WO2001060975A2 - Methodologies de disruption genique destinees a la decouverte de medicaments cibles - Google Patents

Methodologies de disruption genique destinees a la decouverte de medicaments cibles Download PDF

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WO2001060975A2
WO2001060975A2 PCT/US2001/005551 US0105551W WO0160975A2 WO 2001060975 A2 WO2001060975 A2 WO 2001060975A2 US 0105551 W US0105551 W US 0105551W WO 0160975 A2 WO0160975 A2 WO 0160975A2
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gene
cells
seq
genes
nucleotide sequence
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PCT/US2001/005551
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WO2001060975A3 (fr
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Terry Roemer
Bo Jiang
Charles Boone
Howard Bussey
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Elitra Pharmaceuticals, Inc.
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Priority to EP01916144A priority Critical patent/EP1292668A2/fr
Priority to JP2001560347A priority patent/JP2003523197A/ja
Priority to NZ521037A priority patent/NZ521037A/en
Priority to CA002398861A priority patent/CA2398861A1/fr
Priority to KR1020027010815A priority patent/KR20020097180A/ko
Priority to AU2001243204A priority patent/AU2001243204A1/en
Publication of WO2001060975A2 publication Critical patent/WO2001060975A2/fr
Publication of WO2001060975A3 publication Critical patent/WO2001060975A3/fr

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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention is directed toward (1) methods for constructing strains useful for identification and validation of gene products as effective targets for therapeutic intervention, (2) methods for identifying and validating gene products as effective targets for therapeutic intervention, (3) a collection of identified essential genes, and (4) screening methods and assay procedures for the discovery of new drugs.
  • Validation of a cellular target for drug screening purposes generally involves an experimental demonstration that inactivation of that gene product leaves the cell inviable. Accordingly, a drug active against the same essential gene product expressed, for example, by a pathogenic fungus, would be predicted to be an effective therapeutic agent. Similarly, a gene product required for fungal pathogenicity and virulence is also expected to provide a suitable target for drug screening programs. Target validation in this instance is based upon a demonstration that inactivation of the gene encoding the virulence factor creates a fungal strain that is shown to be either less pathogenic or, ideally, avirulent, in animal model studies. Identification and validation of drug targets are critical issues for detection and discovery of new drugs because these targets form the basis for high throughput screens within the pharmaceutical industry.
  • Target discovery has traditionally been a costly, time-consuming process, in which newly-identified genes and gene products have been individually analyzed as potentially-suitable drug targets.
  • DNA sequence analysis of entire genomes has markedly accelerated the gene discovery process. Consequently, new methods and tools are required to analyze this information, first to identify all of the genes of the organism, and then, to discern which genes encode products that will be suitable targets for the discovery of effective, non-toxic drugs.
  • Gene discovery through sequence analysis alone does not validate either known or novel genes as drug targets. Elucidation of the function of a gene from the underlying and a determination of whether or not that gene is essential still present substantial obstacles to the identification of appropriate drug targets. These obstacles are especially pronounced in diploid organisms.
  • C. albicans is a major fungal pathogen of humans.
  • An absence of identified specific, sensitive, and unique drug targets in this organism has hampered the development of effective, non-toxic compounds for clinical use.
  • the recent completion of the DNA sequence analysis of the entire C. albicans genome has rejuvenated efforts to identify new antifungal drug targets.
  • two primary obstacles to the exploitation of this information for the development of useful drug targets remain: the paucity of suitable markers for genetic manipulations in C. albicans and the inherent difficulty in establishing, in this diploid organism, whether a specific gene encodes an essential product.
  • Co-pending provisional patent application filed February 18, 2000, discloses the identification of dominant selectable markers, and the construction of two genes encoding those markers, which are suitable for transformation and gene disruption in C. albicans.
  • Fig.1 Current methods for gene disruption in C. albicans typically involve a multistep process employing a "URA blaster" gene cassette which is recombined into the genome, displacing the target gene of interest.
  • the URA blaster cassette comprises the CaURA3 marker which is selectable in the corresponding auxotrophic host and which is flanked by direct repeats of the Salmonella typhimurium HisG gene.
  • the URA blaster cassette also carries flanking sequences corresponding to the gene to be replaced, which facilitate precise replacement of that gene by homologous recombination.
  • Putative heterozygous transformants which have had one allele of the target gene deleted, are selected as uracil prototrophs, and their identity and chromosomal structure confirmed by Southern blot and PCR analyses. Isolates within which intrachromosomal recombination events have occurred between HisG repeats, leading to excision of the CaURA3 gene and loss of the integrated cassette, are selected on 5-fluoroorotic acid (5-FOA) containing media. This allows a repetition of the entire process, including reuse of the Ura-blaster cassette, for disruption of the second allele of the target gene. In those instances in which the target gene is nonessential, homozygous gene disruptions are produced in the second round gene replacement and identified by Southern blot and PCR analyses.
  • 5-FOA 5-fluoroorotic acid
  • the Ura blaster method precludes direct demonstration of gene essentiality. Therefore, one is unable to critically evaluate the terminal phenotype characteristic of essential target genes. Consequently, establishing whether inactivation of a validated drug target gene results in cell death (i.e., a cidal terminal phenotype) versus growth inhibition (i.e., a static terminal phenotype) is not possible with current approaches, despite the value such information would provide in prioritizing drug targets for suitability in drug development.
  • the present invention provides effective and efficient methods that enable, for each gene in the genome of an organism, the experimental determination as to whether that gene is essential, and for a pathogenic organism, in addition, whether it is required for virulence or pathogenicity.
  • the identification and validation of essential genes and those genes critical to the development of virulent infections provides a basis for the development of high-throughput screens for new drugs against the pathogenic organism.
  • the present invention can be practiced with any organism independent of ploidy, and in particular, pathogenic fungi.
  • the pathogenic fungi are diploid pathogenic fungi, including but not limited to Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans and the like.
  • the present invention is directed toward a method for constructing a diploid fungal strain in which one allele of a gene is modified by insertion of or replacement by a cassette comprising an expressible dominant selectable marker.
  • This cassette is introduced into the chromosome by recombination, thereby providing a heterozygous strain in which the first allele of the gene is inactivated.
  • the other allele of the gene is modified by the introduction, by recombination, of a promoter replacement fragment comprising a heterologous promoter, such that the expression of the second allele of the gene is regulated by the heterologous promoter.
  • Expression from the heterologous promoter can be regulated by the presence of a transactivator protein comprising a DNA-binding domain and transcription-activation domain.
  • the DNA-binding domain of this transactivator protein recognizes and binds to a sequence in the heterologous promoter and increases transcription of that promoter.
  • the transactivator protein can be produced in the cell by expressing a nucleotide sequence encoding the protein.
  • This method for the construction of a diploid fungus having both alleles of a gene modified is carried out, in parallel, with each and every gene of the organism, thereby allowing the assembly a collection of diploid fungal cells each of which comprises the modified alleles of a gene.
  • This collection therefore, comprises modified alleles of substantially all of the genes of the diploid organism.
  • substantially all includes at least 60%, 70%, 80%, 90%, 95% or 99% of the total.
  • every gene in the genome of the diploid organism is represented in the collection.
  • the present invention also encompasses diploid organisms, such as diploid pathogenic fungal strains, comprising modified alleles of a gene, where the first allele of a gene is inactivated by insertion of or replacement by a nucleotide sequence encoding an expressible dominant selectable marker; and where the second allele of the gene has also been modified so that expression of the second allele is regulated by a heterologous promoter.
  • the alleles modified in the mutant diploid pathogenic fungal strain correspond to an essential gene, which is required for growth, viability and survival of the strain.
  • the modified alleles correspond to a gene required for the virulence and pathogenicity of the diploid pathogenic fungal strain against a host organism.
  • the essential gene and the virulence/pathogenicity gene are potential drug targets.
  • the present invention encompasses collections of mutant diploid fungal strains wherein each collection comprises a plurality of strains, each strain containing the modified alleles of a different gene.
  • the collections of strains of the invention include modified alleles for substantially all the different essential genes in the genome of a fungus or substantially all the different virulence genes in the genome of a pathogenic fungus.
  • the present invention is also directed to nucleic acid microarrays which comprise a plurality of defined nucleotide sequences disposed at identifiable positions in an array on a substrate.
  • the defined nucleotide sequences can comprise oligonucleotides complementary to, and capable of hybridizing with, the nucleotide sequences of the essential genes of the diploid pathogenic organism that are required for the growth and survival of the diploid pathogenic organism, the nucleotide sequences of genes contributing to the pathogenicity or virulence of the organism, and/or the unique molecular tags employed to mark each of the mutant strains.
  • the present invention is also directed to methods for the identification of genes essential to the survival of a diploid organism, and of genes that contribute to the virulence and/or pathogenicity of the diploid pathogenic organism.
  • the invention provides mutants of diploid organisms, such as mutant fungal cells, having one allele of a gene inactivated by insertion of or replacement with a disruption cassette, and the other allele modified by a nucleic acid molecule comprising a heterologous regulated promoter, such that expression of that second allele is under the control of the heterologous promoter.
  • mutant cells are cultured under conditions where the second allele of the modified gene is substantially not expressed. The viability or pathogenicity of the cells are then determined.
  • the resulting loss of viability or exhibition of a severe growth defect indicates that the gene that is modified in the mutant cells is essential to the survival of a pathogenic fungus.
  • the resulting loss of virulence and/or pathogenicity of the mutant cells indicates that the gene that is modified contributes to the virulence and/or pathogenicity of the pathogenic fungus.
  • the mutant pathogenic fungal strains constructed according to the methods disclosed are used for the detection of antifungal agents effective against pathogenic fungi.
  • Mutant cells of the invention are cultured under differential growth conditions in the presence or absence of a test compound. The growth rates are then compared to indicate whether or not the compound is active against a target gene product.
  • the second allele of the target gene may be substantially underexpressed to provide cells with enhanced sensitivity to compounds active against the gene product expressed by the modified allele.
  • the second allele may be substantially overexpressed to provide cells with increased resistance to compounds active against the gene product expressed by the modified allele of the target gene.
  • the strains constructed according to the methods disclosed are used for the screening of therapeutic agents effective for the treatment of non-infectious diseases in a plant or an animal, such as a human.
  • active compounds so identified may have therapeutic applications for the treatment of diseases in the plant or animal, in particular, human diseases, such as cancers and immune disorders.
  • the present invention in other embodiments, further encompasses the use of transcriptional profiling and proteomics techniques to analyze the expression of essential and/or virulence genes under a variety of conditions, including in the presence of known drugs.
  • the information yielded from such studies can be used to uncover the target and mechanism of known drugs, to discover new drugs that act in a similar fashion to known drugs, and to delineate the interactions between gene products that are essential to growth and survival of the organism and that are instrumental to virulence and pathogenicity of the organism.
  • a set of genes of a pathogenic organism are identified as potential targets for drug screening.
  • Such genes comprise, genes that have been determined, using the methods and criteria disclosed herein, to be essential for survival of a pathogenic fungus and/or for the virulence and/or pathogenicity of the pathogenic fungus.
  • the polynucleotides of the essential genes or virulence genes of a pathogenic organism (i.e., the target genes) provided by the present invention can be used by various drug discovery purposes.
  • the polynucleotides can be used to express recombinant protein for characterization, screening or therapeutic use; as markers for host tissues in which the pathogenic organisms invade or reside (either permanently or at a particular stage of development or in a disease states); to compare with DNA sequences of other related or distant pathogenic organisms to identify potential orthologous essential or virulence genes; for selecting and making oligomers for attachment to a nucleic acid array for examination of expression patterns; to raise anti- protein antibodies using DNA immunization techniques; as an antigen to raise anti-DNA antibodies or elicit another immune response; and as a therapeutic agent (e.g., antisense).
  • a therapeutic agent e.g., antisense
  • polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in assays to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
  • polypeptides or proteins encoded by the essential genes and virulence genes can also be used in assays to determine biological activity, including its uses as a member in a panel or an array of multiple proteins for high-throughput screening; to raise antibodies or to elicit immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as a marker for host tissues in which the pathogenic organisms invade or reside (either permanently or at a particular stage of development or in a disease states); and, of course, to isolate correlative receptors or ligands (also referred to as binding partners) especially in the case of virulence factors.
  • the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction, such as those involved in invasiveness, and pathogenicity of the pathogenic organism.
  • kits may comprise polynucleotides and/or polypeptides corresponding to a plurality of essential genes and virulence genes of the invention, antibodies, and/or other reagents. 4. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 depicts the URA blaster method for gene disruption in Candida albicans.
  • Figure 2 depicts the GRACE method for constructing a gene disruption of one allele of a gene (CaKRE9), and promoter replacement of the second allele of the target gene, placing the second allele under conditional, regulated control by a heterologous promoter.
  • Figure 3 presents conditional gene expression , using GRACE technology, with KRE1, KRE5, KRE6 and KRE9.
  • Figure 4 presents conditional gene expression using GRACE technology with CaKREl, CaTUBl, CaALG7, CaAURl, CaFKSl and CaSAT2.
  • Figure 5 presents a Northern Blot Analysis of CaHIS3, CaALRl, CaCDC24 and CaKRE9 mRNA isolated from GRACE strains to illustrate elevated expression under non-repressing conditions.
  • Figure 6 presents growth of a CaHIS3 heterozygote strain and a tetracycline promoter-regulated CaHIS3 GRACE strain compared to growth of a wild-type diploid CaHIS3 strain in the presence and absence of 3-aminotriazole (3-AT).
  • Figure 6 A depicts growth of a wild-type strain and a CaHIS3 heterozygote strain as compared with a CaHIS3 GRACE strain constitutively expressing the tetracycline promoter-regulated imidazoleglycerol phosphate dehydratase, in the presence of inhibitory levels of 3-aminotriazole.
  • Figure 6B depicts growth of a wild-type strain, a haploinsufficient CaHIS3 heterozygote strain, and a CaHIS3 GRACE strain constitutively expressing the tetracycline promoter-regulated imidazoleglycerol phosphate dehydratase, in the presence of an intermediate level of 3-aminotriazole.
  • Figure 6C depicts growth of a wild-type strain, a haploinsufficient CaHIS3 heterozygote strain, and a CaHIS3 GRACE strain minimally expressing the tetracycline promoter-regulated imidazoleglycerol phosphate dehydratase, in the presence of an intermediate level of 3-aminotriazole.
  • Figure 6D demonstrates the hypersensitivity of the CaHIS3 GRACE strain 5 minimally expressing the tetracycline promoter-regulated imidazoleglycerol phosphate dehydratase, in the presence of an intermediate level of 3-aminotriazole.
  • the present invention provides a systematic and efficient method for drug target identification and validation.
  • the approach is based on genomics information as well as the biological function of individual genes.
  • the methods of the invention generates a collection of genetic mutants in which the dosage of specific genes can be modulated, such that their functions in growth, survival, and/or pathogenicity can be investigated. The information accrued from such investigations allows the identification of individual gene products as potential drug targets.
  • the present invention further provides methods of use of the genetic mutants either 20 individually or as a collection in drug screening and for investigating the mechanisms of drug action.
  • a direct demonstration that a given gene is essential for survival of a cell can be established by disrupting its expression in diploid organisms which have a haploid stage.
  • the invention provides a method for creating a diploid mutant cell of an organism in which the dosage of a specific gene can be modulated. By this method of the invention, one allele of a target gene in a diploid cell of an organism is disrupted while the second allele is modified by having its promoter replaced by a regulated
  • a strain constructed in this manner is said to comprise a modified allelic pair, i.e., a gene wherein both alleles are modified as described above.
  • this process may be repeated with each and every gene of the organism, thereby constructing a collection of mutant organisms each harboring a disrupted allele and an allele which can be conditionally
  • This gene disruption strategy therefore, provides a substantially complete set of potential drug target genes for that organism.
  • This collection of mutant organisms comprising a substantially complete set of modified allelic pairs, forms the basis for the development of high throughput drug screening assays. A collection of such mutant organisms can be made even when the genomic sequences of an organism are not
  • the GRACE method which involves disruption of one allele coupled with conditional expression of the other allele, overcomes limitations relying upon repeated cycles of disruption with the URA blaster cassette followed by counterselection for its loss.
  • the GRACE method permits large scale target validation in a diploid pathogenic
  • microorganism such as a pathogenic fungus.
  • the GRACE method of the invention as applied to a diploid cell involves two steps: (i) gene replacement resulting in disruption of the coding and/or non-coding region(s) of one wild type allele by insertion, truncation, and/or deletion, and (ii) conditional expression of the remaining wild type allele via promoter replacement or conditional protein
  • GRACE strains of the organism Isolated mutant organisms resulting from the application of the GRACE method are referred to herein as GRACE strains of the organism. Such mutant strains of an organism are encompassed by the invention.
  • each strain comprises the modified alleles of a different gene, and substantially all the genes of the organism are represented in the collection. It is intended that a GRACE strain is generated for every gene in an organism of interest. Alternatively, a smaller collection of GRACE strains of an organism can be generated wherein a desired subset of the genes in the organism are modified by the GRACE method.
  • a gene is generally considered essential when viability and/or normal growth of the organism is substantially coupled to or dependent on the expression of the gene.
  • An essential function for a cell depends in part on the genotype of the cell and in part the cell's environment. Multiple genes are required for some essential function, for example, energy metabolism, biosynthesis of cell structure, replication and repair of genetic material, etc. Thus, the expression of many genes in an organism are essential for its growth and/or survival. Accordingly, when the viability or normal growth of a GRACE strain under a defined set of conditions is coupled to or dependent on the conditional expression of the remaining functional allele of a modified allelic gene pair, the gene which has been modified in this strain by the GRACE method is referred to as an "essential gene" of the organism.
  • a gene is generally considered to contribute to the virulence/pathogenicity of an organism when pathogenicity of the organism is associated at least in part to the expression of the gene.
  • Many genes in an organism are expected to contribute to the virulence and/or pathogenicity of the organism. Accordingly, when the virulence and/or pathogenicity of a GRACE strain to a defined host or to defined set of cells from a host is associated with the conditional expression of the remaining functional allele of a modified allelic gene pair, the gene which has been modified in this strain by the GRACE method is referred to as a "virulence gene" of the organism.
  • the present invention provides a convenient and efficient method to identify essential genes of a pathogenic organism, and to validate their usefulness in drug discovery programs. The method of the invention can similarly be used to identify virulence genes of a pathogenic organism. The identities of these essential genes and virulence genes of an organism as identified by the GRACE method are encompassed in the present invention.
  • Substantially all of the essential genes and virulence genes of an organism can be identified and validated by the GRACE method of the invention.
  • each of the essential genes and virulence genes so identified represent a potential drug target for the organism, and can be used individually or as a collection in various methods of drug screening.
  • the essential genes and virulence genes of the invention can be classified and divided into subsets based on the structural features, functional properties, and expression profile of the gene products.
  • the gene products encoded by the essential genes and virulence genes within each subset may share similar biological activity, similar intracellular localization, structural homology, and/or sequence homology. Subsets may also be created based on the homology or similarity in sequence to other organisms in a similar or distant taxonomic group, e.g.
  • the present invention provides a plurality of mutant organisms, such as a collection of GRACE strains, each comprising the modified alleles of a different gene, wherein each gene is essential for the growth and/or survival of the cells.
  • substantially all of the essential genes in the genome of a pathogenic fungus are identified by the GRACE method, and the GRACE strains containing the modified allelic pairs of essential genes are included in a collection of GRACE strains.
  • substantially all of the virulence genes in the genome of a pathogenic fungus are identified by the GRACE method, and the GRACE strains containing the modified allelic pairs of virulence genes are included in a collection of GRACE strains.
  • a GRACE strain collection for the entire genome may comprise approximately 7000 modified allelic pairs of genes based on analysis of the C. albicans genome sequence.
  • the complete set of essential genes of C. albicans is estimated to comprise approximately 1000 genes.
  • the present invention provides the identities of some of these genes in C. albicans, and the various uses of these genes and their products as drug targets. In addition, estimates as to the number of genes participating in the virulence of this pathogen range between 100 and 400 genes. Once the identity of an essential gene is known, various types of mutants containing one or more copies of the mutated essential g ene created by other methods beside the GRACE method are contemplated and encompassed by the invention.
  • the invention also provides biological and computational methods, and reagents that allow the isolation and identification of genes that are homologous to the identified essential and virulence genes of C. albicans.
  • Information obtained from the GRACE strains of diploid organisms can be used to identify homologous sequences in haploid organisms. The identities and uses of such homologous genes are also encompassed by the present invention.
  • the invention is described in the subsections below by way of example for the pathogenic fungus, Candida albicans.
  • the principles may be analogously applied to the essential and virulence genes of other pathogens and parasites, of plants and animals including humans.
  • the GRACE method can be applied to any pathogenic organisms that has a diploid phase in their life cycles.
  • diploid pathogenic organism is not limited to organism that exist exclusively in diploid form, but encompasses also organisms that have both haploid and diploid phases in their life cycle.
  • the GRACE method for drug target identification and validation can be directly applied to other pathogenic fungi.
  • Deuteromycetous fungi i.e. those lacking a sexual cycle and classical genetics, (in which C. albicans is included), represent the majority of human fungal pathogens.
  • Aspergillus fumigatus is another medically-significant member of this phylum, which, more strictly, includes members of the Ascomycota and the Basidiomycota.
  • A. fumigatus, an Ascomycte is the predominant air borne infectious fungal agent causing respiratory infection, or invasive aspergillosis (IA), in immunocompromised patients. While relatively unknown 20 years ago, today the number of IA cases is estimated to be several thousand per year. Moreover, IA exhibits a mortality rate exceeding 50% and neither amphothericin B nor fluconazole are highly efficacious. Compounding these problems is that identification of novel drug targets is limited by the current state of target validation in this organism.
