Background of the Invention Fungal infections (mycoses) may be cutaneous, subcutaneous, or systemic.

Superficial mycoses include tinea capitis, tinea corporis, tinea pedis, perionychomycosis, pityriasis versicolor, oral thrush, and other candidoses such as vaginal, respiratory tract, biliary, eosophageal, and urinary tract candidoses. Systemic mycoses include systemic and mucocutaneous candidosis, cryptococcosis, aspergillosis, mucormycosis (phycomycosis), paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, and sporotrichosis.

Fungal infections can also contribute to meningitis and pulmonary or respiratory tract diseases. Opportunistic fungal infections proliferate, especially in patients afflicted with AIDS or other diseases that compromise the immune system.

Examples of pathogenic fungi include dermatophytes (e. g., Microsporum canis and other M. spp. ; and Trichophyton spp. such as T. rubrum, and T. mentagrophytes), yeasts (e. g., Candida albicans, C. Tropicalis, or other Candida species), Torulopsis glabrata, Epidermophytonfloccosum, Malasseziafurfur (Pityropsporon orbiculare, or P. ovale), Cryptococcus neoformarts, Aspergillus fumigatus, and other Aspergillus sp., Zygomycetes (e. g., Rhizopus, Mucor), Paracoccidioides brasiliensis, Blastomyces dermatitides, Histoplasma capsulatum, Coccidioides immitis, and Sporothrix schenckii.

By way of background, the Fungi Kingdom consists of two divisions, the Eumycota and Myxomycota or the true fungi and slime molds, respectively. The true fungi are those species that are hyphal or are clearly related to species that are hyphal, possess cell walls throughout most or all of their life cycle, and are exclusively absorptive in their function. The slime molds are organisms that do not form hyphae,

lack cell walls during the phase in which they obtain nutrients and grow and are capable of ingesting nutrients in particulate form by phagocytosis.

The two most important classes of true fungi in which most species produce motile cells, known as zoospores, are the Oomycetes, and the Chytridiomycetes. The fungi that lack zoospores are classified according to the sexual phase of the fungal life cycle. The sexual process leads to the production of characteristic spores in the different groups. The fungi that form zygospores are classified as Zygomycetes, those that form ascospores are classified as Ascomycetes, and those forming basidiospores are classified as Basidiomycetes. There are also many species, recognizable as higher fungi through the presence of cell walls in their hyphae, that produce asexual spores but lack a sexual phase. These are known as Deuteromycetes, and details of their asexual sporulation are used to classify them. A representative member of the Deuteromycetes includes Candida albicans. These species are extensively reviewed in"The Fungi" (Ed M J Carlile and SC Watkinson 1994 Acad Press Ltd) and"The Growing Fungus" (Ed. NAR Gow and GM Gadd, 1995, Chapman and Hall).

Yeast are fungi that are normally unicellular and reproduce by budding although some will, under appropriate conditions, produce hyphae, just as some normally hyphal fungi may produce a yeast phase. The best known of all yeasts is Saccharomyces cerevisiae, which is a member of the Ascomycetes species. It is commonly regarded as a diploid yeast since mating usually soon follows ascospore germination. However, single cells can be used to establish permanently haploid cultures.

Fungal and other mycotic pathogens are responsible for a variety of diseases in humans, animals and plants. Fungal infection is also a significant problem in veterinary medicine. Some of the fungi that infect animals can be transmitted from animals to humans. Fungal infections or infestations are also a very serious problem in agriculture with fungicides being employed to protect vegetable and fruit and cereal crops. Fungal attack of wood products is also of major economic importance.

Additional products that are susceptible to fungal infestation include textiles, plastics, paper and paint. Some of these fungal targets are extensively reviewed in WO 95/11969.

Statistics show that the incidence of fungal infections has doubled from the 1980's to the 1990's, and infections of the blood stream have increased fivefold with an observed mortality of 50% (Tally et al., 1997, Int. Conference Biotechnol Microb.

Prods : Novel Pharmacol. Agrobiol. Activities, Williamsburg, VA Abstr S5 pl9).

These include those fungal infections, such as candidiasis, to which all individuals are susceptible, but also infections such as cryptococcosis and aspergillosis, which occur particularly in patients of compromised immune status.

By way of example, the yeast Candida albicans (C. albicans) has been shown to be one of the most pervasive fungal pathogens in humans. It has the capacity to opportunistically infect a diverse spectrum of compromised hosts, and to invade many diverse tissues in the human body. It can in many instances evade antibiotic treatment and the immune system. Although Candida albicans is a member of the normal flora of the mucous membranes in the respiratory, gastrointestinal, and female genital tracts, in such locations, it may gain dominance and be associated with pathologic conditions. Sometimes it produces progressive systematic disease in debilitated or immunosuppressed patients, particularly if cell-mediated immunity is impaired.

Sepsis may occur in patients with compromised cellular immunity, e. g., those undergoing cancer chemotherapy or those with lymphoma, AIDS, or other conditions.

Candida may produce bloodstream invasion, thrombophlebitis, endocarditis, or infection of the eyes and virtually any organ or tissue when introduced intravenously, e. g., via tubing, needles, narcotics abuse etc.

Candida albicans has been shown to be diploid with balanced lethals, and therefore probably does not go through a sexual phase or meiotic cycle. This yeast appears to be able to spontaneously and reversibly switch at high frequency between at least seven general phenotypes. Switching has been shown to occur not only in standard laboratory strains, but also in strains isolated from the mouths of healthy individuals.

Nystatin, ketoconazole, and amphotericin B are drugs that have been used to treat oral and systemic Candida infections. However, orally administered nyastin is limited to treatment within the gut and is not applicable to systemic treatment. Some systemic infections are susceptible to treatment with ketoconazole or amphotericin B, but these drugs may not be effective in such treatment unless combined with additional drugs. Amphotericin B has a relatively narrow therapeutic index and

numerous undesirable side effects, ranging from nausea and vomiting to kidney damage and toxicities occur even at therapeutic concentrations. While ketoconazole and other azole antifungals exhibit significantly lower toxicity, their mechanism of action, through inactivation of cytochrome P450 prosthetic group in certain enzymes (some of which are found in humans) precludes use in patients that are simultaneously receiving other drugs that are metabolized by the body's cytochrome Peso enzymes.

These adverse effects mean that their use is generally limited to the treatment of topical or superficial infections. In addition, resistance to these compounds is emerging and may pose a serious problem in the future. The more recently developed triazole drugs, such as fluconazole, are believed by some to have fewer side effects but are not completely effective against all pathogens.

Invasive aspergillosis, caused by Aspergillus fumigatus (A. fumigatus) has also become an increasingly opportunistic infection. There has been a 14-fold increase in its incidence during the past 12 years as detected by autopsy, and only two drugs are available that are effective in its treatment, neither of which is completely satisfactory.

Amphotericin B needs to be given intravenously and has a number of toxic side effects. Itraconazole, which can be given orally is often prescribed imprudently, encouraging the emergence of resistant strains of A. fumigatus (Dunn-Coleman and Prade, Nature Biotechnology, 1998, 16 : 5). Resistance is also developing to synthetic azoles (such as fluconazole and flucytosine), and the natural polyenes (such as amphotericin B) are limited in use by their toxicity.

Fungicide resistance generally develops when a fungal cell or fungal population that originally was sensitive to a fungicide becomes less sensitive by heritable changes after a period of exposure to the fungicide.

In certain applications, such as agriculture, it is possible to combat resistance through alteration of fungicides or the use of fungicide mixtures. To prevent or delay the build up of a resistant pathogen population, different agents that are effective against a particular disease must be available. One way of increasing the number of available agents is to search for new site-specific inhibitors.

Consequently, antifungal drug discovery efforts have been directed at components of the fungal cell or its metabolism that are unique to fungi, and hence might be used as therapeutic targets of new agents which act on the fungal pathogen without undue toxicity to host cells. Such potential targets include enzymes critical to

fungal cell wall assembly (US-A-5194600) as well as topoisomerases (enzymes required for replication of fungal DNA). Two semisynthetic antifungal agents such as the echinocandins and the related pneumocandins are in late stage clinical trials. Both are cyclic lipopeptides produced by fungi that non-competitively inhibit (1, 3)-glucan synthase and thus interfere with the biosynthesis of the fungal cell wall. These clinical candidates are generally more water-soluble, have improved pharmacokinetics and broader antifungal spectra than their natural parent compounds and have activity spectra that include many Candida species, including Candida albicans, and Aspergilli.

Because no single approach may be effective against all fungal pathogens, however, and because of the possibility of developed resistance to previously effective antifungal compounds, there remains a need for new antifungal agents with novel mechanisms of action and improved or different activity profiles. There is also a need for agents which are active against fungi but are not toxic to mammalian cells, as toxicity to mammalian cells can lead to a low therapeutic index and undesirable side effects in the host (e. g., patient). An important aspect of meeting this need is the selection of an appropriate component of fungal structure or metabolism as a therapeutic target.

Even after a particular intracellular target is selected, the means by which new antifungal agents are identified pose certain challenges. Despite the increased use of rational drug design, a preferred method continues to be the mass screening of compound"libraries"for active agents by exposing cultures of fungal pathogens to the test compounds and assaying for inhibition of growth. In testing thousands or tens of thousands of compounds, however, a correspondingly large number of fungal cultures must be grown over time periods which are relatively long compared to most bacterial culture times. Moreover, a compound which is found to inhibit fungal growth in culture may be acting not on the desired target but on a different, less unique fungal component, with the result that the compound may act against host cells as well and thereby produce unacceptable side effects. Consequently, there is a need for an assay or screening methods which more specifically identifies those agents that are active against a certain intracellular target. Additionally, there is a need for assay methods having greater throughput, that is, assay methods which reduce the time and materials needed to test each compound of interest.

Summary of the Invention The invention is based on the discovery that the YDR416W, YFL024C, YGR090W, YHR074W, YIL026C, YJL049W, YMR077C, YMR258C, YDR325W, YDR434W, YGL101W, YGR113W, YHR122W, YJLOlOC, YLR100W, YMR185W, and YOR166C genes in the fungus Saccharomyces cerevisiae are essential for survival of Saccharomyces. Essential genes are genes which are required for growth (such as metabolism, division, or reproduction) and survival of an organism. These essential genes can be used to identify therapeutic antifungal agents. These therapeutic agents can reduce or prevent growth, or decrease pathogenicity or virulence, and preferably, kill the fungi.

