lysates containing the target gene product or may use purified target gene product.
  • test agents are incubated with reaction mixtures containing target gene-expressing cells under suitable conditions and for a time sufficient to permit the test agent to modulate the activity of the target gene product and wherein a control sample is incubated under identical conditions in the absence of the test agent.
  • modulation of the activity of a target gene product includes, but is not limited to, increasing or decreasing the activity of a target gene product through, for example, direct binding to the gene product and increasing or decreasing the activity of a target gene product by up-regulating or down-regulating transcription or translation of the target gene product.
  • aspects of the invention include high-throughput screening assays designed to identify modulators of the target gene product.
  • candidate drugs are identified by the following method a) exposing a test cell system comprising a cell having a deletion or mutation in a primary gene such as MEC1 to a candidate drug; b) comparing the viability of the test cell system with the viability of a control cell system comprising a cell having a deletion or mutation in a primary gene such as MEC1 or analogs or homologs thereof and a secondary gene which in its mutant form is synthetically lethal in combination with the primary mutation, such as MEC1 or analogs or homologs thereof, wherein a candidate drug that causes the test cell system to be less viable than the control cell system is a therapeutic drug candidate.
  • viability is measured as the ability of a cell to reproduce itself.
  • Non- viable cells are those cells that do not or cannot divide, those that are arrested in any phase of the cell cycle, those that apoptose or proceed along the apoptotic pathway and those that die. Means for determining the presence of viable and non- viable cells will be evident to one skilled in the art and include vital dye staining, measurement of chromosome condensation, and the like.
  • a method of identifying a compound useful in the treatment of proliferative diseases, such as cancer characterized by the a mutation in the MEC1 gene or analog or homolog thereof which comprises the steps of 1) contacting the wild-type gene product of a secondary gene that in its mutant form is synthetically lethal in combination with the mutant MEC1 gene or an analog or homolog thereof with a test compound under conditions and for a time sufficient to permit the test compound to effect the secondary gene product; 2) comparing the activity of the gene product in the presence of the test compound with the activity of the secondary gene product in the absence of the test compound.
  • compounds that can decrease the activity secondary gene product or an analog or homolog thereof are identified as useful for the treatment of cancer or other proliferative diseases characterized by a mutation in the MEC1 gene or analog or homolog thereof.
  • whole cell assays are conducted wherein a cell with a wildtype secondary gene or analog or homolog thereof is contacted with a test compound and the expression and/or activity of the secondary gene product or analog or homolog thereof is assessed and compared with the expression and/or activity of the secondary gene product in cells not exposed to the test compound.
  • those compounds that result in decreased expression or activity of the gene product are useful for the treatment of cancer or other proliferative diseases characterized by a mutation in the MEC1 gene or analog or homolog thereof.
  • assays for modulation of the gene products of the secondary genes are known to those skilled in the art.
  • the secondary gene target will determine the appropriate assay for measuring modulation of protein activity as the artisan will be familiar with the assays used to characterize the genes and their respective mutations.
  • the increase or decrease in enzymatic activity of ribonucleotide reductase (the gene products of RNR1 and RNR2) by the direct reduction of ribonucleotides to deoxyribonucleotides using the methods of Thelander et al. (J. Biol. Chem.
  • the measurement of thymidilate kinase (the gene product of CDC8) activity can be carried out by measuring the phosphorylation or deoxythumidine monophosphate as described, for example, by Yong and Campbell (J. Biol. Chem. 259: 14394-14398, 1984), and the endo- and 5' exonuclease activities of the RAD27 protein and the polymerase activities associated with POL1 can be determined using such assays that follow nuclease and polymerase activities.
  • Activity of a protein can also be determined by measuring the increase or decrease in binding of a gene product to its ligand or substrate by, for example, visualization using antibody staining or by immunoprecipitation. While, structural proteins, such as tubulin (the gene products of TUB 1 and TUB2) can be measured by measuring the quality and quantity of tubulin made by a cell. After mecl ⁇ dependent synthetic lethality is demonstrated the mutants are sorted into complementation groups, which is usually a good indication of the number of alleles of specific genes present in the pool of mutants. This is accomplished through switching the mating type of a number of MSLs and mating them to other MSLs. If complementation is successful the MECl ADE3 URA3 plasmid can be lost and the diploid strain is phenotypically white or sectored and partially or completely deficient for growth on medium lacking uracil.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydrox
  • peptides and compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • Benomyl (BE) plates are prepared by dissolving BE in DMSO to lOOx final concentration (stock solution), adding 0.2 mis of the stock solution to 20 mis of medium (55 degrees C). DMSO did not exceed 1% final concentration. All the experiments, unless otherwise indicated, are performed in drop-out synthetic medium lacking uracil and tryptophan, and containing the appropriate carbon source.
  • NUCLEIC ACID METHODS DNA cloning is performed by standard techniques outlined by Sambrook et al. 1989. Standard procedures are used for restriction analysis and plasmid constructions (Sambrook et al. 1989). Double-stranded plasmid DNA is sequenced with a Sequenase kit (United States Biochemical Corp.; USA).
  • TETRAD ANALYSIS When diploid S. cerevisiae cells undergo meiosis, four haploid spores are produced which are enclosed within a sac called an ascus. Using a micromanipulator, the 4 spores (the "tetrad") can be "dissected" out of the ascus and separated on an agar plate. The spores then germinate, and the colonies that arise can be tested for the presence or absence of phenotypes characteristic of the genetic markers present in the original diploid strain. In this way the segregation pattern of the genetic markers can be assessed, allowing determination of genetic linkage of, and distances between, the markers. When 2 genetic markers are analyzed in this way, the data is called 2 point data.
  • CELL COUNT Two ml samples are fixed with three ml of formalin solution (lxPBS (Phosphate-buffered saline) and 0.74% formaldehyde), sonicated for twelve seconds, and counted in a Coulter Counter.
  • SERIAL DILUTIONS lOx serial dilutions are made from a suspension of cells made in water and 5 ⁇ l are plated and grown to assess viability in different conditions of carbon source and temperature.
  • Cells are resuspended in 250 ⁇ l of TNN extraction buffer (TN buffer + 0.1 % Nonidet P-40) containing 5% aprotinin (Sigma; USA), 0.1 mM phenylmethylsulfonyl fluoride, 10 ⁇ g of leupeptin per ml, 10 ⁇ g of pepstatin per ml, 10 mM NaPPi (Sodium pyrophosphate) (pH 7.4) and 10 mM NaF.
  • TNN extraction buffer TN buffer + 0.1 % Nonidet P-40
  • aprotinin Sigma; USA
  • 0.1 mM phenylmethylsulfonyl fluoride 10 ⁇ g of leupeptin per ml
  • 10 ⁇ g of pepstatin per ml 10 mM NaPPi (Sodium pyrophosphate) (pH 7.4)
  • 10 mM NaF sodium pyrophosphate
  • Immunoprecipitates are washed with extraction buffer three times, once for two minutes with HNN (TNN buffer + 250 mM NaCl), and resuspended in 60 ⁇ l of 2X SDS (sodium dodecyl sulfate) sample buffer, heated at 95° for 5 minutes and used for immunoblot analysis.
  • SDS-polyacrylamide gel electrophoresis (10% and
  • PHOSPHATASE TREATMENT The immunoprecipitation is performed as described above. After the washes in extraction buffer, samples are washed once with kinase buffer (lOmM HEPES (pH 7.4), 10 mM MgC12, 10% glycerol, 1 mM dithiothreitol) and resuspended in 60 ⁇ l of kinase assay buffer.
  • kinase buffer pH 7.4
  • 10 mM MgC12 10% glycerol
  • 1 mM dithiothreitol resuspended in 60 ⁇ l of kinase assay buffer.
  • CIP Calf Intestinal Phosphatase
  • CIP+inhibitors 5% aprotinin (Sigma; USA)
  • 0.1 mM phenylmethylsulfonyl fluoride 10 ⁇ g of leupeptin per ml, 10 ⁇ g of pepstatin per ml, 10 mM NaPPi (pH 7.4) and 10 mM NaF) are added to 20 ⁇ l of suspension, and incubated for one hour at 37°. Reactions are stopped by addition of 20 ⁇ l of 2x SDS sample buffer, heated for 5 minutes at 95° and used for immunoblot analysis as described above.
  • a yeast strain is constructed which bears a wild type copy of the analyzed gene under the control of an inducible promoter from the GAL1 gene. Then the strain is mutagenized to a 10-30% survival rate. Mutagenization is performed either with a UN. light or with a chemical mutagen selected from the group consisting of with ethyl methanesulfonate (EMS).
  • EMS ethyl methanesulfonate
  • mutagenizing agents are equally suitable, i.e., methyl methanesulfonate, methylnitrosoguanidine, 4-nitroquinoline-l-oxide, 2-aminopurine, 5- bromouracil, ICR 191, acridine derivatives, ethidium bromide, nitrous acid, and/or ⁇ - methyl- ⁇ '-mtroso- ⁇ -nitroguanidine.
  • the mutagenized cell titer is then determined by plating 500 colony forming units per 120 mm Petri dish on an agar-based media containing necessary salts, vitamins and galactose and sucrose.
  • the colonies containing a synthetic lethal mutation are then transformed with a plasmid bearing a copy of the gene being analyzed. If the colonies indeed contain a synthetic lethal mutation with the analyzed gene, then introduction of such plasmid into the cells should allow transform cells to grow on dextrose-containing media.
  • the transformed cells are then crossed with the wild type strain of the opposite mating type. If the hybrid strain grow on glucose, it is concluded that the synthetic lethal mutation in the isolate is recessive and, therefore, can be cloned by complementation. If the hybrid strain cannot grow on glucose, the mutation is dominant and it is omitted from the successive analysis.
  • a set of mutants that require MECl function for viability is used to genetically dissect functional relationships among known components of the MEC1/RAD53 pathway in two ways.
  • each mecl synthetic lethal mutant is crossed to a panel of checkpoint mutants defective in one or more checkpoint-related functions, including rad9d, pol2-ll, rad53d, and pdsld.
  • the mecl synthetic lethal mutants show different requirements for upstream and downstream components of the checkpoint pathway, depending on whether genetic alterations impact dNTP synthesis or DNA lagging strand synthesis.
  • dNTP synthesis cells require S-phase sensors, for example POL2, and at least two RAD53 functions: One dependent upon DUNl and another independent of both DUNl and PDS1.
  • RAD53 DNA damage sensors
  • RAD9, PDS1 DNA damage sensors
  • EXAMPLE 5 GENETIC APPROACHES TO MECl FUNCTION AND CHECKPOINT RESPONSE IN Saccharomyces cerevisiae. Key molecular players, among eukaryotes, are critical for maintaining cell viability in response to replication blocks and DNA damage. Amid this group in budding yeast is the checkpoint transducer MECl, a homologue of the human checkpoint kinases ATM and ATR. MECl is required for all DNA-related checkpoint responses in yeast. While much has been uncovered concerning the role of MECl and other genes in checkpoint responses, surprisingly few down stream functions of the MECl pathway have been identified.