  • the GRACE method demonstrated for C. albicans is readily adapted for use with A. fumigatus, for the following reasons. Although, A. fumigatus possesses a haploid genome, the GRACE method could be simplified to one step-conditional promoter replacement of the wild type promoter. Since A. fumigatus, in contrast to Candida albicans, adheres to the universal genetic code, extensive site-directed mutagenesis, like that required to engineer the GRACE method for C. albicans, would not be required. Moreover, essential molecular biology techniques such as transformation and gene disruption via homologous recombination have been developed for A. fumigatus. Selectable markers are available for these techniques in A.
  • the GRACE method for drug target identification and validation is applied to Basidiomycetous pathogenic fungi.
  • Basidiomycetous pathogenic fungi One particular, medically-significant member of this phylum is Cryptococcus neoformans. This air borne pathogen represents the fourth (7-8%) most commonly recognized cause of life- threatening infections in AIDS patients. Transformation and gene disruption strategies exist for C. neoformans and a publically funded genome sequencing project for this organism is in place. C. neoformans possesses a sexual cycle, thus enabling the GRACE method to be employed with both haploid and diploid strains.
  • Other medically-significant Basidiomycetes include Trichosporon beigelii and Schizophylum commune.
  • Examples include the wheat fungal pathogens causing leaf blotch (Septoria tritici, glume blotch (Septoria nodorum), various wheat rusts (Puccinia recondita, Puccinia graminis); powdery mildew (various species), and stem/stock rot (Fusarium spp.)
  • Other particularly destructive examples of plant pathogens include, Phytophthora infestans, the causative agent of the Irish potato famine, the Dutch elm disease causing ascomycetous fungus, Ophiostoma ulmi, the corn smut causing pathogen, Ustilago maydis and the rice-blast-causing pathogen Magnapurtla grisea.
  • the present invention encompasses the application of the GRACE method to identify and validate drug targets in pathogens and parasites of plants and livestock.
  • Table I lists exemplary groups of haploid and diploid fungi of medical, agricultural, or commercial value.
  • Table I Exemplary Haploid and Diploid Fungi
  • Candida spp including Botrytis cinerea Hansenula polymorpha Candida dublinensis Claviceps purpurea Ashbya gossipii Candida glabrata Corticum rolfsii Aspergillus nidulans Candida krusei Endothia parasitica Trichoderma reesei Candida lustaniae Sclerotinia sclerotiorum Aureobasidium pullulans Candida parapsilopsis Erysiphe gramini Yarrowia lipolytica Candida tropicalis Erysiphe triticii Candida utilis Coccidioides immitis Fusarium spp.
  • Kluveromyces lactis Exophalia dermatiditis Magnaporthe grisea Fusarium oxysporum Plasmopara viticola Histoplasma capsulatum Penicillium digitatum Pneumocystis carinii Ophiostoma ulmi
  • Rhizoctonia species including oryzae
  • Plant Pathogens General commercial significance Absidia corymbifera Mucor rouxii ⁇ Rhizomucor pusillus Rhizopus arrhizus
  • Candida glabrata are obligate diploid species that lack a haploid phase in its life cycle, and are thus subject to the application of the GRACE methods.
  • a heterologous promoter is used to provide a range of levels of expression of the second allele.
  • the second allele can be non-expressing, underexpressing, overexpressing, or expressing at a normal level relative to that when the allele is linked to its native promoter.
  • a heterologous promoter is a promoter from a different gene from the same pathogenic organism, or it can be a promoter from a different
  • a gene disruption cassette comprising a selectable marker, preferably a dominant selectable marker, that is expressible in the strain of interest.
  • a selectable marker preferably a dominant selectable marker
  • the present invention encompasses a method for constructing a strain of diploid pathogenic fungal cells, in which both alleles of a gene are modified, the method comprising the steps of (a) modifying a first allele of a gene in diploid pathogenic fungal cells by recombination using a gene disruption cassette comprising a nucleotide
  • JV sequence encoding a selectable marker that is expressible in the cells, thereby providing heterozygous pathogenic fungal cells in which the first allele of the gene is inactivated; and (b) modifying the second allele of the gene in the heterozygous diploid pathogenic fungal cells by recombination with a promoter replacement fragment comprising a heterologous promoter, such that the expression of the second allele of the gene is regulated by the
  • the process can be repeated for a desired subset of the genes such that a collection of GRACE strains is generated wherein each strain comprises a modified allelic pair of a different gene.
  • a complete set of GRACE strains representing the entire genome of the pathogenic fungus can be obtained.
  • the present invention provides a method of assembling a collection of diploid pathogenic fungal cells, each of which comprises the modified alleles of a different gene.
  • the method comprises repeating the steps of modifying pairs of alleles a plurality of times, wherein a different pair of gene alleles is modified with each repetition, thereby providing the collection of diploid pathogenic fungal cells each comprising the modified alleles of a different gene.
  • a preferred embodiment for the construction of GRACE strains uses the following two-step method.
  • C. albicans is used as an example.
  • auxotrophic markers such as but not limited to CaURA3, CaHIS3, CaLEU2, or CaTRPl
  • the preferred method of heterozygote construction in diploid fungi employs a genetically modified dominant selectable marker.
  • C. albicans is sensitive to the nucleoside-like antibiotic streptothricin at a concentration of 200 micrograms per milliliter. The presence of the Escherichia coli SAT1 gene within C.
  • SAT1 acetylation of the drug rendering it nontoxic and permitting the strain to grow in the presence of streptothricin at a concentration of 200 micrograms per milliliter.
  • Expression of the SAT1 gene in C. albicans is made possible by engineering the gene so that its DNA sequence is altered to conform to the genetic code of this organism and by providing a CaACTl promoter (Morschhauser et al. (1998) Mol. Gen. Genet. 257:412-420) and a CaPCKl terminator sequence (Leuker et al. (1997) Gene 192: 235-40).
  • CaSATl This genetically modified marker is referred to as CaSATl which is the subject of a copending United States nonprovisional application, filed February 16, 2001. C.
  • albicans is also sensitive to a second fungicidal compound, blasticidin, whose cognate resistance gene from Bacillus cereus, BSR, has similarly been genetically engineered for expression in C. albicans (CaBSRl), and has been shown to confer a dominant drug resistance phenotype.
  • BSRl Bacillus cereus
  • PCR amplification of either dominant selectable marker so as to include about 65 bp of flanking sequence identical to the sequence 5' and 3' of the C. albicans gene to be disrupted, allows construction of a gene disruption cassette for any given C. albicans gene.
  • a gene disruption event can be obtained following transformation of a C. albicans strain with the PCR-amplified gene disruption cassette and selection for drug resistant transformants that have precisely replaced the wild type gene with the dominant selectable marker.
  • Such mutant strains can be selected for growth in the presence of a drug, such as but not limited to streptothricin.
  • the resulting gene disruptions are generally heterozygous in the diploid C. albicans, with one copy of the allelic pair on one homologous chromosome disrupted, and the other allele on the other homologous chromosome remaining as a wild type allele as found in the initial parental strain.
  • the disrupted allele is non-functional, and expression from this allele of the gene is nil.
  • a set of gene disruptions can be obtained for every gene in the organism.
  • the method can also be applied to a desired subset of genes.
  • conditional expression system used in this embodiment of the invention comprises a regulatable promoter and a means for regulating promoter activity.
  • Conditional expression of the remaining wild type allele in a heterozygote constructed as set forth in Section 5.1.1 is achieved by replacing its promoter with a tetracycline-regulatable promoter system that is developed initially for S. cerevisiae but which is modified for use in
  • conditional expression is achieved by first constructing a transactivation fusion protein comprising the E. coli TetR tetracycline repressor domain or DNA binding domain (amino acids 1-207) fused to the transcription activation domain of S. cerevisiae GAL4 (amino acids 785-881) or HAP 4 (amino acids 424-554). Multiple CTG codon corrections were introduced to comply with the C. albicans genetic code. The nucleotide sequences encoding the transactivation fusion proteins of E. coli TetR (amino acids 1-207) plus S. cerevisiae GAL4 (amino acids 785-881), and of E. coli TetR (amino acids 1-207) plus S.
  • the invention provides haploid or diploid cells that can comprise a nucleotide sequence encoding a transactivation fusion protein expressible in the cells, wherein the transactivation fusion protein comprises a DNA binding domain and a transcription activation domain.
  • Constitutive expression of the transactivation fusion protein in C. albicans can be achieved by providing a CaACTl promoter and CaACTl terminator sequence.
  • any regulatory regions, promoters and terminators, that are functional in C. albicans can be used to express the fusion protein.
  • a nucleic acid molecule comprising a promoter functional in C albicans, the coding region of a transactivation fusion protein, and a terminator functional in C. albicans, are encompassed by the present invention.
  • Such a nucleic acid molecule can be a plasmid, a cosmid, a transposon, or a mobile genetic element.
  • the TetR-Gal4 or TetR-Hap4 transactivators can be stably integrated into a C. albicans strain, by using either ura3 and his3 auxotrophic markers.
  • the invention further provides that a promoter replacement fragment comprising a nucleotide sequence encoding heterologous promoter which comprises at least one copy of a nucleotide sequence which is recognized by the DNA binding domain of the transactivation fusion protein, and wherein binding of the transactivation fusion protein increases transcription of the heterologous promoter.
  • the heterologous tetracycline promoter initially developed for S. cerevisiae gene expression contains an ADH1 3' terminator sequence, variable number of copies of the tetracycline operator sequence (2, 4, or 7 copies), and the CYC1 basal promoter.
  • the tetracycline promoter has been subcloned adjacent to both C ⁇ HIS3 and C ⁇ SATl selectable markers in the orientation favoring tetracycline promoter-dependent regulation when placed immediately upstream the open reading frame of the gene of interest.
  • PCR amplification of the C ⁇ HIS3-Tet promoter cassette incorporates 65bp of flanking sequence homologous to the promoter sequence around nucleotide positions -200 and -1 (relative to the start codon) of the target gene, thereby producing a conditional promoter replacement fragment for transformation. When transformed into a C.
  • albicans strain made heterozygous as described in Section 5.1.1 using the CaSATl disruption cassette homologous recombination between the promoter replacement fragment and the promoter of the wild type allele generates a strain in which the remaining wild type gene is conditionally regulated gene by the tetracycline promoter.
  • Transformants are selected as His prototrophs and verified by Southern blot and PCR analysis.
  • the promoter is induced in the absence of tetracycline, and repressed by the presence of tetracycline.
  • Analogs of tetracycline including but not limited to chlortetracycline, demeclocycline, doxycycline, meclocycline, methocycline, minocycline hydrochloride, anhydrotetracycline, and oxytetracycline, can also be used to repress the expression of the modified gene allele in a GRACE strain.
  • the present invention also encompasses alternative variants of the tetracycline promoter system, based upon a mutated tetracycline repressor (tetR) molecule, designated tetR', which is activated (i.e. binds to its cognate operator sequence) by binding of the antibiotic effector molecule to promote expression, and is repressed (i.e. does not bind to the operator sequence) in the absence of the antibiotic effectors, when the tetR' is used instead of, or in addition to, the wild-type tetR.
  • tetR mutated tetracycline repressor
  • the GRACE method could be performed using tetR' instead of tetR in cases where repression is desired under conditions which lack the presence of tetracycline, such as shut off of a gene participating in drug transport (e.g. CaCDRl, CaPDR5, or CaMDRl).
  • the GRACE method could be adapted to incorporate both the tetR and tetR' molecules in a dual activator/repressor system where tetR is fused to an activator domain and tetR' is fused to a general repressor (e.g.
  • the method may also be applied to haploid pathogenic fungi by modifying the single allele of the gene via recombination of the allele with a promoter replacement fragment comprising a nucleotide sequence encoding a heterologous promoter, such that the expression of the gene is conditionally regulated by the heterologous promoter.
  • a promoter replacement fragment comprising a nucleotide sequence encoding a heterologous promoter, such that the expression of the gene is conditionally regulated by the heterologous promoter.
  • this variation to the method of the invention may be applied to haploid fungal pathogens including, but not limited to, animal fugal pathogens such as Aspergillus fumigatus, Aspergillus niger, Aspergillus flavis,
  • conditional expression are not restricted to the tetracycline promoter system and can be performed using other conditional promoters.
  • conditional promoter may, for example, be regulated by a repressor which repress transcription from the promoter under particular condition or by a transactivator which increases transcription from the promoter, such as, when in the presence of an inducer.
  • the C. albicans CaPCKl promoter is not transcribed in the presence of glucose but has a high level of expression in cells grown on other carbon sources, such as succinate, and therefore could also be adopted for conditional expression of the modified allele in a GRACE strain.
  • both CaHISl and CaSATl are essential for growth on glucose-containing medium using the CaPCKl promoter as an alternative to the tetracycline promoter in the above description.
  • the CaPCKl promoter is heterologous to the gene expressed and not to the organism, and such heterologous promoters are also encompassed in the invention.
  • Alternative promoters that could functionally replace the tetracycline promoter include but are not limited to other antibiotic- based regulatable promoter systems (e.g., pristinamycin-induced promoter or PIP) as well as Candida albicans conditionally-regulated promoters such as MET25, MAL2, PH05, GAL1, 10, STE2, or STE3.
  • performing the gene disruption first enables heterozygous strains to be constructed and separately collected as a heterozygote strain collection during the process of drug target validation.
  • a C. albicans heterozygote strain collection enables drug screening approaches based on haploinsufficiency for validated targets within the collection.
  • haploinsufficiency refers to the phenomenon whereby heterozygous strains for a given gene express approximately half the normal diploid level of a particular gene product. Consequently, these strains provide constructions having a diminished level of the encoded gene product, and they may be used directly in screens for antifungal compounds.
  • differential sensitivity of a diploid parent as compared with its heterozygous derivative, will indicate that a drug is active against the encoded gene product.
  • conditional expression could be achieved by means other than the reliance of conditional promoters.
  • conditional expression could be achieved by the replacement of the wild type allele in heterozygous strains with temperature sensitive alleles derived in vitro, and their phenotype would then be analyzed at the nonpermissive temperature.
  • insertion of a ubiquitination signal into the remaining wild type allele to destabilize the gene product during activation conditions can be adopted to examine phenotypic effects resulting from gene inactivation.
  • a constitutive promoter regulated by an excisable transactivator can be used.
  • the promoter is placed upstream to a target gene to repress expression to the basal level characteristic of the promoter.
  • a heterologous promoter containing lexA operator elements may be used in combination with a fusion protein composed of the lexA DNA binding domain and any transcriptional activator domain (e.g. GAL4, HAP4, VP16) to provide constitutive expression of a target gene.
  • Counterselection mediated by 5-FOA can be used to select those cells which have excised the gene encoding the fusion protein.
  • This procedure enables an examination of the phenotype associated with repression of the target gene to the basal level of expression provided by the lexA heterologous promoter in the absence of a functional transcription activator.
  • the GRACE strains generated by this approach can be used for drug target validation as described in detail in the sections below.
  • the low basal level expression associated with the heterologous promoter is critical.
  • the basal level of expression of the promoter is low to make this alternative shut-off system more useful for target validation.
  • conditional expression of a target gene can be achieved without the use of a transactivator containing a DNA binding, transcriptional activator domain.
  • a cassette could be assembled to contain a heterologous constitutive promoter downstream of, for example, the URA3 selectable marker, which is flanked with a direct repeat containing homologous sequences to the 5' portion of the target gene. Additional homologous sequences upstream of the target, when added to this cassette would facilitate homologous recombination and replacement of the native promoter withe above-described heterologous promoter cassette immediately upstream of the start codon of the target gene or open reading frame.
  • Conditional expression is achieved by selecting strains, by using 5-FOA containing media, which have excised the heterologous constitutive promoter and URA3 marker (and consequently lack those regulatory sequences upstream of the target gene required for expression of the gene) and examining the growth of the resulting strain versus a wild type strain grown under identical conditions.
  • the present invention provides methods for determining whether the gene that has been modified in a GRACE strain is an essential gene or a virulence gene in a pathogenic organism of interest.
  • a GRACE strain containing the modified alleles of the gene is cultured under conditions wherein the second modified allele of the gene which is under conditional expression, is substantially underexpressed or not expressed.
  • the viability and/or growth of the GRACE strain is compared with that of a wild type strain cultured under the same conditions. A loss or reduction of viability or growth indicates that the gene is essential to the survival of a pathogenic fungus.
  • the present invention provides a method for identifying essential genes in a diploid pathogenic organism comprising the steps of culturing a plurality of GRACE strains under culture conditions wherein the second allele of each of the gene modified in the respective GRACE strain is substantially underexpressed or not expressed; determining viability and/or growth indicator(s) of the cells; and comparing that with the viability and/or growth indicator(s) of wild type cells.
  • the level of expression of the second allele can be less than 50% of the non-modified allele, less than 30%, less than 20%, and preferably less than 10%.
  • the level of expression can be controlled by, for example, antibiotics, metal ions, specific chemicals, nutrients, pH, temperature, etc.
  • Candida albicans is used herein as an example which has been analyzed by the GRACE methodology.
  • C. albicans conditional gene expression using the GRACE method was performed using CaKREl, CaKRE5, CaKRE ⁇ , and CaKRE9 (Fig. 3).
  • CaKRE5, CaKRE ⁇ , and CaKRE9 are predicted to be essential or conditionally essential
  • CaKRE9 null strains are nonviable on glucose but viable on galactose
  • C. albicans as demonstrated by gene disruption using the Ura blaster method.
  • CaKREl has been demonstrated as a nonessential gene using the Ura blaster method in C. albicans.
  • Strains heterozygous for the above genes were constructed by PCR-based gene disruption method using the CaSATl disruption cassette followed by tetracycline regulated promoter replacement of the native promoter of the wild type allele. Robust growth of each of these strains suggests expression proceeds no ⁇ nally in the absence of tetracycline. When tetracycline is added to the growth medium, expression of these tetracycline promoter- regulated genes is greatly reduced or abolished.
  • the GRACE strain cells containing each one of the three essential C. albicans genes cited above stop growing.
  • the CaKREl GRACE strain demonstrates robust growth despite repression of CaKREl expression.
  • Target genes were selected as being fungal specific and essential. Such genes are referred to as target essential genes in the screening assays described below.
  • the 13 genes are CaCCT8 (Rademacher et al., Microbiology, UK 144, 2951-2960 (1998)); CaFKSl (Mio et al., J. Bacteriol, 179, 4096-105 (1997); and Douglas, et al., Antimicrob Agents Chemother 41, 2471-9 (1997)); CaHSP90 (Swoboda et al., Infect Immun 63, 4506-14 (1995)); CaKRE ⁇ (Mio et al., J.
  • GRACE strains containing modified alleles of any one of these 17 genes and their uses are encompassed by the invention, for example, the CaTUBl, CaALGl, and CaAURl GRACE strains in Fig. 4 and the CaKRE ⁇ GRACE strain in Fig. 3. Any of these 17 genes may be included as a control for comparisons in the methods of the invention, or as a positive control for essentiality in the collections of essential genes of the invention.
  • the nucleic acid molecules comprising a nucleotide sequence corresponding to any of these 17 genes may be used in the methods of drug discovery of the invention as drug targets, or they may be included individually or in subgroups as controls in a kit or in a nucleic acid microarray of the invention.
  • repression of expression of the modified gene allele within a GRACE strain may be achieved by homologous recombination-mediated excision of the gene encoding the transactivator protein.
  • constitutive expression under nonrepressing conditions
  • TetR-GAL4AD homologous recombination-mediated excision of the transactivator gene
  • Excision of the transactivator gene is made possible by virtue of the selectable marker and integration strategy used in GRACE strain construction.
  • Stable integration of the CaURA J-marked plasmid containing the TetR-GAL4AD transactivator gene into the CaLEU2 locus results in a tandem duplication of CaLEU2 flanking the integrated plasmid.
  • Counterselection on 5-FOA-containing medium can then be performed to select for excision of the CaURA J-marked transactivator gene and to directly examine whether this alternative repression strategy reveals the target gene to be essential.
  • genes defined as essential on 5-FOA containing medium but lacking any detectable growth impairment on tetracycline supplemented medium are the genes, CaYCL052c, CaYNL194c and CaYJR046c. Presumably, this is due to the target gene exhibiting a lower basal level of expression under conditions where the transactivator gene has been completely eliminated than its gene product incompletely inactivated by addition of tetracycline.
  • the GRACE method offers two independent approaches for the determination of whether or not a given gene is essential for viability of the host strain.
  • the present invention also provides methods of using the GRACE strains of a diploid pathogenic organism to identify virulence/pathogenicity genes.
  • the GRACE methodology enables the identification of other genes and gene products potentially relevant to the screening of drugs useful for the treatment of diseases caused by the pathogenic organism. Nonessential genes and their gene products of a pathogen which nevertheless display indispensable roles in the pathogenesis process, may therefore serve as potential drug targets for prophylactic drug development and could be used in combination with existing cidal therapeutics to improve treatment strategies.
  • genes and their products implicated in virulence and/or pathogenicity represent another important class of potential drug targets.
  • genes implicated in virulence and pathogenicity may be species-specific, and unique to a particular strain of pathogen. It has been estimated that approximately 6-7% of the genes identified through the C albicans sequencing project are absent in S. cerevisiae. This represents as many as 420 Candida albicans-specific genes which potentially participate in the process of pathogenesis or virulence. Such a large scale functional evaluation of this gene set can only be achieved using the GRACE methodology of the invention.
  • nonessential C. albicans-specific genes provide preferred targets, value would also be placed on those nonessential C. albicans specific genes identified.
  • the potential role of nonessential C. albicans-specific genes in pathogenesis may be evaluated and prioritized according to virulence assays (e.g. buccal epithelial cell adhesion assays and macrophage assays) and various C. albicans infection studies (e.g. oral, vaginal, systemic) using mouse or other animal models.