As summarized in Table 1, the nucleic acid sequences and amino acid sequences of these genes and the polypeptides encoded by these genes are shown in Figs. 1-17. Accordingly, these nucleic acid sequences of the invention, and the polypeptides of the invention, are useful targets for identifying compounds that are inhibitors of Saccharomyces. Such inhibitors attenuate fungal growth by inhibiting the activity of the YDR416W, YFL024C, YGR090W, YHR074W, YIL026C, YJL049W, YMR077C, YMR258C, YDR325W, YDR434W, YGL101W, YGR113W, YHR122W, YJL010C, YLR100W, YMR185W, or YOR166C polypeptide, or by inhibiting transcription or translation.

Table 1 : Essential Genes and Polypeptides of the Invention Gene or Figure No. SEQ ID NO. OF CODING SEQ ID NO. OF NON-SEQ ID NO. OF Polypeptide STRAND OF NUCLEIC CODING STRAND OF AMINO ACID ACID SEQUENCE NUCLEIC ACID SEQUENCE | YDR416W 1 1 3 2 YFL024C 2 4 6 5 YGR090W 3 7 9 8 YHR074W 4 10 12 11 YIL026C 5 13 14 YJL049W 6 15 16 YMR077C 7 17 19 18 YMR258C 8 20 22 21 YDR325W 9 23 24 YDR434W 10 25 27 26 YGL101W 11 28 30 29 YGR113W 12 31 33 32 YHR122W 13 34 36 35 YJLOIOC 14 37 38 YLR100W 15 39 40 YMR185W 16 41 43 42 YOR166C 17 44 45 Since these Saccharomyces genes have been identified as being essential for

survival, these nucleic acids and the polypeptides that they encode can be used to identify antifungal agents. Such antifungal agents can be readily identified with high throughput assays to detect inhibition of the essential gene or polypeptide. Such inhibition can be caused by small molecules binding directly to the essential polypeptide or by binding of small molecules to other essential polypeptides in a biochemical pathway shared with the essential polypeptide described herein.

The invention also provides methods of identifying agents (such as compounds, other substances or compositions including same) that affect, or selectively affect, (such as inhibit or otherwise modify) the activity of and/or

expression of the essential polypeptide, by contacting the essential polypeptide or the nucleotide sequence coding for same with the agent and then measuring the activity of the essential polypeptide and/or the expression thereof. In a related aspect, the invention features a method of identifying agents (such as compounds, other substances or compositions including same) that affect (such as inhibit or otherwise modify) the activity of and/or expression of the essential polypeptide, the method entailing measuring the activity of and/or expression of the essential polypeptide in the presence of the agent or after the addition of the agent in : (a) a cell line into which has been incorporated a recombinant construct including the nucleotide sequence of the essential gene (e. g., SEQ ID NO : I) or an allelic variation thereof, or (b) a cell population or cell line that naturally selectively expresses the essential polypeptide, and then measuring the activity of the essential polypeptide and/or the expression thereof.

In one embodiment, the invention features a method for identifying a compound for the treatment of a fungal infection, wherein the method entails, in sequence, (i) preparing a first cell and a second cell, the first and second cells being capable of expressing an essential gene, as described herein, (ii) contacting the first cell with a test compound, (iii) determining the level of expression of the essential gene in the first and second cells, (iv) comparing the level of expression in the first cell with the second cell, and (v) selecting the test compound for treatment of a fungal infection where expression of the essential gene in the first cell is less than expression of the essential gene in the second cell, and wherein the essential gene is a first nucleic acid molecule which encodes a polypeptide including the amino acid sequence of SEQ ID NO : 2, SEQ ID NO : 5, SEQ ID NO : 8, SEQ ID NO : 11, SEQ ID NO : 14, SEQ ID NO : 16, SEQ ID NO : 18, SEQ ID NO : 21, SEQ ID N0 : 24, SEQ ID NO : 26, SEQ ID NO : 29, SEQ ID NO : 32, SEQ ID NO : 35, SEQ ID NO : 38, SEQ ID NO : 40, SEQ ID N0 : 42, or SEQ ID NO : 45, or a naturally occurring allelic variant thereof, and wherein the first nucleic acid molecule hybridizes under stringent conditions to a second nucleic acid molecule, the second nucleic acid molecule consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO : 3, SEQ ID N0 : 6, SEQ ID N0 : 9, SEQ ID NO : 12, the complement of SEQ ID NO : 13, the complement of SEQ ID NO : 15, SEQ ID NO : 19, SEQ ID NO : 22, the complement of SEQ ID NO : 23, SEQ ID NO : 27, SEQ ID NO : 30, SEQ ID N0 : 33,

SEQ ID NO : 36, the complement of SEQ ID NO : 37, the complement of SEQ ID NO : 39, SEQ ID NO : 43, and the complement of SEQ ID NO : 44. The level of expression of the essential gene can be determined by measuring the amount of mRNA transcribed from the essential gene. Alternatively, the level of the essential polypeptide encoded by the essential gene can be measured.

Another suitable method for identifying antifungal compounds involves screening for small molecules that specifically bind to one of the essential polypeptides described herein. A variety of suitable binding assays are known in the art as described, for example, in U. S. Patent Nos. 5, 585, 277 and 5, 679, 582, hereby incorporated herein by reference. For example, in various conventional assays, test compounds can be assayed for their ability to bind to a polypeptide by measuring the ability of the small molecule to stabilize the polypeptide in its folded, rather than unfolded, state. More specifically, one can measure the degree of protection against unfolding that is afforded by the test compound. Test compounds that bind to the essential polypeptide with high affinity cause, for example, a significant shift in the temperature at which the polypeptide is denatured. Test compounds that stabilize the polypeptide in a folded state can be further tested for antifungal activity in a standard susceptibility assay.

In a related method for identifying antifungal compounds, an essential polypeptide described herein is used to isolate peptide or nucleic acid ligands that specifically bind to the polypeptide. These peptide or nucleic acid ligands are then used in a displacement screen to identify small molecules that bind to the essential polypeptide described. Such binding assays can be carried out essentially as described above.

The essential polypeptides described herein also can be used, in assays to identify test compounds that bind to the polypeptides. Test compounds that bind to the essential polypeptides then can be tested, in conventional assays, for their ability to inhibit fungal growth. Test compounds that bind to the essential polypeptides are candidate antifungal agents, in contrast to compounds that do not bind to the essential polypeptides. As described herein, any of a variety of art-known methods can be used to assay for binding of test compounds to the essential polypeptides. Typically, the test compound will be a small organic molecule. Alternatively, the test compound can be a test polypeptide (e. g., a polypeptide having a random or predetermined

amino acid sequence ; or a naturally-occurring or synthetic polypeptide) or a nucleic acid, such as a DNA or RNA molecule. The test compound can be a naturally- occurring compound or it can be synthetically produced, if desired. Synthetic libraries, chemical libraries, and the like can be screened to identify compounds that bind to the essential polypeptides described herein.

The invention includes, for example, a method for identifying a compound useful for treating a fungal infection, wherein the method entails (a) measuring the level of expression of an essential gene in a cell in the presence of a test compound ; (b) comparing the level of expression measured in step (a) to the level of expression of an essential gene in a cell in the absence of the test compound ; and (c) selecting the test compound as being useful for treating a fungal infection when the level of expression of the essential gene in the presence of the test compound is less than the level expression of the essential gene in the absence of the test compound, and wherein the essential gene is selected from the group consisting of SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID N0 : 7, SEQ ID NO : 10, SEQ ID NO : 13, SEQ ID NO : 15, SEQ ID NO : 17, SEQ ID NO : 20, SEQ ID NO : 23, SEQ ID NO : 25, SEQ ID NO : 28, SEQ ID NO : 31, SEQ ID NO : 34, SEQ ID N0 : 37, SEQ ID NO : 39, SEQ ID N0 : 41, and SEQ ID NO : 44. If desired, the level of expression can be measured by measuring the amount of mRNA from an essential gene described herein, or by measuring the amount of protein encoded by an essential gene described herein. Typically, the cell is Saccharomyces (e. g., Saccharomyces cerevisiae).

In a variation of the above method, the invention features a method for identifying a compound useful for treating a fungal infection, wherein the method entails (a) measuring the activity of an essential gene in a cell in the presence of a test compound ; (b) comparing the activity measured in step (a) to the level activity of an essential gene in a cell in the absence of the test compound ; and (c) selecting the test compound as being useful for treating fungal infections when the level of activity of the essential gene measured in the presence of the test compound is less than the level of activity of the essential gene measured in the absence of the test compound, wherein the essential gene is selected from the group consisting of SEQ ID NO : 1, SEQ ID N0 : 4, SEQ ID N0 : 7, SEQ ID NO : 10, SEQ ID NO : 13, SEQ ID NO : 15, SEQ ID NO : 17, SEQ ID N0 : 20, SEQ ID N0 : 23, SEQ ID N0 : 25, SEQ ID N0 : 28, SEQ

ID NO : 31, SEQ ID N0 : 34, SEQ ID N0 : 37, SEQ ID N0 : 39, SEQ ID N0 : 41, and SEQ ID NO : 44.

In an alternative method, the invention features a method for identifying a compound useful for treating a fungal infection, wherein the method entails (a) measuring, in the presence of a test compound, the growth of a sample of cells which have been engineered to express an essential gene ; (b) comparing the growth measured in step (a) to the growth of a sample of the cells in the absence of the test compound ; and (c) selecting the test compound as being useful for treating a fungal infection when the growth of the sample of cells in the presence of the test compound is slower than the growth of a sample of cells in the absence of the test compound, wherein the essential gene is selected from the group consisting of SEQ ID NO : 1,, SEQ ID NO : 4, SEQ ID NO : 7, SEQ ID NO : 10, SEQ ID NO : 13, SEQ ID NO : 15, SEQ ID NO : 17, SEQ ID NO : 20, SEQ ID NO : 23, SEQ ID N0 : 25, SEQ ID N0 : 28, SEQ ID N0 : 31, SEQ ID N0 : 34, SEQ ID N0 : 37, SEQ ID N0 : 39, SEQ ID N0 : 41, and SEQ ID NO : 44. Typically, the cell sample contains fungal cells (e. g., Saccharomyces).