  • a synthetic lethal screen with a mecl mutant reveals two basic scenarios in which cells require MECl function for viability: (a) deficiencies in dNTP synthesis, where cells probably experience chronic replication blocks; and (b) deficiencies in lagging strand DNA synthesis, where cells likely generate chronic DNA damage.
  • triButyrate 4- phenylbutyrtic acid sodium salt or sodium phenylbutyrate
  • various benzopyran drivatives as prepared by known in the art means. See, for example, U.S. Pat. Nos. 5,359,115; 5,362,899; 5,288,514; 5,733,920 or PCT publications WO 94/08051; WO92/10092; WO93/09668; WO91/07087; WO93/20242.
  • Drugs that are screened out as positive in yeast based assays are then tested for treating diseases caused by excessive cell growth.
  • malignant diseases i.e., cancers of any of a wide variety of types, including without limitation, solid tumors and leukemias such as apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), histiocytic disorders, leukemia (e.g., B-cell, mixed-cell, null-cell, T-cell, T-cell chronic, HTLV-II-associated, lymphocytic acute, lymphocytic chronic, mast
  • Some diseases occur due to excessive but benign cell proliferation (i.e. non- malignant).
  • diseases are fibrosis, benign prostate hyperplasia, atherosclerosis, restenosis, glomulerosclerosis, cheloid, psoriasis, lentigo, keratosis, achrochordon, molluscum contagiosum, venereal warts, sebaceous hyperplasia, condylomata acuminatum, angioma, venous lakes, chondrodermatitis, granuloma pyogenicum, hidradenitis suppurativa, keloids, keratoacanthoma, leukoplakia, steatocystoma multiplex, trichiasis, superficial epithelial nevus, polyp, junctional nevus, pyogenic granuloma, prurigo nodularis, dermatofibroma, adenoma sebaceum,
  • the effect of sodium phenylbutyrate or benzopyran derivatives is then determined on the tumorigenic phenotype of human glioblastoma cells in vivo.
  • Fisher 344 rats received an inoculation of syngeneic 9 L gliosarcoma cells (4x10 4 ) into the deep white matter of the right cerebral hemisphere.
  • the animals are then subjected to two weeks of continuous treatment with sodium phenylbutyrate (550 mg/kg/day, s.c), using osmotic minipumps transplanted subcutaneously. In control rats the minipumps are filled with saline.
  • Phenylbutyrate which is an intermediate metabolite of phenylacetate inhibited tumor cell replication, while the end metabolite, phenylacetylglutamine, is inactive.
  • both phenylacetate and phenylbutyrate promoted cell maturation and reversion to a nonmalignant phenotype, manifested by an altered pattern of cytoskeletal intermediate filaments, loss of anchorage-independence, and reduced tumorigenicity in athymic mice.
  • these compounds can be used effectively by women to control yeast infection without upsetting the microfloral balance of the vagina.
  • the compounds may similarly be used to control Candida microbes around wounds.
  • Other yeast organisms are equally suitable as targets of treatment including but not limited to Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus or combination thereof.
  • isolation and characterization of a secondary gene is exemplary of a general approach for isolating genes of the present invention. Isolation of the gene allows one of ordinary skill to readily isolate homologous genes in yeast and other microbial species. The isolated genes can then be used to construct recombinant vectors for altering the gene of interest.
  • DNA adjacent to the transposon in the isolated mutant gene as a probe the normal wild type allele of the target gene can be isolated.
  • a particularly useful transposon tagging system is disclosed, for example, in U.S. Pat. No. 5,013,658.
  • An alternative method uses oligonucleotide probes to identify the desired gene in a cDNA or genomic DNA library.
  • genomic libraries large segments of genomic DNA are generated by random fragmentation, e.g., using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector.
  • cDNA library mRNA is isolated, and a cDNA library which contains the gene transcript is prepared from the mRNA.
  • cDNA may be prepared from mRNA extracted from other cells in which secondary genes or homologs are expressed.
  • Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species.
  • the use of such hybridization techniques for identifying homologous genes is well known in the art and need not be described further.
  • DNA polymerase with an appropriate primer sequence.
  • a polyadenylation region at the 3'-end of the coding region should be included.
  • the polyadenylation region can be derived from the natural gene, or from a variety of other genes.
  • the vector comprising the sequences from a secondary gene will typically comprise a marker gene which confers a selectable phenotype on yeast cells.
  • the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or other conventional resistance.
  • Such DNA constructs may be introduced into the genome of the desire host by a variety of conventional techniques.
  • Cross Came out of candidate cross.
  • Literature Previously published interaction with mecl mutation.

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Abstract

The present invention relates to composition and methods of identifying one or more secondary drug targets and their use in the identification of drugs or drug candidates, particularly for the treatment of cancer or cell replication disorders. The yeast-based synthetic lethal screens are used to functionally identify and validate new gene targets to kill cells with defects in cell cycle pathways. These newly identified gene targets can be used to develop new therapeutics to treat cancer, benign cell growth, and yeast infections.

Description

MUTATION OF MEC1 -RELATED GENES IN TREATMENT FOR DISEASE
1. FIELD OF THE INVENTION
This invention relates to composition and methods of identifying eukaryote genes, which are important in cell life cycle. More specifically it relates to yeast genes and their mammalian counterparts such as human genes that are essential in cell growth and death. Methods and compositions are provided for identifying and treating cell cycle related gene dysfunctions resulting in diseases and clinical conditions such as malignancy and other diseases associated therewith.
2. BACKGROUND OF THE INVENTION
Genetic manipulation of the yeast genome provides a convenient model for identifying essential genes required for eukaryotic cell replication, growth, and death. The complete DNA sequence of the yeast Saccharomyces cerevisiae strain S288C was determined through an international collaboration of more than 100 laboratories on April
1996. General information and databases containing the yeast genome of about 6000 genes are available publicly and can be found for example on public websites such as for example: http://www.ncbi.nlm.nih.gov/Yeast; http://genome- www.stanford.edu/Saccharomyces/. Other databases exist as well and these databases and links therein to other websites are equally suitable for the purposes of this invention.
The examples include but are not limited to Yeast GenBank (A collection of all GenBank sequences that are derived from Saccharomyces cerevisiae); Yeast Swiss-Prot (The collection of Swiss-Prot protein sequences that are derived from Saccharomyces cerevisiae); YPD (The Yeast Protein Database maintained by Proteome, Inc.), and periodic updates thereof the content of which is incorporated herein by way of reference.
Methods of manipulating yeast are well established and are well known to those skilled in the art and can be found in publicly available web sites by using appropriate keywords, e.g., yeast and protocol, among many others.
While the sequence of the majority of all yeast genes is known and mapped on their respective chromosomes it is still difficult to predict the biological function of many of these genes especially those that have no counterparts in other species or fail to reveal a sufficient sequence homology with known genes. In other words determining the sequence of a gene is easier then ascribing a meaningful function to a gene. The function of only half of the 6,000 genes of the yeast is known. Furthermore, even with genes having known functions, it is not obvious how the function or regulation of such a gene may be modified when combined with another gene's regulatory mechanism or product.
Thus, the art is still highly unpredictable when it comes to a problem of identifying a gene's known or unknown function in combination with another gene.
In the past this task was accomplished on a case by case basis, whereby investigators used known gene manipulation techniques and screening methods and applied such methods or techniques to each specific gene of interest or to each particular gene combination. Yeast is often selected as a model due to the ease of manipulation and possibility of screening a large number of candidates in a relatively short period of time. Yeast have the highest rate of recombination and gene conversion among organisms tested, which is several orders of magnitude higher than in mammals. In the post- genomic era, serial gene-knockout programs in yeast (which you can tell by tetrad analysis, e.g., http:/ bioinformatics.weizmann.ac.il/pub/software/mac/mactetrad 69.readme) confirm that about 1 in 6 gene products are essential to the life of that cell (their deletion is lethal) under tested conditions. While a reasonable fraction of tested gene products are enzymes, other genes have either unknown function or their function does not fit into apriori postulation.
Several US patents exist which provide an insight into means of screening and identifying yeast genes and their respective functions. For example, incorporated by reference, U.S. Pat. Nos. 5,916,752 and 5,698,686 disclose telomerase compositions and screening methods; U.S. Pat. Nos. 5,821,076 and 5,756,305 disclose essential survival genes; and U.S. Pat. Nos. 5,585,245 and 5,503,977 deal with ubiquitin split protein sensor. A commonly used approach is screening by gene knockout and complementation. The term "complementation" is used herein as a genetic term intended to mean that the subject genetic element is homologous to a mutant genetic element (homologous by at least 40%) such that when introduced into a cell it rescues the cell from the effects of the mutation (see e.g., U.S. Pat. No. 5,527,896). For example, MEC-1
DNA rescues the mec-1 defect in a mec-1, cdc9-8 cell or RAD9 rescues the rad9 defect in a rad9, cdc9-8 cell through complementation process, and the MEC-1 (or RAD9) DNA so capable is referred to as a "complementing cDNA." Similarly, human CDC34 is homologous with yeast CDC34 and able to complement the mutation in single (or double mutant) cells, e.g., single mutant cells of yeast strain cdc34. For further details and examples see U. S. Pat. No. 5,866,338 to Hartwell, et al., incorporated herein by way of reference. Means of cross-referencing the yeast and human genes are now achievable and can be for example performed using "XREF2" program as found on http://www.ncbi.nlm.nih.gov/XREFdb/, which is incorporated herein by way of reference. These strategies resulted for example in identification DNA replication accessory factors from the yeast Saccharomyces cerevisiae such as DNA polymerase alpha and the human counterpart gene thereof.
In the setting of anti-cancer drug target identification, the primary defect would be a mutation in a gene conserved from yeast to humans that is frequently deregulated in tumors (e.g., overexpression of cyclin). Gene products with mutations that specifically kill cells with the primary defect would constitute putative "secondary drug targets" (that is, secondary to the primary defect) whose modulation in tumors may yield great therapeutic advantage. In principle, synthetic lethality can also result when there are two mutations that have an additive negative effect on a single essential biological pathway, or when the mutations inactivate two different but functionally overlapping pathways. Using genetic methods DNA damage checkpoint is identified, a signal transduction system that detects DNA damage and coordinates repair, replication and cell cycle progression. One of these genes in this pathway is MEC1 (MEC stands for mitosis entry checkpoint). MEC1, also known as ESR1, is required for the following: mitotic growth, DNA repair and mitotic recombination, regulation phosphorylation of Rad53p, dmcl arrest and meiotic recombination. The MEC1 gene product is similar to phosρhatidylinositol(PI)3-kinases required for DNA damage induced checkpoint responses in Gl, S/M, intra S, and G2/M in mitosis. The MEC1 null mutant is inviable, however the overproduction of Rad53p rescues some esrl alleles. MEC1 is a homolog of the human ATR gene (Cliby et al, EMBO J. 17:159-169, 1998). ATR is a phosphatidylinositol kinase-related (PIK-related) protein homologous to ATM, which is mutated in ataxia-telangiectasia patients. 3. SUMMARY OF THE INVENTION
The instant invention provides a recombinant eukaryotic cell comprising at least one secondary gene and at least one primary gene MEC1, an analog thereof, or a homolog thereof. In this recombinant eukaryotic cell at least one secondary gene is mutated such that the elimination or disruption of at least one primary gene results in synthetic lethality. The elimination can take any of several forms including truncation by mutation to form a stop codon. The disruption can take any of several forms including a loss of function or activity in the gene expression product. The recombinant eukaryotic cell of the invention can have the secondary gene selected from cdc8-l, rad27. mbpl-1 It, slmld/YOR080w, rnrl-77, rnr2-72, rnr4-106, cdc21-l, pril-62, poll-101, 141/154, 87, 240, and others.
