Field of the Invention

The present invention relates generally to phosphatidylserine synthase enzymes, particularly Candida albicans and Aspergillus fumigates enzymes, and more specifically to methods of identifying inhibitors of the enzymes for use as antifungal agents.

Background Phosphatidylserine is a phospholipid that is a major component of cell membranes of many organisms, from bacteria to fungi to mammals. The synthesis of phosphatidylserine is accomplished in the yeast S. cerevisiae by the enzyme CDP- diacylglyceroLL-serine O-phosphatidyl transferase otherwise known as phosphatidylserine synthase EC2.7.8.8. [Letts, et al., Proc NatlAcadSci USA, 80:7279-83 (1983)]. The yeast enzyme catalyzes the formation of phosphatidylserine from CDP-diacylglycerol and serine. In Chinese hamster ovary cells, the synthesis of phosphatidylserine is carried out by two different enzymes PSS I and PSS II. [Kuge and Nishijima, Biochim Biophys Acta, 4:1-2 (1997)]. PSS I converts phosphatidylcholine to phosphatidylserine and PSS II converts phosphatidylethanolarnine to phosphatidylserine. Phosphatidylserine synthases are thus important participants in the generation and maintenance of cell membranes and in overall lipid metabolism in cells.

Disruption of the fungal cell membrane has been a useful therapeutic strategy against fungi and parasites. For example, amphotericin B and fluconazole exert their anti-fungal activity by affecting membrane steroids. Despite the existence of antifungal therapeutics, fungal infections of humans have increasingly become responsible for life-threatening disorders. See, Georgopapadakou et al., Trends Microbiol., 3:98-104 (1995). The fungal species and parasites responsible for these diseases are mainly Candida, Aspergillus, Cryptococcus, Histoplasma, Coccidioides and Pneumocystis. These pathogens are particularly dangerous in immunocompromised individuals, such as patients with AIDS, patients undergoing chemotherapy, and immunosuppressed organ transplant patients.

Summary of the Invention

Noting, for example, the prevalence of fungal infections and the increasing incidence of life-threatening fungal infection in immunocompromised individuals, the present inventor recognized a need in the art to identify and isolate Candida albicans and Aspergillus fumigatus polynucleotides encoding phosphatidylserine synthases, to develop materials and methods useful for the recombinant production of the enzymes, and to identify inhibitors of the Candida albicans and Aspergillus fumigatus enzymes for use as antifungal agents. As fungal phosphatidylserine and human phosphatidylserine are made by different enzymes, it is contemplated that the inhibitors would not be toxic to humans or other animals.

The present invention provides purified, isolated Candida albicans and Aspergillus fumigatus polynucleotides (i.e., DNA and RNA, both sense and antisense strands) encoding phosphatidylserine synthases and analogs thereof; methods for the recombinant production of Candida albicans and Aspergillus fumigatus phosphatidylserine synthases, fragments and analogs thereof; purified, isolated

Candida albicans and Aspergillus fumigatus phosphatidyl synthase fragments and analogs; antibodies to such Candida albicans and Candida albicans phosphatidylserine synthases, fragments and analogs; and to methods of identifying inhibitors of such Candida albicans and Aspergillus fumigatus phosphatidylserine synthases.

Specifically provided are: purified, isolated polynucleotides encoding the phosphatidylserine synthase amino acid sequence of SEQ ID NOS: 2 {Candida albicans) or 4 (Aspergillus fumigatus); purified, isolated DNAs comprising the protein coding nucleotides of SEQ ID NOS: 1 {Candida albicans) or 3 {Aspergillus fumigatus); purified, isolated polynucleotides encoding phosphatidylserine synthase selected from the group consisting of: (a) a double-stranded DNA comprising the protein coding portions of the sequence set out in either SEQ ID NO: 1 or SEQ ID NO: 3, (b) a DNA which hybridizes under stringent conditions to a non-coding strand of the DNA of (a), and (c) a DNA which, but for the redundancy of the genetic code, would hybridize under stringent conditions to a non-coding strand of DNA sequence of (a) or (b); vectors comprising such DNAs, particularly expression vectors wherein the DNA is operatively linked to an expression control DNA sequence; host cells stably transformed or transfected with such DNAs in a manner allowing the expression in said host cell of phosphatidylserine synthase; a method for producing

phosphatidylserine synthase comprising culturing such host cells in a nutrient medium and isolating phosphatidylserine synthase from said host cell or said nutrient medium; purified, isolated polypeptides produced by this method; purified, isolated enzymes comprising the phosphatidylserine synthase amino acid sequence of SEQ ID NOS: 2 or 4; hybridoma cell lines producing a monoclonal antibody that is specifically reactive with enzymes comprising the phosphatidylserine synthase amino acid sequence of SEQ ID NOS: 2 or 4; and monoclonal antibodies produced by such hybridomas.

DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences. The nucleotide sequence of genomic DNAs encoding Candida albicans and Aspergillus fumigatus phosphatidylserine synthases are respectively set forth in SEQ ID NO: 1 and SEQ ID NO: 3. These DNA sequences and DNA sequences which hybridize to the non- coding strand thereof under standard stringent conditions or which would hybridize but for the redundancy of the genetic code, are contemplated by the invention. Certain embodiments of DNAs of the present invention comprise the phosphatidylserine synthase coding region (corresponding to nucleotides 968 to 1789 of SEQ ID NO: 1 or nucleotides 1001 to 1180 and 1240 to 1608 of SEQ ID NO: 3). Exemplary stringent hybridization conditions are as follows: hybridization at 42°C in 50% formamide and washing at 60 ⁰ C in 0.1 x SSC, 0.1% SDS. It is understood by those of skill in the art that variation in these conditions occurs based on the length and GC nucleotide base content of the sequences to be hybridized. Formulas standard in the art are appropriate for determining exact hybridization conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

Polynucleotides according to the invention can have, e.g., at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94% and even more typically at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 or 3. Among the uses for the polynucleotides of the present invention is use as a hybridization probe, to identify and isolate ^" cDNA encoding phosphatidylserine synthase; to identify and isolate non-human and human genes encoding proteins homologous to phosphatidylserine synthase; to identify non-human and human proteins having similarity to phosphatidylserine synthase; and to identify those cells

which express phosphatidylserine synthase and the biological conditions under ^, which this protein is expressed. Also made available by the invention are anti-sense polynucleotides relevant to regulating expression of phosphatidylserine synthase by those cells which ordinarily express it. Accordingly, in one embodiment the invention provides for the use of antisense oligonucleotides which negatively regulate phosphatidylserine synthase expression via hybridization to messenger RNA (mRNA) encoding phosphatidylserine synthase. In one aspect, oligonucleotides that decrease phosphatidylserine synthase expression may be used in the methods of the invention. Antisense oligonucleotides at least 5 to about 50 nucleotides in length, including all lengths (measured in number of nucleotides) in between, which specifically hybridize to mRNA encoding phosphatidylserine synthase and inhibit mRNA expression, and as a result phosphatidylserine synthase protein expression, are contemplated for use in the methods of the invention. Antisense oligonucleotides include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo. It is understood in the art that, while antisense oligonucleotides that are perfectly complementary to a region in the target polynucleotide possess the highest degree of specific inhibition, antisense oligonucleotides that are not perfectly complementary, i.e., those which include a limited number of mismatches with respect to a region in the target polynucleotide, also retain high degrees of hybridization specificity and therefore also can inhibit expression of the target mRNA. Accordingly, the invention contemplates methods using antisense oligonucleotides that are perfectly complementary to a target region in a polynucleotide encoding phosphatidylserine synthase, as well as methods that utilize antisense oligonucleotides that are not perfectly complementary (i.e., include mismatches) to a target region in the target polynucleotide to the extent that the mismatches do not preclude specific hybridization to the target region in the target polynucleotide. Methods for designing and optimizing antisense nucleotides are described in Lima et ah, J Biol. Chem.,

272:626-38 (1997); Kurreck et al., Nucleic Acids Res., 30:1911-1918 (2002) and U.S. Patent No. 6,277,981, which are incorporated herein by reference.

The invention further contemplates methods utilizing ribozyme inhibitors which, as is known in the art, include a nucleotide region which specifically

hybridizes to a target polynucleotide and an enzymatic moiety that digests the target polynucleotide. Specificity of ribozyme inhibition is related to the length the antisense region and the degree of complementarity of the antisense region to the target region in the target polynucleotide. The methods of the invention therefore contemplate ribozyme inhibitors comprising antisense regions from 5 to about 50 nucleotides in length, including all nucleotide lengths in between, that are perfectly complementary, as well as antisense regions that include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target phosphatidylserine synthase-encoding polynucleotide. Ribozymes useful in methods of the invention include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo, to the extent that the modifications do not alter the ability of the ribozyme to specifically hybridize to the target region or diminish enzymatic activity of the molecule. Because ribozymes are enzymatic, a single molecule is able to direct digestion of multiple target molecules thereby offering the advantage of being effective at lower concentrations than non-enzymatic antisense oligonucleotides. Preparation and use of ribozyme technology is described in U.S. Patent Nos. 6,696,250, 6,410,224 and 5,225,347, which are incorporated herein by reference.