In yet another embodiment, the invention features a method for identifying a compound useful for treating a fungal infection, wherein the method entails (a) contacting an essential polypeptide with a test compound ; (b) detecting binding of the test compound to the essential polypeptide ; and (c) selecting a compound useful for treating a fungal infection as one that binds to the essential polypeptide, wherein the essential polypeptide is encoded by a gene selected from the group consisting of SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID N0 : 7, SEQ ID NO : 10, SEQ ID NO : 13, SEQ ID NO : 15, SEQ ID NO : 17, SEQ ID NO : 20, SEQ ID NO : 23, SEQ ID NO : 25, SEQ ID NO : 28, SEQ ID NO : 31, SEQ ID NO : 34, SEQ ID N0 : 37, SEQ ID NO : 39, SEQ ID NO : 41, and SEQ ID NO : 44. Optionally, this method can also include (d) determining whether a test compound that binds to the essential polypeptide inhibits growth of fungi, relative to growth of fungi cultured in the absence of the test compound, wherein inhibition of growth indicates that the test compound is an antifungal agent.

If desired, the test compound can be immobilized on a substrate, and binding of the test compound to the essential polypeptide is detected as immobilization of the essential polypeptide on the immobilized test compound. Immobilization of the essential polypeptide on the test compound can be detected in an immunoassay with

an antibody that specifically binds to the essential polypeptide. In various embodiments, the test compound can be a polypeptide, ribonucleic acid, small molecule, or deoxyribonucleic acid.

The invention also features a pharmaceutical formulation containing an antifungal agent identified by this method, and a pharmaceutically acceptable excipient. The antifungal agent can be, without limitation, an antibody (e. g., a monoclonal antibody), a ribozyme, or an antisense nucleic acid. Such a pharmaceutical formulation can be administered in a therapeutically effective amount to an organism (e. g., a human) having a fungal infection in a method for treating the fungal infection.

In the aforementioned method for identifying a compound useful for treating a fungal infection, the essential polypeptide can be provided as a first fusion protein including the essential polypeptide fused to (i) a transcription activation domain of a transcription factor or (ii) a DNA-binding domain of a transcription factor. The test compound can be an test polypeptide that is provided as a second fusion protein including the test polypeptide fused to (i) a transcription activation domain of a transcription factor or (ii) a DNA-binding domain of a transcription factor, to interact with the first fusion protein. Binding of the test compound to the essential polypeptide is detected as reconstitution of a transcription factor. Reconstitution of the transcription factor can be detected, for example, by detecting transcription of a gene that is operably linked to a DNA sequence bound by the DNA-binding domain of the reconstituted transcription factor (See, for example, White, 1996, Proc. Natl.

Acad. Sci. 93 : 10001-10003 and references cited therein and Vidal et al., 1996, Proc.

Natl. Acad. Sci. 93 : 10315-10320).

The invention also features a method for identifying an antifungal agent, where the method entails (a) contacting an essential polypeptide with a test compound ; (b) detecting a decrease in activity of the essential polypeptide the contacted with test compound ; (c) selecting a compound useful for treating a fungal infection as one that decreases the activity of the essential polypeptide ; and, optionally, (d) determining whether a test compound that decreases activity of a contacted essential polypeptide inhibits growth of fungi, relative to growth of fungi cultured in the absence of a test compound that decreases activity of a contacted essential polypeptide, wherein inhibition of growth indicates that the test compound is

an antifungal agent, and wherein the essential polypeptide is encoded by a gene selected from the group consisting of SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 7, SEQ ID NO : 10, SEQ ID NO : 13, SEQ ID NO : 15, SEQ ID NO : 17, SEQ ID NO : 20, SEQ ID N0 : 23, SEQ ID N0 : 25, SEQ ID NO : 28, SEQ ID NO : 31, SEQ ID NO : 34, SEQ ID NO : 37, SEQ ID NO : 39, SEQ ID NO : 41, and SEQ ID NO : 44. The test compound can be, without limitation, a polypeptide, ribonucleic acid, small molecule, deoxyribonucleic acid, antisense oligonucleotide, or ribozy.

In another variation, the invention features a method for identifying a compound useful for treating a fungal infection, wherein the method entails (a) contacting an essential gene with a test compound ; (b) detecting binding of the test compound to the essential gene ; and (c) selecting a compound useful for treating a fungal infection as one that binds to the essential gene, wherein the essential gene is selected from the group consisting of SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID NO : 7, SEQ ID NO : 10, SEQ ID NO : 13, SEQ ID NO : 15, SEQ ID NO : 17, SEQ ID NO : 20, SEQ ID NO : 23, SEQ ID NO : 25, SEQ ID NO : 28, SEQ ID N0 : 31, SEQ ID N0 : 34, SEQ ID N0 : 37, SEQ ID NO : 39, SEQ ID NO : 41, and SEQ ID NO : 44. Optionally, this method can also include (d) determining whether a test compound that binds to the essential gene inhibits growth of fungi, relative to growth of fungi cultured in the absence of a test compound that binds to the essential gene, wherein inhibition of growth indicates that the test compound is an antifungal agent. Without limitation, the test compound can be selected from the group consisting of polypeptides, small molecules, ribonucleic acids, deoxyribonucleic acids, antisense oligonucleotides and ribozymes.

In yet another embodiment, the invention features a method for identifying a compound useful for treating a fungal infection, wherein the method entails (a) contacting a variant, homolog, or ortholog of an essential polypeptide with a test compound ; (b) detecting binding of the test compound to the variant, homolog, or ortholog of the essential polypeptide ; and (c) selecting a compound useful for treating a fungal infection as one that binds to the variant, homolog, or ortholog of the essential polypeptide, wherein the essential polypeptide is encoded by a gene selected from the group consisting of SEQ ID NO : 1, SEQ ID NO : 4, SEQ ID N0 : 7, SEQ ID NO : 10, SEQ ID N0 : 13, SEQ ID NO : 15, SEQ ID NO : 17, SEQ ID N0 : 20, SEQ ID NO : 23, SEQ ID N0 : 25, SEQ ID N0 : 28, SEQ ID NO : 31, SEQ ID NO : 34, SEQ ID

NO : 37, SEQ ID NO : 39, SEQ ID NO : 41, and SEQ ID NO : 44. Optionally, the method can also include (d) determining whether a test compound that binds to the variant, homolog, or ortholog of the essential polypeptide inhibits growth of fungi, relative to growth of fungi cultured in the absence of a test compound that binds to the variant, homolog, or ortholog of the essential polypeptide, wherein inhibition of growth indicates that the test compound is an antifungal agent. The variant, homolog, or ortholog can be derived from a non-pathogenic, or pathogenic, fungus. If desired, the test compound can immobilized on a substrate, and binding of the test compound to the variant, homolog, or ortholog can be detected as immobilization of the variant, homolog, or ortholog on the immobilized test compound, e. g., in an immunoassay with an antibody that specifically binds to the variant, homolog, or ortholog of the essential polypeptide. Without limitation, the test compound can be selected from the group consisting of polypeptides, ribonucleic acids, small molecules, and deoxyribonucleic acids.

Some specific embodiments of the present invention relate to assay methods for the identification of antifungal agents using assays for antifungal agents which may be carried out both in whole cell preparations and in ex vivo cell-free systems. In each instance, the assay target is the target nucleotide sequence-which is essential for fungal viability-and/or its essential polypeptide. Candidate agents which are found to inhibit the target nucleotide sequence and/or the essential polypeptide in any assay method of the present invention are thus identified as potential antifungal agents. It is expected that the assay methods of the present invention will be suitable for both small and large-scale screening of test compounds as well as in quantitative assays such as serial dilution studies wherein the target nucleotide sequence or its essential polypeptide thereof are exposed to a range of candidate agent concentrations.

When the assay methods of the present invention are carried out as a whole- cell assay, the target nucleotide sequence and/or its essential polypeptide and the entire living fungal cell may be exposed to the candidate agent under conditions normally suitable for growth. Optimal conditions including essential nutrients, optimal temperatures and other parameters, depend upon the particular fungal strain being used and suitable conditions are well known in the art. Inhibition of expression of the target nucleotide sequence and/or the activity of the essential polypeptide may be determined in a number of ways including observing the cell culture's growth or

lack thereof. Such observation may be made visually, by optical densitometric or other light absorption/scattering means or by yet other suitable means, whether manual or automated.

In the above whole-cell assay, an observed lack of cell growth may be due to inhibition of the target nucleotide sequence and/or the essential polypeptide or may be due to an entirely different effect of the candidate agent, and further evaluation may be required to establish the mechanism of action and to determine whether the candidate agent is a specific inhibitor of the target. Accordingly and in a preferred embodiment of the present invention, the method may be performed as a paired-cell assay in which each test compound is separately tested against two different fungal cells, the first fungal cells having a target with altered properties making it more susceptible to inhibition compared with that of the second fungal cells.

One manner of achieving differential susceptibility is by using mutant strains expressing a modified target essential polypeptide. A particularly useful strain is one having a temperature sensitive ("ts") mutation as a result of which the target is more prone than the wild type target to loss of functionality at high temperatures (that is, temperatures higher than optimal, but still permitting growth in wild type cells).

When grown at semi-permissive temperatures, the activity of a ts mutant target may be attenuated but sufficient for growth.

Alternatively or in conjunction with the above, differential susceptibility to target inhibitors may be obtained by using a second fungal cell which has altered properties making it less susceptible to inhibition compared with that of wild type cells as for example, a fungal cell which has been genetically manipulated to cause overexpression of the target. Such overexpression can be achieved by placing into a wild type cell a plasmid carrying the nucleotide sequence for the target. The techniques for generating temperature sensitive mutants, for preparing specific plasmids and for transforming cell lines with such plasmids are well known in the art.

Alternatively or in conjunction with the above, the access of candidate agents to a cell or an organism, may be enhanced by mutating or deleting a gene or genes which encode a protein or proteins responsible for providing a permeability barrier for a cell or an organism.

The present invention also relates to a method for identifying antifungal agents utilizing fungal cell systems that are sensitive to perturbation to one or several transcriptional/translational components.

By way of example, the present invention relates to a method of constructing mutant fungal cells in which one or more of the transcriptional/translational components is present in an altered form or in a different amount compared with a corresponding wild-type cell. This method further involves examining a candidate agent for its ability to perturb transcription/translation by assessing the impact it has on the growth of the mutant and wild-type cells. Agents that perturb transcription/translation by acting on a particular component that participates in transcription/translation may cause a mutant fungal cell which has an altered form or amount of that component to grow differently from the corresponding wild-type cell, but do not affect the growth relative to the wild type cell of other mutant cells bearing alterations in other components participating in transcription/translation. This method thus provides not only a means to identify whether a candidate agent perturbs transcription/translation but also an indication of the site at which it exerts its effects.

The transcriptional/translational component which is present in altered form or amount in a cell whose growth is affected by a candidate agent is likely to be the site of action of the agent.