  • virulence assays e.g. buccal epithelial cell adhesion assays and macrophage assays
  • C. albicans infection studies e.g. oral, vaginal, systemic
  • GRACE strains that fail to cause fungal infection in mice under conditions of gene inactivation by tetracycline define the GRACE virulence/pathogenicity subset of genes. More defined subsets of pathogenicity genes, for example those genes required for particular steps in pathogenesis (e. g. adherence or invasion) can be determined by applying the GRACE pathogenicity subset of strains to in vitro assays which measure the corresponding process. For example, examining GRACE pathogenicity strains in a buccal adhesion or macrophage assay by conditional expression of individual genes would identify those pathogenicity factors required for adherence or cell invasion respectively. Moreover, essential genes that display substantially reduced virulence and growth rate when only partially inactivated represent "multifactorial" drug targets for which even minimally inhibitory high specificity compounds would display therapeutic value.
  • a GRACE strain of the pathogen containing the modified alleles of the gene is allowed to infect host cells or animals under conditions wherein the second modified allele of the gene which is under conditional expression, is substantially underexpressed or not expressed.
  • the condition of the cells and/or animals is compared with cells and/or animals infected by a wild type strain under the same conditions.
  • Various aspects of the infected cell's morphology, physiology, and/or biochemistry can be measured by methods known in the art.
  • any loss or reduction of virulence or pathogenicity displayed by the GRACE strain indicates that the gene modified in the strain contributes to or is critical to the virulence and/or pathogenicity of the virus.
  • Such genes are referred to as target virulence genes in the screening assays described below.
  • GRACE methodology can be used for the identification and delineation of genetic pathways known to be essential to the development of pathogenicity.
  • extensive work in S. cerevisiae has uncovered a number of processes including cell adhesion, signal transduction, cytoskeletal assembly, that play roles in the dimorphic transition between yeast and hyphal morphologies.
  • Deletion of orthologous genes participating in functionally homologous cellular pathways in pathogenic fungi such as C. albicans, A. fumigatus, and C. neoformans, has clearly demonstrated a concomitant loss of virulence. Therefore, the use of GRACE strains of orthologous genes found in C. albicans and other pathogenic fungi could rapidly validate potential antifungal drug target genes whose inactivation impairs hyphal development and pathogenicity.
  • Target gene validation refers to the process by which a gene product is identified as suitable for use in screening methods or assays in order to find modulators of the function or structure of that gene product. Criteria used for validation of a gene product as a target for drug screening, however, may be varied depending on the desired mode of action that the compounds sought will have, as well as the host to be protected. In one aspect of the present invention, a set of GRACE strains identified and grouped as having only modified alleles of essential genes can be used directly for drug screening.
  • the initial set of essential genes is further characterized using, for example, nucleotide sequence comparisons, to identify a subset of essential genes which include only those genes specific to fungi - that is, a subset of genes encoding essential genes products which do not have homologs in a host of the pathogen, such as humans.
  • Modulators, and preferably inhibitors, of such a subset of genes in a fungal pathogen of humans would be predicted to be much less likely to have toxic side effects when used to treat humans.
  • subsets of the larger essential gene set could be defined to include only those GRACE strains carrying modified allele pairs that do not have a homologous sequence in one or more host (e.g., mammalian) species to allow the detection of compounds expected to be used in veterinary applications.
  • host e.g., mammalian
  • a subset of GRACE strains could be identified that would be used for the detection of anti-fungal compounds active against agricultural pathogens, inhibiting targets that do not have homologs in the crop to be protected.
  • one or more target genes can be directly evaluated as displaying either a cidal or static null phenotype. This is determined by first incubating GRACE strains under repressing conditions for the conditional expression of the second allele for varying lengths of time in liquid culture, and measuring the percentage of viable cells following plating a defined number of cells onto growth conditions which relieve repression. The percentage of viable cells that remain after return to non-repressing conditions reflects either a cidal (low percent survival) or static (high percent survival) phenotype.
  • vital dyes such as methylene blue or propidium iodide could be used to quantify percent viability of cells for a particular strain under repressing versus inducing conditions.
  • known fungicidal drug targets are included in the GRACE strain collection (e.g CaAURl)
  • direct comparisons can be made between this standard fungicidal drug target and novel targets comprising the drug target set. In this way each member of the target set can be immediately ranked and prioritized against an industry standard cidal drug target to select appropriate drug targets and screening assays for the identification of the most rapid-acting cidal compounds.
  • the essentiality and the contribution to virulence of substantially all the genes in the genome of an organism can be determined.
  • the identities of essential genes and virulence genes of a diploid pathogenic organism, such as Candida albicans, once revealed by the methods of the invention, allow the inventors to study their functions and evaluate their usefulness as drug targets.
  • Information regarding the structure and function of the gene product of the individual essential gene or virulence gene allows one to design reagents and assays to find compounds that interfere with its expression or function in the pathogenic organism.
  • the present invention provides information on whether a gene or its product(s) is essential to growth, survival, or proliferation of the pathogenic organism, or that a gene or its product(s) contributes to virulence or pathogenicity of the organism with respect to a host. Based on this information, the invention further provides, in various embodiments, novel uses of the nucleotide and/or amino acid sequences of genes that are essential and/or that contributes to virulence or pathogenicity of a pathogenic organism, for purpose of discovering drugs that act against the pathogenic organism.
  • the present invention provides specifically the use of this information to identify orthologs of these essential genes in a non-pathogenic yeast, such as Saccharomyces cerevisiae, and the use of these orthologs in drug screening methods.
  • a non-pathogenic yeast such as Saccharomyces cerevisiae
  • the nucleotide sequence of the orthologs of these essential genes in S. cerevisiae may be known, it was not appreciated that these S. cerevisiae genes can be useful for discovering drugs against pathogenic fungi.
  • the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising a nucleotide sequence encoding a polypeptide or a biologically active ribonucleic acid (RNA).
  • the term can further include nucleic acid molecules comprising upstream, downstream, and/or intron nucleotide sequences.
  • the term "open reading frame (ORF),” means a series of nucleotide triplets coding for amino acids without any termination codons and the triplet sequence is translatable into protein using the codon usage information appropriate for a particular organism.
  • target gene refers to either an essential gene or a virulence gene useful in the invention, especially in the context of drug screening.
  • target essential gene and “target virulence gene” will be used where it is appropriate to refer to the two groups of genes separately. However, it is expected that some genes will contribute to virulence and be essential to the survival of the organism.
  • the target genes of the invention may be partially characterized, fully characterized, or validated as a drug target, by methods known in the art and/or methods taught hereinbelow.
  • target organism refers to a pathogenic organism, the essential and/or virulence genes of which are useful in the invention.
  • nucleotide sequence refers to a heteropolymer of nucleotides, including but not limited to ribonucleotides and deoxyribonucleotides, or the sequence of these nucleotides.
  • nucleic acid and “polynucleotide” are also used interchangeably herein to refer to a heteropolymer of nucleotides, which may be unmodified or modified DNA or RNA.
  • polynucleotides can be single-stranded or double- stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA with a mixture of single-stranded and double- stranded regions.
  • polynucleotide can be composed of triple-stranded regions comprising DNA, RNA, or both.
  • a polynucleotide can also contain one or modified bases, or DNA or RNA backbones modified for nuclease resistance or other reasons.
  • nucleic acid segments provided by this invention can be assembled from fragments of the genome and short oligonucleotides, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid.
  • recombinant when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e. g. , microbial or mammalian) expression systems.
  • Microbial refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g. , yeast) expression systems.
  • recombinant microbial defines a polypeptide or protein essentially unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e. g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will be glycosylated.
  • expression vehicle or vector refers to a plasmid or phage or virus, for expressing a polypeptide from a nucleotide sequence.
  • An expression vehicle can comprise a transcriptional unit, also referred to as an expression construct, comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and which is operably linked to the elements of (1); and (3) appropriate transcription initiation and termination sequences.
  • “Operably linked” refers to a link in which the regulatory regions and the DNA sequence to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation. In the case of C.
  • Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • a recombinant protein may include an N-terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • recombinant host cells means cultured cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry stably the recombinant transcriptional unit extrachromosomally.
  • Recombinant host cells as defined herein will express heterologous polypeptides or proteins, and RNA encoded by the DNA segment or synthetic gene in the recombinant transcriptional unit.
  • This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers.
  • Recombinant expression systems as defined herein will express RNA, polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed.
  • the cells can be prokaryotic or eukaryotic.
  • polypeptide refers to the molecule form by joining amino acids to each other by peptide bonds, and may contain amino acids other than the twenty commonly used gene-encoded amino acids.
  • active polypeptide refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide.
  • naturally occurring polypeptide refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, proteolytic processing, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • isolated refers to a nucleic acid or polypeptide separated from at least one macromolecular component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source.
  • the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99.8% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).
  • Table II lists a set of fungal specific genes that are demonstrated to be essential in C. albicans when conditionally expressed under the tetracycline repression system in the respective GRACE strains or when the gene encoding the transactivator protein is excised in the respective GRACE strain.in a 5-FOA assay.
  • the present invention provides the identities of 61 essential genes. Although the nucleotide sequence and the reading frame of a number of these genes are known, the fact that these genes are essential to the growth and/or survival
  • SEQ ID NO:l through to SEQ ID NO:62 each identifies a nucleotide sequence of the opening reading frame (ORF) of an identified essential gene.
  • the nucleotide sequences labeled as SEQ ID NO: 1-62 were obtained from a Candida • albicans genomic sequence database version 6 assembled by the Candida albicans ,., ⁇ - Sequencing Project and is accessible by internet at the web sites of Stanford University and University of Minnesota (See http://www-sequence.stanford.edu:8080/ and http://alces.med.umn.edu/Candida.html).
  • the predicted amino acid sequence of the identified essential genes are set forth in SEQ ID NO:63 through to SEQ ID NO: 123 which are obtained by conceptual , ⁇ translation of the nucleotide sequences of SEQ ID NO: 1 through to 61 once the reading frame is determined.
  • the codon CTG is translated to a serine residue in C. albicans, instead of the usual leucine in other organisms. Accordingly, the conceptual translation of the ORF is performed using the codon usage of C. albicans.
  • DNA sequences were generated by sequencing reactions and may contain minor errors which may exist as misidentified nucleotides, insertions, and/or
  • conceptual translation of the nucleotide sequence of SEQ ID NO: 62 leads to an apparently premature termination of the opening reading frame when compared to its ortholog in S. cerevisiae.
  • four nucleotides were added to create SEQ ID NO: 58 which results in the amino acid sequence of SEQ ID NO: 120.
  • the invention provides the genomic sequence of an identified essential gene, wherein the genomic sequence as set forth in SEQ ID NO: 490 contains an intron.
  • the unpublished nucleotide sequence which does not contain intron sequence and encodes a protein is set forth in SEQ ID NO: 39.
  • SEQ ID NO: 124-486 refers to oligonucleotide primers and probes that were designed for and used in the construction of the GRACE strain for the corresponding identified essential gene, (i.e., SEQ ID NO: 124-184 knockout upstream primer (KO-UP); SEQ ID NO: 185-245 knockout downstream primer (KO-Down); SEQ ID NO:246-306 tetracycline promoter upstream primer (Tet-Up); SEQ ID NO: 307-367 Tetracycline promoter downstream primer (Tet-Down); and SEQ ID NO:368-489 primers for identification of the respective GRACE strains (primers A and B). Therefore, each set of oligonucleotides can be used to identify a unique essential gene and a unique GRACE strain, e.g. by hybridization, or PCR.
  • the essential genes listed in Table II can be obtained using cloning methods well known to those of skill in the art, and include but are not limited to the use of appropriate probes to detect the genes within an appropriate cDNA or gDNA (genomic DNA) library. (See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, which is incorporated herein by reference in its entirety.) Probes for the sequences identified herein can be synthesized based on the DNA sequences disclosed herein in SEQ ID NO: 1-62.
  • target gene i.e. essential and/or virulence gene refers to (a) a gene containing at least one of the DNA sequences and/or fragments thereof that are set forth in SEQ ID NO: 1 through to SEQ ID NO:62; (b) any DNA sequence or fragment thereof that encodes the amino acid sequence that are set forth in SEQ ID NO:63 through to SEQ ID NO: 123 using the universal genetic code or the codon usage of C.
  • any DNA sequence that hybridizes to the complement of the nucleotide sequences set forth in SEQ ID NO:l through to SEQ ID NO:62 under stringent conditions e.g., hybridization to filter-bound DNA in 6x sodium chloride/sodium citrate (SSC) at about 45°C followed by one or more washes in 0.2xSSC/0.1% SDS at about 50-65°C, or under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6xSSC at about 45°C followed by one or more washes in O.lxSSC/0.2% SDS at about 68°C, or under other hybridization conditions which are apparent to those of skill in the art (see, for example, Ausubel, F.M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing
  • the polynucleotides that hybridize to the complements of the DNA sequences disclosed herein encode gene products, e.g., gene products that are functionally equivalent to a gene product encoded by a target gene.
  • target gene sequences include not only degenerate nucleotide sequences that encode the amino acid sequences of SEQ ID NO:63 to 123 in C. albicans, but also degenerate nucleotide sequences that when translated in organisms other than C.
  • Albicans would yield a polypeptide comprising one of the amino acid sequences of SEQ ID NO:63 to 123, or a fragment thereof.
  • One of skill in the art would know how to select the appropriate codons or modify the nucleotide sequences of SEQ ID NO: 1 to 62 when using the target gene sequences in C. albicans or in other . organisms.
  • target gene encompasses genes that are naturally occurring in Saccharomyces cerevisiae or variants thereof, that share extensive nucleotide sequence homology with C. albicans genes having one of the DNA sequences that axe, set forth in SEQ ID NO:l through to SEQ ID NO:62, i.e., the orthologs in S. cerevisiae. It is contemplated that methods for drug screening that can be applied to C. albicans genes can also be applied to orthologs of the same genes in the non-pathogenic S. cerevisiae.
  • the invention also encompasses the following polynucleotides, host cells expressing such polynucleotides and the expression products of such nucleotides: (a) polynucleotides that encode portions of target gene product that corresponds to its functional domains, and the polypeptide products encoded by such nucleotide sequences, and in which, in the case of receptor-type gene products, such domains include, but are not limited to signal sequences, extracellular domains (ECD), transmembrane domains (TM) and cytoplasmic domains (CD); (b) polynucleotides that encode mutants of a target gene product, in which all or part of one of its domains is deleted or altered, and which, in the case of receptor-type gene products, such mutants include, but are not limited to, mature proteins in which the signal sequence is cleaved, soluble receptors in which all or a portion of the TM is deleted, and nonfunctional receptors in which all or a portion of CD is deleted; and
  • the invention also includes polynucleotides, preferably DNA molecules, that hybridize to, and are therefore the complements of, the DNA sequences of the target gene sequences.
  • Such hybridization conditions can be highly stringent or less highly stringent, as described above and known in the art.
  • the nucleic acid molecules of the invention that hybridize to the above described DNA sequences include oligodeoxynucleotides ("oligos") which hybridize to the target gene under highly stringent or stringent conditions.
  • hybridization is carried out at about 20-25 degrees below Tm (for DNA-DNA hybrids) or about 10-15 degrees below Tm (for RNA-DNA hybrids).
  • Other exemplary highly stringent conditions may refer, e.g. , to washing in 6xSSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17- base oligos), 55°C (for 20-base oligos), and 60°C (for 23-base oligos). Examples of such oligos are set forth in SEQ ID NO: 124-489.
  • nucleic acid molecules can encode or act as target gene antisense molecules, useful, for example, in target gene regulation and/or as antisense primers in amplification reactions of target gene nucleotide sequences. Further, such sequences can be used as part of ribozyme and/or triple helix sequences, also useful for target gene regulation. Still further, such molecules can be used as components of diagnostic methods whereby the presence of the pathogen can be detected. The uses of these nucleic acid molecules are discussed in detail below.
  • Fragments of the target genes of the invention can be at least 10 nucleotides in length.
  • the fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more contiguous nucleotides in length.
  • the fragments can comprise nucleotide sequences that encode at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450 or more contiguous amino acid residues of the target gene products.
  • Fragments of the target genes of the invention can also refer to exons or introns of the above described nucleic acid molecules, as well as portions of the coding regions of such nucleic acid molecules that encode functional domains such as signal sequences, extracellular domains (ECD), transmembrane domains (TM) and cytoplasmic domains (CD).
  • ECD extracellular domains
  • TM transmembrane domains
  • CD cytoplasmic domains
  • homologs or orthologs of these target gene sequences can be identified and isolated by molecular biological techniques well known in the art, and without undue experimentation, used in the methods of the invention.
  • yeasts in the genera oi Candida Saccharomyces, Schizosaccharomyces, Sporobolomyces, Torulopsis, Trichosporon, Tricophyton, Dermatophytes, Microsproum, Wickerhamia, Ashbya.
  • Rhizopus arrhizus Mucor rouxii, Rhizomucor pusillus, or Absidia corymbigera
  • the plant fungal pathogens such as Alternaria solanii, Botrytis cinerea, Erysiphe graminis
  • Magnaporthe grisea Puccinia recodita, Sclerotinia sclerotiorum, Septoria triticii, Tilletia controversa, Ustilago maydis, Venturia inequalis, Verticullium dahliae or any species falling within the genera of any of the above species.
  • the present invention provides nucleotide sequences that are hybridizable to the polynucleotides of the target genes, and that are of a species other than
  • the present invention encompasses an isolated nucleic acid comprising a nucleotide sequence that is at least 50% identical to a nucleotide sequence selected from the group consisting of SEQ ID No. 1 through to SEQ ID NO:62.
  • the present invention encompasses an isolated nucleic acid comprising a nucleotide sequence that hybridizes under medium stringency conditions to a second nucleic acid that consists of a nucleotide sequence selected from the group consisting of SEQ ID NO:l through to SEQ ID NO:62.
  • the present invention includes an isolated nucleic acid
  • 5 acid comprising a nucleotide sequence that encodes a polypeptide the amino acid sequence of which is at least 50%) identical to an amino acid sequence selected from the group consisting of SEQ ID No.63 through to 123, wherein the polypeptide is that of a species other than Saccharomyces cerevisiae and Candida albicans.
  • Proteome (www.proteome.com ' ) may be used to identify and retrieve the sequences.
  • the name of the S. cerevisiae gene is indicated in parenthesis in column 1 of Table I.
  • Orthologs of S. cerevisiae can also be identified by hybridization assays using nucleic acid probes consisting of any one of the nucleotide sequences of SEQ ID NO: 1 to 61, and 490.
  • nucleotide sequences of the invention still further include nucleotide sequences that have at least 40%, 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more nucleotide sequence identity to the nucleotide sequences set forth in SEQ ID NO:l through to SEQ ID NO: 62.
  • the nucleotide sequences of the invention also include nucleotide sequences that encode polypeptides having at least 25%, 30%, 40%, 50%, 55%,
  • the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in the sequence of a first amino acid or nucleotide sequence for optimal 0 alignment with a second amino acid or nucleotide sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a
  • % identity number of identical overlapping positions/total number of positions x 100%.
  • the two sequences are the same length.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. U.S.A. ⁇ _?7:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-0.
  • Gapped BLAST can be utilized as described in Altschul et al. , 1997, Nucleic Acids Res. 25:3389-3402.
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id).
  • the default parameters of the respective programs e.g., of XBLAST and NBLAST
  • Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • the C. albicans target gene sequence described above can be labeled and used to screen a cDNA library constructed from mRNA obtained from the organism of interest.
  • Hybridization conditions should be of a lower stringency when the cDNA library was derived from an organism different from the type of organism from which the labeled sequence was derived.
  • cDNA screening can also identify clones derived from alternatively spliced transcripts in the same or different species.
  • the labeled fragment can be used to screen a genomic library derived from the organism of interest, again, using appropriately stringent conditions.
  • Low stringency conditions will be well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology, (Green Publishing Associates and Wiley Interscience, N.Y.). Further, a homologous target gene sequence can be isolated by performing a polymerase chain reaction (PCR) using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the target gene of interest.
  • PCR polymerase chain reaction
  • the template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from the organism of interest.
  • the PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a homologous target gene sequence.
  • the PCR fragment can then be used to isolate a full length cDNA clone by a variety of methods well known to those of ordinary skill in the art. -Alternatively, the labeled fragment can be used to screen a genomic library.
  • RNA can be isolated, following standard procedures, from an organism of interest.
  • a reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.
  • the resulting RNA/DNA hybrid can then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid can be digested with RNAase H, and second strand synthesis can then be primed with a poly-C primer.
  • cDNA sequences upstream of the amplified fragment can easily be isolated.
  • an expression library can be constructed utilizing DNA isolated from or cDNA synthesized from the organism of interest.
  • gene products made by the homologous target gene can be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the C. albicans gene product, as described, below.
  • Standard antibody screening techniques see, for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A Laboratory Manual," Cold Spring Harbor Press, Cold Spring Harbor).
  • Library clones detected via their reaction with such labeled antibodies can be purified and subjected to sequence analysis by well known methods.
  • homologous target genes or polypeptides may be identified by searching a database to identify sequences having a desired level of homology to a target gene or polypeptide involved in proliferation, virulence or pathogenicity.
  • databases are available to those skilled in the art, including GenBank and GenSeq.
  • the databases are screened to identify nucleic acids with at least 97%, at least 95%, at least 90%, at least 85%, at least 80%, at least 70%, at least 60%, at least 50%), or at least 40% identity to a target nucleotide sequence, or a portion thereof.
  • the databases are screened to identify polypeptides having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40% or at least 25%) identity or similarity to a polypeptide involved in proliferation, virulence or pathogenicity or a portion thereof.
  • functionally homologous target sequences or polypeptides may be identified by creating mutations that have phenotypes by removing or altering the function of a gene. This can be done for one or all genes in a given fungal species including, for example: Saccharomyces cerevisiae, Candida albicans, and Aspergillus fumigatus. Having mutants in the genes of one fungal species offers a method to identify functionally similar genes (orthologs) or related genes (paralogs) in another species, by use of a functional complementation test.
  • a library of gene or cDNA copies of messenger RNA of genes can be made from a given species, e.g. Candida albicans, and the library cloned into a vector permitting expression (for example, with the Candida albicans promoters or a Saccharomyces cerevisiae promoter) of the genes in a second species, e.g. Saccharomyces cerevisiae.