The recombinant eukaryotic cell of the invention can have the primary gene impacted or altered to produce a loss of function. The recombinant cells of the invention can, among other cells, be yeast cells or yeast mutants and human cells or human neoplastic cells. One embodiment of the invention is the synthetic lethal mutant resulting from a point mutation in the mecl gene, which impacts the kinase domain of MEC 1. Another embodiment of the invention is the synthetic lethality resulting from mutations in mecl in combination with deficiencies in lagging strand DNA synthesis. Until the present invention no reports exist in the prior art suggesting or teaching the synthetic lethality in yeast when MEC1 is not expressed in combination with secondary gene mutants of genes in the deoxynucleotide synthesis pathway or where there are deficiencies in lagging strand DNA synthesis.
Mass screening and genetic analysis compounds or drug targets are identified that could specifically kill target cells, such as tumor cells. In this context, examples of compounds that simulate the activity of found lethal mutants are provided. Accordingly, therapeutic agents are contemplated, which are developed from the identification of essential genes of eukaryotic organisms. Such an identified gene or product thereof serves as a novel target for therapeutics based on a mechanism which is distinct or similar to the mechanisms of existing drugs. Such a compound inhibits the function of a gene product identified by methods disclosed herein, for example, by producing a phenotype or morphology similar to that found in the original mutant strain.
According to one aspect of the invention, a mutant collection is systematically screened to identify genes and/or gene products, which are targets for drugs. For example, a drug may act as an antagonist by binding reversibly, or preferably irreversibly, to the identified gene or gene product target, and thereby impairing its function. Loss of the function (or the synthesis or the complete processing) of the gene product target will result in inhibition of the cell growth, and preferably will result in death of the cells. This aspect includes a method for identifying anti-cancer agents, including the step of exposing a gene product corresponding to the wildtype sequence of a mutant sequence identified by methods disclosed herein to the test agent; and selecting agents which impair (preferably, selectively) the function of the gene product.
The present invention provides for a method of identifying a drug that inhibits the growth or replication of a cell having a mutated MEC 1 gene or an analog or homolog thereof, by contacting, a cell having a mutated MEC1 gene or an analog or homolog thereof with the drug; and determining whether the drug modulates the activity of a wildtype secondary gene which is synthetically lethal when it is mutated and is present in combination with mutated MEC1 gene or an analog or homolog thereof.
In a particular embodiment of the invention, the primary gene defect is preferably one found in or associated with a tumor cell or cell affected with cell cycle deregulation.
Alternatively, the primary gene defect in the cell provided by the instant method is analogous or homologous to a defect found in or associated with a mammalian or human tumor cell or chromosomally aberrant cell. By "homologous" is meant a direct relationship among a "family" of genes in which certain sequences or domains are strongly conserved among the members of the family. For instance, the yeast MEC1 gene is homologous to mammalian genes encoding AT-related kinase (Cliby et al., ibid., 1998). On the other hand, "analogous" genes may serve similar or "analogous" functions, but they are not directly related (i.e., sequences are not conserved among analogous genes). Within this context, such "analogous" gene are referred to as "functionally homologous" by which is meant that the gene product functions in an analogous manner in another system, such as a gene product that has a function in a yeast cell that is analogous to a function in a mammalian cell. MEC1 for example codes for phosphatidyl inositol kinase-related kinase. Analogs of MEC 1 include the family of kinases and particularly the phosphatidyl inositol kinases.
In the present method, the primary gene defect may result in the alteration, loss, or inhibition of a function, for example, a cellular function. However, the primary gene defect may also result in the enhancement or gain of a function
Generally, the functions affected can vary widely. The affected functions may include, but are not limited to, the suppression of tumor growth, DNA damage checkpoint, DNA mismatch repair, nucleotide excision repair, O6-methylguanine reversal, double-strand break repair, DNA helicase function, signaling, cell cycle control, or apoptosis. Such defects may be effectively modeled by primary gene defects in other organisms such as Drosophila.
By the methods of the present invention, it has been found that certain secondary site mutations can be effected, which may turn out to be lethal to the cell harboring the primary gene defect. Such secondary site mutations may be effected, for example, within a gene selected from the group cdc8-l thymidilate ldnase activity: deoxythymidine monophosphate kinase; rad27, endo and 5' exonuclease; mbpl-1 It, transcription factor slmd/(YOR080w); rnrl-77, ribonucleotide reductase large subunit; rnr2~72, ribonucleoptide reductase small subunit; rnr4-106, ribonucleotide reductase small subunit; cdc21-l, thymidylate synthase; pril-62, DNA primase; POLalpha, poll-101,
DNA polymerase alpha.
Double mutants that involve MEC1 and a secondary gene and/or ORF (open reading frame) are also contemplated within the scope of this invention. Most preferably, the double mutation of yeast is effected within a gene having a mammalian analog or homolog so that mammalian models can be built which will rely on discoveries made in yeast. These may include but are not limited to yeast CDC8, RAD27, MBP1, SLM1, R R1, RNR2, RNR4, CDC21, PRIl, POL1 among many others. In a preferred embodiment of the invention, the homologous (by at least 40%) or analogous mammalian gene is selected from this group. A further object of the invention includes the identification of drug or drug candidates. Hence, after the secondary drug target is elucidated, the secondary drug target can be used to screen for a drug or drug candidate that can potentially interact with the secondary drug target, for example to disable its physiological activity. Accordingly, the present invention may provide a drug or drug candidate that interacts with, binds to, or inhibits a particular gene product. Such gene products may include, but are not limited to examples listed supra. It is desirable that the drug or drug candidate exhibit the capacity to inhibit or arrest the growth of a human tumor or benign neoplastic growth.
The preferred pharmaceutical composition of the drug comprises oligonucleotide, gene product, homologs or analogs of oligonucleotide or gene product, a small molecule, or a peptide mimetic. Thus an object of this invention is to provide means of controlling unwanted proliferation or differentiation of eukaryotic cells. The term "unwanted proliferation" refers to proliferation of cells which is undesired, be it due to transformation of the cells, e.g., neoplastic or hyperplastic, for purposes of wound healing for example keloid, treatment of restenosis, infection by eukaryotic pathogens and other unwanted smooth muscle proliferation, cosmetic applications, retinopathy resulting from diabetes, etc.
Likewise, the term " unwanted differentiation" refers to an undesirable change in the differentiation of a cell, such as where a differentiated cell reverts to an earlier state and recovers or retains an ability to proliferate un-naturally or in an "unwanted" manner.
The present invention provides methods of treating malignant conditions, such as prostatic cancer, melanoma, adult and pediatric tumors, e.g., brain tumors of glial. origin, astrocytoma, Kaposi's sarcoma, lung adenocarcinoma and leukemias, as well as hyperplastic lesions, e.g., benign hyperplastic prostate and papillomas by administering a therapeutically effective amount of subject drug or a pharmaceutically acceptable derivative thereof. Most preferably, administration of the drug or drug candidate results in death of the tumor cell, reduction in neoplastic tissue and a therapy for the cancer. Without limiting to cancer, other clinical conditions are anticipated as being treatable by compounds of the present invention. These conditions include, for example, cardiovascular conditions like restenosis, in which smooth muscle cells proliferate excessively in blood vessels after angioplasty, potentially leading to restricted blood flow and death. Without limiting to aforementioned diseases, this invention also provides a means for treating various mycotic or yeast pathogens selected from a group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus or combination thereof.
Thus, the object of this invention is to provide a means of controlling unwanted proliferation or differentiation of eukaryotic cells. The term "unwanted proliferation" refers to proliferation of cells which is undesired, be it due to transformation of the cells, e.g., neoplastic or hyperplastic, for purposes of wound healing, treating of restenosis and other unwanted smooth muscle proliferation, cosmetic applications, etc. Likewise, the term "unwanted differentiation" refers to an undesirable change in the differentiation of a cell, such as senescence. The present invention also embodies diagnostic and prognostic assays, which assess the phenotype and aggressiveness of a disorder by detecting the expression of a cyclin protein or expression of regulating genes or gene products thereof.
A further object of this invention includes a pharmaceutical composition comprising a drug, in a pharmaceutically acceptable carrier or diluent, which drug selectively interacts with the production of at least one gene product in a cell population that contains at least one primary gene defect, wherein the exposure of the cell population to the drug arrests cell division selectively in the cell population. Such gene products are encoded or regulated by a human gene analogous or homologous to a yeast gene.
A further object of this invention includes means of using of gene therapy to induce synthetic lethality in a cell containing at least one defect in a primary gene such as
MEC1 or a homolog or analog thereof. Within the context of this invention, methods of inducing synthetic lethality include introducing a polynucleotide into a cell containing at least one defect in a primary gene such as MEC1 or a homolog or analog thereof wherein the action or expression of the polynucleotide results in the perturbation of a secondary gene that results in synthetic lethality of the cell. Perturbation of the secondary gene includes but is not limited to the up-regulation, down-regulation, elimination or disruption of the secondary gene. Polynucleotide suitable for use in this invention include, but are not limited to triplex forming oligonucleotides, antisense polynucleotides, RNA-I (for review see Nature 402: 128-129, 1999; which is incorporated herein by reference in its entirety) and ribozymes that specifically target the secondary gene. Secondary genes useful in this regard include analogs and homologs of
CDC8, RAD27, MBP1, SLMl/YOR080w, RNR1, RNR2, RNR4, CDC21, PRIl, POL1, 141/154, 87, and 240, and combinations thereof. It should be noted that findings in yeast are readily translatable to human gene(s) regulation since many of yeast genes are homologous to human counterparts. Means of cross-referencing the yeast and human genes are now achievable and can be for example performed using "XREF2" program as found on http://www.ncbi.nlm.nih.gov/XREFdb/, which is incorporated herein by way of reference.
The use of antisense polynucleotides and their applications are described generally in, for example, Mol and Van der rul, eds., Antisense Nucleic Acids and Proteins Fundamentals and Applications, New York, NY, 1992, incorporated by reference herein in its entirety. Suitable antisense oligonucleotides are at least 15 nucleotides in length and up to and including the upstream untranslated and associated coding sequences of the secondary gene of choice. As will be evident to one skilled in the art, the optimal length of antisense oligonucleotides is dependent on the strength of the interaction between the antisense oligonucleotides and their complementary sequence on the mRNA, the temperature and ionic environment in which translation takes place, the base sequence of the antisense oligonucleotide, the presence of secondary and tertiary structure in the mRNA and/or in the antisense oligonucleotide and the preferred delivery mode. For example, soluble antisense oligonucleotides have been used to inhibit transcription/translation of a target gene (Ching et al., Proc. Natl. Acad. Sci. USA
86:10006-10010, 1989; Broder et al., Ann. Int. Med. 113: 604-618 (1990); Loreau et al., FEBS Letters 274:53-56 1990; Holcenberg et al., WO91/11535; WO91/09865; WO91/04753; WO90/13641; and EP 386563, each incorporated herein by reference). Suitable target sequences for antisense polynucleotides include intron-exon junctions (to prevent proper splicing), regions in which DNA/RNA hybrids will prevent transport of mRNA from the nucleus to the cytoplasm, initiation factor binding sites, ribosome binding sites, and sites that interfere with ribosome progression. A particularly preferred target region for antisense polynucleotides is the 5' untranslated region of the secondary gene of choice.