By way of example, the present invention provides a method for identifying antifungal agents which interfere with steps in translational accuracy, such as maintaining a proper reading frame during translation and terminating translation at a stop codon. This method involves constructing mutant fungal cells in which a detectable reporter polypeptide can only be produced if the normal process of staying in one reading frame or of terminating translation at a stop codon has been disrupted.

This method further involves contacting the mutant fungal cells with a candidate agent to examine whether it increases or decreases the production of the reporter polypeptide.

The present invention also provides a method of screening an agent for specific binding affinity with the essential polypeptide (or a derivative, homolog, variant or fragment thereof) or the nucleotide sequence coding for same (including a derivative, homolog, variant or fragment thereof) ; the method entails : a) providing a candidate agent ; b) combining the essential polypeptide (or the derivative, homolog,

variant or fragment thereof) or the nucleotide sequence coding for same (or the derivative, homolog, variant or fragment thereof) with the candidate agent for a time sufficient to allow binding under suitable conditions ; such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the essential polypeptide or the nucleotide sequence encoding same with the agent ; and c) determining whether the agent binds to or otherwise interacts with and activates or inhibits an activity of the essential polypeptide (or the derivative, homolog, variant or fragment thereof) or the expression of the nucleotide sequence coding for same (or the derivative, homolog, variant or fragment thereof) by detecting the presence or absence of a signal generated from the binding and/or interaction of the agent with the essential polypeptide (or the derivative, homolog, variant or fragment thereof) or the nucleotide sequence coding for same (or the derivative, homolog, variant or fragment thereof).

In other embodiments, the cell system is an extract of a fungal cell that is grown under defined conditions, and the method involves measuring transcription or translation in vitro. Such defined conditions are selected so that transcription or translation of the reporter is increased or decreased by the addition of a transcription inhibitor or a translation inhibitor to the cell extract.

One such method for identifying antifungal agents relies upon a transcription- responsive gene product. This method involves constructing a fungal cell in which the production of a reporter molecule, measured as a percentage of over-all transcription, increases or decreases under conditions in which overall fungal cell transcription is reduced. Specifically, the reporter molecule is encoded by a nucleic acid transcriptionally linked to a sequence constructed and arranged to cause a relative increase or decrease in the production of the reporter molecule when overall transcription is reduced. Typically, the overall transcription is measured by the expression of a second indicator gene whose expression, when measured as a percentage of overall transcription, remains constant when the overall transcription is reduced. The method further involves contacting the fungal cell with a candidate agent, and determining whether the agent increases or decreases the production of the first reporter molecule in the fungal cell.

In one embodiment, the reporter molecule is itself the transcription-responsive gene product whose production increases or decreases when overall transcription is

reduced. In another embodiment, the reporter is a different molecule whose production is linked to that of the transcription-responsive gene product. Such linkage between the reporter and the transcription-responsive gene product can be achieved in several ways. A gene sequence encoding the reporter may, for example, be fused to part or all of the gene encoding the transcription-responsive gene product and/or to part or all of the genetic elements which control the production of the gene product. Alternatively, the transcription-responsive gene product may stimulate transcription of the gene encoding the reporter, either directly or indirectly.

Alternatively, the method for identifying antifungal agents relies upon a translation-responsive gene product. This method involves constructing a fungal cell in which the production of a reporter molecule, measured as a percentage of over-all translation, increases or decreases under conditions in which overall fungal cell translation is reduced. Specifically, the reporter molecule is encoded by nucleic acid either translationally linked or transcriptionally linked to a sequence constructed and arranged to cause a relative increase or decrease in the production of the reporter molecule when overall translation is reduced. The overall translation can be measured by the expression of å second indicator gene whose expression, when measured as a percentage of overall translation, remains constant when the overall translation is reduced. The method further involves contacting the fungal cell with a candidate agent, and determining whether the agent increases or decreases the production of the first reporter molecule in the fungal cell.

In another embodiment, the reporter molecule is itself the translation- responsive gene product whose production increases or decreases when overall translation is reduced. In another embodiment, the reporter is a different molecule whose production is linked to that of the translation-responsive gene product. Such linkage between the reporter and the translation-responsive gene product can be achieved in several ways. A gene sequence encoding the reporter may, for example, be fused to part or all of the gene encoding the translation-responsive gene product and/or to part or all of the genetic elements which control the production of the gene product. Alternatively, the translation-responsive gene product may stimulate translation of the gene encoding the reporter, either directly or indirectly.

A wide variety of reporters may be used, with typical reporters providing conveniently detectable signals (e. g., by spectroscopy). By way of example, a

reporter gene may encode an enzyme which catalyses a reaction which alters light absorption properties.

Examples of reporter molecules include but are not limited to-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, beta-glucuronidase, exo-glucanase and glucoamylase. Alternatively, radiolabeled or fluorescent tag-labeled nucleotides can be incorporated into nascent transcripts which are then identified when bound to oligonucleotide probes. For example, the production of the reporter molecule can be measured by the enzymatic activity of the reporter gene product, such as-galactosidase.

In another embodiment of the present invention, a selection of hybridization probes corresponding to a predetermined population of genes of the selected fungal organism may be used to specifically detect changes in gene transcription which result from exposing the selected organism or cells thereof to a candidate agent. In this embodiment, one or more cells derived from the organism is exposed to the candidate agent in vivo or ex vivo under conditions wherein the agent effects a change in gene transcription in the cell to maintain homeostasis. Thereafter, the gene transcripts, primarily mRNA, of the cell or cells are isolated by conventional means. The isolated transcripts or cDNAs complementary thereto are then contacted with an ordered matrix of hybridization probes, each probe being specific for a different one of the transcripts, under conditions wherein each of the transcripts hybridizes with a corresponding one of the probes to form hybridization pairs. The ordered matrix of probes provides, in aggregate, complements for an ensemble of genes of the organism sufficient to model the transcriptional responsiveness of the organism to a candidate agent. The probes are generally immobilized and arrayed onto a solid substrate such as a microtiter plate. Specific hybridization may be effected, for example, by washing the hybridized matrix with excess non-specific oligonucleotides. A hybridization signal is then detected at each hybridization pair to obtain a transcription signal profile. A wide variety of hybridization signals may be used. In one embodiment, the cells are pre-labeled with radionucleotides such that the gene transcripts provide a radioactive signal that can be detected in the hybridization pairs. The transcription signal profile of the agent-treated cells is then compared with a transcription signal profile of negative control cells to obtain a specific transcription response profile to the candidate agent.

A variety of protocols for detecting and measuring the expression of the essential polypeptide, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the essential polypeptide is suitable, although a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R et al. (1990, Serological Methods, A Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al. (1983, J. Exp.

Med. 15 8 : 121).

In an embodiment of the present invention, the essential polypeptide or a variant, homolog, fragment or derivative thereof and/or a cell line that expresses the essential polypeptide or variant, homolog, fragment or derivative thereof may be used to screen for antibodies, peptides, or other agents, such as organic or inorganic molecules, that act as modulators of the essential polypeptide activity, thereby identifying a therapeutic agent capable of modulating the activity of the essential polypeptide. For example, antibodies that specifically bind to an essential polypeptide and are capable of neutralizing the activity of the essential polypeptide may be used to inhibit the essential polypeptide activity. Alternatively, screening of peptide libraries or organic libraries made by combinatorial chemistry with recombinantly expressed essential polypeptide or a variant, homolog, fragment or derivative thereof or cell lines expressing the essential polypeptide or a variant, homolog, fragment or derivative thereof may be useful for identification of therapeutic agents that function by modulating the essential polypeptide activity.

Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in a number of ways deemed to be routine to those of skill in the art. For example, nucleotide sequences encoding the N-terminal region of the essential polypeptide can be expressed in a cell line and used for screening of allosteric modulators, either agonists or antagonists, of the essential polypeptide activity. Alternatively, nucleotide sequences encoding the conserved catalytic domain of the essential polypeptide can be expressed in cell lines and used to screen for inhibitors of the essential polypeptide activity.

Accordingly, the present invention provides a method for screening a plurality of agents for specific binding affinity with the essential polypeptides, or a portion, variant, homolog, fragment or derivative thereof, by providing a plurality of agents ; combining the essential polypeptide or a portion, variant, homolog, fragment or derivative thereof with each of a plurality of agents for a time sufficient to allow binding under suitable conditions ; and detecting binding of the essential polypeptide, or portion, variant, homolog, fragment or derivative thereof, to each of the plurality of agents, thereby identifying the agent or agents which specifically bind to the essential polypeptide. In such an assay, the plurality of agents may be produced by combinatorial chemistry techniques known to those of skill in the art.

Another technique for screening provides for high throughput screening of agents having suitable binding affinity to the essential polypeptide polypeptides and is based upon the method described in detail in WO 84/03564. In summary, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test agents are reacted with the essential polypeptide fragments and washed. A bound essential polypeptide is then detected-such as by appropriately adapting methods well known in the art. A purified essential polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

Typically, in an antifungal discovery process, potential new antifungal agents are tested for their ability to inhibit the in vitro catalytic activity of the purified expression product of the present invention in a biochemical assay. Agents with inhibitory activity can then progress to an in vitro antifungal activity screening using a standard MIC (Minimum Inhibitory Concentration) test (based on the M27-A NCCLS approved method). Antifungal active agents identified at this point are then tested for antifungal efficacy in vivo, such as by using rodent systemic candidiasis/aspergillosis models. Efficacy is measured by measuring the agent's ability to increase the host animal's survival rate against systemic infection, and/or reduce the fungal burden in infected tissues, compared to control animals receiving no administered agent (which can be by oral or intravenous routes).

The present invention also provides a pharmaceutical composition for treating an individual in need of such treatment of a disease caused by the essential

polypeptide activity (or that can be treated by inhibiting such activity) ; the treatment method entails administering a therapeutically effective amount of an agent that affects (such as inhibits) the activity and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

The pharmaceutical compositions can be used for humans or animals and will typically include any one or more of a pharmaceutically acceptable diluent, carrier, excipient, or adjuvant. The choice of pharmaceutical carrier, excipient, or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent any suitable binder (s), lubricant (s), suspending agent (s), coating agent (s), solubilizing agent (s).

The invention also features a method for treating a fungal infection in a patient by administering to the patient a therapeutically effective amount of a compound identified by any of the aforementioned methods, i. e., an amount sufficient to ameliorate signs and/or symptoms of the fungal infection. Such antifungal agents inhibit the growth of, or kill, pathogenic fungal strains. In particular, such pharmaceutical formulations can be used to treat fungal infections in mammals such as humans and domesticated mammals (e. g., cows, pigs, dogs, and cats), and in plants. The efficacy of such antifungal agents in humans can be estimated in an animal model system well known to those of skill in the art (e. g., mouse systems of fungal infections).