  • Such a library is referred to as a "heterologous library.” Transformation of the Candida albicans heterologous library into a defined mutant oi Saccharomyces cerevisiae that is functionally deficient with respect to the identified gene, and screening or selecting for a gene in the heterologous library that restores phenotypic function in whole or in part of the mutational defect is said to be
  • heterologous functional complementation permits identification of gene in Candida albicans that are functionally related to the mutated gene in Saccharomyces cerevisiae.
  • Inherent in this functional-complementation method is the ability to restore gene function without the requirement for sequence similarity of nucleic acids or polypeptides; that is, this method permits interspecific identification of genes with conserved biological function, even where sequence similarity comparisons fail to reveal or suggest such conservation.
  • the mutation in the essential gene can be a conditional allele, including but not limited to, a temperature-sensitive allele, an allele conditionally expressed from a regulatable promoter, or an allele that has been rendered the mRNA transcript or the encoded gene product conditionally unstable.
  • the strain carrying a mutation in an essential gene can be propagated using a copy of the native gene (a wild type copy of the gene mutated from the same species) on a vector comprising a marker that can be selected against, permitting selection for those strains carrying few or no copies of the vector and the included wild type allele.
  • a strain constructed in this manner is transformed with the heterologous library, and those clones in which a heterologous gene can functionally complement the essential gene mutation, are selected on medium non-permissive for maintenance of the plasmid carrying the wild type gene.
  • Candida albicans homolog of a Saccharomyces cerevisiae gene, KRE 9 is described.
  • the host strain was a Saccharomyces cerevisiae haploid null mutant in KRE 9, kre 9::HIS3, which has a severe growth defect phenotype.
  • the host strain carried a wild type copy of the native Saccharomyces cerevisiae KRE 9 gene on a LYS-2 based pRS317 shuttle vector and was transformed with a Candida albicans genomic library.
  • This heterologous library was constructed using, as a vector, the multicopy plasmid YEp352, which carries the URA3 gene as a selectable marker.
  • a heterologous functional complementation test is not restricted to the exchange of genetic information between Candida albicans and Saccharomyces cerevisiae; functional complementation tests can be performed, as described above, using any pair of fungal species.
  • the CRE1 gene of the fungus Sclerotininia sclerotiorum can functionally complement the creAD30 mutant of the CREA gene oi Aspergillus nidulans (see Vautard et al.
  • the results allow rapid identification of homologous genes in the two species.
  • the invention also encompasses (a) DNA vectors that contain a nucleotide sequence comprising any of the foregoing coding sequences of the target gene and/or their complements (including antisense); (b) DNA expression vectors that contain a nucleotide sequence comprising any of the foregoing coding sequences operably linked with a regulatory element that directs the expression of the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing coding sequences of the target gene operably linked with a regulatory element that directs the expression of the coding sequences in the host cell.
  • regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression.
  • Such regulatory elements include but are not limited to the lac system, the trp system, the tet system and other antibiotic-based repression systems (e.g.PIP), the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, and the fungal promoters for 3-phosphoglycerate kinase, acid phosphatase, the yeast mating pheromone responsive promoters (e.g. STE2 and STE3), and promoters isolated from genes involved in carbohydrate metabolism (e.g. GAL promoters), phosphate-responsive promoters (e.g. PHO5), or amino acid metabolism (e.g. MET genes).
  • the invention includes fragments of any of the DNA vector sequences disclosed herein.
  • nucleotide sequence of the identified genes can be used to reveal homologies to one or more known sequence motifs which can yield information regarding the biological function of the identified gene product. Computer programs well known in the art can be employed to identify such relationships.
  • sequences of the identified genes can be used, utilizing standard techniques such as in situ hybridization, to place the genes onto chromosome maps and genetic maps which can be correlated with similar maps constructed for another organism, e.g., Saccharomyces cerevisiae. The information obtained through such characterizations can suggest relevant methods for using the polynucleotides and polypeptides for discovery of drugs against Candida albicans and other pathogens.
  • the target gene products used and encompassed in the methods and compositions of the present invention include those gene products (e.g. , RNA or proteins) that are encoded by the target essential gene sequences as described above, such as, the target gene sequences set forth in SEQ ID NO:l through to 62.
  • the amino acid sequences of SEQ ID NO: 63 to 123 are deduced using the codon usage of C. albicans from the respective nucleotide sequences of SEQ ID NO: 1 to 61.
  • protein products of the target genes having the amino acid sequences of SEQ ID NO: 63 to 123 may be encoded by nucleotide sequences that are translated using the universal genetic code.
  • One of skill in the art would know the modifications that are necessary to accommodate for such a difference in codon usage.
  • the methods and compositions of the invention also use and encompass proteins and polypeptides that represent functionally equivalent gene products.
  • functionally equivalent gene products include, but are not limited to, natural variants of the polypeptides having an amino acid sequence set forth in SEQ ID NO:63 through to 123.
  • Such equivalent target gene products can contain, e.g., deletions, additions or substitutions of amino acid residues within the amino acid sequences encoded by the target gene sequences described above, but which result in a silent change, thus producing a functionally equivalent target gene product.
  • Amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved.
  • nonpolar (i.e., hydrophobic) amino acid residues can include alanine (Ala or A), leucine (Leu or L), isoleucine (He or I), valine (Val or V), proline (Pro or P), phenylalanine (Phe or F), tryptophan (Trp or W) and methionine (Met or M);
  • polar neutral amino acid residues can include glycine (Gly or G), serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N) and glutamine (Gin or Q); positively charged (i.e., basic) amino acid residues can include arginine (Arg or R), lysine (Lys or K) and histidine (His or H); and negatively charged (i.e., acidic) amino acid residues can include aspartic acid (Asp or D) and gluta
  • a composition comprising a mixture of natural variants of the polypeptides having one of SEQ ID NO:63 through to 123 is provided. Since it is known in the art that, in C. albicans, 99% of the tRNA molecules that recognize the codon CTG is charged with a serine residue, and 1% are charged with a leucine residue, there is a possibility that during biosynthesis, a leucine is incorporated into a growing polypeptide chain. Accordingly, when a nucleotide sequence comprising the codon CTG is translated in C.
  • a small percentage of the resulting polypeptides may have a leucine residue in positions where a serine residue encoded by CTG (conforming to the codon usage of C. albicans) is expected.
  • the product of translation of such a nucleotide sequence may comprise a mixture of polypeptides with minor leucine/serine variations at positions that correspond to a CTG codon in the nucleotide sequence.
  • “Functionally equivalent,” as the term is utilized herein, refers to a polypeptide capable of exhibiting a substantially similar in vivo activity as the Candida albicans target gene product encoded by one or more of the target gene sequences described in Table II.
  • the term “functionally equivalent” can refer to peptides or polypeptides that are capable of interacting with other cellular or extracellular molecules in a manner substantially similar to the way in which the corresponding portion of the target gene product would interact with such other molecules.
  • the functionally equivalent target gene products of the invention are also the same size or about the same size as a target gene product encoded by one or more of the target gene sequences described in Table II.
  • target gene products that are RNA or proteins oi Saccharomyces cerevisiae are provided.
  • Peptides and polypeptides corresponding to one or more domains of the target gene products e.g. , signal sequence, TM, ECD, CD, or ligand-binding domains
  • truncated or deleted target gene products e.g., polypeptides in which one or more domains of a target gene product are deleted
  • fusion target gene proteins e.g., proteins in which a full length or truncated or deleted target gene product, or a peptide or polypeptide corresponding to one or more domains of a target gene product is fused to an unrelated protein
  • fusion proteins can include, . but are not limited to, epitope tag-fusion proteins which facilitates isolation of the target gene product by affinity chromatography using reagents that binds the epitope.
  • Other exemplary fusion proteins include fusions to any amino acid sequence that allows, e.g.
  • the fusion protein to be anchored to a cell membrane, thereby allowing target gene polypeptides to be exhibited on a cell surface; or fusions to an enzyme (e.g., ⁇ -galactosidase encoded by the LAC4 gene of Kluyveronmyces lactis (Leuker et al., 1994, Mol. Gen. Genet., 245:212-217)), to a fluorescent protein (e.g., from Renilla reniformis (Srikantha et al., 1996, J. Bacteriol. 178:121-129), or to a luminescent protein which can provide a marker function.
  • an enzyme e.g., ⁇ -galactosidase encoded by the LAC4 gene of Kluyveronmyces lactis (Leuker et al., 1994, Mol. Gen. Genet., 245:212-217)
  • a fluorescent protein e.g., from Renilla reniformis (Srikantha
  • the invention provides a fusion protein comprising a fragment of a first polypeptide fused to a second polypeptide, said fragment of the first polypeptide consisting of at least 6 consecutive residues of an amino acid sequence selected from one of SEQ ID NO: 63 to 123.
  • target gene product coding sequences described above can be made to generate polypeptides that are better suited, e.g. , for expression, for scale up, etc. in a chosen host cell.
  • cysteine residues can be deleted or substituted with another amino acid in order to eliminate disulfide bridges.
  • the target gene products of the invention preferably comprise at least as many contiguous amino acid residues as are necessary to represent an epitope fragment (that is, for the gene products to be recognized by an antibody directed to the target gene product).
  • protein fragments or peptides can comprise at least about 8 contiguous amino acid residues from a full length differentially expressed or pathway gene product.
  • the protein fragments and peptides of the invention can comprise about 6, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450 or more contiguous amino acid residues of a target gene product.
  • target gene products used and encompassed in the methods and compositions of the present invention also encompass amino acid sequences encoded by one or more of the above-described target gene sequences of the invention wherein domains often encoded by one or more exons of those sequences, or fragments thereof, have been deleted.
  • the target gene products of the invention can still further comprise post translational modifications, including, but not limited to, glycosylations, acetylations and myristylations.
  • the target gene products of the invention can be readily produced, e.g., by synthetic techniques or by methods of recombinant DNA technology using techniques that are well known in the art. Thus, methods for preparing the target gene products of the invention are discussed herein.
  • the polypeptides and peptides of the invention can be synthesized or prepared by techniques well known in the art. See, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman and Co., N.Y., which is incorporated herein by reference in its entirety.
  • Peptides can, for example, be synthesized on a solid support or in solution.
  • recombinant DNA methods which are well known to those skilled in the art can be used to construct expression vectors containing target gene protein coding sequences such as those set forth in SEQ ID NO: 1 through to 61, and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., Pla et al., Yeast 12:1677-1702 (1996), which are incorporated by reference herein in their entireties, and Ausubel, 1989, supra.
  • RNA capable of encoding target gene protein sequences can be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in Oligonucleotide Synthesis, 1984, Gait, M.J. ed., IRL Press, Oxford, which is incorporated by reference herein in its entirety.
  • host-expression vector systems can be utilized to express the target gene coding sequences of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the target gene protein of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing target gene protein coding sequences; yeast (e.g., Saccharomyces, Schizosaccarhomyces, Neurospora, Aspergillus, Candida, Pichia) transformed with recombinant yeast expression vectors containing the target gene protein coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the target gene protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g.
  • Ti plasmid containing target gene protein coding sequences
  • mammalian cell systems e.g. COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • the nucleotide sequences of coding regions may be * modified according to the codon usage of the host such that the translated product has the correct amino acid sequence.
  • a number of expression vectors can be advantageously selected depending upon the use intended for the target gene protein being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of antibodies or to screen peptide libraries, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified can be desirable.
  • vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBOJ.
  • pG ⁇ X vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione- agarose beads followed by elution in the presence of free glutathione.
  • the pG ⁇ X vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene protein can be released from the GST moiety.
  • a target gene When a target gene is to be expressed in mammalian host cells, a number of viral-based expression systems can be utilized.
  • the target gene coding sequence of interest can be ligated to an adenovirus transcription/translation control complex, e.g. , the late promoter and tripartite leader sequence. This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination.
  • Insertion in a non-essential region of the viral genome will result in a recombinant virus that is viable and capable of expressing target gene protein in infected hosts, (e.g., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA S7:3655-3659).
  • Specific initiation signals can also be required for efficient translation of inserted target gene coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire target gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals can be needed.
  • exogenous translational control signals including, perhaps, the ATG initiation codon
  • the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
  • exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544).
  • a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used.
  • cell lines which stably express the target gene protein can be engineered.
  • Host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells can be allowed to grow for 1 -2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method can advantageously be used to engineer cell lines which expiess the target gene protein.
  • Such engineered cell lines can be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the target gene protein.
  • a number of selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk " , hgprt " or aprt " cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Proc. Natl. Acad Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J Mol. Biol. 150:1); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147) genes.
  • any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed.
  • a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cells lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-8976).
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni 2+ nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. Fusions at the carboxy terminal of the target gene product are also contemplated.
  • 5 target gene protein can be labeled, either directly or indirectly, to facilitate detection of a complex formed between the target gene protein and a test substance.
  • Any of a variety of suitable labeling systems can be used including but not limited to radioisotopes such as l25 I; enzyme labeling systems that generate a detectable colorimetric signal or light when exposed to substrate; and fluorescent labels.
  • Indirect labeling involves the use of a protein, such as a labeled antibody, which specifically binds to either a target gene product.
  • a protein such as a labeled antibody
  • Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any
  • Protein purification techniques are well known in the art. Proteins encoded and expressed from identified exogenous nucleotide sequence 17 s can be partially purified using precipitation techniques, such as precipitation with polyethylene
  • epitope tagging of the protein can be used to allow simple one step purification of the protein.
  • chromatographic methods such as ion-exchange chromatography, gel filtration, use of hydroxyapaptite columns, immobilized reactive dyes, chromatofocusing, and use of high-performance liquid chromatography, may also be used to purify the protein. Electrophoretic methods such as one-dimensional gel electrophoresis, high- 5 resolution two-dimensional polyacrylamide electrophoresis, isoelectric focusing, and others are contemplated as purification methods.
  • affinity chromatographic methods comprising solid phase bound- antibody, ligand presenting columns and other affinity chromatographic matrices are contemplated as purification methods in the present invention.
  • the purified target gene products, fragments thereof, or derivatives -in thereof may be administered to an individual in a pharmaceutically acceptable carrier to induce an immune response against the protein or polypeptide.
  • the immune response is a protective immune response which protects the individual.
  • antibodies capable of specifically recognizing epitopes of one or more of the target gene products described above.
  • Such antibodies can include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • various host animals can be immunized by injection with a target gene protein, or a portion thereof.
  • host animals can include but are not limited to rabbits, mice, and rats, to name but a few.
  • Various adjuvants can be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • the invention provides a method of eliciting an immune response in an animal, comprising introducing into the animal an immunogenic composition comprising an isolated polypeptide, the amino acid sequence of which comprises at least 6 consecutive residues of one ofSEQ ID NO: 63 to l23.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as target gene product, or an antigenic functional derivative thereof.
  • host animals such as those described above, can be immunized by injection with differentially expressed or pathway gene product supplemented with adjuvants as also described above.
  • the antibody titer in the immunized animal can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules can be isolated from the animal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction.
  • Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Patent No.
  • Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAb of this invention can be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
  • a monoclonal antibody directed against a polypeptide of the invention can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest.
  • Kits for generating and screening phage display libraries are commercially available (e.g. , the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • Humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule.
  • CDRs complementarily determining regions
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671 ; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No.
  • Fully human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non- human monoclonal antibody e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.
  • Antibody fragments which recognize specific epitopes can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries can be constructed (Huse et al., 1989, Sc ence.246: 1275- 1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • Antibodies of the present invention may also be described or specified in terms of their binding affinity to a target gene product.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5 X lO ⁇ M, lO ⁇ M, 5 X 10 '7 M, 10 "7 M, 5 X 1Q- 8 M, IO “8 M, 5 X 10 "9 M, 10 "9 M, 5 X 10- ,0 M, 10 ,0 M, 5 X 10- U M, 10- U M, 5 X 10 "12 M, 10 "12 M, 5 X 10 "13 M, 10 ' 13 M, 5 X 10 "14 M, 10 "14 M, 5 X 10 ",5 M, or 10 ",5 M.
  • Antibodies directed against a target gene product or fragment thereof can be used to detect the a target gene product in order to evaluate the abundance and pattern of expression of the polypeptide under various environmental conditions, in different morphological forms (mycelium, yeast, spores) and stages of an organism's life cycle.
  • Antibodies directed against a target gene product or fragment thereof can be used diagnostically to monitor levels of a target gene product in the tissue of an infected host as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and
  • suitable radioactive material include 125l 5 131i 5 35 ⁇ or 3R
  • Antibodies can also be used to modify a biological activity of a target gene product. Antibodies to gene products related to virulence or pathogenicity can also be used to prevent infection and alleviate one or more symptoms associated with infection by the organism.
  • an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a toxin or fungicidal agent. Techniques for conjugating a therapeutic moiety to antibodies are well known, see, e.g., Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119- 58 (1982).
  • An antibody with or without a therapeutic moiety conjugated to it can be used as a therapeutic that is administered alone or in combination with chemotherapeutic agents. 5.4.5 Antisense Molecules
  • antisense molecules as inhibitors of gene expression may be a specific, genetically based therapeutic approach (for a review, see Stein, in Ch. 69, Section 5 "Cancer: Principle and Practice of Oncology", 4th ed., ed. by DeVita et al., J.B. Lippincott, Philadelphia 1993).
  • the present invention provides the therapeutic or prophylactic use of nucleic acids of at least six nucleotides that are antisense to a target essential or virulence gene or a portion thereof.
  • An "antisense" target nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a portion of a target gene RNA (preferably mRNA) by virtue of some sequence complementarity.
  • the invention further provides pharmaceutical compositions comprising an effective amount of the antisense nucleic acids of the invention in a pharmaceutically acceptable carrier, as described infra.
  • the invention is directed to methods for inhibiting the expression of a target gene in an organism of interest, such as C. albicans in vitro or in vivo comprising providing the cell with an effective amount of a composition comprising an antisense nucleic acid of the invention.
  • a target gene in an organism of interest, such as C. albicans in vitro or in vivo
  • providing the cell with an effective amount of a composition comprising an antisense nucleic acid of the invention comprising an antisense nucleic acid of the invention.
  • Multiple antisense polynucleotides hybridizable to different target genes may be used in combinations, sequentially or simultaneously.
  • the present invention is directed toward methods for modulating expression of an essential gene which has been identified by the methods described supra, in which an antisense RNA molecule, which inhibits translation of mRNA transcribed from an essential gene, is expressed from a regulatable promoter.
  • an antisense RNA molecule which inhibits translation of mRNA transcribed from an essential gene, is expressed from a regulatable promoter.
  • the antisense RNA molecule is expressed in a GRACE strain oi Candida albicans or another GRACE strain constructed from another diploid pathogenic organism.
  • the antisense RNA molecule is expressed in a wild-type or other non-GRACE strain oi Candida albicans or another diploid pathogenic organism, including animal fiigal pathogens such as Aspergillus fumigatus, Aspergillus niger, Aspergillus flavis, Candida tropical is, Candida parapsilopsis, Candida krusei, Cryptococcus neoformans, Coccidioides immitis, Exophalia dermatiditis, Fusarium oxysporum, Histoplasma capsulatum, Phneumocystis carinii, Trichosporon beigelii, Rhizopus arrhizus, Mucor rouxii, Rhizomucor pusillus, or Absidia corymbigera, or the plant fungal pathogens, such as Botrytis cinerea, Erysiphe graminis, Magnaporthe grisea, Puccinia recodita, Septori
  • the nucleic acid molecule comprising an antisense nucleotide sequence of the invention may be complementary to a coding and/or noncoding region of a target gene mRNA.
  • the antisense molecules will bind to the complementary target gene mRNA transcripts and reduce or prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • Nucleic acid molecules that are complementary to the 5' end of the message should work most efficiently at inhibiting translation.
  • sequences complementary to the 3' untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335.
  • Nucleic acid molecules comprising nucleotide sequences complementary to the 5' untranslated region of the mRNA can include the complement of the AUG start codon.
  • Antisense nucleic acid molecules complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5'-, 3'- or coding region of target gene mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, at least 50 nucleotides, or at least 200 nucleotides.
  • in vitro studies are first performed to quantitate the ability of the antisense molecule to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • the antisense molecule can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the antisense molecule can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the antisense molecule may include other appended groups such as peptides (e.g., for targeting cell receptors in vivo), hybridization- triggered cleavage agents. (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549).
  • the antisense molecule may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross- linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the antisense molecule may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiourid_ne, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyammomethyl-2-thiouracil, beta- D-mannosylqueosine
  • the antisense molecule may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the antisense molecule comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the antisense molecule is an ⁇ -anomeric oligonucleotide.
  • An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641).
  • the oligonucleotide is a 2'-0- methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA- DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
  • Antisense molecules of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209)
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
  • antisense nucleotides complementary to the coding region of a target gene could be used, those complementary to the transcribed untranslated region are also preferred.
  • compositions of the invention comprising an effective amount of an antisense nucleic acid in a pharmaceutically acceptable carrier, can be administered to a subject infected with the pathogen of interest.
  • the amount of antisense nucleic acid which will be effective in the treatment of a particular disease caused by the pathogen will depend on the site of the infection or condition,
  • antisense molecules can be injected directly into the tissue site in which the
  • pathogens are residing, or modified antisense molecules, designed to target the desired cells (e.g., antisense molecule linked to peptides or antibodies that specifically bind receptors or antigens expressed on the pathogen's cell surface) can be administered systemically.
  • Antisense molecules can be delivered to the desired cell population via a delivery complex.
  • pharmaceutical compositions comprising antisense nucleic acids of the target
  • genes are administered via biopolymers (e.g., poly- ⁇ -1 -4-N-acetylglucosamine polysaccharide), liposomes, microparticles, or microcapsules.
  • biopolymers e.g., poly- ⁇ -1 -4-N-acetylglucosamine polysaccharide
  • liposomes e.g., liposomes, microparticles, or microcapsules.
  • liposomes targeted via antibodies to specific identifiable pathogen antigens Leonetti et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2448-
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA (For a review see, for example Rossi, J., 1994, Current Biology 4:469-471).