Antisense polynucleotides targeted to the secondary gene of choice may also be prepared by inserting a DNA molecule containing the target polynucleotide sequence into a suitable expression vector such that the DNA molecule is inserted downstream of a promoter in a reverse orientation as compared to the gene itself. The expression vector may then be transduced, transformed or transfected into a suitable cell resulting in the expression of antisense polynucleotides. Alternatively, antisense oligonucleotides may be synthesized using standard manual or automated synthesis techniques. Synthesized oligonucleotides may be introduced into suitable cells by a variety of means including electroporation (e.g., as described in Yang et al., Nucl. Acids. Res. 23: 2803-2810, 1995), calcium phosphate precipitation, microinjection, poly-L-ornithine/DMSO (Dong et al., Nucl. Acids. Res. 21: 771-772, 1993). The selection of a suitable antisense oligonucleotide administration method will be evident to one skilled in the art. With respect to synthesized oligonucleotides, the stability of antisense oligonucleotide-mRNA hybrids may be increased by the addition of stabilizing agents to the oligonucleotide. Stabilizing agents include intercalating agents that are covalently attached to either or both ends of the oligonucleotide. Oligonucleotides may be made resistant to nucleases by, for example, modifications to the phosphodiester backbone by the introduction of phosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates, phosphoramidates or phosphorodithioates. Oligonucleotides may also be made nuclease resistant by the synthesis of the oligonucleotides with alpha-anomers of the deoxyribonucleotides, as generally described in Mol and Van der Krul (ibid.). Within another embodiment, polynucleotide-based inhibitors of the present invention include the triplex forming oligonucleotides, sequence-specific DNA binding drugs that interfere with target gene transcription. Triplex-forming oligonucleotides are generally described in Maher, Bioessays 14: 807-815, 1992; Gee et al., Gene 149: 109- 114, 1994; Noonberg et al, Gene 149: 123-126, 1994; Song et al, Ann. NY Acad. Sci. 761: 97-108, 1995; Westin et al., Nuc. Acids. Res. 23: 2184-2191, 1995; and Wand and
Glazer, J. Biol. Chem. 207: 22595-22901, 1995, each incorporated herein by reference in its entirety. These oligonucleotides form triple helical complexes under physiological conditions on double-stranded DNA, selectively inhibiting gene transcription by physically blocking RNA polymerase or transcription factor access to the DNA template. See also, e.g., WO 95/25818; WO 95/20404; WO 94/15616; WO 94/04550; and WO 93/09788, each of which is incorporated herein by reference. The triplex forming oligonucleotides may contain either a nucleotide or non-nucleotide tail to enhance the inhibition of transcription factor binding. Within one example, the triplex forming oligonucleotides are targeted to the secondary genes: CDC8, RAD27, MBP1, SLMl/YOR080w, RNR1, RNR2, RNR4, CDC21, PRIl, POL1, 141/154, 87, and 240 and their respective analogs and homologs.
For polynucleotide-based inhibitors, the choice of a suitable sequence will be guided by, for example, the type of inhibitor (i.e., triplex forming oligonucleotide or antisense polynucleotide) and the species to be treated. It may be preferable to choose sequences that are conserved between species to permit use in readily available animal models.
The present invention also provides compositions and methods for using ribozyme inhibitors for inhibiting secondary gene expression. The ribozymes can be administered in a variety of ways, including by gene therapy targeted to a desired cell. The ribozyme of the invention may target the RNA transcripts of the gene of interest in a sequence-specific manner. For example, ribozymes may be designed to specifically inhibit the transcription of the secondary gene Each ribozyme molecule is designed to contain a catalytically active segment capable of cleaving secondary gene RNA, and further comprises flanking sequences having a nucleotide sequence complementary to portions of the targeted RNA. The flanking sequences to the target sequence do not need to be completely complimentary, however, as the flanking sequences need only be sufficiently complimentary to form a duplex with the target RNA and to allow the catalytically active segment of the ribozyme to cleave at the target sites. Thus, the flanking sequences need only be sufficiently complimentary to pemiit the ribozyme to be hybridizable with the target RNA under physiological conditions. As used herein, the term "ribozyme" means an RNA molecule having an enzymatic activity that is able to cleave or splice other separate RNA molecules in a nucleotide base sequence specific manner. By reference to catalytic or enzymatic RNA molecule is meant an RNA molecule which has complementarity in a substrate binding region to a specific RNA target (e.g. CDC8, RAD27, MBP1, SLMl/YOR080w, RNR1, RNR2, RNR4, CDC21, PRIl, POL1, 141/154, 87 or 240 RNA), and also has enzymatic activity that is active to cleave and/or splice RNA in that target, thereby altering the target molecule. In preferred embodiments of the present invention the enzymatic RNA molecule is formed in a hammerhead motif, but the ribozyme may also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RNAse P RNA (in association with an RNA guide sequence). Examples of hammerhead motifs are described by Rossi et al, AIDS Res. Hum. Retrovir. 8: 183, 1992, hairpin motifs are described by Hampel et al., Biochem. 28:4929, 1989 and Hampel et al., Nucl. Acids Res. 18: 299, 1990, the hepatitis delta virus motif is exemplified in Perrotta and Been, Biochem. 31: 16, 1992, an RNAseP motif is described in Guerrier-Takada et al., Cell 35: 849, 1983, and examples of the group I intron motif are described in Cech et al., U.S. Patent 4,987,071, each of the foregoing disclosures being incorporated herein by reference. These specific motifs are not limiting in the present invention and those of skill in the art will recognize that an enzymatic RNA molecule of the invention has a specific substrate binding site which is complementary to one or more of the target RNA regions and that it has nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule. The flanking sequences upstream and downstream of the ribozyme catalytic site may comprise segments of any length that effectively imparts the desired degree of targeting specificity for the ribozyme. Preferably a flanking sequence comprises from about 4 to about 24 nucleotides, more preferably from about 6 to about 15 nucleotides, and typically about 9 to 12 nucleotides, and results in base pairing to the substrate sequence immediately upstream and downstream of the RNA sequences which comprise the cleavage site.
Polynucleotide inhibitors, e.g., triplex forming oligonucleotides, antisense oligonucleotide, ribozyme, etc., or a combination of such inhibitors targeted to different portions of the target DNA or corresponding RNA can be delivered in a wide variety of ways to targeted cells to facilitate inhibition of the gene of interest. The oligonucleotides can be administered as synthetic oligonucleotides or expressed from an expression vector. The oligonucleotide can be administered ex vivo, i.e., contacted with target cells that have been removed from an individual or other cell source, treated and returned, or the oligonucleotide molecule can be administered in vivo. When administered ex vivo typically the target cells are exposed to mitogens, e.g., serum mitogens (SCF, IL-3, EPO, TPO, etc.) or the like depending on the particular cell population.
A synthetic lethal screening method is disclosed based on lethality of yeast mutants having a mutation in a primary gene of interest and secondary target genes, which when present in a mutant form and in combination with the primary gene mutation, determine cell proliferation and death regulation. The subject screening assay uses single and double mutants that are capable of complementing or suppressing the function of the primary gene of interest. Experimental examples enabling this invention are provided which identify novel genes involved in human cell cycle control. These genes include but are not limited to yeast CDC8, RAD27, MBP1, SLMl/YOR080w, RNR1, RNR2, RNR4, CDC21, PRIl, POL1, 141/154, 87, 240, and others and also include analogs and homologs of these genes. These genes encode proteins involved in aspects of the cell cycle including, but not limited to aspects of DNA replication, DNA repair, transcription control and nucleotide cycling. POL1, for example is required for mitotic DNA synthesis, premeiotic DNA synthesis, recombination, and full sporulation and its null mutant is inviable. Poll(ts) mutants show blocked cell division at 36 degrees C. CDC8 and its homologs and analogs encode proteins with thymidilate kinase activity.
RAD27 and its homologs and analogs encode proteins with endo and 5' exonuclease activity. MBP1 and its homologs and analogs encode proteins that act as transcriptions factors. RNR proteins act as subunits of ribonuclease reductase. CDC21 and its homologs and analogs encode proteins that act as thymidilate synthases. PRIl and its homologs and analogs encode proteins that act as a DNA primase.
These and other objects of the present invention will be evident from the disclosure provided herewith.
DETAILED DESCRIPTION OF THE INVENTION While scientists have known for centuries that cells divide, only recently have researchers really begun to make progress in investigating the mechanism of the division. Understanding the driving mechanisms involved and the chemical regulators thereof will help to determine why this process sometimes goes wrong (as in the case of cancer cell growth) and may aid in the development of methods to prevent and reverse such malfunctions.
This invention teaches a synthetic lethal screen carried out in Saccharomyces cerevisiae strain A364a to find mutants that require MEC1 function for viability. The screening method included an initial mutagenesis of a MEC1 deletion strain (yEF611) harboring a MEC1 plasmid (pEF208) containing ADE3 and URA3 genes. The presence of the ADE3 gene allows for an initial red/white selection in the strain background used, while maintaining URA3 on the plasmid allows for a secondary test for plasmid dependent growth. A tertiary test included transforming meclΔ synthetic lethal mutants (MSLs) with another MEC1 plasmid containing HIS3. The identification of genes that are synthetic lethal in combination with mutations in the primary gene MEC1 permit the development of screens for agents that can mimic the secondary gene mutation. Such agents are useful therapeutics within the context of this invention for the treatment of diseases associated with aberrant proliferation. The secondary genes of the present invention are used, for example, within assays to identify therapeutic agents for the treatment of proliferative diseases such as cancer wherein the disease is characterized by a mutation in the primary gene, such as mutations in MEC lor an analog or homolog thereof. Within these aspects of the invention, screening assays may be carried out to identify agents, including candidate drugs, that modulate the activity of the secondary gene or the secondary gene product or an analog or homolog thereof, wherein such modulation results in the inhibition or reduction in activity of secondary gene product, the up-regulation, down-regulation, elimination or disruption of said secondary gene. Assays in this context may use whole cells expressing the target gene, cell lysates containing the target gene product or may use purified target gene product. Within certain assays of the invention, test agents are incubated with reaction mixtures containing target gene-expressing cells under suitable conditions and for a time sufficient to permit the test agent to modulate the activity of the target gene product and wherein a control sample is incubated under identical conditions in the absence of the test agent. As used herein, modulation of the activity of a target gene product includes, but is not limited to, increasing or decreasing the activity of a target gene product through, for example, direct binding to the gene product and increasing or decreasing the activity of a target gene product by up-regulating or down-regulating transcription or translation of the target gene product. Aspects of the invention include high-throughput screening assays designed to identify modulators of the target gene product.