The invention also includes (i) a method of treating a mycosal and/or fungal infection in a target (which target can be a living organism, such as a mammal, e. g., a human, or an inanimate target, such as a textile piece, paper, plastic etc.), by delivering (such as administering or exposing) an effective amount of an agent capable of modulating the expression pattern of the nucleotide sequence of the present invention or the activity of the expression product thereof ; and (ii) a method of treating a mycosal and/or fungal infection in a target (which target can be a living organism, such as a plant or a mammal, e. g., a human, or an inanimate target, such as a textile piece, paper, plastic etc.), by delivering (such as administering or exposing) an effective amount of an agent identified by an assay according to the present

invention. As used herein, the terms"treating,""treat,"or"treatment"include, inter alia, preventative (e. g., prophylactic), palliative, and curative treatment of fungal infections.

The invention also features a method for inducing an immunological response in an individual, particularly a mammal, by inoculating the individual with one or more of the essential genes or polypeptides described herein, and typically in an amount adequate to produce an antibody and/or T cell immune response to protect the individual from mycoses, fungal infection, or infestations. In another aspect, the present invention relates to a method of inducing an immunological response in an individual which entails delivering to the individual a vector that includes an essential gene described herein or a variant, homolog, fragment, or derivative thereof in vivo to induce an immunological response, such as to produce antibody and/or a T-cell immune response to protect the individual from disease whether that disease is already established within the individual or not.

Various affinity reagents that are permeable to the microbial membrane (i. e., antibodies and antibody fragments) are useful in practicing the methods of the invention. For example polyclonal and monoclonal antibodies that specifically bind to the essential polypeptides described herein can facilitate detection of such polypeptides in various fungal strains (or extracts thereof). These antibodies also are useful for detecting binding of a test compound to the essential polypeptides (e. g., using the assays described herein). In addition, monoclonal antibodies that specifically bind to the essential polypeptides described herein can themselves be used as antifungal agents.

In all of the foregoing methods, homologs, orthologs, or variants of the essential genes and polypeptides described herein can be substituted for the essential genes. While"homologs"are structurally similar genes contained within a species, "orthologs"are functionally equivalent genes from other species (within or outside of a given genus, e. g., from E. coli). The terms"variant,""homolog,"or"fragment"in relation to the amino acid sequences of the essential polypeptides of the invention include any substitution, variation, modification, replacement, deletion, or addition of one or more amino acids from or to the sequence providing the resultant essential

polypeptide. The homolog, variant, or fragment has an activity that typically is at least as biologically active as the referenced essential polypeptide (e. g., as represented by SEQ ID NO : 2).

The terms"variant","homolog"or"fragment"in relation to the nucleotide sequence coding for the essential polypeptide of the present invention include any substitution, variation, modification, replacement, deletion, or addition of one or more nucleotides from or to the sequence of an essential gene. Typically, the resultant nucleotide sequence encodes or is capable of encoding an essential polypeptide that is at least as biologically active as the referenced essential polypeptide (e. g., as represented by SEQ ID NO : 2). In particular, the term"homolog"covers homology with respect to structure and/or function providing the resultant nucleotide sequence encodes or is capable of encoding an essential polypeptide. Preferably, the essential polypeptide is at least as biologically active as the referenced essential polypeptide encoded by the sequences shown herein. With respect to sequence homology, there is at least 75% (e. g., 85%, 90%, 95%, 98%, or 100%) homology to a sequence shown herein.

The term"homology"as used herein may be equated with the term"identity".

Relative sequence homology (i. e., sequence identity) can be determined by commercially available computer programs that can calculate the percent homology between two or more sequences. A typical example of such a computer program is CLUSTAL.

"Substantial homology,"where homology indicates sequence identity, means at least 80% sequence identity (e. g., 85%, 90% or more), as judged by direct sequence alignment and comparison."Substantial homology"when assessed by BLAST equates to sequences which match with an EXPECT value of at least 7 (e. g., 9, 10, or more). The default threshold for EXPECT in BLAST searching is usually 10.

Also included within the scope of the present invention are alleles of the essential polypeptides. As used herein, an"allele"or"allelic sequence"is an alternative form of the essential polypeptide. Alleles result from a mutation, i. e., a change in the nucleotide sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or more than one allelic form. Common mutational changes which give rise to alleles are generally ascribed to deletions, additions or substitutions

of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. The invention offers several advantages. For example, the methods for identifying antifungal agents can be configured for high throughput screening of numerous candidate antifungal agents.

Because the essential genes disclosed herein are thought to be highly conserved, antifungal drugs targeted to these gene or their gene products are expected to have a broad spectrum of antifungal activity.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the case of a conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative and are not intended to limit the scope of the invention, which is defined by the claims.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Brief Description of the Drawings Fig. 1 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 1), non-coding strand (SEQ ID NO : 3) and predicted amino acid sequence (SEQ ID NO : 2) of Saccharomyces cerevisiae YDR416W.

Fig. 2 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 4), non-coding strand (SEQ ID NO : 6) and predicted amino acid sequence (SEQ ID NO : 5) of Saccharomyces cerevisiae YFL024C.

Fig. 3 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 7), non-coding strand (SEQ ID NO : 9) and predicted amino acid sequence (SEQ ID NO : 8) of Saccharomyces cerevisiae YGR090W.

Fig. 4 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 10), non-coding strand (SEQ ID NO : 12) and predicted amino acid sequence (SEQ ID NO : 11) of Saccharomyces cerevisiae YHR074W.

Fig. 5 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 13) and predicted amino acid sequence (SEQ ID NO : 14) of Saccharomyces cerevisiae YIL026C Fig. 6 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 15) and predicted amino acid sequence (SEQ ID NO : 16) of Saccharomyces cerevisiae YJL049W.

Fig. 7 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 17), non-coding strand (SEQ ID NO : 19) and predicted amino acid sequence (SEQ ID NO : 18) of Saccharomyces cerevisiae YMR077C.

Fig. 8 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 20), non-coding strand (SEQ ID N0 : 22) and predicted amino acid sequence (SEQ ID NO : 21) of Saccharomyces cerevisiae YMR258C.

Fig. 9 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 23) and predicted amino acid sequence (SEQ ID N0 : 24) of Saccharomyces cerevisiae YDR325W.

Fig. 10 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 25), non-coding strand (SEQ ID NO : 27) and predicted amino acid sequence (SEQ ID NO : 26) of Saccharomyces cerevisiae YDR434W.

Fig. 11 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 28), non-coding strand (SEQ ID NO : 30) and predicted amino acid sequence (SEQ ID N0 : 29) of Saccharomyces cerevisiae YGL101W.

Fig. 12 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 31), non-coding strand (SEQ ID NO : 33) and predicted amino acid sequence (SEQ ID NO : 32) of Saccharomyces cerevisiae YGR1 13W.

Fig. 13 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 34), non-coding strand (SEQ ID NO : 36) and predicted amino acid sequence (SEQ ID NO : 35) of Saccharomyces cerevisiae YHR122W.

Fig. 14 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 37) and predicted amino acid sequence (SEQ ID N0 : 38) of Saccharomyces cerevisiae YJLOlOC.

Fig. 15 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 39) and predicted amino acid sequence (SEQ ID NO : 40) of Saccharomyces cerevisiae YLR100W.

Fig. 16 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 41), non-coding strand (SEQ ID NO : 43) and predicted amino acid sequence (SEQ ID NO : 42) of Saccharomyces cerevisiae YMR185W.

Fig. 17 is a listing of the nucleotide sequence of the coding strand (SEQ ID NO : 44) and predicted amino acid sequence (SEQ ID NO : 45) of Saccharomyces cerevisiae YOR166C.

Detailed Description of the Invention Seventeen genes of Saccharomyces cerevisiae have been identified as essential for survival of Saccharomyces. These 17 genes and polypeptides, as set forth in Table 1, are useful targets for identifying compounds that are inhibitors of the fungi in which the polypeptides are expressed.

Nucleic acids described herein include both RNA and DNA, including genomic DNA and synthetic (e. g., chemically synthesized) DNA. Nucleic acids can be double-stranded or single-stranded. Where single-stranded, the nucleic acid may be a sense strand or an antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (e. g., inosine or phosphorothioate nucleotides).

Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.

An isolated nucleic acid is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5'end and one on the 3'end) in the naturally occurring genome of the organism from which it is derived. Thus, an isolated nucleic acid includes some or all of the 5' non-coding (e. g., promoter) sequences that are immediately contiguous to the coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e. g., a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide

sequence. The term"isolated"refers to a nucleic acid or polypeptide that is substantially free of cellular or viral material with which they are naturally associated, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an isolated nucleic acid fragment is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.

A nucleic acid sequence that is substantially identical to an essential nucleotide sequence described herein is at least 80% identical to a nucleotide sequence as set forth in Table 1 and depicted in any of Figs. 1-17. For purposes of comparison of nucleic acids, the length of the reference nucleic acid sequence will generally be at least 40 nucleotides, e. g., at least 60 nucleotides or more nucleotides.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e. g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid 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 function of the number of identical positions shared by the sequences (i. e., % identity = # of identical positions/total # of overlapping positions x 100). Typically, the two sequences are the same length.

The determination of percent homology between two sequences can 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. USA 87 : 2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl Acad. Sci. USA 90 : 5873-5877.

Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. Mol. Biol. 215 : 403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the essential nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the essential

protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25 : 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e. g., XBLAST and NBLAST) can be used. See http ://www. ncbi. nlm. nih. gov. Another preferred, non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2. 0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.

The essential polypeptides useful in practicing the invention include, but are not limited to, recombinant polypeptides and natural polypeptides. Also included are nucleic acid sequences that encode forms of the essential polypeptides in which naturally occurring amino acid sequences are altered or deleted. Preferred nucleic acids encode polypeptides that are soluble under normal physiological conditions.

Also within the invention are nucleic acids encoding fusion proteins in which a portion of the essential polypeptide is fused to an unrelated polypeptide (e. g., a marker polypeptide or a fusion partner) to create a fusion protein. For example, the polypeptide can be fused to a hexa-histidine tag to facilitate purification of bacterially expressed polypeptides, or to a hemagglutinin tag to facilitate purification of polypeptides expressed in eukaryotic cells. The invention also includes, for example, isolated polypeptides (and the nucleic acids that encode these polypeptides) that include a first portion and a second portion ; the first portion includes, e. g., an essential polypeptide as described herein, and the second portion includes an immunoglobulin constant (Fc) region or a detectable marker.