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage.
  • the composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated by reference
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding target gene proteins.
  • Ribozyme molecules designed to catalytically cleave specific target gene mRNA transcripts can also be used to prevent translation of target gene mRNA and expression of target genes. While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy target gene mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target gene mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
  • ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the target gene mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
  • the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”) such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-433; published International patent application No. WO 88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216).
  • Cech-type ribozymes such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-5
  • the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
  • the invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in a target gene.
  • the ribozymes can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the target gene in vivo. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency. Multiple ribozyme molecules directed against different target genes can also be used in combinations, sequentially or simultaneously.
  • Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention can be prepared by any method known in the art for the synthesis of DNA and RNA molecules.
  • RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.
  • DNA sequences can be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. These nucleic acid constructs can be administered selectively to the desired cell population via a delivery complex.
  • DNA molecules can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phospho- diesterase linkages within the oligodeoxyribonucleotide backbone.
  • the following assays are designed to identify compounds that bind to target gene products, bind to other cellular proteins that interact with the target gene product, and to compounds that interfere with the interaction of the target gene product with other cellular proteins.
  • Compounds identified via such methods can include compounds which modulate the activity of a polypeptide encoded by a target gene of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound).
  • compounds identified via such methods can include compounds which modulate the expression of the polynucleotide (that is, increase or decrease expression relative to expression levels observed in the absence of the compound), or increase or decrease the stability of the expressed product encoded by that polynucleotide.
  • the present invention provides a method for identifying an antimycotic compound comprising screening a plurality of compounds to identify a compound that modulates the activity or level of a gene product, said gene product being encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 61, or a nucleotide sequence that is naturally occurring in Saccharomyces cerevisiae and that is the ortholog of a gene having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 61.
  • In vitro systems are designed to identify compounds capable of binding the target gene products of the invention.
  • Compounds identified in this manner are useful, for example, in modulating the activity of wild type and/or mutant target gene products, are useful in elucidating the biological function of target gene products, are utilized in screens for identifying other compounds that disrupt normal target gene product interactions, or are useful themselves for the disruption of such interactions.
  • the principle of the assays used to identify compounds that bind to the target gene product involves preparing a reaction mixture comprising the target gene product and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which is removed and/or detected within the reaction mixture.
  • These assays are conducted in a variety of ways. For example, one method involves anchoring target gene product or the test substance onto a solid phase and detecting target gene product/test compound complexes anchored, via the intermolecular binding reaction, to the solid phase at the end of the reaction.
  • the target gene product is anchored onto a solid surface, and the test compound, which is not anchored, is labeled, either directly or indirectly.
  • microtiter plates are conveniently utilized as the solid phase.
  • the anchored component is immobilized by non-covalent or covalent attachments.
  • Non-covalent attachment can be accomplished by simply coating the solid surface with a solution of the protein and drying the coated surface.
  • an immobilized antibody preferably a monoclonal antibody, specific for the protein to be immobilized is used to anchor the protein to the solid surface.
  • the surfaces are prepared in advance and stored.
  • the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e. g, by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface is accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label is used to detect complexes anchored on the surface; e ⁇ g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, is directly labeled or indirectly labeled with a labeled anti-Ig antibody).
  • a reaction is conducted in a liquid phase, the reaction products are separated from unreacted components, and complexes are detected; e.g., using an immobilized antibody specific for the target gene product or for the test compound, to anchor complexes formed in solution, and a second labeled antibody, specific for the other component of the complex to allow detection of anchored complexes.
  • an immobilized antibody specific for the target gene product or for the test compound to anchor complexes formed in solution
  • a second labeled antibody specific for the other component of the complex to allow detection of anchored complexes.
  • Any method suitable for detecting protein-protein interactions can be employed for identifying novel target protein-cellular or extracellular protein interactions.
  • the target gene products of the invention interact, in vivo, with one or more cellular or extracellular macromolecules, such as proteins.
  • macromolecules include, but are not limited to, nucleic acid molecules and proteins identified via methods such as those described above.
  • binding partners such cellular and extracellular macromolecules are referred to herein as "binding partners.”
  • binding partners Compounds that disrupt such interactions can be useful in regulating the activity of the target gene protein, especially mutant target gene proteins.
  • Such compounds include, but are not limited to molecules such as antibodies, peptides, and the like, as described.
  • the basic principle of the assay systems used to identify compounds that interfere with the interaction between the target gene product and its cellular or extracellular binding partner or partners involves preparing a reaction mixture containing the target gene product and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex.
  • the reaction mixture is prepared in the presence and absence of the test compound.
  • the test compound is initially included in the reaction mixture, or added at a time subsequent to the addition of target gene product and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test compound. The formation of complexes between the target gene protein and the cellular or extracellular binding partner is then detected.
  • complex formation within reaction mixtures containing the test compound and normal target gene protein can also be compared to complex formation within reaction mixtures containing the test compound and a mutant target gene protein. This comparison can be important in those cases wherein it is desirable to identify compounds that disrupt intermolecular interactions involving mutant but not normal target gene proteins.
  • the assay for compounds that interfere with the interaction of the target gene products and binding partners is conducted in either a heterogeneous or a homogeneous format.
  • Heterogeneous assays involve anchoring either the target gene product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants is varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the target gene products and the binding partners, e.g., by competition, are identified by conducting the reaction in the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the target gene protein and an interacting cellular or extracellular binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the components from the complex, are tested by adding the test compound to the reaction mixture after complexes have been formed.
  • the various formats are described briefly below.
  • either the target gene protein or the interactive cellular or extracellular binding partner is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly.
  • microtiter plates are conveniently utilized.
  • the anchored species is immobilized either by non-covalent or covalent attachment. Non-covalent attachment is accomplished simply by coating the solid surface with a solution of the target gene product or binding partner and drying the coated surface. Alternatively, an immobilized antibody specific for the species to be anchored is used to anchor the species to the solid surface. The surfaces can be prepared in advance and stored.
  • the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface is accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, is directly labeled or indirectly labeled with a labeled anti-Ig antibody).
  • the antibody in turn, is directly labeled or indirectly labeled with a labeled anti-Ig antibody.
  • test compounds which inhibit complex formation or which disrupt preformed complexes are detected.
  • the reaction is conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a second, labeled antibody specific for the other partner to detect anchored complexes.
  • test compounds which inhibit complex or which disrupt preformed complexes are identified.
  • a homogeneous assay can be used.
  • a preformed complex of the target gene protein and the interacting cellular or extracellular binding partner is prepared in which either the target gene product or its binding partner is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g., U.S. Patent No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays).
  • the addition of a test substance that competes with and displaces one of the species from the preformed complex results in the generation of a signal above background. In this way, test substances which disrupt target gene protein/cellular or extracellular binding partner interaction are identified.
  • the target gene product is prepared for immobilization using recombinant DNA techniques described above.
  • the target gene coding region is fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-l , in such a manner that its binding activity is maintained in the resulting fusion protein.
  • GST glutathione-S-transferase
  • the interactive cellular or extracellular binding partner is purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and as described above.
  • This antibody is labeled with the radioactive isotope 125 I, for example, by methods routinely practiced in the art.
  • the GST-target gene fusion protein is anchored to glutathione-agarose beads.
  • the interactive cellular or extracellular binding partner is then added in the presence or absence of the test compound in a manner that allows interaction and binding to occur.
  • unbound material can be washed away, and the labeled monoclonal antibody is added to the system and allowed to bind to the complexed components.
  • the interaction between the target gene protein and the interactive cellular or extracellular binding partner is detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound results in a decrease in measured radioactivity.
  • the GST-target gene fusion protein and the interactive cellular or extracellular binding partner are mixed together in liquid in the absence of the solid glutathione- agarose beads.
  • the test compound is added either during or after the species are allowed to interact. This mixture is added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the target gene product/binding partner interaction is detected by adding the labeled antibody and measuring the radioactivity associated with the beads.
  • these same techniques are employed using peptide fragments that correspond to the binding domains of the target gene product and/or the interactive cellular or extracellular binding partner (in cases where the binding partner is a protein), in place of one or both of the full length proteins.
  • Any number of methods routinely practiced in the art are used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex are then selected. Sequence analysis of
  • the genes encoding the respective proteins reve ⁇ ds the mutations that correspond to the region of the protein involved in interactive binding.
  • one protein is anchored to a solid surface using methods described above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain remains associated with the
  • a target gene product is anchored to a
  • the cellular or extracellular binding partner binding domain is eluted, purified, and analyzed for amino acid sequence by well known methods.
  • Peptides so identified are produced synthetically or fused to appropriate facilitative proteins using well known recombinant DNA technology.
  • the proteins encoded by the fungal genes identified using the methods of the present invention are isolated and expressed. These recombinant proteins are then used as targets in assays to screen libraries of compounds for potential drug candidates.
  • the generation of chemical libraries is well known in the art. For example, combinatorial chemistry is used to generate a library of compounds to be screened in
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building block" reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining amino acids in every possible combination to yield peptides of a given length. Millions of chemical compounds theoretically
  • JJ can be synthesized through such combinatorial mixings of chemical building blocks.
  • one commentator observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds.
  • Other chemical libraries known to those in the art may also be used, including natural product libraries.
  • combinatorial libraries are screened for compounds that possess desirable biological properties.
  • compounds which may be useful as drugs or to develop drugs would likely have the ability to bind to the target protein identified, expressed and purified as discussed above.
  • candidate compounds would likely interfere with the enzymatic properties of the target protein.
  • the enzymatic function of a target protein may be to serve as a protease, nuclease, phosphatase, dehydrogenase, transporter protein, transcriptional enzyme, replication component, and any other type of enzyme known or unknown.
  • the present invention contemplates using the protein products described above to screen combinatorial chemical libraries.
  • the biochemical activity of the protein, as well as the chemical structure of a substrate on which the protein acts is known.
  • the biochemical activity of the target protein is unknown and the target protein has no known substrates.
  • libra ⁇ es of compounds are screened to identify compounds that function as inhibitors of the target gene product.
  • a library of small molecules is generated using methods of combinatorial library formation well known in the art.
  • the target gene product, an enzyme, and chemical compounds of the library are combined and permitted to interact with one another.
  • a labeled substrate is added to the incubation.
  • the label on the substrate is such that a detectable signal is emitted from metabolized substrate molecules.
  • the emission of this signal permits one to measure the effect of the combinatorial library compounds on the enzymatic activity of target enzymes by comparing it to the signal emitted in the absence of combinatorial library compounds.
  • the characteristics of each library compound are encoded so that compounds demonstrating activity against the enzyme can be analyzed and features common to the various compounds identified can be isolated and combined into future iterations of libraries.
  • screening methodology is exemplary only.
  • Other methods are well known to those skilled in the art.
  • a wide variety of screening techniques are known for a large number of naturally-occurring targets when the biochemical function of the target protein is known.
  • some techniques involve the generation and use of small peptides to probe and analyze target proteins both biochemically and genetically in order to identify and develop drug leads.
  • Such techniques include the methods described in PCT publications No. WO9935494, WO9819162, WO9954728, the disclosures of which are incorporated herein by reference in their entireties.
  • the proteins may be from animal fugal pathogens such as Aspergillus fumigatus, Aspergillus niger, Aspergillus flavis, Candida tropicalis, Candida parapsilopsis, Candida krusei, Cryptococcus neoformans, Coccidioides immitis, Exophalia dermatiditis, Fusarium oxysporum, Histoplasma capsulatum, Phneumocystis carinii, Trichosporon beigelii, Rhizopus arrhizus, Mucor rouxii, Rhizomucor pusillus, or Absidia corymbigera, or the plant fungal pathogens, such as Botrytis cinerea, Erysiphe graminis,
  • the proteins are from an organism other than Saccharomyces cerevisiae.
  • GRACE methods and strains are used to develop in vitro assays for biochemical activities that are shown to be essential to cell viability.
  • a number of essential genes identified by the GRACE conditional expression methodologies display statistically significant similarity to biochemically characterized gene products from other organisms. For example, based on amino acid sequence similarity, a number of essential and fungal specific genes listed in Table II are predicted to possess the following biochemical activities:
  • CaRHOl GTPase involved in (l,3)- ⁇ -glucan synthesis and polarity CaYHR118c Origin of replication complex subunit CaYPL128c (TBP1) Telomere binding protein CaYNL256w Dihydropteroate synthase CaYKL004w (AUR1) Phosphatidylinositol: ceramide phosphoinositol transferase CaYJL090c (DPB11) DNA polB subunit CaYOL149w (DCPl) mRNA decapping enzyme CaYNL151c (RPC31) RNA polIII subunit CaYOR148c (SPP2) RNA splicing CaYER026c (CHOI) Phosphatidylserine synthase
  • a number of well characterized standard in vitro biochemical assays are readily adapted for these validated drug targets.
  • the validated target, CaRHOl is used within a in vitro-based drug screen by adapting standard GTPase assays developed for a wide range of such proteins.
  • novel assays are developed using biochemical information pertaining to validated drug targets within our GRACE strain collection. Any assays known in the art for enzymes with similar biochemical activities (e.g., mechanism of action, class of substrate) are adapted for screening for inhibitors of the enzymes encoded by these essential C. albicans genes.
  • CaTBFl C. albicans gene
  • TBF1 S. cerevisiae counterpart
  • CaTBFlp recognizes is known and is relatively short (Koering et al., Nucleic Acid Res. 28:2519-2526, which is incorporated herein by reference in its entirety), enabling inexpensive synthesis of oligonucleotides corresponding to this element.
  • this assay only requires the target protein and a DNA fragment containing the nucleotide sequence it recognizes, only purification of CaTBFlp protein is necessary in order to develop an in vitro binding assay.
  • This in vitro assay involves crosslinking the DNA element to the bottom of a well, incubation of radiolabeled CaTBFlp to facilitate protein-DNA binding, a series of washes to remove unbound material, and determination of the percentage of bound radiolabeled CaTBFlp.
  • purified CaTBFlp is attached to the well and radiolabeled oligonucleotides added.
  • Drug screening including the use of high throughput screening technique, is performed by searching for compounds that inhibit the protein-DNA binding measured in this assay.
  • a second validated drug target, CaORC ⁇ is used in this type of assay since its S. cerevisiae homolog, ORC6, directly binds a DNA element within the origin of replication of yeast chromosomes (Mizushima et al., 2000, Genes & Development 14:1631 - 1641, which is incorporated herein by reference in its entirety). Biochemical purification of any of these targets could be achieved, for example, by PCR-based construction of C. albicans heterozygous strains in which the gene encoding the CaORC ⁇ protein has been modified to include a carboxy-terminal hexahistidine tag enabling purification of the chimeric protein using standard Ni +2 affinity column chromatography techniques.
  • the present invention also provides cell extracts useful in establishing in vitro assays for suitable biochemical targets.
  • GRACE-derived C. albicans strains are grown either under constitutive expression conditions or transcription repression conditions to either overproduce or deplete a particular gene product.
  • Cellular extracts resulting from strains incubated under these two conditions are compared with extracts prepared from identically-grown wild type strains. These extracts are then used for the rapid evaluation of targets using existing in vitro assays or new assays directed toward novel gene products, without having to purify the gene product.
  • Such a whole cell extract approach to in vitro assay development is typically necessary for targets involved in cell wall biosynthetic pathways (e. g.
  • target molecules also include other molecules such as DNAs, lipids, carbohydrates and RNAs including messenger RNAs, ribosomal RNAs, tRNAs and the like.
  • a number of highly sensitive cell-based assay methods are available to those of skill in the art to detect binding and interaction of test compounds with specific target molecules. However, these methods are generally not highly effective when the test compound binds to or otherwise interacts with its target molecule with moderate or low affinity.
  • the target molecule may not be readily accessible to a test compound in solution, such as when the target molecule is located inside the cell or within a cellular compartment such as the periplasm of a bacterial cell.
  • current cell-based assay methods are limited in that they are not effective in identifying or characterizing compounds that interact with their targets with moderate to low affinity or compounds that interact with targets that are not readily accessible.
  • the cell-based assay methods of the present invention have substantial advantages over current cell-based assays. These advantages derive from the use of sensitized cells in which the level or activity of at least one gene product required for fungal proliferation, virulence, or pathogenicity (the target molecule) has been specifically reduced to the point where the presence or absence of its function becomes a rate-determining step for fungal growth, survival, proliferation, virulence, or pathogenicity. Such sensitized cells become much more sensitive to compounds that are active against the affected target molecule.
  • sensitized cells are obtained by growing a GRACE strain in the presence of a concentration of inducer or repressor which provides a level of a gene product required for fungal growth, survival, proliferation, virulence, or pathogenicity such that the presence or absence of its function becomes a rate-determining step for fungal growth, survival, proliferation, virulence, or pathogenicity.
  • cell-based assays of the present invention are capable of detecting compounds exhibiting low or moderate potency against the target molecule of interest because such compounds are substantially more potent on sensitized cells than on non-sensitized cells.
  • the effect may be such that a test compound may be two to several times more potent, at least 10 times more potent, at least 20 times more potent, at least 50 times more potent, at least 100 times more potent, at least 1000 times more potent, or even more than 1000 times more potent when tested on the sensitized cells as compared to the non-sensitized cells.
  • expression of a gene encoding a ribosomal protein at a level such that the function of the ribosomal protein becomes rate limiting for fungal growth, survival, proliferation, virulence, or pathogenicity is expected to sensitize the cell to compounds acting at that ribosomal protein to compounds acting at any of the ribosomal components (proteins or rRNA) or even to compounds acting at any target which is part of the protein synthesis pathway.
  • an important advantage of the present invention is the ability to reveal new targets and pathways that were previously not readily accessible to drug discovery methods.
  • Sensitized cells of the present invention are prepared by reducing the activity or level of a target molecule.
  • the target molecule may be a gene product, such as an RNA or polypeptide produced from the nucleic acids required for fungal growth, survival, proliferation, virulence, or pathogenicity described herein.
  • the target may be an RNA or polypeptide in the same biological pathway as the nucleic acids required for fungal growth, survival, proliferation, virulence, or pathogenicity as described herein.
  • biological pathways include, but are not limited to, enzymatic, biochemical and metabolic pathways as well as pathways involved in the production of cellular structures such as the cell membrane.
  • This information is used to design subsequent directed libraries containing compounds with enhanced activity against the target molecule. After one or several iterations of this process, compounds with substantially increased activity against the target molecule are identified and may be further developed as drugs. This process is facilitated by use of the sensitized cells of the present invention since compounds acting at the selected targets exhibit increased potency in such cell-based assays, thus; more compounds can now be characterized providing more useful information than would be obtained otherwise.
  • cell-based assays of the present invention identify or characterize compounds that previously would not have been readily identified or characterized including compounds that act at targets that previously were not readily exploited using cell-based assays.
  • the process of evolving potent drug leads from initial hit compounds is also substantially improved by the cell-based assays of the present invention because, for the same number of test compounds, more structure-function relationship information is likely to be revealed.
  • the method of sensitizing a cell entails selecting a suitable gene.
  • a suitable gene is one whose expression is required for the growth, survival, proliferation, virulence, or pathogenicity of the cell to be sensitized.
  • the next step is to obtain a cell in which the level or activity of the target can be reduced to a level where it is rate limiting for growth, survival, proliferation, virulence or pathogenicity.
  • the cell may be a GRACE strain in which the selected gene is under the control of a regulatable promoter.
  • the amount of RNA transcribed from the selected gene is limited by varying the concentration of an inducer or repressor which acts on the regulatable promoter, thereby varying the activity of the promoter driving transcription of the RNA.
  • cells are sensitized by exposing them to an inducer or repressor concentration that results in an RNA level such that the function of the selected gene product becomes rate limiting for fungal growth, survival, proliferation, virulence, or pathogenicity.
  • GRACE strains in which the sequences required for fungal growth, survival, proliferation, virulence, or pathogenicity of Candida albicans described herein are under the control of a regulatable promoter, are grown in the presence of a concentration of inducer or repressor which causes the function of the gene products encoded by these sequences to be rate limiting for fungal growth, survival, proliferation, virulence, or pathogenicity.
  • inducer or repressor is calculated by plotting various doses of inducer or repressor against the corresponding growth inhibition caused by the limited levels of the gene product required for fungal proliferation. From this dose-response curve, conditions providing various growth rates, from 1 to 100% as compared to inducer or repressor-free growth, can be determined. For example, if the regulatable promoter is repressed by tetracycline, the GRACE strain may be 0 grown in the presence of varying levels of tetracyline. Similarly, inducible promoters may be used. In this case, the GRACE strains are grown in the presence of varying concentrations of inducer.
  • the highest concentration of the inducer or repressor that does not reduce the growth rate significantly can be estimated from the dose-response curve.
  • Cellular proliferation can be monitored by growth medium turbidity via OD measurements.
  • the concentration of inducer or repressor that reduces growth by 25% can be predicted from the dose-response curve.
  • a concentration of inducer or repressor that reduces growth by 50% can be calculated from the dose-response curve. Additional parameters such as colony forming units (cfu) are also used to measure cellular growth, survival and/or viability.
  • cfu colony forming units
  • an individual haploid strain may similarly be used as the basis for detection of an antifungal or therapeutic agent.
  • the test organism e.g. Aspergillus, fumigatus, Cryptococcus neoformans,
  • Magnaportha grisea or any other haploid organisms represented in Table I is a strain constructed by modifying the single allele of the target gene in one step by recombination with a 25 promoter replacement fragment comprising a heterologous regulatable promoter, such that the expression of the gene is conditionally regulated by the heterologous promoter.
  • sensitized haploid cells may similarly be used in whole cell-based assay methods to identify compounds displaying a preferential activity against the affected target.