Within one assay of the invention, candidate drugs are identified by the following method a) exposing a test cell system comprising a cell having a deletion or mutation in a primary gene such as MEC1 to a candidate drug; b) comparing the viability of the test cell system with the viability of a control cell system comprising a cell having a deletion or mutation in a primary gene such as MEC1 or analogs or homologs thereof and a secondary gene which in its mutant form is synthetically lethal in combination with the primary mutation, such as MEC1 or analogs or homologs thereof, wherein a candidate drug that causes the test cell system to be less viable than the control cell system is a therapeutic drug candidate. Within the context of this invention, viability is measured as the ability of a cell to reproduce itself. Non- viable cells are those cells that do not or cannot divide, those that are arrested in any phase of the cell cycle, those that apoptose or proceed along the apoptotic pathway and those that die. Means for determining the presence of viable and non- viable cells will be evident to one skilled in the art and include vital dye staining, measurement of chromosome condensation, and the like.
Within another embodiment is a method of identifying a compound useful in the treatment of proliferative diseases, such as cancer, characterized by the a mutation in the MEC1 gene or analog or homolog thereof which comprises the steps of 1) contacting the wild-type gene product of a secondary gene that in its mutant form is synthetically lethal in combination with the mutant MEC1 gene or an analog or homolog thereof with a test compound under conditions and for a time sufficient to permit the test compound to effect the secondary gene product; 2) comparing the activity of the gene product in the presence of the test compound with the activity of the secondary gene product in the absence of the test compound. Within this exemplary assay, compounds that can decrease the activity secondary gene product or an analog or homolog thereof are identified as useful for the treatment of cancer or other proliferative diseases characterized by a mutation in the MEC1 gene or analog or homolog thereof. Within another embodiment of the invention, whole cell assays are conducted wherein a cell with a wildtype secondary gene or analog or homolog thereof is contacted with a test compound and the expression and/or activity of the secondary gene product or analog or homolog thereof is assessed and compared with the expression and/or activity of the secondary gene product in cells not exposed to the test compound. Within this aspect, those compounds that result in decreased expression or activity of the gene product are useful for the treatment of cancer or other proliferative diseases characterized by a mutation in the MEC1 gene or analog or homolog thereof.
For purposes of the assays described herein, assays for modulation of the gene products of the secondary genes are known to those skilled in the art. As would be evident to one skilled in the art, the secondary gene target will determine the appropriate assay for measuring modulation of protein activity as the artisan will be familiar with the assays used to characterize the genes and their respective mutations. For example, the increase or decrease in enzymatic activity of ribonucleotide reductase (the gene products of RNR1 and RNR2) by the direct reduction of ribonucleotides to deoxyribonucleotides using the methods of Thelander et al. (J. Biol. Chem. 255: 7426-7432, 1980) and Engstrom et al., (Biochemistry 18: 2941-2948, 1979), the measurement of thymidilate kinase (the gene product of CDC8) activity can be carried out by measuring the phosphorylation or deoxythumidine monophosphate as described, for example, by Yong and Campbell (J. Biol. Chem. 259: 14394-14398, 1984), and the endo- and 5' exonuclease activities of the RAD27 protein and the polymerase activities associated with POL1 can be determined using such assays that follow nuclease and polymerase activities. Activity of a protein can also be determined by measuring the increase or decrease in binding of a gene product to its ligand or substrate by, for example, visualization using antibody staining or by immunoprecipitation. While, structural proteins, such as tubulin (the gene products of TUB 1 and TUB2) can be measured by measuring the quality and quantity of tubulin made by a cell. After meclΔ dependent synthetic lethality is demonstrated the mutants are sorted into complementation groups, which is usually a good indication of the number of alleles of specific genes present in the pool of mutants. This is accomplished through switching the mating type of a number of MSLs and mating them to other MSLs. If complementation is successful the MECl ADE3 URA3 plasmid can be lost and the diploid strain is phenotypically white or sectored and partially or completely deficient for growth on medium lacking uracil.
Successful complementation indicates that MSLs maintain mutations that are not allelic, while unsuccessful complementation, in most cases, indicates that mutations occur in the same gene. Once MSLs have been sorted into complementation groups, plasmids containing genomic libraries are transformed into selected MSLs in order to find yeast ORF/genes which alleviate the synthetic interaction. Once a candidate gene has been found allelism is minimally demonstrated by:
(1) determining that the MSL mutation is linked to the candidate ORF (eg segregates with the ORF during meiosis.)
(2) showing that the candidate ORF can singly complement the synthetic phenotype on low copy number plasmid.
As a last confirmation, MSLs are backcrossed three to four times to a wild type strain and then crossed to a MECl kinase inactive mutant.
One MSL mutant contains a mutation in CDC17/POLl/POLalpha. This allele has been designated poll-101 (strain yPP210). The poll-101 mutant requires MECl function between 30 degrees and 36 degrees. A poll-101 medld double mutant, however, is capable of growth below 28 degrees. Thus, synthetic lethal interaction occurs at a temperature where poll-101 function is compromised presumably due to the temperature sensitive nature of the lesion.
This invention provides recombinant eukaryotic cells, e.g. yeast mutants, as models of cancer and other diseases associated with deregulation of cell growth and replication.
Yeast is an extremely useful model for studying human diseases. For example, as of January 20, 1996 the following human diseases and clinically important conditions have been identified as having matches between human genes and S. cerevisiae genes/proteins: Hereditary Non-polyposis Colon Cancer; Cystic Fibrosis; Wilson Disease; Glycerol Kinase Deficiency; Adrenoleukodystrophy; Ataxia Telangiectasia; Amyotrophic Lateral
Sclerosis; Myotonic Dystrophy; Lowe Syndrome; Neurofibromatosis, Type 1; Choroideremia; Diastrophic Dysplasia; Lissencephaly; Thomsen Disease; Wilms Tumor; Achondroplasia; Menkes Syndrome; Multiple Endocrine Neoplasia 2A; Wiskott-Aldrich Syndrome; Duchenne Muscular Dystrophy; Aniridia; Gonadal Dysgenesis; Breast and Ovarian Cancer, Early Onset; Chronic Granulomatous Disease; Epidermolytic Palmoplantar Keratoderma; Waardenburg Syndrome; Adenomatous Polyposis Coli;
Neurofibromatosis, Type 2; Kallmann Syndrome; Tuberous Sclerosis; Polycystic Kidney Disease; Aarskog-Scott Syndrome; Marfan Syndrome; Huntington Disease; Spinocerebellar Ataxia; Long QT Syndrome, Type 1; Fragile X Syndrome; Emery- Dreifuss Muscular Dystrophy; Retinoblastoma; McLeod Syndrome; Norrie Disease; Von Hippel-Lindau Disease; Alzheimer Disease; Hyperekplexia; Agammaglobulinemia, X- linked (for detailed information see http://www.ncbi.nlm.nih.gov/Bassett/Yeast/ and updates thereof as incorporated herein by way of reference). Means of cross-referencing the yeast and human genes are now achievable and can be for example performed using "XREF2" program as found on http://www.ncbi.nlm.nih.gov/XREFdb/, which is incorporated herein by way of reference. Abnormalities in components of the cell cycle surveillance system have been identified in human cancers and other diseases such as listed above. These abnormalities include alterations in cyclin (80-90% of tumors), p53 (50-60% of tumors), and DNA mismatch repair (10-20% of some tumor types such as colon and pancreatic). Often the primary genetic alteration is a loss of function and so a drug discovery program focused on these defects would require restoring the lost function. An alternative approach is to identify which other protein(s) when inhibited selectively kill cells that have the primary defect.
General information and databases containing yeast genome are available publicly and can be found for example on public websites such as for example: http://bioinformatics.weizmann.ac.il; http://ourworld.compuserve.coin/homepages/C_Velten/yeast.htiri; http://www.ncbi.nlm.nih.gov/Yeast; http://genome-www.stanford.edu/Saccharomyces/; genome-ftp.stanford.edu (directory /yeast/genome_seq); http ://vectordb . atcg.com/vectordb/; http ://www.mpimg-berlin- dahlem.mpg.de/~andy/GN/S.cerevisiae/; or http://www.mips. biochem.mpg.de/proj/yeast, the content of which and links therein are incorporated herein by way of reference. Other databases exist as well and these databases and links therein to other websites are equally suitable for the purposes of this invention. The examples include but are not limited to Yeast GenBank (A collection of all GenBank sequences that are derived from Saccharomyces cerevisiae); Yeast Swiss-Prot (The collection of Swiss-Prot protein sequences that are derived from Saccharomyces cerevisiae); YPD (The Yeast Protein Database maintained by Proteome, Inc.), and periodic updates thereof the content of which is incorporated herein by way of reference. Methods of manipulating yeast are well established and are well known to those skilled in the art and can be found in publicly available web sites such as for example www.goshen.edu/bio/yeast, www.fhcrc.org/~gottschling, and www.sacs.ucsf.edu/home/HerskowitzLab /protocols/protocol, among many others.
The invention features a method of identifying mutant organisms having conditional- sensitive lethal mutations, and subsequently gene products thereof. The disclosed methods are useful for high-throughput screening of genomic or mutant libraries to rapidly identify genes, and corresponding gene products, which are essential for survival.
A lethal mutation results in a gene or a protein that is not functional under restrictive conditions (i.e., in a tumor cell). A non-functional gene can have a defect in the promoter resulting in a reduced or abnormal gene expression. A non-functional protein may have a conformational defect causing improper protein folding or abnormal protein degradation. Improper protein folding can result in partial or total failure to fold, to recognize a native substrate, and/or to bind and release the substrate.
There are a wide variety of pathological cell proliferative conditions for which the compounds of the present invention can provide therapeutic benefits, with the general strategy being the inhibition of an anomalous cell proliferation. To illustrate, cell types which exhibit pathological or abnormal growth include various cancers and leukemias, psoriasis, bone diseases, fibroproliferative disorders such as involving connective tissues, atherosclerosis and other smooth muscle proliferative disorders, as well as chronic inflammation.
In addition to proliferative disorders, the treatment of differentiative disorders which result from, for example, de-differentiation of tissue which may (optionally) be accompanied by abortive reentry into mitosis. Such degenerative disorders include chronic neurodegenerative diseases of the nervous system, including Alzheimer's disease, Parkinson's disease, Huntington's chorea, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations. Other differentiative disorders include, for example, disorders associated with connective tissue, such as may occur due to de- differentiation of chondrocytes or osteocytes, as well as vascular disorders which involve de-differentiation of endothelial tissue and smooth muscle cells, gastric ulcers characterized by degenerative changes in glandular cells, and renal conditions marked by failure to differentiate, e.g. Wilm's tumors.
In addition to therapeutic applications (e.g., for both human and veterinary uses) it will be apparent the subject compounds can be used as a cell culture additive for controlling proliferative and/or differentiation states of cells in vitro.