The fusion partner can be, for example, a polypeptide which facilitates secretion, e. g., a secretory sequence. Such a fused polypeptide is typically referred to as a preprotein. The secretory sequence can be cleaved by the host cell to form the mature protein. Also within the invention are nucleic acids that encode an essential

polypeptide fused to a polypeptide sequence to produce an inactive preprotein.

Preproteins can be converted into the active form of the protein by removal of the inactivating sequence.

Useful nucleic acids for practicing the invention include nucleic acids that hybridize, e. g., under stringent hybridization conditions (as defined herein) to all or a portion of the nucleotide sequence represented by SEQ ID NO : 1, SEQ ID N0 : 4, SEQ ID NO : 7, SEQ ID NO : 10, SEQ ID NO : 13, SEQ ID NO : 15, SEQ ID N0 : 17, SEQ ID NO : 20, SEQ ID N0 : 23, SEQ ID NO : 25, SEQ ID NO : 28, SEQ ID NO : 31, SEQ ID NO : 34, SEQ ID N0 : 37, SEQ ID N0 : 39, SEQ ID N0 : 41, or SEQ ID N0 : 44, or their complements. The hybridizing portion of the hybridizing nucleic acids is typically at least 15 (e. g., 20, 30, or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 60%, e. g., at least 70%, 80%, 95%, or at least 98%, identical to the sequence of a portion or all of a nucleic acid encoding an essential polypeptide or its complement. Hybridizing nucleic acids of the type described herein can be used as a cloning probe, a primer (e. g., a PCR primer), or a diagnostic probe. Nucleic acids that hybridize to the coding strands of the nucleic acids described herein are considered"antisense oligonucleotides." Also useful in the invention are various engineered cells, e. g., transformed host cells, that contain an essential nucleic acid described herein. A transformed cell is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid encoding an essential polypeptide. Both prokaryotic and eukaryotic cells are included, e. g., fungi, and bacteria, such as E. coli, and the like.

Also useful in the invention are genetic constructs (e. g., vectors and plasmids) that include a nucleic acid of the invention which is operably linked to a transcription and/or translation sequence to enable expression, e. g., expression vectors. A selected nucleic acid, e. g., a DNA molecule encoding an essential polypeptide as described herein, is"operably linked"when it is positioned adjacent to one or more sequence elements, e. g., a promoter, which direct transcription and/or translation of the sequence such that the sequence elements can control transcription and/or translation of the selected nucleic acid.

Also useful in the invention are purified or isolated polypeptides encoded by the coding strands of the essential nucleic acid sequences disclosed herein. The terms

"protein"and"polypeptide"both refer to any chain of amino acids, regardless of length or post-translational modification (e. g., glycosylation or phosphorylation).

Thus, the term essential polypeptide includes full-length, naturally occurring, isolated essential proteins as described herein, as well as recombinantly or synthetically produced polypeptides that correspond to the full-length, naturally occurring proteins, or to a portion of the naturally occurring or synthetic polypeptide.

A purified or isolated compound is a composition that is at least 60% by weight the compound of interest, e. g., an essential polypeptide or antibody. Generally the preparation is at least 75% (e. g., at least 90%, 95%, or even 99%) by weight the compound of interest. Purity can be measured by any appropriate standard method, e. g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

Suitable essential polypeptides for use in the invention include a sequence substantially identical to all or a portion of a naturally occurring essential Saccharomyces polypeptide as shown in Figs. 1-17. Polypeptides"substantially identical"to the essential polypeptide sequences described herein have an amino acid sequence that is at least 65% identical to an amino acid sequence shown in Figs. 1-17 (measured as described herein). The new polypeptides can also have a greater percentage identity, e. g., 85%, 90%, 95%, or even higher. For purposes of comparison, the length of the reference essential Saccharomyces polypeptide sequence will generally be at least 16 amino acids, e. g., at least 20 or 25 amino acids.

In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups : glycine and alanine ; valine, isoleucine, and leucine ; aspartic acid and glutamic acid ; asparagine and glutamine ; serine and threonine ; lysine and arginine ; and phenylalanine and tyrosine.

Where a particular polypeptide is to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference polypeptide. Thus, a polypeptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It also might be a 100 amino acid long polypeptide which is 50% identical to the

reference polypeptide over its entire length. Of course, other polypeptides also will meet the same criteria.

The methods of the invention can utilize purified or isolated antibodies that specifically bind to an essential Saccharomyces polypeptide as described herein. An antibody"specifically binds"to a particular antigen when it binds to that antigen, e. g., an essential polypeptide described herein, but does not substantially recognize and bind to other molecules in a sample, e. g., a biological sample, that naturally includes an essential polypeptide described herein.

Identifying the Essential Saccharomyces Genes Disclosed Herein The essential Saccharomyces genes disclosed herein were identified as being essential as follows. First, transposon-tagged mutagenesis was used to mutate each gene, according to the method of Chun and Goebl (Genetics 142 : 39-50 (1996), incorporated herein by reference). Each of the resulting mutated genes was then rescued by plasmid rescue. For plasmid rescue, plasmid pJJ282 was linearized with ScaI and then used to transform each diploid, selecting for growth on leucine minus media. Five to ten transformants were then grown together for DNA isolation. The genomic DNA was digested with SacI, religated, and used to transform E. coli. Five to ten of the E. coli transformants were pooled for the preparation of plasmid DNA.

Plasmids shown by restriction digest to contain a URA3 gene were sequenced with a Tn3R primer. The essential genes disclosed herein were then cloned and sequenced using a mass cloning method. Briefly, 96 strains from a 96-well plate were grown together, and DNA was isolated from the culture. The isolated DNA was then digested with MunI and EcoRI and ligated into ZAP (Stratagene ; LaJolla, CA) at the EcoRI site. The ligation was used as a template to amplify by PCR the transposon flanking region of each strain. Two rounds of PCR were performed using a first primer in the URA3 gene (SCURA2) and T3 on the arms. The reaction was further amplified using Tn3R and T3. The PCR reaction was purified using Qiaprep columns (Qiagen, Inc.) and cloned into pT7blue T vector (Novogen). Individual E. coli colonies were cultured, and the plasmids were isolated and sequenced using Tn3R as a primer. The essential nature of the genes disclosed herein was verified by direct gene deletion (see Baudin et al., Nucl. Acids. Res. 21 : 3329-3330 (1993), incorporated herein by reference).

Identification of Homologous Genes in Additional Fungal Strains Since the Saccharomyces genes described herein have been identified as being essential, these genes, or fragments thereof, can be used to detect homologous genes in yet other organisms. Fragments of a nucleic acid (DNA or RNA) encoding an essential polypeptide described herein (or sequences complementary thereto) can be used as probes in conventional nucleic acid hybridization assays of various organisms.

For example, nucleic acid probes (which typically are 8-30, or usually 15-20, nucleotides in length) can be used to detect homologous genes in art-known molecular biology methods, such as Southern blotting, Northern blotting, dot or slot blotting, PCR amplification methods, colony hybridization methods, and the like. Typically, an oligonucleotide probe based on the nucleic acid sequences described herein, or fragment thereof, is labeled and used to screen a genomic library constructed from mRNA obtained from a fungal strain of interest. A suitable method of labeling involves using polynucleotide kinase to add 32P-labeled ATP to the oligonucleotide used as the probe. This method is well known in the art, as are several other suitable methods (e. g., biotinylation and enzyme labeling).

Hybridization of the oligonucleotide probe to the library, or other nucleic acid sample, typically is performed under stringent to highly stringency conditions.

Nucleic acid duplex or hybrid stability is expressed as the melting temperature or Tm, which is the temperature at which a probe dissociates from a target DNA. This melting temperature is used to define the required stringency conditions. If sequences are to be identified that are related and substantially identical to the probe, rather than identical, then it is useful to first establish the lowest temperature at which only homologous hybridization occurs with a particular concentration of salt (e. g., SSC or SSPE). Then, assuming that 1% mismatching results in a 1 °C decrease in the Tm, the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if sequences having > 95% identity with the probe are sought, the final wash temperature is decreased by 5 °C). In practice, the change in Tm can be between 0. 5 ° and 1. 5°C per 1% mismatch. High stringency conditions include are hybridizing at 68 °C in 5x SSC/5x Denhardt's solution/1. 0% SDS, or in 0. 5 M NaHP04 (pH 7. 2)/1 mM EDTA/7% SDS, or in 50% formamide/0. 25 M NaHP04 (pH 7. 2)/0. 25 M NaCI/1 mM EDTA/7% SDS ; and washing in 0. 2x SSC/0. 1% SDS at room temperature or at

42°C, or in O. lx SSC/0. 1% SDS at 68°C, or in 40 mM NaHP04 (pH 7. 2)/1 mM EDTA/5% SDS at 50°C, or in 40 mM NaHP04 (pH 7. 2) 1 mM EDTA/1% SDS at 50°C. Stringent conditions include washing in 3x SSC at 42°C. The parameters of salt concentration and temperature can be varied to achieve the optimal level of identity between the probe and the target nucleic acid. Additional guidance regarding such conditions is readily available in the art, for example, by Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N. Y. ; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N. Y.) at Unit 2. 10.

In one approach, libraries constructed from pathogenic or non-pathogenic fungal strains can be screened. For example, such strains can be screened for expression of the essential genes described herein by Northern blot analysis. Upon detection of transcripts of the essential genes thereof, libraries can be constructed from RNA isolated from the appropriate strain, utilizing standard techniques well known to those of skill in the art. Alternatively, a total genomic DNA library can be screened using an essential gene probe.

New gene sequences can be isolated, for example, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences within the essential genes as depicted herein. The template for the reaction can be DNA obtained from strains known or suspected to express an essential gene as described herein. The PCR product can be subcloned and sequenced.

Synthesis of the various essential polypeptides (or an antigenic fragment thereof) for use as antigens, or for other purposes, can be readily accomplished using any of the various art-known techniques. For example, an essential polypeptide, or an antigenic fragment (s), can be synthesized chemically in vitro, or enzymatically (e. g., by in vitro transcription and translation). Alternatively, the gene can be expressed in, and the polypeptide purified from, a cell (e. g., a cultured cell) by using any of the numerous, available gene expression systems. For example, the polypeptide antigen can be produced in a prokaryotic host (e. g., E. coli) or in eukaryotic cells, such as yeast cells.