  • the modified strain is grown under a first set of conditions where the heterologous promoter is expressed at a relatively low level (i.e. partially repressed) and the extent of growth determined. This experiment is repeated in the presence of a test compound and a second measurement of growth obtained. The extent of growth in the presence and in the absence of the test compound are then compared to provide a first indicator
  • Whole-cell assays comprising collections or subsets of multiple sensitized strains may also be screened, for example, in a series of 96-well, 384-well, or even 1586-well microtiter plates, with each well containing individual strains sensitized to identify 0 compounds displaying a preferential activity against each affected target comprising a target set or subset selected from, but not limited to the group consisting of fungal-specific, pathogen- specific, desired biochemical-function, human-homolog, cellular localization, and signal transduction cascade target sets.
  • Cells to be assayed are exposed to the above-determined concentrations of inducer or repressor.
  • the presence of the inducer or repressor at this sub-lethal concentration reduces the amount of the proliferation-required gene product to the lowest amount in the cell that will support growth.
  • Cells grown in the presence of this concentration of inducer or repressor are therefore specifically more sensitive to inhibitors of the proliferation-required protein or RNA of interest as well as to inhibitors of proteins or RNAs in the same biological pathway as the proliferation-required protein or RNA of interest but not specifically more sensitive to inhibitors of unrelated proteins or RNAs.
  • the sub-lethal concentration of inducer or repressor may be any concentration consistent with the intended use of the assay to identify candidate compounds to which the cells are more sensitive than are control cells in which this gene product is not rate-limiting.
  • the sub-lethal concentration of the inducer or repressor may be such that growth inhibition is at least about 5%, at least about 8%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%), at least about 60%> at least about 75%, , at least 80%, at least 90%), at least
  • Cells which are pre-sensitized using the preceding method are more sensitive to inhibitors of the target protein because these cells contain less target protein to inhibit than wild-type cells.
  • virulence or pathogenicity may be used to identify compounds which inhibit virulence or pathogenicity.
  • the virulence or pathogenicity of cells exposed to the candidate compound which express rate limiting levels of a gene product involved in virulence or pathogenicity is compared to the virulence or pathogenicity of cells exposed to the candidate compound in which the levels of the gene product are not rate limiting. Virulence or pathogenicity may be measured using the techniques described herein.
  • the level or activity of a gene product required for fungal growth, survival, proliferation, virulence, or pathogenicity is reduced using a mutation, such as a temperature sensitive mutation, in the sequence required for fungal growth, survival, proliferation, virulence, or pathogenicity and an inducer or repressor level which, in conjunction with the temperature sensitive mutation, provides levels of the gene product required for fungal growth, survival, proliferation, virulence, or pathogenicity which are rate limiting for proliferation.
  • a mutation such as a temperature sensitive mutation
  • the concentration of inducer or repressor is chosen so as to further reduces the activity of the gene product required for fungal growth, survival, proliferation, virulence, or pathogenicity.
  • Drugs that may not have been found using either the temperature sensitive mutation or the inducer or repressor alone may be identified by determining whether cells in which expression of the nucleic acid encoding the proliferation-required gene product has been reduced and which are grown at a temperature between the permissive temperature and the restrictive temperature are substantially more sensitive to a test compound than cells in which expression of the gene product required for fungal growth, survival, proliferation, virulence, or pathogenicity has not been reduced and which are grown at a permissive temperature.
  • drugs found previously from either the use of the inducer or repressor alone or the temperature sensitive mutation alone may have a different sensitivity profile when used in cells combining the two approaches, and that sensitivity profile may indicate a more specific action of the drug in inhibiting one or more activities of the gene product.
  • Temperature sensitive mutations may be located at different sites within a gene and may lie within different domains of the protein.
  • Escherichia coli encodes the replication fork DNA helicase.
  • DnaB has several domains, including domains for oligomerization, ATP hydrolysis, DNA binding, interaction with primase, interaction with DnaC, and interaction with DnaA.
  • Temperature sensitive mutations in different domains of DnaB confer different phenotypes at the restrictive temperature, which include either an abrupt stop or a slow stop in DNA replication either with or without DNA breakdown (Wechsler, J.A. and Gross, J.D. 1971 Escherichia coli mutants temperature-sensitive for DNA synthesis. Mol. Gen. Genetics 113:273-284) and termination of growth or cell death.
  • temperature sensitive mutations in different domains of the protein may be used in conjunction with GRACE strains in which expression of the protein is under the control of a regulatable promoter.
  • the above method may be performed with any mutation which reduces but does not eliminate the activity or level of the gene product which is required for fungal growth, survival, proliferation, virulence, or pathogenicity.
  • growth inhibition, virulence or pathogenicity of cells containing a limiting amount of that gene product can be assayed. Growth inhibition can be measured by directly comparing the amount of growth, measured by the optical density of the culture relative to uninoculated growth medium, between an experimental sample and a control sample.
  • GFP green fluorescent protein
  • enzymatic activity assays include measuring green fluorescent protein (GFP) reporter construct emissions, various enzymatic activity assays, and other methods well known in the art. Virulence and pathogenicity may be measured using the techniques described herein. It will be appreciated that the above method may be performed in solid phase, liquid phase, a combination of the two preceding media, or in vivo. For example, cells grown on nutrient agar containing the inducer or repressor which acts on the regulatable promoter used to express the proliferation required gene product may be exposed to compounds spotted onto the agar surface. A compound's effect may be judged from the diameter of the resulting killing zone, the area around the compound application point in which cells do not grow.
  • GFP green fluorescent protein
  • Multiple compounds may be transferred to agar plates and simultaneously tested using automated and semi-automated equipment including but not restricted to multi-channel pipettes (for example the Beckman Multimek) and multi-channel spotters (for example the Genomic Solutions Flexys). In this way multiple plates and thousands to millions of compounds may be tested per day.
  • automated and semi-automated equipment including but not restricted to multi-channel pipettes (for example the Beckman Multimek) and multi-channel spotters (for example the Genomic Solutions Flexys). In this way multiple plates and thousands to millions of compounds may be tested per day.
  • the compounds are also tested entirely in liquid phase using microtiter plates as described below.
  • Liquid phase screening may be performed in microtiter plates containing 96, 384, 1536 or more wells per microtiter plate to screen multiple plates and thousands to millions of compounds per day.
  • Automated and semi-automated equipment are used for addition of reagents (for example cells and compounds) and for determination of cell density.
  • the compounds are also tested in vivo using the methods described herein. It will be appreciated that each of the above cell-based assays may be used to identify compounds which inhibit the activity of gene products from organisms other than Candida albicans which are homologous to the Candida albicans gene products described herein.
  • the target gene products may be from animal fugal pathogens such as Aspergillus fumigatus, Aspergillus niger, Aspergillus flavis, Candida tropicalis, Candida parapsilopsis, Candida krusei, Cryptococcus neoformans, Coccidioides immitis, Exophalia dermatiditis, Fusarium oxysporum, Histoplasma capsulatum, Phneumocystis carinii, Trichosporon beigelii, Rhizopus arrhizus, Mucor rouxii, Rhizomucor pusillus, or Absidia corymbigera, or the plant fungal pathogens, such as Botrytis cinerea, Erysiphe graminis, Magnaporthe grisea, Puccinia recodita, Septoria triticii, Tilletia controversa, Ustilago maydis, or any species falling within the genera of any of the above species.
  • GRACE strains in which one allele of a gene required for fungal growth, survival, proliferation, virulence, or pathogenicity is inactivated while the other allele is under the control of a regulatable promoter are constructed using the methods described herein.
  • the regulatable promoter may be the tetracycline regulated promoter described herein, but it will be appreciated that any regulatable promoter may be used.
  • an individual GRACE strain is used as the basis for detection of a therapeutic agent active against a diploid pathogenic fungal cell.
  • the test organism is a GRACE strain having a modified allelic gene pair, where the first allele of the gene has been inactivated by the insertion of, or replacement by, a nucleotide sequence encoding an expressible, dominant selectable marker and the second allele has been modified, by recombination, to place the second allele under the controlled expression of a heterologous promoter.
  • This test GRACE strain is then grown under a first set of conditions where the heterologous promoter is expressed at a relatively low level (“repressing") and the extent of growth determined.
  • This measurement may be carried out using any appropriate standard known to those skilled in the art including optical density, wet weight of pelleted cells, total cell count, viable count, DNA content, and the like.
  • This experiment is repeated in the presence of a test compound and a second measurement of growth obtained.
  • the extent of growth in the presence and in the absence of the test compound, which can conveniently be expressed in terms of indicator values, are then compared. A dissimilarity in the extent of growth or indicator values provides an indication that the test compound may interact with the target essential gene product.
  • the extent of growth in the first and in the second set of growth conditions can also be compared. If the extent of growth is essentially the same, the data suggest that the test compound does not inhibit the gene product encoded by the modified allelic gene pair carried by the GRACE strain tested. However, if the extent of growth are substantially different, the data indicate that the level of expression of the subject gene product may determine the degree of inhibition by the test compound and, therefore, it is likely that the subject gene product is the target of that test compound.
  • each GRACE strain can be tested individually, it will be more efficient to screen entire sets or subsets of a GRACE strain collection at one time.
  • arrays may be established, for example in a series of 96-well microtiter plates, with each well containing a single GRACE strain.
  • four microtiter plates are used, comprising two pairs where the growth medium in one pair supports greater expression of the heterologous promoter controlling the remaining active allele in each strain, than the medium in the other pair of plates.
  • One member of each pair is supplemented with a compound to be tested and measurements of growth of each GRACE strain is determined using standard procedures to provide indicator values for each isolate tested.
  • the collection of diploid pathogenic GRACE strains used in such a method for screening for therapeutic agents may comprise, for example, a substantially complete set of all the modified allelic gene pairs of the organism, the substantially complete set of all the modified allelic essential gene pairs of the organism or the collection may be selected from a subset of GRACE strains selected from, but not limited to the group consisting of fungal-specific, pathogen-specific, desired biochemical-function, human-homolog, cellular localization, and signal transduction cascade target sets.
  • the GRACE strains are grown in medium comprising a range of tetracycline concentrations to obtain the growth inhibitory dose-response curve for each strain. First, seed cultures of the GRACE strains are grown in the appropriate medium.
  • the GRACE strains may be grown in duplicate cultures containing two-fold serial dilutions of tetracycline.
  • control cells are grown in duplicate without tetracycline. The control cultures are started from equal amounts of cells derived from the same initial seed culture of a GRACE strain of interest. The cells are grown for an appropriate period of time and the extent of growth is determined using any 0 appropriate technique. For example, the extent of growth may be determined by measuring the optical density of the cultures.
  • the percent growth (relative to the control culture) for each of the tetracycline containing cultures is plotted against the log concentrations of tetracycline to produce a growth inhibitory dose response curve for tetracycline.
  • concentration of tetracycline that inhibits cell growth to 50% (IC 50 ) as compared to the 0 mM tetracyline control (0% growth inhibition) is then calculated from the curve.
  • Alternative methods of measuring growth are also contemplated. Examples of these methods include measurements of proteins, the expression of which is engineered into the cells being tested and can readily be measured.
  • GFP green fluorescent protein
  • Cells are pretreated with the selected concentration of tetracycline and then used to test the sensitivity of cell populations to candidate compounds.
  • the cells may be pretreated with a concentration of tetracycline which inhibits growth by at least about 5%, at least about 8%, at least about 10%), at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% at least about 75%, at least 80%, at 5 least 90%, at least 95% or more than 95%.
  • the cells are then contacted with the candidate compound and growth of the cells in tetracycline containing medium is compared to growth of the control cells in medium which lacks tetracycline to determine whether the candidate compound inhibits growth of the sensitized cells (i.e.
  • the growth of the cells in tetracycline containing medium may be compared to the growth of the cells in medium lacking tetracycline to determine whether the candidate compound inhibits the growth of the sensitized cells (i.e. the cells grown in the presence of tetracyline) to a greater extent than the candidate compound inhibits the growth of cells grown in the absence of tetracycline. For example, if a significant difference
  • the candidate compound may be used to inhibit the proliferation of the organism or may be further optimized to identify compounds which have an even greater ability to inhibit the growth, survival, or proliferation of the organism.
  • test animals are challenged with the GRACE strain and fed a diet containing the desired amount of tetracycline and the
  • the GRACE strain infecting the test animals expresses a rate limiting amount of a gene product required for virulence or pathogenicity (i.e. the GRACE cells in the test animals are sensitized).
  • Control animals are challenged with the GRACE strain and are fed a diet containing the candidate compound but lacking tetracycline.
  • the virulence or pathogenicity of the GRACE strain in the test animals is compared to that in the control animals.
  • the virulence or pathogenicity of the GRACE strain in the test animals may be compared to that in the control animals to determine whether the candidate compound inhibits the virulence or pathogenicity of the sensitized GRACE cells (i.e. the cells in the animals whose diet included tetracyline) to a greater extent than the candidate compound inhibits the growth of the GRACE cells in animals whose diet lacked
  • the candidate compound may be used to inhibit the virulence or pathogenicity of the organism or may be further optimized to identify compounds which have an even greater 25 ability to inhibit the virulence or pathogenicity of the organism. Virulence or pathogenicity may be measured using the techniques described therein.
  • Candida albicans which are homologous to the Candida albicans gene products described herein.
  • the gene products may be from animal fugal pathogens such as
  • the gene products are from an organism other than Saccharomyces cerevisae
  • the cell-based assay described above may also be used to identify the biological pathway in which a nucleic acid required for fungal proliferation, virulence or pathogenicity or the gene product of such a nucleic acid lies.
  • cells expressing a rate limiting level of a target nucleic acid required for fungal proliferation, virulence or pathogenicity and control cells in which expression of the target nucleic acid is not rate limiting are contacted with a panel of antibiotics known to act in various pathways. If the antibiotic acts in the pathway in which the target nucleic acid or its gene product lies, cells in which expression of target nucleic acid is rate limiting will be more sensitive to the antibiotic than cells in which expression of the target nucleic acid is not rate limiting.
  • the results of the assay may be confirmed by contacting a panel of cells in which the levels of many different genes required for proliferation, virulence or pathogenicity, including the target gene, is rate limiting. If the antibiotic is acting specifically, heightened sensitivity to the antibiotic will be observed only in the cells in which the target gene is rate limiting (or cells in which genes in the same pathway as the target gene is rate limiting) but will not be observed generally in which a gene product required for proliferation, virulence or pathogenicity is rate limiting.
  • nucleic acids from organisms other than Candida albicans which are homologous to the Candida albicans nucleic acids described herein.
  • the nucleic acids may be from animal fugal pathogens such as Aspergillus fumigatus,
  • Rhizopus arrhizus a Rhizopus arrhizus, Mucor rouxii, Rhizomucor pusillus, or Absidia corymbigera
  • the plant fungal pathogens such as Botrytis cinerea, Erysiphe graminis, Magnaporthe grisea,
  • nucleic acids are from an organism other than Saccharomyces cerevisae
  • the above method may be used to determine the pathway on which a test compound, such as a test antibiotic acts.
  • a panel of cells each of which expresses a rate limiting amount of a gene product required for fungal proliferation, virulence or pathogenicity where the gene product lies in a known pathway, is contacted with a compound for which it is desired to determine the pathway on which it acts.
  • the sensitivity of the panel of cells to the test compound is determined in cells in which expression of the nucleic acid encoding the gene product required for proliferation, virulence or pathogenicity
  • test compound 5 is at a rate limiting level and in control cells in which expression of the gene product required for proliferation, virulence or pathogenicity is not at a rate limiting level. If the test compound acts on the pathway in which a particular gene product required for proliferation, virulence, or pathogenicity lies, cells in which expression of that particular gene product is at a rate limiting level will be more sensitive to the compound than the cells in which gene
  • control cells in which expression of the particular gene required for fungal proliferation, virulence or pathogenicity is not rate limiting will not exhibit heightened sensitivity to the compound. In this way, the pathway on which the test compound acts may be determined.
  • the above method for determining the pathway on which a test compound acts may be applied to organisms other than Candida albicans by using panels of cells in which the activity or level of gene products which are homologous to the Candida albicans gene products described herein is rate limiting.
  • the gene products may be from animal fugal pathogens such as Aspergillus fumigatus,
  • Rhizopus arrhizus a Rhizopus arrhizus, Mucor rouxii, Rhizomucor pusillus, or Absidia corymbigera
  • the plant fungal pathogens such as Botrytis cinerea, Erysiphe graminis, Magnaporthe grisea,
  • Puccinia recodita Septoria triticii, Tilletia controversa, Ustilago maydis, or any species 25 falling within the genera of any of the above species.
  • the gene products are from an organism other than Saccharomyces cerevisiae.
  • Example 6.4, infra, provided below describes one method for performing such assays.
  • assay conditions such as the concentration of inducer or repressor used to produce rate limiting levels of a gene product required for fungal proliferation, virulence or pathogenicity and or the growth conditions used for the assay (for example incubation temperature and medium components) may further increase the selectivity and/or magnitude of the antibiotic sensitization exhibited.
  • the gene products may be from animal fugal pathogens such as Aspergillus fumigatus, Aspergillus niger, Aspergillus flavis, Candida tropicalis, Candida parapsilopsis, Candida krusei, Cryptococcus neoformans, Coccidioides immitis, Exophalia dermatiditis, Fusarium oxysporum, Histoplasma capsulatum, Pneumocystis carinii, Trichosporon beigelii, Rhizopus arrhizus, Mucor rouxii, Rhizomucor pusillus, or Absidia corymbigera, or the plant fungal pathogens, such as Botrytis cinerea, Erysiphe graminis, Magnaporthe grisea, Puccinia recodita, Septoria triticii, Tilletia controversa, Ustilago maydis, or any species falling within the genera of any of the above species.
  • panels of GRACE strains may be used to characterize the point of intervention of any compound affecting an essential biological pathway including antibiotics with no known mechanism of action.
  • Another embodiment of the present invention is a method for determining the pathway against which a test antibiotic compound is active, in which the activity of proteins or nucleic acids involved in pathways required for fungal growth, survival, proliferation, virulence or pathogenicity is reduced by contacting cells with a sub-lethal concentration of a known antibiotic which acts against the protein or nucleic acid.
  • the method is similar to those described above for determining which pathway a test antibiotic acts against, except that rather than reducing the activity or level of a gene product required for fungal proliferation, virulence or pathogenicity by expressing the gene product at a rate limiting amount in a GRACE strain, the activity or level of the gene product is reduced using a sub-lethal level of a known antibiotic which acts against the gene product.
  • Growth inhibition resulting from the presence of sub-lethal concentration of the known antibiotic may be at least about 5%, at least about 8%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%), or at least about 75%, at least 80%, at least 90%, at least 95% or more than 95%.
  • the sub-lethal concentration of the known antibiotic may be determined by measuring the activity of the target proliferation-required gene product rather than by measuring growth inhibition.
  • Cells are contacted with a combination of each member of a panel of known antibiotics at a sub-lethal level and varying concentrations of the test antibiotic. As a control, the cells are contacted with varying concentrations of the test antibiotic alone.
  • the IC 50 of the test antibiotic in the presence and absence of the known antibiotic is determined. If the IC 50 s in the presence and absence of the known drug are substantially similar, then the test drug and the known drug act on different pathways. If the IC 50 s are substantially different, then the test drug and the known drug act on the same pathway. Similar methods may be performed using known antibiotics which act on a gene product homologous to the Candida albicans sequences described herein.
  • the homolgous gene product may be from animal fugal pathogens such as Aspergillus fumigatus, Aspergillus niger, Aspergillus flavis, Candida tropicalis, Candida parapsilopsis, Candida krusei, Cryptococcus neoformans, Coccidioides immitis, Exophalia dermatiditis, Fusarium oxysporum, Histoplasma capsulatum, Pneumocystis carinii, Trichosporon beigelii, Rhizopus arrhizus, Mucor rouxii, Rhizomucor pusillus, or Absidia corymbigera, or the plant fungal pathogens, such as Botrytis cinerea, Erysiphe graminis, Magnaporthe grisea, Puccinia recodita, Septoria triticii, Tilletia controversa, Ustilago maydis, or any species falling within the genera of any of the above species
  • Another embodiment of the present invention is a method for identifying a candidate compound for use as an antibiotic in which the activity of target proteins or nucleic acids involved in pathways required for fungal proliferation, virulence or pathogenicity is reduced by contacting cells with a sub-lethal concentration of a known antibiotic which acts against the target protein or nucleic acid.
  • the method is similar to those described above for identifying candidate compounds for use as antibiotics except that rather than reducing the activity or level of a gene product required for proliferation, virulence or pathogenicity using GRACE strains which express a rate limiting level of the gene product, the activity or level of the gene product is reduced using a sub-lethal level of a known antibiotic which acts against the proliferation required gene product.
  • the growth inhibition from the sub-lethal concentration of the known antibiotic may be at least about 5%, at least about 8%, at least about 10%, at least about
  • the sub-lethal concentration of the known antibiotic may be determined by measuring the activity of the target proliferation-required gene product rather than by measuring growth inhibition.
  • test compounds of interest In order to characterize test compounds of interest, cells are contacted with a panel of known antibiotics at a sub-lethal level and one or more concentrations of the test compound. As a control, the cells are contacted with the same concentrations of the test compound alone. The IC 50 of the test compound in the presence and absence of the known antibiotic is determined. If the IC 50 of the test compound is substantially different in the presence and absence of the known drug then the test compound is a good candidate for use as an antibiotic. As discussed above, once a candidate compound is identified using the above methods its structure may be optimized using standard techniques such as combinatorial chemistry.
  • the homolgous gene product may be from animal fugal pathogens such as Aspergillus fumigatus, Aspergillus niger, Aspergillus flavis, Candida tropicalis, Candida parapsilopsis, Candida krusei, Cryptococcus neoformans, Coccidioides immitis, Exophalia dermatiditis, Fusarium oxysporum, Histoplasma capsulatum, Pneumocystis carinii, Trichosporon beigelii, Rhizopus arrhizus, Mucor rouxii, Rhizomucor pusillus, or Absidia corymbigera, or the plant fungal pathogens, such as Botrytis cinerea, Erysiphe graminis, Magnaporthe grisea, Puccinia recodita, Septoria triticii
  • An exemplary target gene product is encoded by CaTBFl.