It will also be apparent that differential screening assays can be used to select for those compounds of the present invention with specificity for non-human yeast enzymes. Thus, compounds that act specifically on eukaryotic pathogens, e.g., are anti-fungal or anti-parasitic agents, can be selected from the subject inhibitors. To illustrate, inhibitors of the invention can be used in the treatment of candidiasis- an opportunistic infection that commonly occurs in debilitated and immunosuppressed patients. These same inhibitors could be used to treat these infections in patients with leukemias and lymphomas, in people who are receiving immunosuppressive therapy, and in patients with such predisposing factors as diabetes mellitus or AIDS, where fungal infections are a particular problem.
By way of illustration, the assays described in the art can be used to screen for agents which may ultimately be useful for inhibiting at least one fungus implicated in such mycosis as candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, coccidiodomycosis, conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis, nocaidiosis, para-actinomycosis, penicilliosis, monoliasis, or sporotrichosis.
In addition to such therapeutic uses, anti-fungal agents developed with such differential screening assays can be used, for example, as preservatives in foodstuff, feed supplement for promoting weight gain in livestock, or in disinfectant formulations for treatment of non-living matter, e.g., for decontaminating hospital equipment and rooms. In similar fashion, side by side comparison of inhibition of a mammalian gene and an insect gene, such as the Drosophilia, will permit selection amongst the subject derivatives of inhibitors which discriminate between the human/mammalian and insect enzymes. Accordingly, the present invention expressly contemplates the use and formulations of the subject in insecticides, such as for use in management of insects like the fruit fly.
In yet another embodiment, certain of the subject inhibitor compounds can be selected on the basis of inhibitory specificity for plant gene relative to the mammalian counterpart. For example, a plant gene can be disposed in a differential screen with one or more of the human enzymes to select those compounds of greatest selectivity for inhibiting the plant enzyme. Thus, the present invention specifically contemplates formulations of the subject inhibitors for agricultural applications, such as in the form of a defoliant or the like.
In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the compounds described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam. The phrase "therapeutically-effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect by modulating the activity of expression of a target secondary gene in at least a sub-population of cells in an animal containing a mutation in the primary gene (MECl or an analog or homolog thereof) and thereby blocking the biological consequences of that protein activity in the treated cells, at a reasonable benefit/risk ratio applicable to any medical treatment. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
As set out above, certain embodiments of the drugs or agents of the present invention may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically- acceptable acids. The term "pharmaceutically-acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like.
In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term "pharmaceutically-acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfϊte, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Formulations of the present invention include those suitable for oral, nasal, topical
(including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent. Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.
In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients. Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and poly amide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, eye drops, powders, implants and the like, are also contemplated as being within the scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
The preparations of the present invention may be given orally, parenterally, topically, or rectally . They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred.
The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
These peptides and compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compounds employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
Generally, intravenous, intracerebro ventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects will range from about 0.0001 to about 100 mg per kilogram of body weight per day. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.
EXAMPLE 1. GENERAL YEAST HANDLING PROCEDURES, MATERIALS, AND
METHODS
STRAINS AND MEDIA: Extrachromosomally replicating vectors are either vectors containing autonomously replicating sequences (ars vectors are usually present in high copy numbers in the cell, however, they are frequently lost during cell division) or vectors containing a DNA sequence acting as a centromere during cell division (cen vectors, though very stable, are present only in a few copies in the cell). Standard methods are used for all strain constructions and transformations as well as for the media used. Yeast nitrogen base (USB Biologicals; USA) without amino acids and ammonium sulfate (Sc) is used when a selection for an auxotrophy is required (Guthrie and Fink 1991). Various yeast growing and drop-out media are known in the art (see e.g., www.clontech.com or www.biol01.com/yeast/). YPD medium and drop out synthetic media containing either 2% glucose (SD medium), 2% galactose (SGAL medium) or 2% raffmose (SRAF medium) are according to Rose et al. (Methods in Yeast Genetics, 1990, CSH Lab. Press). Hydroxyurea (HU) plates are prepared by dissolving directly in the medium (55 degrees C) the appropriate amount of HU. Benomyl (BE) plates are prepared by dissolving BE in DMSO to lOOx final concentration (stock solution), adding 0.2 mis of the stock solution to 20 mis of medium (55 degrees C). DMSO did not exceed 1% final concentration. All the experiments, unless otherwise indicated, are performed in drop-out synthetic medium lacking uracil and tryptophan, and containing the appropriate carbon source. NUCLEIC ACID METHODS: DNA cloning is performed by standard techniques outlined by Sambrook et al. 1989. Standard procedures are used for restriction analysis and plasmid constructions (Sambrook et al. 1989). Double-stranded plasmid DNA is sequenced with a Sequenase kit (United States Biochemical Corp.; USA).
TETRAD ANALYSIS: When diploid S. cerevisiae cells undergo meiosis, four haploid spores are produced which are enclosed within a sac called an ascus. Using a micromanipulator, the 4 spores (the "tetrad") can be "dissected" out of the ascus and separated on an agar plate. The spores then germinate, and the colonies that arise can be tested for the presence or absence of phenotypes characteristic of the genetic markers present in the original diploid strain. In this way the segregation pattern of the genetic markers can be assessed, allowing determination of genetic linkage of, and distances between, the markers. When 2 genetic markers are analyzed in this way, the data is called 2 point data.
MORPHOLOGICAL ANALYSIS: Tubulin is visualized using anti-tubulin antibody (1:200) (Wente et al. 1992) followed by Cy-3 donkey conjugated anti-Rat IgG (Jackson Immuno Research Laboratories; USA). DNA is visualized by DAPI (4', 6- diamino-2-phenylindoIe). Contrast and brightness are further adjusted using the Adobe PhotoShop computer program.
FACS (Fluorescence Activated Cell Sorting) ANALYSIS: DNA flow cytometry is carried out as described (Epstein and Cross 1992). Results are always graphed by plotting fluorescence (X axis) against number of events (Y axis) and overlaying them to align and compare the different results.
CELL COUNT: Two ml samples are fixed with three ml of formalin solution (lxPBS (Phosphate-buffered saline) and 0.74% formaldehyde), sonicated for twelve seconds, and counted in a Coulter Counter. SERIAL DILUTIONS: lOx serial dilutions are made from a suspension of cells made in water and 5 μl are plated and grown to assess viability in different conditions of carbon source and temperature.
PROTEIN EXTRACTION, IMMUNOPRECIPITATION AND IMMUNOBLOTTING: The procedures are adapted from Levine et al. 1996. Yeast cultures (lOOml at an optical density at 660nm of 0.9) are collected by filtration and resuspended in ice-cold TN (50 mM Tris (pH 7.5), 250 mM NaCl, 5 mM EDTA (Ethylenediamine tetraacetic acid), 10% glycerol). Cells are resuspended in 250 μl of TNN extraction buffer (TN buffer + 0.1 % Nonidet P-40) containing 5% aprotinin (Sigma; USA), 0.1 mM phenylmethylsulfonyl fluoride, 10 μg of leupeptin per ml, 10 μg of pepstatin per ml, 10 mM NaPPi (Sodium pyrophosphate) (pH 7.4) and 10 mM NaF.
Four hundred micro liters of acid- washed glass beads (Sigma; USA) are added, and cells are broken by two cycles of two minutes of vortexing in a Vortex-Genie (VWR Scientific; USA) sleeve at top speed. Breakage is performed at 4°. Between cycles, extracts are spun briefly in a microcentrifuge to condense foam, and unbroken cells are resuspended by stirring with a micropipettor tip. Extracts are microcentrifuged, 250 μl are removed, and another 250 μl of extraction buffer is added to cells and beads. After one minute of vortexing, the sample is microcentrifuged and the extraction buffers are combined. The extracts are clarified by two minutes of microcentrifugation and incubated with a 1:10 dilution of the monoclonal antibody HA11 (ascites; Babco; USA) for one hour on ice. Samples are microcentrifuged for two minutes, and supernatant is added to 30 μl of slurry of protein A-agarose (Sigma; USA) washed in extraction buffer. Samples are rotated for one hour at 4°. Immunoprecipitates are washed with extraction buffer three times, once for two minutes with HNN (TNN buffer + 250 mM NaCl), and resuspended in 60 μl of 2X SDS (sodium dodecyl sulfate) sample buffer, heated at 95° for 5 minutes and used for immunoblot analysis. SDS-polyacrylamide gel electrophoresis (10% and
9%) and transfer to Immobilon (Millipore; USA) are done as described previously (Cross and Blake 1993). Following the transfer, blots are blocked in PBS containing 0.5% on Nonidet P-40, 0.1% Tween 20, 0.5% bovine serum albumin and 5% dry powered nonfat milk (Carnation; USA). Antibody incubations are in PBS-Tween (0.2%), with 1% milk included to reduce background. Antibodies used are a polyclonal rabbit anti-HA peptide antibody (1:15 dilution of the commercial antibody in the PBS-Tween-milk buffer) (Babco; USA) to detect the HA tag followed by an incubation with the polyclonal antibody donkey anti-rabbit:. -HRP (Amersham; USA) (1:10 dilution of the commercial antibody in the PBS-Tween-milk buffer). Detection is by enhanced chemiluminescence (ECL) using an ECL kit (Amersham; USA).
PHOSPHATASE TREATMENT: The immunoprecipitation is performed as described above. After the washes in extraction buffer, samples are washed once with kinase buffer (lOmM HEPES (pH 7.4), 10 mM MgC12, 10% glycerol, 1 mM dithiothreitol) and resuspended in 60 μl of kinase assay buffer. Five units of Calf Intestinal Phosphatase (CIP) (Boehringer Mannheim; USA) or CIP+inhibitors (5% aprotinin (Sigma; USA), 0.1 mM phenylmethylsulfonyl fluoride, 10 μg of leupeptin per ml, 10 μg of pepstatin per ml, 10 mM NaPPi (pH 7.4) and 10 mM NaF) are added to 20 μl of suspension, and incubated for one hour at 37°. Reactions are stopped by addition of 20 μl of 2x SDS sample buffer, heated for 5 minutes at 95° and used for immunoblot analysis as described above.
EXAMPLE 2. SYNTHETIC LETHAL SCREENING BASED ON INDUCIBLE EXPRESSION OF THE ANALYZED GENE.
A yeast strain is constructed which bears a wild type copy of the analyzed gene under the control of an inducible promoter from the GAL1 gene. Then the strain is mutagenized to a 10-30% survival rate. Mutagenization is performed either with a UN. light or with a chemical mutagen selected from the group consisting of with ethyl methanesulfonate (EMS). Other mutagenizing agents are equally suitable, i.e., methyl methanesulfonate, methylnitrosoguanidine, 4-nitroquinoline-l-oxide, 2-aminopurine, 5- bromouracil, ICR 191, acridine derivatives, ethidium bromide, nitrous acid, and/or Ν- methyl-Ν'-mtroso-Ν-nitroguanidine. The mutagenized cell titer is then determined by plating 500 colony forming units per 120 mm Petri dish on an agar-based media containing necessary salts, vitamins and galactose and sucrose. In two to three days, when the colonies have grown, the Petri dishes containing mutagenized colonies are replica-plated on two similar plates, having either glucose, or galactose as a carbon source. Colonies containing mutations synthetically lethal with the analyzed gene will grow on galactose but will not grow on glucose-containing media. Such isolates are re- screened for the absence of growth on dextrose-containing media.