Proteins and polypeptides can also be produced in plant cells, if desired. For plant cells, viral expression vectors (e. g., cauliflower mosaic virus and tobacco mosaic virus) and plasmid expression vectors (e. g., Ti plasmid) are suitable. Such

cells are available from a wide range of sources (e. g., the American Type Culture Collection ; also, see, e. g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994). The optimal methods of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e. g., in Ausubel et al., supra ; expression vehicles may be chosen from those provided, e. g., in Cloning Vectors : A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987). The host cells harboring the expression vehicle can be cultured in conventional nutrient media, adapted as needed for activation of a chosen gene, repression of a chosen gene, selection of transformants, or amplification of a chosen gene.

If desired, the essential polypeptides described herein can be produced as fusion proteins. For example, the expression vector pUR278 (Ruther et al., EMBO J., 2 : 1791, 1983) can be used to create lacZ fusion proteins. The art-known pGEX vectors can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an exemplary expression system, a baculovirus such as Autographa californica nuclear polyhedrosis virus (AcNPV), which grows in Spodoptera frugiperda cells, can be used as a vector to express foreign genes. A coding sequence encoding an essential polypeptide can be cloned into a non-essential region (for example the polyhedrin gene) of the viral genome and placed under control of a promoter, e. g., the polyhedrin promoter or an exogenous promoter. Successful insertion of a gene encoding an essential polypeptide can result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i. e., virus lacking the proteinaceous coat encoded by the polyhedrin gene). These recombinant viruses are then typically used to infect insect cells (e. g., Spodopterafrugiperda cells) in which the inserted gene is expressed see, e. g., Smith et al., J Virol., 46 : 584, 1983 ; Smith, U. S. Patent No. 4, 215, 051). If desired, mammalian cells can be used in lieu of insect cells, provided that the virus is engineered such that the gene encoding the

Essential polypeptide is placed under the control of a promoter that is active in mammalian cells.

In mammalian host cells, a number of viral-based expression systems can be utilized. When an adenovirus is used as an expression vector, the nucleic acid sequence encoding the essential polypeptide 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 into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e. g., region El or E3) will result in a recombinant virus that is viable and capable of expressing an Essential gene product in infected hosts (see, e. g., Logan, Proc. Natl. Acad. Sci. USA, 81 : 3655, 1984).

Specific initiation signals may be required for efficient translation of inserted nucleic acid sequences. These signals include the ATG initiation codon and adjacent sequences. In general, exogenous translational control signals, including, perhaps, the ATG initiation codon, should be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire sequence. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, or transcription terminators (Bittner et al., Methods in Enzymol., 153 : 516, 1987).

The essential polypeptides described herein can be expressed individually or as a fusion with a heterologous polypeptide, such as a signal sequence or other polypeptide having a specific cleavage site at the N-and/or C-terminus of the protein or polypeptide. The heterologous signal sequence selected should be one that is recognized and processed, i. e., cleaved by a signal peptidase, by the host cell in which the fusion protein is expressed.

A host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion.

Such modifications and processing (e. g., cleavage) of protein products may facilitate optimal functioning of the protein. Various host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those of skill in the art of

molecular biology can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.

If desired, the essential polypeptides described herein can be produced by a stably-transfected mammalian cell line. A number of vectors suitable for stable transection of mammalian cells are available to the public, see, e. g., Pouwels et al.

(supra) ; methods for constructing such cell lines are also publicly known, e. g., in Ausubel et al. (supra). In one example, DNA encoding the protein is cloned into an expression vector that includes the dihydrofolate reductase (DHFR) gene. Integration of the plasmid and, therefore, the essential polypeptide-encoding gene into the host cell chromosome is selected for by including 0. 01-300 AM methotrexate in the cell culture medium (as described in Ausubel et al., supra). This dominant selection can be accomplished in most cell types.

Recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described in Ausubel et al. (supra) ; such methods generally involve extended culture in medium containing gradually increasing levels of methotrexate.

DHFR-containing expression vectors commonly used for this purpose include pCVSEII-DHFR and pAdD26SV (A) (described in Ausubel et al., supra).

A number of other selection systems can be used, including but not limited to, herpes simplex virus thymidine kinase genes, hypoxanthine-guanine phosphoribosyl- transferase genes, and adenine phosphoribosyltransferase genes, which can be employed in tk, hgprt, or aprt cells, respectively. In addition, gpt, which confers resistance to mycophenolic acid (Mulligan et al., Proc. Natl. Acad. Sci. USA, 78 : 2072, 1981) ; neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., J. Mol. BioL, 150 : 1, 1981) ; and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30 : 147, 1981), can be used.

Alternatively, any fusion protein can be readily purified by utilizing an antibody or other molecule that specifically binds the fusion protein being expressed.

For example, a system described in Janknecht et al., Proc. Natl. Acad. Sci. USA,

88 : 8972 (1981), allows for the ready purification of non-denatured fusion proteins expressed in human cell lines. In this system, 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 Ni2+ nitriloacetic acid-agarose columns, and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.

Alternatively, an essential polypeptide as described herein, or a portion thereof, can be fused to an immunoglobulin Fc domain. Such a fusion protein can be readily purified using a protein A column, for example. Moreover, such fusion proteins permit the production of a chimeric form of the essential polypeptide having increased stability in vivo.

Once the recombinant polypeptide is expressed, it can be isolated (i. e., purified). Secreted forms of the polypeptides can be isolated from cell culture media, while non-secreted forms must be isolated from the host cells. Polypeptides can be isolated by affinity chromatography. For example, an anti-YDR416W antibody (e. g., produced as described herein) can be attached to a column and used to isolate the protein. Lysis and fractionation of cells harboring the protein prior to affinity chromatography can be performed by standard methods (see, e. g., Ausubel et al., supra). Alternatively, a fusion protein can be constructed and used to isolate an essential polypeptide (e. g., a YDR416W-maltose binding fusion protein, a YDR416W--galactosidase fusion protein, or a YDR416W-trpE fusion protein ; see, e. g., Ausubel et al., supra ; New England Biolabs Catalog, Beverly, MA). The recombinant protein can, if desired, be further purified, e. g., by high performance liquid chromatography using standard techniques (see, e. g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, eds., Work and Burdon, Elsevier, 1980).

Given the amino acid sequences described herein, polypeptides useful in practicing the invention, particularly fragments of essential polypeptides, can be produced by standard chemical synthesis (e. g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., The Pierce Chemical Co., Rockford, IL, 1984) and used as antigens, for example.

Antibodies The essential polypeptides described herein (or antigenic fragments or analogs of such polypeptides) can be used to raise antibodies useful in the invention, and such polypeptides can be produced by recombinant or peptide synthetic techniques (see, e. g., Solid Phase Peptide Synthesis, supra ; Ausubel et al., supra). In general, the polypeptides can be coupled to a carrier protein, such as KLH, as described in Ausubel et al., supra, mixed with an adjuvant, and injected into a host mammal. A "carrier"is a substance that confers stability on, and/or aids or enhances the transport or immunogenicity of, an associated molecule. Antibodies can be purified, for example, by affinity chromatography methods in which the polypeptide antigen is immobilized on a resin.

In particular, various host animals can be immunized by injection of a polypeptide of interest. Examples of suitable host animals include rabbits, mice, guinea pigs, and rats. 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 adjuvant), adjuvant mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of the immunized animals.

Antibodies useful in the invention include monoclonal antibodies, polyclonal antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab') 2 fragments, and molecules produced using a Fab expression library.

Monoclonal antibodies (mAbs), which are homogeneous populations of antibodies to a particular antigen, can be prepared using the essential polypeptides disclosed herein, and standard hybridoma technology (see, e. g., Kohler et al., Nature, 256 : 495, 1975 ; Kohler et al., Eur. J. Immunol., 6 : 511, 1976 ; Kohler et al., Eur. J.

Immunol., 6 : 292, 1976 ; Hammering et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981 ; Ausubel et al., supra).

In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture, such as those described in Kohler et al., Nature, 256 : 495, 1975, and U. S. Patent

No. 4, 376, 110 ; the human B-cell hybridoma technique (Kosbor et al., Immunology Today, 4 : 72, 1983 ; Cole et al., Proc. Natl. Acad. Sci. USA, 80 : 2026, 1983) ; and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1983). Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridomas producing the mAbs of this invention can be cultivated in vitro or in vivo.

Once produced, polyclonal or monoclonal antibodies are tested for specific recognition of the essential polypeptides disclosed herein in an immunoassay, such as a Western blot or immunoprecipitation analysis using standard techniques, e. g., as described in Ausubel et al., supra. Antibodies that specifically bind to the essential polypeptides described herein, or conservative variants or homologs are useful in the invention. For example, such antibodies can be used in an immunoassay to detect an YDR416W polypeptide in pathogenic or non-pathogenic strains of fungi.

Antibodies of the invention can be produced using fragments of the essential polypeptides that appear likely to be antigenic, by criteria such as high frequency of charged residues. In one specific example, such fragments are generated by standard techniques of PCR, and are then cloned into the pGEX expression vector (Ausubel et al., supra). Fusion proteins are expressed in E. coli and purified using a glutathione agarose affinity matrix as described in Ausubel, et al., supra.

If desired, several (e. g., two or three) fusions can be generated for each protein, and each fusion can be injected into at least two rabbits. Antisera can be raised by injections in a series, typically including at least three booster injections.

Typically, the antisera is checked for its ability to immunoprecipitate a recombinant essential polypeptide, or unrelated control proteins, such as glucocorticoid receptor, chloramphenicol acetyltransferase, or luciferase.

Techniques developed for the production of"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci., 81 : 6851, 1984 ; Neuberger et al., Nature, 312 : 604, 1984 ; Takeda et al., Nature, 314 : 452, 1984) can be used to splice the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity. 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.

Alternatively, techniques described for the production of single chain antibodies (U. S. Patent 4, 946, 778 ; and U. S. Patents 4, 946, 778 and 4, 704, 692) can be adapted to produce single chain antibodies against an essential polypeptide described herein. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can be generated by known techniques. For example, such fragments can include but are not limited to F (ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of F (ab') 2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., Science, 246 : 1275, 1989) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

Polyclonal and monoclonal antibodies that specifically bind to an essential polypeptide can be used, for example, to detect expression of Essential in another strain of fungi. For example, an Essential polypeptide can be readily detected in conventional immunoassays of fungal cells or extracts. Examples of suitable assays include, without limitation, Western blotting, ELISAs, radioimmune assays, and the like.

Assay for Antifungal Agents The invention provides a method for identifying an antifungal agent (s).