  • CaTBFl A number of features make this C. albicans gene product a valuable drug target.
  • the protein encoded by CaTBFl is compatible with in vitro high throughput screening of compounds that inhibit its activity. Modulated expression of this gene product in whole cell assays could be performed in parallel with in vitro assays to broaden the spectrum of possible inhibitory compounds identified.
  • demonstration of the predicted physical interaction between CaTbflp and chromosomal telomerases could be used to develop two- hybrid assays for drug screening purposes.
  • CaTBFl is a fungal specific gene, its nucleotide sequence could serve in designing PCR-based diagnostic tools for fungal infection.
  • C. albicans genes CaRHOl, CaERG8, CaAURl, and CaCHOl, as well as those encoded by SEQ ID NOs.:l-62.
  • GRACE methods of the present invention will improve drug screening practices now in use that are designed to identify inhibitors of these critical gene products.
  • all potential drug targets of a pathogen could be screened simultaneously against a library of compounds using, for example a 96 well microtiter plate format, where growth, measured by optical density or pellet size after centrifugation, may be determined for each well.
  • a genomic approach to drug screening eliminates reliance upon potentially arbitrary and artificial criteria used in evaluating which target to screen and instead allows all potential targets to be screened. This approach not only offers the possibility of identifying specific compounds which inhibit a preferred process (e. g. cell wall biosynthetic gene products) but also the possibility of identifying all fungicidal compounds within that library and linking them to their cognate cellular targets.
  • GRACE strains could be screened to identify synthetic lethal mutations, and thereby uncover a potentially novel class of drug targets of significant therapeutic value.
  • two separate genes may encode homologous proteins that participate in a common and essential cellular function, where the essential nature of this function will only become apparent upon inactivation of both family members. Accordingly, examination of the null phenotype of each gene separately would not reveal the essential nature of the combined gene products, and consequently, this potential drug target would not be identified.
  • the gene products are highly homologous to one another, compounds found to inhibit one family member are likely to inhibit the other and are therefore predicted to approximate the synthetic growth inhibition demonstrated genetically.
  • a directed approach to uncovering synthetic lethal interactions with essential and nonessential drug targets is now performed where a GRACE strain or heterozygote strain is identified as displaying an enhanced sensitivity to the tested compound, not because it expresses a reduced level of activity for the drug target, but because its mutation is synthetically lethal in combination with inhibition of a second drug target.
  • Discerning whether the compound specifically inhibits the drug target in the sensitized GRACE strain or heterozygote strain or a second target may be achieved by screening the entire GRACE or heterozygote strain sets for additional mutant strains displaying equal or greater sensitivity to the compound, followed by genetic characterization of a double mutant strain demonstrating synthetic lethality between the two mutations.
  • biochemical similarity existing between pathogenic fungi and the mammalian hosts they infect limits the range of clinically useful antimycotic compounds.
  • this similarity can be exploited using a GRACE strain collection to facilitate the discovery of therapeutics that are not used as antimycotics, but are useful for treatment a wide-range of diseases, such as cancer, inflammation, etc.
  • fungal genes that are homologous to disease-causing genes in an animal or plant are selected and GRACE strains of this set of genes are used for identification of compounds that display potent and specific bioactivity towards the products of these genes, and therefore have potential medicinal value for the treatment of diseases.
  • Essential and non-essential genes and the corresponding GRACE strains carrying modified allelic pairs of such genes are useful in this embodiment of the invention. It has been predicted that as many as 40% of the genes found within the C. albicans genome share human functional homologs. It has also been predicted that as many as 1% of human genes are involved in human diseases and therefore may serve as potential drug targets.
  • the invention provides a pluralities of GRACE strains in which the modified alleles are fungal genes that share sequence, structural and/or functional similarities to genes that are associated with one or more diseases of the animal or plant.
  • the taxol family of anti-cancer compounds which hold promise as therapeutics for breast and ovarian cancers, bind tubulin and promote microtubule assembly, thereby disrupting normal microtubule dynamics.
  • Yeast tubulin displays similar sensitivity to taxol, suggesting that additional compounds affecting other fundamental cellular processes shared between yeast and man could similarly be identified and assessed for antitumor activity.
  • pathogenesis extends far beyond the taxonomic borders of microbes and ultimately reflects the underlying physiology.
  • the phenomenon of cancer is analogous to the process of pathogenesis by an opportunistic pathogen such as C. albicans.
  • C. albicans Both are non-infectious diseases caused by either the body's own cells, or microbes from its natural fauna. These cells grow in a manner unchecked by the immune system and in both cases disease manifests itself by colonization of vital organs and eventual tissue damage resulting in death.
  • Effective drug-based treatment is also elusive for both diseases primarily because the causative agent in both cases is highly related to the host.
  • Cyclosporin A and FK506 form distinct drug-prolyl isomerase complexes (CyPA- Cyclosporin A and FKBP12-FK506 respectively) which bind and inactivate the regulatory subunit of the calcium and calmodulin-dependent phosphatase, calcineurin.
  • Rapamycin also complexes with FKBP12, but this drug-protein complex also binds to the TOR family of phosphatidylinositol kinases to inhibit translation and cell cycle progression. In each case, both the mechanism of drug action, and the drug targets themselves are highly conserved from yeast to humans.
  • C. albicans drug targets and grouping the targets into essential-gene, fungal-specific, and pathogen-specific target sets provide the basis for the development of whole-cell screens for compounds that interact with and inhibit individual members of any of these targets. Therefore, similar analyses can be used to identify other sets of GRACE strains having modified allelic pairs of genes encoding drug targets with other specific common functions or attributes.
  • GRACE strain subsets can be established which comprise gene targets that are highly homologous to human genes, or gene targets that display a common biochemical function, enzymatic activity, or that are involved in carbon compound catabolism, bosynthesis, transport of molecules (transporter activity), )cellular localization, signal transduction cascades, cell cycle control, cell adhesion, transcription, translation, DNA replication, etc. 5.5.2.3 Target Gene Dosage-Based Whole Cell Assays
  • the principle of drug-target-level variation in drug screening involves modulating the expression level of a drug target to identify specific drug resistance or drug sensitivity phenotypes, thereby linking a drug target to a particular compound. Often, these phenotypes are indicative of the target gene encoding the bona fide drug target of this compound. In examples where this is not the case, the candidate target gene may
  • Variation of the expression levels of the target protein is also incorporated within both drug screening and drug target identification procedures.
  • the total, cellular expression level of a gene product in a diploid organism is modified by disrupting one allele of the gene encoding that product, thereby reducing its functional activity in half, creating a "haploinsufficient" phenotype.
  • a heterozygous S. cerevisiae strain collection has been used in such a haploinsufficiency screen to link drug-based resistance and hypersensitive
  • Nonessential genes are screened directly using a haploid deletion strain collection against a compound library for specific phenotypes or
  • chemotypes However, this procedure cannot be used in a haploid organism where the target gene is an essential one.
  • the expression level of a given gene product is also elevated by cloning the 25 gene into a plasmid vector that is maintained at multiple copies in the cell. Overexpression of the encoding gene is also achieved by fusing the corresponding open reading frame of the gene product to a more powerful promoter carried on a multicopy plasmid. Using these strategies, a number of overexpression screens have been successfully employed in S. cerevisiae to discover novel compounds that interact with characterized drug targets as well as to identify the protein targets bound by existing therapeutic compounds.
  • the GRACE strain collection replaces the surrogate use of S. cerevisiae in whole cell drug screening by providing a dramatic range in gene expression levels for drug targets directly within the pathogen (Fig. 5). In one embodiment of the invention, this is
  • the GRACE strain collections of the invention are not only useful in target validation under repressing conditions, but are also useful as a collection of strains overexpressing these same validated drug targets under nonrepressing conditions for whole cell assay development and drug screening.
  • Variation in the level of expression of a target gene product in a GRACE strain is also used to explore resistance to antimycotic compounds.
  • Resistance to existing antifungal therapeutic agents reflects both the limited number of antifungal drugs available and the alarming dependence and reliance clinicians have in prescribing them. For example, dependence on azole-based compounds such as fluconazole for the treatment of fungal infections, has dramatically undermined the clinical therapeutic value for this compound.
  • the GRACE strain collection is used to combat fluconazole resistance by identifying gene products that interact with the cellular target of fluconazole. Such products are used to identify drug targets which, when inactivated in concert with fluconazole, provide a synergistic effect and thereby overcome resistance to fluconazole seen when this compound is used alone. This is accomplished, for example, by using the GRACE strain collection to overexpress genes that enhance drug resistance.
  • genes include novel or known plasma membrane exporters including ATP-binding cassette (ABC) transporters and multidrug resistance (MDR) efflux pumps, pleiotropic drug resistance (PDR) transcription factors, and protein kinases and phosphatases.
  • genes specifically displaying a differential drug sensitivity are identified by screening GRACE strains expressing reduced levels
  • overexpression of the target gene for whole cell assay purposes is supported with promoters other than the tetracycline promoter system, (see Section 5.3.1)
  • the CaPGKl promoter is used to overexpress C. albicans drug targets genes.
  • the PGK1 promoter is known to provide strong constitutive expression in the presence of glucose. See, Guthrie, C, and G. R. Fink. 1991. Guide to yeast genetics and molecular biology. Methods Enzymol. 194:373- 398. A preliminary analysis of five C.
  • albicans genes placed under the control of the CaPGKl promoter revealed dramatic overexpression versus wild type as judged by Northern blot analysis.
  • CaAURl which was not overexpressed significantly when constitutively expressed using the tetracycline promoter, was overexpressed 5-fold relative to wild type CaAURl expression levels, suggesting that the CaPGKl promoter is useful in overexpressing genes normally not overexpressed by the tetracycline promoter.
  • intermediate expression levels of individual drug targets within the GRACE strain collection may be engineered to provide strains tailored for the development of unique whole cell assays.
  • GRACE strains are grown in a medium containing a tetracycline concentration determined to provide only a partial repression of transcription. Under these conditions, it is possible to maintain an expression level between that of the constitutively expressed overproducing strain and that of wild type strain, as well as levels of expression lower than that of the wild-tvpe strain. That is, it is possible to titrate the level of expression to the minimum required for cell viability.
  • novel phenotypes By repressing gene expression to this critical state, novel phenotypes, resembling those produced by a partial loss of function mutation (i.e. phenocopies of hypomorphic mutants) may be produced and offer additional target expression levels applicable for whole cell assay development and drug screening.
  • Repressing expression of the remaining allele of an essential gene to the threshold level required for viability therefore will provide a strain with enhanced sensitivity toward compounds active against this essential gene product.
  • unique oligonucleotide sequence tags or "bar codes” are incorporated into individual mutant strains included within a heterozygous strain collection of validated targets.
  • the presence of these sequence tags enables an alternative whole cell assay approach to drug screening.
  • Multiple target strains may be screened simultaneously in a mixed population (rather than separately) to identify phenotypes between a particular drug target and its inhibitory agent.
  • the mutant strains are GRACE strains
  • each of the GRACE strains of the set comprises a unique molecular tag, which, generally, is incorporated within the cassette used to replace the first allele of the gene pair to be modified.
  • Each molecular tag is flanked by primer sequences which are common to all members of the set being tested. Growth is carried out in repressive and non-repressive media, in the presence and absence of the compound to be tested. The relative growth of each strain is assessed by carrying out simultaneous PCR amplification of the entire collection of embedded sequence tags.
  • the PCR amplification is performed in an asymmetric manner with fluorescent primers and the resulting single stranded nucleic acid product hybridized to an oligonucleotide array fixed to a surface and comprises the entire corresponding set of complementary sequences. Analysis of the level of each fluorescent molecular tag sequence is then determined to estimate the relative amount of growth of GRACE strain of the set, in those media, in the presence and absence of the compound tested.
  • each potential drug target gene in this heterozygous tagged or bar-coded collection may be overexpressed by subsequently introducing either the Tet promoter or another strong, constitutively expressed promoter (e. g. CaACTl, CaADHl and CaPGKl) upstream of the remaining non-disrupted allele.
  • the Tet promoter or another strong, constitutively expressed promoter e. g. CaACTl, CaADHl and CaPGKl
  • screens for antifungal compounds can be carried out using complex mixtures of compounds that comprise at least one compound active against the target strain.
  • Tagging or bar-coding the GRACE strain collection facilitates a number of large scale analyses necessary to identify gene sets as well as evaluate and ultimately evaluate individual targets within particular gene sets.
  • mixed-population drug screening using a bar-coded GRACE strain collection effectively functions as a comprehensive whole cell assay.
  • Minimal amounts of a complex compound library are sufficient to identify compounds that act on individual essential target genes within the collection. This is done without the need to array the collection. Also, strong predictions as to the 'richness' of any particular compound library could be made before committing to it in drug screening.
  • 15 drug target genes deemed nonessential under standard laboratory conditions may be examined within an animal model, for example, by testing the pathogenicity of a strain homozygous for a deletion in the target gene versus wild type.
  • essential drug targets are precluded from animal model studies. Therefore, the most desirable drug targets are omitted from the most pertinent conditions to their target evaluation.
  • conditional expression provided by the
  • GRACE essential strain collection overcomes this longstanding limitation to target validation within a host environment.
  • Animal studies can be performed using mice inoculated with GRACE essential strains and examining the effect of gene inactivation by conditional expression. In a preferred embodiment of the invention, the effect on mice
  • mice 25 injected with a lethal inoculum of a GRACE essential strain could be determined depending on whether the mice were provided with an appropriate concentration of tetracycline to inactivate expression of a drug target gene.
  • the lack of expression of a gene demonstrated to be essential under laboratory conditions can thus be correlated with prevention of a terminal C. albicans infection. In this type of experiment, only mice "treated" with
  • conditional expression could be achieved using a temperature-responsive promoter to regulate expression of the target gene or a temperature sensitive allele of a particular drug target, such that the gene is functional at
  • GRACE pathogenicity subset a subset of genes that are required for pathogenicity, i.e., GRACE pathogenicity subset. More defined subsets of pathogenicity genes, for example those genes required for particular steps in pathogenesis (e.g. adherence or invasion) may be determined by applying the GRACE pathogenicity subset of strains to in vitro assays which measure the corresponding process. For example, examining GRACE pathogenicity strains in a buccal adhesion or macrophage assay by conditional expression of individual genes would identify those pathogenicity factors required for adherence or cell invasion respectively.
  • the GRACE strain collection or a desired subset thereof is also well suited for evaluating acquired resistance/suppression or distinguishing between fungicidal/fungistatic phenotypes for an inactivated drug target within an animal model system.
  • GRACE strains repressed for expression of different essential drug target genes would be inoculated into mice raised on tetracycline-supplemented water. Each of the GRACE strains would then be compared according to the frequency of death associated with the different mice populations they infected. It is expected that the majority of infected mice will remain healthy due to fungal cell death caused by tetracycline-dependent inactivation of the essential gene in the GRACE strain.
  • a GRACE strain harboring a drug target more likely to develop extragenic suppressors because it is a fungistatic target rather than fungicidal one, or suppressed by an alternative physiological process active within a host environment can be identified by the higher incidence of lethal infections detected in mice infected with this particular strain. By this method, it is possible to evaluate/rank the likelihood that individual drug target genes may develop resistance within the host environment.
  • Binding compounds can also include, but are not limited to, peptides such as, for example, soluble peptides, comprising, for example, extracellular portions of target gene product transmembrane receptors, and members of random peptide libraries (see, e.g., Lam et al., 1991, Nature 354:82-84; Houghten et al., 1991, Nature 354:84-86) made of D-and/or L-configuration amino acids, rationally-designed antipeptide peptides, (see e.g., Hurby et al., Application of Synthetic Peptides: Antisense Peptides," In Synthetic Peptides, A User 's Guide, W.H.
  • antibodies including, but not limited to polyclonal, monoclonal, human, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab') 2 and FAb expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.
  • such compounds can include organic molecules (e.g., peptidomimetics) that bind to the ECD and either mimic the activity triggered by the natural ligand (i.e., agonists): as well as peptides, antibodies or fragments thereof, and other organic compounds that mimic the ECD (or a portion thereof) and bind to a "neutralize" natural ligand.
  • organic molecules e.g., peptidomimetics
  • the active sites or regions are preferably identified.
  • such active sites might typically be ligand binding sites, such as the interaction domains of ligand with receptor itself.
  • the active site is identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods are used to find the active site by finding where on the factor the complexed ligand is found.
  • the three-dimensional geometric structure of the active site is then preferably determined. This is done by known methods, including X-ray crystallography, which determines a complete molecular structure. Solid or liquid phase NMR is also used to determine certain intra-molecular distances within the active site and/or in the ligand binding complex. Other experimental methods of structure determination known to those of skill in the art, are also used to obtain partial or complete geometric structures.
  • the geometric structures are measured with a complexed ligand, natural or artificial, which increases the accuracy of the active site structure determined. Methods of computer based numerical modeling are used to complete the structure (e.g., in embodiments wherein an incomplete or insufficiently accurate structure is determined) or to improve its accuracy.
  • candidate modulating compounds are identified by searching databases containing compounds along with information on their molecular structure.
  • Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site.
  • Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential target or pathway gene product modulating compounds.
  • these methods ire used to identify improved modulating compounds from an already known modulating compound or ligand.
  • the composition of the known compound is modified and the structural effects of modification are determined using the experimental and computer modeling methods described above applied to the new composition.
  • the altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, are quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.
  • Gene expression profiling techniques are important tools for the identification of suitable biochemical targets, as well as for the determination of the mode of action of known compounds. Completion of the C. albicans genome sequence and development of nucleic acid microarrays incorporating this information, will enable genome-wide gene expression analyses to be carried out with this diploid pathogenic fungus. Therefore, the present invention provides methods for obtaining the transcriptional response profiles for both essential and virulence/pathogenicity genes of Candida albicans. Conditional expression of essential genes serves to delineate, for example, regulatory interactions valuable for the design of drug screening programs focused upon C. albicans. In an embodiment of the present invention, the GRACE strain collection is used for the analysis of expression of essential genes within this pathogen.
  • One particularly powerful application of such a strain collection involves the construction of a comprehensive transcriptional profile database for the entire essential gene set or a desired subset of essential genes within a pathogen.
  • a comprehensive transcriptional profile database for the entire essential gene set or a desired subset of essential genes within a pathogen.
  • Such a database is used to compare the response profile characteristic of lead antimycotic compounds with the profile obtained with new anti-fungal compounds to distinguish those with similar from those with distinct modes of action.
  • Matching (or even partially overlapping) the transcriptional response profiles determined after treatment of the strain with the lead compound with that obtained with a particular essential target gene under repressing conditions is used to identity the target and possible mode of action of the drug.
  • Gene expression analysis of essential genes also permits the biological function and regulation of those genes to be examined within the pathogen, and this information is incorporated within a drug screening program.
  • transcriptional profiling of essential drug targets in C. albicans permits the identification of novel drug targets which participate in the same cellular process or pathway uncovered for the existing drug target and which could not otherwise be identified without direct experimentation within the pathogen.
  • pathogen-specific pathways may be uncovered and exploited for the first time.
  • the gene expression profile of GRACE-derived strains under nonrepressing or induced conditions is established to evaluate the overexpression response profile for one or more drug targets.
  • overexpression of genes functioning in signal transduction pathways often display unregulated activation of the pathway under such conditions.
  • several signaling pathways have been demonstrated to function in the pathogenesis process.
  • Transcriptional response profiles generated by overexpressing C albicans GRACE strains provide information concerning the set of genes regulated by such pathways; any of which may potentially serve an essential role in pathogenesis and therefore representing promising drug targets.
  • analysis of the expression profile may reveal one or more genes whose expression is critical to the subsequent expression of an entire regulatory cascade.
  • genes are particularly important targets for drug discovery and mutants carrying the corresponding modified allelic pair of genes form the basis of a mechanism-of-action based screening assays.
  • Presently such an approach is not possible.
  • Current drug discovery practices result in an exceedingly large number of "candidate” compounds and little understanding of their mode of action.
  • a transcriptional response database comprising both gene shut-off and overexpression profiles generated using the
  • the invention provides a method for evaluating a compound against a target gene product encoded by a nucleotide sequence comprising one of SEQ ID NO: 1
  • said method comprising the steps of (a) contacting wild type diploid fungal cells or control cells with the compound and generating a first transcription profile; (b) determining the transcription profile of mutant diploid fungal cells, such as a GRACE strain, which have been cultured under conditions wherein the second allele of the target gene is substantially underexpressed, not expressed or overexpressed and generating a second transcription profile for the cultured cells; and comparing the first transcription profile with the second transcription profile to identify similarities in the profiles. For comparisons, similarities of profiles can be expressed as an indicator value; and the higher the indicator value, the more desirable is the compound.
  • Secondary target refers to a gene whose gene product exhibits the ability to interact with target gene products involved in the growth and/or survival of an organism (i.e., target essential gene products), under a set of defined conditions, or in the pathogenic mechanism of the organism, (i.e., target virulence gene products) during infection of a host.
  • target essential gene products i.e., target essential gene products
  • target virulence gene products i.e., target virulence gene products
  • Any method suitable for detecting protein-protein interactions can be employed for identifying secondary target gene products by identifying interactions between gene products and target gene products.
  • Such known gene products can be cellular or extracellular proteins. Those gene products which interact with such known gene products represent secondary target gene products and the genes which encode them represent secondary targets.
  • a secondary target gene product is used, in conjunction with standard techniques, to identify its corresponding secondary target. For example, at least a portion of the amino acid sequence of the secondary target gene product is ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton, 1983, "Proteins: Structures and Molecular Principles," W.H. Freeman & Co., N.Y., pp.34-49).
  • the amino acid sequence obtained can be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for secondary target gene sequences. Screening can be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and for screening are well-known. (See, e.g., Ausubel, supra., and PCR Protocols: A Guide to Methods and Applications, 1990, Innis, M. et al., eds. Academic Press, Inc., New York).
  • methods are employed which result in the simultaneous identification of secondary targets which encode proteins interacting with a protein involved in the growth and/or survival of an organism under a set of defined conditions, or in the pathogenic mechanism of the organism during infection of a host.