The colonies containing a synthetic lethal mutation are then transformed with a plasmid bearing a copy of the gene being analyzed. If the colonies indeed contain a synthetic lethal mutation with the analyzed gene, then introduction of such plasmid into the cells should allow transform cells to grow on dextrose-containing media. The transformed cells are then crossed with the wild type strain of the opposite mating type. If the hybrid strain grow on glucose, it is concluded that the synthetic lethal mutation in the isolate is recessive and, therefore, can be cloned by complementation. If the hybrid strain cannot grow on glucose, the mutation is dominant and it is omitted from the successive analysis.
The hybrids that contain recessive synthetic lethal mutations are crossed with the wild type strain, sporulated and dissected, and the spore tetrads are checked for the appropriate single mutation pattern of segregation of the synthetic lethal phenotype. The mutants that show such pattern of segregation are cloned by complementation. Variations of this and other screening yeast methods are known (see e.g., U.S. Pat. Νos. 5,912,154; 5,908,752; 5,876,951; 5,869,287; 5,866,338; 5,789,184; 5,674,996; 5,578,477; 5,527,896; 5,352,581; 5,175,091; and 5,139,936) and one skilled in the art readily knows which one select for a specific purpose. EXAMPLE 3. BIOCHEMICAL CHARACTERIZATION OF A mecl MUTATION Streakout assays for growth of mutants at various temperatures (28°C, 30°C, 32°C, 34°C) are performed. Isolates are picked from crosses between poll-101 (yPP888) and a mecl kinase dead mutant (yPP175) and between poll-101 (yPP888) and dunldl- lOOd (yPP130). Double mutants are scored by temperature sensitivity and sensitivity to hydroxyurea. Strains containing single mutations in mecl or dunl grow similar to wild type at all temperatures. Additionally, the meclkd poll-101 mutant contains a deletion of SML1, which is required to suppress the essentiality of mecl. Deletion of smll does not affect the temperature sensitivity profile of poll-101 or wild type proliferation at any temperature tested (19-38 degrees).
Thus, a poll-101 mutant is isolated from a synthetic lethal screen with meclΔ carried out at 30 degrees. Upon backcrossing to wildtype and recrossing to meclΔ it is revealed that the genetic interaction occurs between 28 and 30 degrees, but not at lower temperatures. Thus, meclΔ lowers the maximum permissive temperature of poll-101 by 4-5 degrees, as the poll-101 strain normally becomes temperature sensitive between 33 and 34 degrees. A similar interaction is seen with a kinase inactive allele of mecl and with a rad53d mutation.
Furthermore, it is important to know whether DUNl would also be required for sustaining poll-101 mutants at 30 degrees. The results demonstrate that DUNl is not singly important for maintaining poll-101 mutants at any temperature. Thereby, although
DUNl transcriptional response has been shown to be active in POLl mutants, its absence does not appear to impact MECl or RAD53's ability to sustain poll-101 mutants.
EXAMPLE 4. MUTANTS SYNTHETIC LETHAL WITH MECl HAVE DISTINCT FUNCTIONAL REQUIREMENTS FOR THE MEC1/RAD53 PATHWAY
A set of mutants that require MECl function for viability is used to genetically dissect functional relationships among known components of the MEC1/RAD53 pathway in two ways. First, each mecl synthetic lethal mutant is crossed to a panel of checkpoint mutants defective in one or more checkpoint-related functions, including rad9d, pol2-ll, rad53d, and pdsld. Second, an attempt is made to ameliorate MECl dependency in mecl synthetic lethal mutants by overproducing pathway players know to be downstream of MECl, that is, RAD53, DUNl, and PDS1. The mecl synthetic lethal mutants show different requirements for upstream and downstream components of the checkpoint pathway, depending on whether genetic alterations impact dNTP synthesis or DNA lagging strand synthesis. When dNTP synthesis is affected, cells require S-phase sensors, for example POL2, and at least two RAD53 functions: One dependent upon DUNl and another independent of both DUNl and PDS1. When lagging strand synthesis is affected, cells rely on DNA damage sensors, for example RAD9, PDS1, and a RAD53 activity suppressible by PDS1. Thus, different dependencies on pathway inputs/ reflect two functionally distinct forms of the mECl/RAD53 pathway, one specified under conditions of replicative stress and a second specifiiecd by the presence of DNA damage.
EXAMPLE 5. GENETIC APPROACHES TO MECl FUNCTION AND CHECKPOINT RESPONSE IN Saccharomyces cerevisiae. Key molecular players, among eukaryotes, are critical for maintaining cell viability in response to replication blocks and DNA damage. Amid this group in budding yeast is the checkpoint transducer MECl, a homologue of the human checkpoint kinases ATM and ATR. MECl is required for all DNA-related checkpoint responses in yeast. While much has been uncovered concerning the role of MECl and other genes in checkpoint responses, surprisingly few down stream functions of the MECl pathway have been identified.
A synthetic lethal screen with a mecl mutant reveals two basic scenarios in which cells require MECl function for viability: (a) deficiencies in dNTP synthesis, where cells probably experience chronic replication blocks; and (b) deficiencies in lagging strand DNA synthesis, where cells likely generate chronic DNA damage.
Initial testing of mecl synthetic lethal mutants against a panel of strains partially defective for checkpoint responses indicates that the transcriptional regulator DUNl plays a key role in sustaining cells with dNTP requirements but has little or no impact on the viability of DNA synthesis mutants. In order to pursue new targets of MECl important for regulation when dNTPs are limiting, two overexpression lethality screens are used, where lethality is sought in the presence of hydroxyurea (a dNTP synthesis inhibitor) or mutation in RNR1 (the large subunit of ribonucleotide reductase). Several plasmids are identified that differentially sensitize cells to rnrl and cdc8 (dTMP kinase) deficiencies, but not to cdc9 (DNA ligase) mutation. These three approaches uncover important aspects of checkpoint as biological contingencies and also teach more about budding yeast's approach to checkpoint arrest which precludes the regulation of Cdkl.
EXAMPLE 6. COMPLEMENTATION OF A YEAST STRAIN WITH A HUMAN cDNA LIBRARY
In this example the principle is provided whereby human cDNAs are identified, which encode proteins that substitute above described synthetic lethality in yeast with MECl proteins. A yeast strain is constructed which contains the MECl gene under the control of the GAL1 promoter. As MECl protein is required for passage through the check point, this strain grows on galactose, where the GAL1 promoter is induced; when this strain is grown for example on glucose the GAL1 promoter is repressed, no MECl protein is made, and the cells either arrest or die. A cDNA library using mRNA prepared from the human fetal fϊbroblasts is constructed in a S. cerevisae vector containing the constitutive yeast promoter such as ADH for expression of the human cDNAs (see for details e.g., U. S. Pat. No.5,783,661; 5,952,195). The library is transfected into host
MECl yeast strain and 105 independent transformants are screened, by replica plating, for their ability to grow on glucose or galactose. Several transformants are isolated as a result whose growth patterns are rescued by expression of a human cDNA. Some of them are already known genes.
EXAMPLE 7. SCREENING FOR PHARMACEUTICAL ANTAGONISTS AND AGONISTS
Drugs useful in treating cell cycle or DNA replication disorders identified in the present invention can be now screened using established yeast models as described hereinabove. Several classes of drugs can be screened such as chemical, organic and inorganic compounds, peptides or peptide mimetics, antisense molecules, antibodies, etc. Methods of generating and performing high-throughput screening of chemical libraries are well established and are well known to those skilled in the art. Using the insights gained in the cell cycle regulation a drug discovery program is established to screen selected candidates in yeast-based assays and evaluate their therapeutic potential by further testing mammalian cells or cell lines. These pharmaceutical agents would ideally inhibit aspects of the cell cycle in a predictable fashion, thereby enabling the determination of their future promise in cancer or other cell cycle related clinical conditions.
Among tested compounds several are found as being active at pharmaceutically acceptable concentrations. These include but are not limited to: triButyrate (4- phenylbutyrtic acid sodium salt or sodium phenylbutyrate), various benzopyran drivatives as prepared by known in the art means. See, for example, U.S. Pat. Nos. 5,359,115; 5,362,899; 5,288,514; 5,733,920 or PCT publications WO 94/08051; WO92/10092; WO93/09668; WO91/07087; WO93/20242. Accordingly, a variety of libraries on the order of 1000 to 100,000 or more diversomers of the subject compounds can be synthesized, and, by use of a high throughput assay for detecting inhibitors, such as described in PCT publication WO 94/09135, rapidly screened for biological activity. For a review of methods of combinatorial synthesis, and methods of library screening and deconvolution, see, e.g., E. M. Gordon et al, (1994) J. Med. Chem. 37:1385-1401, and references cited therein.
EXAMPLE 8. TREATMENT OF CLINICAL CONDITIONS DUE TO BENIGN OR MALIGNANT CELL PROLIFERATION
Drugs that are screened out as positive in yeast based assays are then tested for treating diseases caused by excessive cell growth. Most of such diseases are malignant diseases, i.e., cancers of any of a wide variety of types, including without limitation, solid tumors and leukemias such as apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), histiocytic disorders, leukemia (e.g., B-cell, mixed-cell, null-cell, T-cell, T-cell chronic, HTLV-II-associated, lymphocytic acute, lymphocytic chronic, mast-cell, and myeloid), histiocytosis malignant, Hodgkin's disease, immunoproliferative small, non-Hodgkin's lymphoma, plasmacytoma, reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, craniopharyngioma, dysgerminoma, hamartoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, Leydig cell tumor, papilloma, sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, meduUoblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffm, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angioma sclerosing, angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcomas, neoplasms (e.g., bone, breast, digestive system, colorectal, liver, pancreatic, pituitary, testicular, orbital, head and neck, central nervous system, acoustic, pelvic, respiratory tract, and urogenital), neurofibromatosis, and cervical dysplasia.
Some diseases occur due to excessive but benign cell proliferation (i.e. non- malignant). Examples of such diseases are fibrosis, benign prostate hyperplasia, atherosclerosis, restenosis, glomulerosclerosis, cheloid, psoriasis, lentigo, keratosis, achrochordon, molluscum contagiosum, venereal warts, sebaceous hyperplasia, condylomata acuminatum, angioma, venous lakes, chondrodermatitis, granuloma pyogenicum, hidradenitis suppurativa, keloids, keratoacanthoma, leukoplakia, steatocystoma multiplex, trichiasis, superficial epithelial nevus, polyp, junctional nevus, pyogenic granuloma, prurigo nodularis, dermatofibroma, adenoma sebaceum, and other diseases of the skin and non-malignant neoplastic diseases such as for example Kaposi's sarcoma, papilloma. The effect of sodium phenylbutyrate or benzopyran derivatives is then determined on the tumorigenic phenotype of human glioblastoma cells in vivo. Fisher 344 rats received an inoculation of syngeneic 9 L gliosarcoma cells (4x104) into the deep white matter of the right cerebral hemisphere. The animals are then subjected to two weeks of continuous treatment with sodium phenylbutyrate (550 mg/kg/day, s.c), using osmotic minipumps transplanted subcutaneously. In control rats the minipumps are filled with saline.