Although the inventor is not bound by any particular theory as to the biological mechanism involved, the new antifungal agents are thought to inhibit specifically (1) the function of the essential polypeptides described herein or (2) expression of the essential genes described herein. Screening for antifungal agents can be accomplished by identifying those compounds (e. g., small organic molecules) that inhibit the activity of an essential polypeptide or the expression of an essential gene described herein.

Screening for antifungal agents can be accomplished by (i) identifying those compounds that bind to the essential polypeptides described herein and (ii) further testing such compounds for their ability to inhibit fungal growth in vitro or in vivo.

Specific binding of a test compound to a polypeptide can be detected, for example, in vitro by reversibly or irreversibly immobilizing the test compound (s) on a substrate, e. g., the surface of a well of a 96-well polystyrene microtitre plate.

Methods for immobilizing polypeptides and other small molecules are well known in the art. For example, the microtitre plates can be coated with a test polypeptide (s), for example, by adding the polypeptide (s) in a solution (typically, at a concentration of 0. 05 to 1 mg/ml in a volume of 1-100 » 1) to each well, and incubating the plates at room temperature to 37°C for 0. 1 to 36 hours. Polypeptides that are not bound to the plate can be removed by shaking the excess solution from the plate, and then washing the plate (once or repeatedly) with water or a buffer. Typically, the polypeptide is in water or a buffer. The plate is then washed with a buffer that lacks the bound polypeptide. To block the free protein-binding sites on the plates, the plates are blocked with a protein that is unrelated to the bound polypeptide. For example, 300 121 of bovine serum albumin (BSA) at a concentration of 2 mg/ml in Tris-HCl is suitable. Suitable substrates include those substrates that contain a defined cross- linking chemistry (e. g., plastic substrates, such as polystyrene, styrene, or polypropylene substrates from Coming Costar Corp. (Cambridge, MA), for example).

If desired, a beaded particle, e. g., beaded agarose or beaded sepharose, can be used as the substrate.

The essential polypeptide (typically 0. 05 to 1 mg/ml in 1-100 ßQ) then is allowed to contact the bound substrate at room temperature 37°C for 0. 1 to 36 hours.

Excess polypeptide can be removed as described above. Interaction of the test compound with polypeptide can be detected by any of a variety of art-known methods. For example, an antibody that specifically binds a YDR416W polypeptide can be used in an immunoassay to detect the YDR416W polypeptide. If desired, the antibody can be labeled (e. g., fluorescently or with a radioisotope) and detected directly (see, e. g., West and McMahon, J. Cell Biol. 74 : 264, 1977). Alternatively, a second antibody can be used for detection (e. g., a labeled antibody that binds the Fc portion of an anti-YDR416W antibody). In an alternative detection method, the YDR416W polypeptide is labeled, and the label is detected (e. g., by labeling a YDR416W polypeptide with a radioisotope, fluorophore, chromophore, or the like).

In still another method, the YDR416W polypeptide is produced as a fusion protein with a protein that can be detected optically, e. g., green fluorescent protein (which can

be detected under W light). In an alternative method, the polypeptide (e. g., YDR416W) can be produced as a fusion protein with an enzyme having a detectable enzymatic activity, such as horse radish peroxidase, alkaline phosphatase,- galactosidase, or glucose oxidase. Genes encoding all of these enzymes have been cloned and are readily available for use by those of skill in the art. If desired, the fusion protein can include an antigen, and such an antigen can be detected and measured with a polyclonal or monoclonal antibody using conventional methods.

Suitable antigens include enzymes (e. g., horse radish peroxidase, alkaline phosphatase, and-galactosidase) and non-enzymatic polypeptides (e. g., serum proteins, such as BSA and globulins, and milk proteins, such as caseins).

In various in vivo methods for identifying polypeptides that bind to the essential polypeptide, the conventional two-hybrid assays of protein/protein interactions can be used (see e. g., Chien et al., Proc. Natl. Acad. Sci. USA, 88 : 9578, 1991 ; Fields et al., U. S. Pat. No. 5, 283, 173 ; Fields and Song, Nature, 340 : 245, 1989 ; Le Douarin et al., Nucleic Acids Research, 23 : 876, 1995 ; Vidal et al., Proc. Natl.

Acad Sci. USA, 93 : 10315-10320, 1996 ; and White, Proc. Natl. Acad. Sci. USA, 93 : 10001-10003, 1996). Generally, the two-hybrid methods involve in vivo reconstitution of two separable domains of a transcription factor. One fusion protein contains the essential polypeptide fused to either a transactivator domain or DNA binding domain of a transcription factor (e. g., of Gal4). The other fusion protein contains a test polypeptide fused to either the DNA binding domain or a transactivator domain of a transcription factor. Once brought together in a single cell (e. g., a yeast cell or mammalian cell), one of the fusion proteins contains the transactivator domain and the other fusion protein contains the DNA binding domain. Therefore, binding of the essential polypeptide to the test polypeptide (i. e., candidate antifungal agent) reconstitutes the transcription factor. Reconstitution of the transcription factor can be detected by detecting expression of a gene (i. e., a reporter gene) that is operably linked to a DNA sequence that is bound by the DNA binding domain of the transcription factor. Kits for practicing various two-hybrid methods are commercially available (e. g., from Clontech ; Palo Alto, CA).

The methods described above can be used for high throughput screening of numerous test compounds to identify candidate antifungal (or anti-fungal) agents.

Having identified a test compound as a candidate antifungal agent, the candidate

antifungal agent can be further tested for inhibition of fungal growth in vitro or in vivo (e. g., using an animal, e. g., rodent, model system) if desired. Using other, art-known variations of such methods, one can test the ability of a nucleic acid (e. g., DNA or RNA) used as the test compound to bind to an essential polypeptide.

In vitro, further testing can be accomplished by means known to those in the art such as an enzyme inhibition assay or a whole-cell fungal growth inhibition assay.

For example, an agar dilution assay identifies a substance that inhibits fungal growth.

Microtiter plates are prepared with serial dilutions of the test compound, adding to the preparation a given amount of growth substrate, and providing a preparation of fungi.

Inhibition of fungal growth is determined, for example, by observing changes in optical densities of the fungal cultures.

Inhibition of fungal growth is demonstrated, for example, by comparing (in the presence and absence of a test compound) the rate of growth or the absolute growth of fungal cells. Inhibition includes a reduction of one of the above measurements by at least 20%. Particularly potent test compounds may further reduce the growth rate (e. g., by at least 25%, 30%, 40%, 50%, 75%, 80%, or 90%).

Animal (e. g., rodent such as murine) models of fungal infections are known to those of skill in the art, and such animal model systems are accepted for screening antifungal agents as an indication of their therapeutic efficacy in human patients. In a typical in vivo assay, an animal is infected with a pathogenic strain of fungi, e. g., by inhalation of fungi, and conventional methods and criteria are used to diagnose the mammal as being afflicted with a fungal infection. The candidate antifungal agent then is administered to the mammal at a dosage of 1-100 mg/kg of body weight, and the mammal is monitored for signs of amelioration of disease. Alternatively, the test compound can be administered to the mammal prior to infecting the mammal with the fungi, and the ability of the treated mammal to resist infection is measured. Of course, the results obtained in the presence of the test compound should be compared with results in control animals, which are not treated with the test compound.

Administration of candidate antifungal agents to the mammal can be carried out as described below, for example.

Pharmaceutical Formulations Treatment includes administering a pharmaceutically effective amount of a composition containing an antifungal agent to a subject in need of such treatment, thereby inhibiting fungal growth in the subject. Such a composition typically contains from about 0. 1 to 90% by weight (such as 1 to 20% or 1 to 10%) of an antifungal agent of the invention (e. g., a polypeptide, ribonucleic acid, small molecule, deoxyribonucleic acid, antisense molecule, or ribozyme) in a pharmaceutically acceptable carrier.

Solid formulations of the compositions for oral administration may contain suitable carriers or excipients, such as corn starch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodium chloride, or alginic acid. Disintegrators that can be used include, without limitation, micro-crystalline cellulose, corn starch, sodium starch glycolate and alginic acid. Tablet binders that may be used include acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Povidone), hydroxypropyl methylcellulose, sucrose, starch, and ethylcellulose. Lubricants that may be used include magnesium stearates, stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica.

Liquid formulations of the compositions for oral administration prepared in water or other aqueous vehicles may contain various suspending agents such as methylcellulose, alginates, tragacanth, pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone, and polyvinyl alcohol. The liquid formulations may also include solutions, emulsions, syrups and elixirs containing, together with the active compound (s), wetting agents, sweeteners, and coloring and flavoring agents. Various liquid and powder formulations can be prepared by conventional methods for inhalation into the lungs of the mammal to be treated.

Injectable formulations of the compositions may contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injections, water soluble versions of the compounds may be administered by the drip method, whereby a pharmaceutical formulation containing the antifungal agent and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5%

dextrose, 0. 9% saline, Ringer's solution or other suitable excipients. Intramuscular preparations, a sterile formulation of a suitable soluble salt form of the compounds can be dissolved and administered in a pharmaceutical excipient such as Water-for- Injection, 0. 9% saline, or 5% glucose solution. A suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, such as an ester of a long chain fatty acid, (e. g., ethyl oleate).

A topical semi-solid ointment formulation typically contains a concentration of the active ingredient from about 1 to 20%, e. g., 5 to 10% in a carrier such as a pharmaceutical cream base. Various formulations for topical use include drops, tinctures, lotions, creams, solutions, and ointments containing the active ingredient and various supports and vehicles.

The optimal percentage of the antifungal agent in each pharmaceutical formulation varies according to the formulation itself and the therapeutic effect desired in the specific pathologies and correlated therapeutic regimens. Appropriate dosages of the antifungal agents can be readily determined by those of ordinary skill in the art of medicine by monitoring the mammal for signs of disease amelioration or inhibition, and increasing or decreasing the dosage and/or frequency of treatment as desired. The optimal amount of the antifungal compound used for treatment of conditions caused by or contributed to by fungal infection may depend upon the manner of administration, the age and the body weight of the subject, and the condition of the subject to be treated. Generally, the antifungal compound is administered at a dosage of 1 to 100 mg/kg of body weight, and typically at a dosage of 1 to 10 mg/kg of body weight.

Other Embodiments It is to be understood that, while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. For example, other art-known assays to detect interactions of test compounds with proteins, or to detect inhibition of fungal growth also can be used with the essential genes described herein. The invention also

includes methods of making a pharmaceutical composition for use in inhibiting fungi. Specifically, the method includes formulating a pharmaceutically acceptable excipient with an antifungal agent, such as those described herein.

What is claimed is :