  • These methods include, for example, probing expression libraries with labeled primary target gene protein known or suggested to be involved in or critical to these mechanisms, using this protein in a manner similar to the well known technique of antibody probing of ⁇ gtl 1 phage libraries.
  • plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to a known protein, in this case, a protein known to be involved in growth of the organism, or in pathogenicity, and the other consists of the activator protein's activation domain fused to an unknown protein that is encoded by a cDNA which has been recombined into this plasmid as part of a cDNA library.
  • the plasmids are transformed into a strain of the yeast S. cerevisiae that contains a reporter gene (e.g., lacZ) whose regulatory region contains the transcription activator's binding sites.
  • a reporter gene e.g., lacZ
  • the two-hybrid system or related methodology is used to screen activation domain libraries for proteins that interact with a known "bait" gene product.
  • target essential gene products and target virulence gene products are used as the bait gene products.
  • Total genomic or cDNA sequences encoding the target essential gene product, target virulence gene product, or portions thereof, are fused to the DNA encoding an activation domain.
  • This library and a plasmid encoding a hybrid of the bait gene product fused to the DNA-binding domain are cotransformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene.
  • the bait gene is cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein.
  • These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing-is then used to identify the proteins encoded by the library plasmids.
  • a cDNA library of the cell line from which proteins that interact with bait gene product are to be detected is made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments are inserted into a vector such that they are translationally fused to the activation domain of GAL4.
  • This library is co-transformed along with the bait gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence.
  • a cDNA encoded protein, fused to GAL4 activation domain, that interacts with bait gene product reconstitutes an active GAL4 protein and thereby drive expression of the lacZ gene. Colonies which express lacZ are detected by their blue color in the presence of X-gal.
  • the cDNA can then be purified from these strains, and used to produce and isolate the bait gene-interacting protein using techniques routinely practiced in the art.
  • Gene expression arrays are high density arrays of DNA samples deposited at specific locations on a glass surface, silicon, nylon membrane, or the like. Such arrays are used by researchers to quantify relative gene expression under different conditions. An example of this technology is found in U.S. Patent No. 5807522, which is hereby incorporated by reference.
  • the arrays may consist of 12 x 24 cm nylon filters containing PCR products corresponding to ORFs from Candida albicans. 10 ngs of each PCR product are spotted every 1.5 mm on the filter.
  • Single stranded labeled cDNAs are prepared for hybridization to the array (no second strand synthesis or amplification step is done) and placed in contact with the filter. Thus the labeled cDNAs are of "antisense" orientation. Quantitative analysis is done using a phosphorimager.
  • Hybridization of cDNA made from a sample of total cell mRNA to such em array followed by detection of binding by one or more of various techniques known to those in the art provides a signal at each location on the array to which cDNA hybridized.
  • the intensity of the hybridization signal obtained at each location in the array thus reflects the amount of mRNA for that specific gene that was present in the sample. Comparing the results obtained for mRNA isolated from cells grown under different conditions thus allows for a comparison of the relative amount of expression of each individual gene during growth under the different conditions.
  • Gene expression arrays are used to analyze the total mRNA expression pattern at various time points after reduction in the level or activity of a gene product required for fungal proliferation, virulence or pathogenicity. Reduction of the level or activity of the gene product is accomplished by growing a GRACE strain under conditions in which the product of the nucleic acid linked to the regulatable promoter is rate limiting for fungal growth, survival, proliferation, virulence or pathogenicity or by contacting the cells with an agent which reduces the level or activity of the target gene product. Analysis of the expression pattern indicated by hybridization to the array provides information on other genes whose expression is influenced by reduction in the level or activity of the gene product.
  • levels of other mRNAs may be observed to increase, decrease or stay the same following reduction in the level or activity of the gene product required for growth, survival, proliferation, virulence or pathogenicity.
  • the mRNA expression pattern observed following reduction in the level or activity of a gene product required for growth, survival, proliferation, virulence or pathogenicity identifies other nucleic acids required for growth, survival, proliferation, virulence or pathogenicity.
  • the mRNA expression patterns observed when the fungi are exposed to candidate drug compounds or known antibiotics are compared to those observed when the level or activity of a gene product required for fungal growth, survival, proliferation, virulence or pathogenicity is reduced. If the mRNA expression pattern observed with the candidate drug compound is similar to that observed when the level of the gene product is reduced, the drug compound is a promising therapeutic candidate.
  • the assay is useful in assisting in the selection of promising candidate drug compounds for use in drug development.
  • a GRACE strain collection provides an invaluable resource for the analysis of the expressed protein complement of a genome.
  • a correlation between the pattern of protein expression of a cell can be made with the non- expression or the level of expression of an essential gene.
  • the invention provides a pattern of expression of a set of proteins in a GRACE strain as determined by methods well known in the art for establishing a protein expression pattern, such as two- dimensional gel electrophoresis. A pluralities of protein expression patterns will be generated for a GRACE strain when the strain is cultured under different conditions and different levels of expression of one of the modified allele.
  • defined genetic mutations can be constructed to create strains exhibiting protein expression profiles comparable to those observed upon treatment of the strain with a previously uncharacterized compound. In this way, it is possible to distinguish between antimycotic compounds that act on multiple targets in a complicated manner from other potential lead compounds that act on unique fungal-specific targets and whose mode of action can be determined.
  • the present invention provides a method of quantitative analysis of the expressed protein complement of a diploid pathogenic fungal cell: a first protein expression profile is developed for a control diploid pathogenic fungus, which has two, unmodified alleles for the target gene. Mutants of the control strain, in which one allele of the target gene is inactivated, for example, in a GRACE strain, by insertion by or replacement with a disruption cassette, is generated. The other allele is modified such that expression of that second allele is under the control of a heterologous regulated promoter.
  • a second protein expression profile is developed for this mutant fungus, under conditions where the second allele is substantially overexpressed as compared to the expression of the two alleles of the gene in the control strain.
  • a third protein expression profile is developed, under conditions where the second allele is substantially underexpressed as compared to the expression of the two alleles of the gene in the control strain.
  • the first protein expression profile is then compared with the second expression profile, and if applicable, a third protein expression profile to identify an expressed protein detected at a higher level in the second profile, and if applicable, at a lower level in the third profile, as compared to the level in first profile.
  • the invention provides a method for evaluating a compound against a target gene product encoded by a nucleotide sequence comprising one of SEQ ID NO: 1 to 61, said method comprising the steps of (a) contacting wild type diploid fungal cells or control cells with the compound and generating a first protein expression profile; (b) determining the protein expression profile of mutant diploid fungal cells, such as a GRACE strain, which have been cultured under conditions wherein the second allele of the target gene is substantially underexpressed, not expressed or overexpressed and generating a second protein expression profile for the cultured cells; and comparing the first protein expression profile with the second protein expression profile to identify similarities in the profiles. For comparisons, similarities of profiles can be expressed as an indicator value; and the higher the indicator value, the more desirable is the compound.
  • Compounds including nucleic acid molecules that are identified by the methods of the invention as described herein can be administered to a subject at therapeutically effective doses to treat or prevent infections by a pathogenic organism, such as Candida albicans.
  • the compounds may also be useful for treatment of a non-infectious disease in a subject, such as but not limited to, cancer.
  • a therapeutically effective dose refers to that amount of a compound (including nucleic acid molecules) sufficient to result in a healthful benefit in the treated subject.
  • the compounds act by reducing the activity or level of a gene product encoded by a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 1 to 62.
  • the subject to be treated can be a plant, a vertebrate, a mammal, an avian, or a human. These compounds can also be used for preventing or containing contamination of an object by Candida albicans, or used for preventing or inhibiting formation on a surface of a biofilm comprising Candida albicans. Biofilm comprising C. albicans are found on surfaces of medical devices, such as but not limited to surgical tools, implanted devices, catheters and stents. 5.8.1 Effective Dose
  • Toxicity and therapeutic efficacy of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50%) of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma can be measured, for example, by high performance liquid chromatography.
  • a useful dosage can range from 0.001 mg/kg body weight to 10 mg/kg body weight.
  • compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvents can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • the preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration can be suitably formulated to give controlled release of the active compound.
  • compositions can take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
  • the compounds can be formulated for parenteral administration (i.e., intravenous or intramuscular) by injection, via, for example, bolus injection or continuous infusion.
  • parenteral administration i.e., intravenous or intramuscular
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds can also be formulated as a depot preparation.
  • Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange 5 resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Oligonucleotide primers for PCR amplification of the SAT selectable marker used in Step 1 contain 25 nucleotides complementary to the SAT disruption cassette in pRC18-ASP, and 65 nucleotides homologous to regions flanking the CaKRE9 open reading frame.
  • Figure 2 illustrates the 2.2 kb cakre9 ⁇ ::SAT disruption fragment produced after PCR amplification and resulting gene replacement of the first wild
  • PCR conditions were as follows: 5-50 ng pRC18-ASP, 100 pmol of each primer, 200 ⁇ M dNTPs, 10 mM Tris- pH 8.3, 1.5 mM MgC12, 50 mM KC1, 1 unit Taq DNA polymerase (Gibco).
  • PCR amplification times were: 5 min 94°C, 1 min 54°C, 2 min 72°C, for 1 cycle; 45 sec 94 °C, 45 sec 54°C, 2 min 72 °C, for 30 cycles. Transformation was performed using the
  • Oligonucleotide primers for PCR amplification of the conditional promoter used in Step 2 contain 25 nucleotides complementary to the C ⁇ H/SJ-marked tetracycline regulated promoter cassette in pBSK-HT4 and 65 nucleotides of homologous sequence corresponding to promoter regions -270 to -205, relative to the 5 point of transcription initiation, and nucleotides 1-65 of the CaKRE9 open reading frame.
  • the resulting 2.2 kb PCR product was transformed into the cakre9 ⁇ ::SAT/CaKRE9 heterozygous strain produced in step 1 , and His + transformants selected on YNB agar.
  • Bonafide CaKRE9 GRACE strains containing both a cakre9 ⁇ ::SAT allele and CaHIS3- ⁇ et- CaKRE9 allele were determined by PCR analysis.
  • 2 independent GRACE strains are constructed and evaluated to provide a reliable determination of the terminal phenotype of any given drug target.
  • Terminal phenotype is that phenotype caused by the absence of the gene product of an essential gene
  • the terminal phenotype of the resulting GRACE strains was evaluated in three independent methods. In the first, rapid determination of the CaKRE9 GRACE strain terminal phenotype was achieved by streaking approximately 1.0 X IO 6 cells onto both a YNB plate and YNB plate containing 1 OO ⁇ g/ml tetracycline and comparing growth rate after 48 hr at room temperature. For essential genes, such as CaKRE9, no significant growth
  • the essential nature of a gene may be determined by streaking CaKRE9 GRACE cells onto a casamino acid plate containing 625 ⁇ g/ml 5-fluroorotic acid (5FOA) and 100 ⁇ g/ml uridine to select for ura " cells which have excised (via recombination between CaLEU2 sequence duplications
  • Overexpression achieved by the constitutive expression level of CaHIS3 maintained by the tetracycline promoter, confers 3-AT resistance at concentrations sufficient to completely inhibit growth of both wild type and CaHIS3 heterozygote strains (Fig 6A).
  • the phenotype observed is consistent with that expected in light of the predicted 7.5 fold overexpression of CaHIS3 determined by Northern bolt analysis (see Fig 5).
  • a heterozygous CaHIS3 strain demonstrates enhanced sensitivity (i.e. haploinsufficient phenotype) to an intermediate 3-AT concentration unable to effect either wild type or tetracycline promoter-based overproducing CaHIS3 strains noticeably (Fig 6B).
  • a third CaHIS3 expression level evaluated for differential sensitivity to 3-AT - was produced by partial repression of the GRACE C ⁇ HlS3 strain using a threshold concentration of tetracycline 0.1% that normally is used to achieve complete shut-off.
  • This level of C ⁇ HIS3 expression represents the minimum expression level required for viability and as predicted, demonstrates an enhanced drug sensitivity relative the heterozygous C ⁇ HIS3 strain at an intermediate 3-AT concentration (Fig 6C).
  • GRACE strain-specific drug resistance and sensitivity phenotypes to fluconazole and tunicamycin have been demonstrated by increasing and decreasing the level of expression of their respective known drug targets, CaERGll and CaALG7.
  • these results demonstrate that three different levels of expression are achieved using the C. albicans GRACE strain collection, and that they exhibit the predicted drug sensitivity phenotypes between known drugs and their known drug target.
  • these experiments clearly indicate how distinct levels of target gene products synthesized within the pathogen could be directly applied in whole cell assay based drug screens to identify novel antifungal compounds against those novel drug targets validated using the GRACE method.
  • a target pathway is a genetic or biochemical pathway wherein one or more of the components of the pathway (e.g., enzymes, signaling molecules, etc) is a drug target as determined by the methods of the invention.
  • the components of the pathway e.g., enzymes, signaling molecules, etc.
  • frozen stocks of host GRACE strains are prepared using standard microbiological techniques. For example, a single clone of the microorganism can be isolated by streaking out a sample of the original stock onto an agar plate containing nutrients for cell growth and an antibiotic for which the GRACE strain contains a gene which confers resistance. After overnight growth an isolated colony is picked from the plate with a sterile needle and transferred to an appropriate liquid growth medium containing the antibiotic to which the GRACE strain is resistant. The cells are incubated under appropriate growth conditions to yield a culture in exponential growth. Cells are frozen using standard techniques.
  • a stock vial Prior to performing an assay, a stock vial is removed from the freezer, rapidly thawed and a loop of culture is streaked out on an agar plate containing nutrients for cell growth and an antibiotic for which the GRACE strain contains a gene which confers resistance. After overnight growth, randomly chosen, isolated colonies are transferred from the plate (sterile inoculum loop) to a sterile tube containing medium containing the antibiotic to which the GRACE strain contains a gene which confers resistance. After vigorous mixing to form a homogeneous cell suspension, the optical density of the suspension is measured and if necessary an aliquot of the suspension is diluted into a second tube of medium plus antibiotic. The culture is then incubated until the cells reach an optical density suitable for use in the assay.
  • Two-fold dilution series of the inducer or repressor for the regulatable promoter which is linked to the gene required for the fungal proliferation, virulence or pathogenicity of the GRACE strain are generated in culture medium containing the appropriate antibiotic for which the GRACE strain contains a gene which confers resistance.
  • Several medium are tested side by side and three to four wells are used to evaluate the effects of the inducer or repressor at each concentration in each media.
  • Equal volumes of test media-inducer or repressor and GRACE cells are added to the wells of a 384 well microtiter plate and mixed. The cells are prepared as described above and diluted in the appropriate medium containing the test antibiotic immediately prior to addition to the microtiter plate wells.
  • cells are also added to several wells of each medium that do not contain inducer or repressor.
  • Cell growth is monitored continuously by incubation by monitoring the optical density of the wells.
  • the percent inhibition of growth produced by each concentration of inducer or repressor is calculated by comparing the rates of logarithmic growth against that exhibited by cells growing in medium without inducer or repressor. The medium yielding greatest sensitivity to inducer or repressor is selected for use in the assays described below.
  • Cells are prepared as described above using the medium selected for assay development supplemented with the antibiotic required to maintain the GRACE strain and are diluted in identical medium immediately prior to addition to the microtiter plate wells. For a control, cells are also added to several wells that lack antibiotic, but contain the solvent used to dissolve the antibiotics. Cell growth is monitored continuously by incubation in a microtiter plate reader monitoring the optical density of the wells. The percent inhibition of growth produced by each concentration of antibiotic is calculated by comparing the rates of logarithmic growth against that exhibited by cells growing in medium without antibiotic. A plot of percent inhibition against log [antibiotic concentration] allows extrapolation of an IC 50 value for each antibiotic.
  • the culture medium selected for use in the assay is supplemented with inducer or repressor at concentrations shown to inhibit cell growth by a desired amount as described above, as well as the antibiotic used to maintain the GRACE strain.
  • Two fold dilution series of the panel of test antibiotics used above are generated in each of these media.
  • Several antibiotics are tested side by side in each medium with three to four wells being used to evaluate the effects of an antibiotic on cell growth at each concentration.
  • Equal volumes of test antibiotic and cells are added to the wells of a 384 well microtiter plate and mixed. Cells are prepared as described above using the medium selected for use in the assay supplemented with the antibiotic required to maintain the GRACE strain.
  • the cells are diluted 1 :100 into two aliquots of identical medium containing concentrations of
  • the gene product encoded by the gene linked to the regulatable promoter in the GRACE strain is involved in the pathway on which the antibiotic acts.
  • the cell-based assay may also be used to determine the pathway against which a test antibiotic acts.
  • the pathways against in which the gene under the control of the regulatable promoter in each member of a panel of GRACE strains lies is identified as described above.
  • a panel of cells, each containing a - . regulatable promoter which directs transcription of a proliferation, virulence or pathogenicity-required nucleic acid which lies in a known biological pathway required for fungal proliferation, virulence or pathogenicity is contacted with a test antibiotic for which it is desired to determine the pathway on which it acts under conditions in which the gene product of the nucleic acid is rate limiting or is not rate limiting.

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Abstract

L'invention concerne des procédés et compositions permettant de déterminer de manière expérimentale si un quelconque gène du génome d'un organisme pathogène diploïde est essentiel et s'il est nécessaire à la virulence ou au pouvoir pathogène. Ces procédés consistent à construire des mutants génétiques dans lesquels un allèle d'un gène spécifique est inactivé tandis que l'autre allèle du gène est placé dans des conditions d'éventuelle expression. L'identification de gènes essentiels et de gènes critiques quant au développement d'infections virulentes constitue une base de développement du criblage de nouveaux médicaments dirigés contre ces organismes pathogènes. L'invention concerne encore des gènes ∫i⊃Candida albicans∫/i⊃ qui se sont révélés être essentiels dans le criblage de médicaments et constituent des cibles potentielles à cette fin. On peut utiliser la séquence nucléotidique de ces gènes cibles à des fins de découverte de médicaments, telle que l'expression de la protéine de recombinaison, le dosage d'hybridation et la construction d'ensembles d'acides nucléiques. L'invention concerne enfin l'utilisation de protéines, codées par les gènes essentiels, et de cellules modifiées génétiquement et comprenant des allèles modifiés de gènes essentiels, dans divers procédés de criblage.
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* Cited by examiner, † Cited by third party
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WO2003010333A2 (fr) * 2001-07-24 2003-02-06 Affinium Pharmaceuticals Inc. Methodes de disruption genique et leurs utilisations
WO2004053486A1 (fr) * 2002-12-09 2004-06-24 Oxford Glycosciences (Uk) Ltd Cca1 utilise en tant que cible antifongique
WO2004053145A2 (fr) * 2002-12-09 2004-06-24 Oxford Glycosciences (Uk) Ltd Trl1 en tant que cible antifongique
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WO2004053485A1 (fr) * 2002-12-09 2004-06-24 Oxford Glycosciences (Uk) Ltd Bpl1 utile en tant qu'agent antifongique
EP1466974A1 (fr) * 2003-04-11 2004-10-13 Max-Delbrück-Centrum Für Molekulare Medizin Mutagenèse dirigé inductible grâce au sauvetage conditionel d'un gène
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Cited By (15)

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Publication number Priority date Publication date Assignee Title
WO2002053728A3 (fr) * 2000-12-29 2003-03-20 Elitra Pharmaceuticals Inc Procedes de disruption de genes pour identification de cible de medicament
WO2002053728A2 (fr) * 2000-12-29 2002-07-11 Elitra Pharmaceuticals, Inc. Procedes de disruption de genes pour identification de cible de medicament
WO2003010333A2 (fr) * 2001-07-24 2003-02-06 Affinium Pharmaceuticals Inc. Methodes de disruption genique et leurs utilisations
WO2003010333A3 (fr) * 2001-07-24 2003-10-30 Affinium Pharm Inc Methodes de disruption genique et leurs utilisations
US7442532B2 (en) 2002-04-26 2008-10-28 Bristol-Myers Squibb Company Essential fungal CaYLR100w polypeptides, and methods of use
US7790428B2 (en) 2002-04-26 2010-09-07 Bristol-Myers Squibb Company Essential fungal polypeptide, CaYDR341c, and methods of use thereof
US7465568B2 (en) 2002-04-26 2008-12-16 Bristol-Myers Squibb Company Essential fungal polynucleotides, polypeptides, and methods of use
WO2004053146A2 (fr) * 2002-12-09 2004-06-24 Oxford Glycosciences (Uk) Ltd Sec14 en tant que cible antifongique
WO2004053145A3 (fr) * 2002-12-09 2004-08-05 Oxford Glycosciences Uk Ltd Trl1 en tant que cible antifongique
WO2004053146A3 (fr) * 2002-12-09 2004-08-05 Oxford Glycosciences Uk Ltd Sec14 en tant que cible antifongique
WO2004053485A1 (fr) * 2002-12-09 2004-06-24 Oxford Glycosciences (Uk) Ltd Bpl1 utile en tant qu'agent antifongique
WO2004053145A2 (fr) * 2002-12-09 2004-06-24 Oxford Glycosciences (Uk) Ltd Trl1 en tant que cible antifongique
WO2004053486A1 (fr) * 2002-12-09 2004-06-24 Oxford Glycosciences (Uk) Ltd Cca1 utilise en tant que cible antifongique
EP1466974A1 (fr) * 2003-04-11 2004-10-13 Max-Delbrück-Centrum Für Molekulare Medizin Mutagenèse dirigé inductible grâce au sauvetage conditionel d'un gène
WO2004090131A1 (fr) * 2003-04-11 2004-10-21 Max-Delbrück-Centrum Für Molekulare Medizin (Mdc) Berlin-Buch Mutagenese dirigee inductible par sauvetage de gene conditionnel

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EP1292668A2 (fr) 2003-03-19
CA2398861A1 (fr) 2001-08-23
JP2003523197A (ja) 2003-08-05

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