Statistical analysis of data employed the Fisher's Exact Test. Treatment of glioblastoma cells with the drug results in time-and dose-dependent growth arrest accompanied by similarly diminished DNA synthesis. After 4-6 days of continuous treatment with 4 mM phenylbutyrate, there is approximately 50% inhibition of growth.
Phenylbutyrate, which is an intermediate metabolite of phenylacetate inhibited tumor cell replication, while the end metabolite, phenylacetylglutamine, is inactive. In addition to inducing selective tumor cytostasis, both phenylacetate and phenylbutyrate promoted cell maturation and reversion to a nonmalignant phenotype, manifested by an altered pattern of cytoskeletal intermediate filaments, loss of anchorage-independence, and reduced tumorigenicity in athymic mice. These profound changes in tumor behavior are accompanied by alterations in the expression of cyclin genes implicated in growth control, angiogenesis, and immunosuppression.
EXAMPLE 9. METHOD OF TREATING YEAST INFECTION
As described above, these compounds can be used effectively by women to control yeast infection without upsetting the microfloral balance of the vagina. The compounds may similarly be used to control Candida microbes around wounds. Other yeast organisms are equally suitable as targets of treatment including but not limited to Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus or combination thereof.
In this study 5 affected patients applied topically drug-containing vaginal suppositories once a day. As a result no yeast infection is observed in any of the patient after period of three days. Also, no untoward reactions of any kind observed in any of these patients. It should be understood in this regard, that topical use in accordance with the present invention is not limited to the areas to be treated. The pharmaceutical composition of the present invention is equally suitable for treating or preventing yeast infections when delivered by other means, e.g., orally, intravenously, etc.
EXAMPLE 10. ISOLATION OF SECONDARY GENE
The example below, which describes the isolation and characterization of a secondary gene is exemplary of a general approach for isolating genes of the present invention. Isolation of the gene allows one of ordinary skill to readily isolate homologous genes in yeast and other microbial species. The isolated genes can then be used to construct recombinant vectors for altering the gene of interest.
Generally, the nomenclature and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for cloning, DNA and RNA isolation, amplification and purification. Generally, enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases, sequencing and the like are performed according to the manufacturer's specifications. These techniques and various other techniques are generally performed according to Sambrook et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989).
The isolation of secondary genes may be accomplished by a number of techniques. For instance, transposon tagging of a target gene can assist in the isolation of the relevant gene. Transposon tagging involves introducing a transposon into the host which leads to a mutation of the target gene and a detectable phenotypic change in the host. Using a probe for the transposon, the mutant gene can then be isolated. Using the
DNA adjacent to the transposon in the isolated mutant gene as a probe, the normal wild type allele of the target gene can be isolated. A particularly useful transposon tagging system is disclosed, for example, in U.S. Pat. No. 5,013,658.
An alternative method uses oligonucleotide probes to identify the desired gene in a cDNA or genomic DNA library. To construct genomic libraries, large segments of genomic DNA are generated by random fragmentation, e.g., using restriction endonucleases, and are ligated with vector DNA to form concatemers that can be packaged into the appropriate vector. To prepare a cDNA library, mRNA is isolated, and a cDNA library which contains the gene transcript is prepared from the mRNA.
Alternatively, cDNA may be prepared from mRNA extracted from other cells in which secondary genes or homologs are expressed.
The cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned secondary gene such as that shown in a yeast gene bank database.
Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different plant species. The use of such hybridization techniques for identifying homologous genes is well known in the art and need not be described further.
Alternatively, polynucleotides may be synthesized by well-known techniques as described in the technical literature. Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using
DNA polymerase with an appropriate primer sequence.
If proper polypeptide expression is desired, a polyadenylation region at the 3'-end of the coding region should be included. The polyadenylation region can be derived from the natural gene, or from a variety of other genes. The vector comprising the sequences from a secondary gene will typically comprise a marker gene which confers a selectable phenotype on yeast cells. For example, the marker may encode biocide resistance, particularly antibiotic resistance, such as resistance to kanamycin, G418, bleomycin, hygromycin, or other conventional resistance. Such DNA constructs may be introduced into the genome of the desire host by a variety of conventional techniques. For example, the DNA construct may be introduced directly into the genomic DNA of host cell using techniques such as electroporation and microinjection, or the DNA constructs can be introduced directly using ballistic methods, such as DNA particle bombardment. Alternatively, the DNA constructs may be combined with suitable DNA flanking regions and introduced into a conventional host vector. Microinjection techniques are known in the art and well described in the scientific and patent literature.
EXAMPLE 10 MECl SYNTHETIC LETHAL SCREEN: REPRESENTATIVE ALLELES
Figure imgf000044_0001
Genetic = Came out of the meclΔ synthetic lethal screen.
Cross = Came out of candidate cross.
Literature = Previously published interaction with mecl mutation.
All of the above-cited internet sources, patents, publications, and references within are hereby expressly incorporated by way of reference in their respective entireties.
The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method of identifying a drug that inhibits the growth or replication of a cell having a mutated MECl gene or an analog or homolog thereof, said method comprising the steps of:
(a) contacting a cell having a mutated MECl gene or an analog or homolog thereof with the drug; and
(b) determining whether the drug modulates the activity of a wildtype secondary gene which is synthetically lethal when it is mutated and is present in combination with mutated MECl gene or an analog or homolog thereof.
2. The method of claiml in which the cell is a tumor cell.
3. The method of claim 1, comprising the further step of comparing the results of step (b) with a control cell grown without the drug.
4. The method of claim 3 in which the control cell is a normal cell.
5. The method of claim 3 in which the control cell is a tumor cell.
6. The method of claim 3 in which the control cell has a mutated MECl gene or an analog or homolog thereof.
7. A method according to claim 1 in which the secondary gene is selected from the group consisting of CDC8, RAD27, MBPl, SLMl/YOR080w, RNRl, RNR2, RNR4,
CDC21, PRIl, POLl, 141/154, 87, and 240 and analogs and homologs thereof.
8. A screening assay system for identifying a candidate drug, comprising:
(a) a yeast cell having a deletion in MECl gene, analog or homolog thereof and a wildtype secondary gene, which in its mutant form is synthetically lethal in combination with a deletion of MECl, an analog or homolog thereof; (b) a test yeast cell system comprising the candidate drug brought in contact with the yeast cell; and
(c) a detector for comparing the viability of the yeast cell in the test system compared to the control system comprising the yeast cell in the absence of drug where a decrease in viability of the test cell system identifies positive drug candidates.
9. A method of screening for the presence of benign or malignant cell growth in a tissue sample comprising the steps of: a) providing a tissue sample from an individual suspected of having cancer; b) assessing the expression of the MECl gene or gene product, analog or homolog thereof in the cells of the tissue sample; and c) comparing the expression of the MECl gene or gene product, analog or homolog thereof in the tissue sample with the expression of the MECl gene or gene product, analog or homolog thereof in a control sample, wherein the presence of aberrant expression of MECl gene or gene product, analog or homolog thereof in the test sample as compared with expression in the control sample is indicative of the presence of cancer.
10. A method of identifying a compound useful in the treatment of tumor cells comprises the steps of: (a) contacting a secondary gene product, which in its mutant form is synthetically lethal in combination with a mutated MECl gene or homolog or analog thereof, with a test compound; and
(b) determining the activity of the secondary gene product.
11. The method of claim 10, comprising the further step of comparing the activity of the secondary gene product in the presence of the test compound with the activity of the gene product in the absence of the secondary gene product.
12. The method of claim 10 in which the secondary gene is selected from the group consisting of analogs and homologs of CDC8, RAD27, MBPl, SLMl/YOR080w, RNRl,
RNR2, RNR4, CDC21, PRIl, POLl, 141/154, 87, and 240 and combinations thereof.
13. A pharmaceutical composition comprising an effective amount of a drug and a pharmaceutically acceptable carrier or diluent, said drug capable of selectively interacting with at least one secondary gene or gene product in a target cell which comprises a primary MECl gene or gene product, whereby the exposure of the target cell to the drug arrests cell division or selectively kills said target cell.
14. The pharmaceutical composition of claim 13 wherein said drug comprises oligonucleotide, gene product, homologs or analogs of oligonucleotide or gene product, a small molecule, or a peptide mimetic.
15. A method for treating a human or animal hosting or susceptible of hosting a disease associated with mutation in a MECl gene, analog or homolog thereof which comprises providing the pharmaceutical composition of claim 12 and treating said human or animal with an effective amount of the pharmaceutical composition.
16. The method of claim 15 in which the disease is selected from the group consisting of apudoma, choristoma, branchioma, malignant carcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell), histiocytic disorders, leukemia (e.g., B-cell, mixed-cell, null-cell, T-cell, T-cell chronic, HTLV-II- associated, lymphocytic acute, lymphocytic chronic, mast-cell, and myeloid), histiocytosis malignant, Hodgkin's disease, immunoproliferative small, non-Hodgkin's lymphoma, plasmacytoma, reticuloendotheliosis, melanoma, chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing's sarcoma, synovioma, adenofibroma, adenolymphoma, carcinosarcoma, chordoma, craniopharyngioma, dysgerminoma, hamartoma, mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma, trophoblastic tumor, adenocarcinoma, adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor, Leydig cell tumor, papilloma, sertoli cell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, meduUoblastoma, meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma, paraganglioma nonchromaffin, angiokeratoma, angiolymphoid hyperplasia with eosinophilia, angioma sclerosing, -angiomatosis, glomangioma, hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma, carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma, neurofibromatosis, cervical dysplasia, fibrosis, benign prostate hyperplasia, atherosclerosis, restenosis, glomulerosclerosis, cheloid, psoriasis, lentigo, keratosis, achrochordon, molluscum contagiosum, venereal warts, sebaceous hyperplasia, condylomata acuminatum, angioma, venous lakes, chondrodermatitis, granuloma pyogenicum, hidradenitis suppurativa, keloids, keratoacanthoma, leukoplakia, steatocystoma multiplex, trichiasis, superficial epithelial nevus, polyp, junctional nevus, pyogenic granuloma, prurigo nodularis, dermatofibroma, adenoma sebaceum, papilloma, and combinations thereof.
17. The method of claim 15 in which the disease comprises yeast infection.
18. A recombinant eukaryotic cell comprising at least one secondary gene and at least one primary gene which is MECl, or analog or homolog thereof, wherein said primary gene is mutated such that the upregulation, downregulation, elimination, or disruption of said secondary gene results in synthetic lethality.
19. A recombinant eukaryotic cell according to claim 18 wherein the mutated primary gene MECl is defective MECl.
20. The recombinant eukaryotic cell of claim 18 in which the secondary gene is selected from CDC8, RAD27, MBPl, SLMl/YOR080w, RNRl, RNR2, RNR4, CDC21, PRIl, POLl, 141/154, 87, and 240 and analogs and homologs thereof.
21. The recombinant eukaryotic cell of claim 18 in which the primary gene is eliminated or disrupted.
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