The invention also contemplates use of methods in which RNAi technology is utilized for inhibiting phosphatidylserine synthase expression. In one aspect, the invention provides double-stranded RNA (dsRNA) wherein one strand is complementary to a target region in a target phosphatidylserine synthase -encoding polynucleotide. In general, dsRNA molecules of this type are less than 30 nucleotides in length and referred to in the art as short interfering RNA (siRNA). The invention also contemplates, however, use of dsRNA molecules longer than 30 nucleotides in length, and in certain aspects of the invention, these longer dsRNA molecules can be about 30 nucleotides in length up to 200 nucleotides in length and longer, and including all length dsRNA molecules in between. As with other RNA inhibitors, complementarity of one strand in the dsRNA molecule can be a perfect match with the target region in the target polynucleotide, or may include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the

target phosphatidylserine synthase -encoding polynucleotide. As with other RNA inhibition technologies, dsRNA molecules include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo.

Preparation and use of RNAi using double stranded (dsRNA) [Fire et ah, Nature, 391: 806-811 (1998) incorporated by reference herein] or short-interfering RNA (siRNA) sequences [Yu et ai, Proc. Natl. Acad. Sci. U S A. , 99: 6047-6052 (2002) and compounds are further described in U.S. Patent Application No. 20040023390, both of which are incorporated by reference herein] .

The invention further contemplates methods wherein inhibition of phosphatidylserine synthase is effected using RNA lasso technology. Circular RNA lasso inhibitors are highly structured molecules that are inherently more resistant to degradation and therefore do not, in general, include or require modified internucleotide linkage or modified nucleotides. The circular lasso structure includes a region that is capable of hybridizing to a target region in a target polynucleotide, the hybridizing region in the lasso being of a length typical for other RNA inhibiting technologies. As with other RNA inhibiting technologies, the hybridizing region in the lasso may be a perfect match with the target region in the target polynucleotide, or may include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target phosphatidylserine synthase-encoding polynucleotide. Because RNA lassos are circular and form tight topological linkage with the target region, inhibitors of this type are generally not displaced by helicase action unlike typical antisense oligonucleotides, and therefore can be utilized as dosages lower than typical antisense oligonucleotides. Preparation and use of RNA lassos is described in U.S. Patent 6,369,038, incorporated herein by reference.

The inhibitors of the invention may be covalently or noncovalently associated with a carrier molecule, such as a linear polymer (e.g. , polyethylene glycol, polylysine, dextran, etc.), a branched-chain polymer (see U.S. Patents 4,289,872 and 5,229,490; PCT Publication WO 93/21259 published 28 October 1993); a lipid; a cholesterol group (such as a steroid); or a carbohydrate or oligosaccharide. Specific examples of carriers for use in the pharmaceutical compositions of the invention include carbohydrate-based polymers, such as trehalose, mannitol, xylitol, sucrose,

lactose, sorbitol, dextrans, such as cyclodextran, cellulose, and cellulose derivatives. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.

Other carriers include one or more water soluble polymer attachments such as polyoxyethylene glycol, or polypropylene glycol as described U.S. Patent Nos: 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337. Still other useful carrier polymers known in the art include monomethoxy-polyethylene glycol, poly-(N- vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethyl ene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of these polymers.

In another aspect, autonomously replicating recombinant constructions such as plasmid and viral DNA vectors incorporating phosphatidylserine synthase polynucleotides, including any of the DNAs described above, are provided. Preferred vectors include expression vectors in which the incorporated phosphatidylserine synthase-encoding DNA is operatively linked to an endogenous or heterologous expression control sequence and a transcription terminator. Such expression vectors may further include polypeptide-encoding DNA sequences operably linked to the phosphatidylserine synthase-encoding DNA sequences, which vectors may be expressed to yield a fusion protein comprising the polypeptide of interest. According to another aspect of the invention, prokaryotic or eukaryotic host cells are stably transformed or transfected with DNA sequences of the invention in a manner allowing the desired phosphatidylserine synthase product to be expressed therein. Host cells expressing phosphatidylserine synthase products can serve a variety of useful purposes. Such cells constitute a valuable source of immunogen for the development of antibody substances specifically immunoreactive with phosphatidylserine synthase. Host cells of the invention are useful in methods for the large scale production of phosphatidylserine synthase wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated, e.g., by immunoaffinity purification, from the cells or from the medium in which the cells are grown.

Phosphatidylserine synthase products may be obtained as isolates from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving prokaryotic or eukaryotic host cells of the

invention. Phosphatidylserine synthase products of the invention may be full length polypeptides, fragments or analogs thereof. Phosphatidylserine synthase products having part or all of the amino acid sequence set out in SEQ ID NO:, 2 or SEQ ID NO: 4 are contemplated. Analogs may comprise phosphatidylserine synthase analogs wherein one or more of the specified (i.e., naturally encoded) amino acids is deleted or replaced or wherein one or more non-specified amino acids are added: (1) without loss of one or more of the enzymatic activities or immunological characteristics specific to phosphatidylserine synthase; or (2) with specific disablement of a particular biological activity of phosphatidylserine synthase. In addition to compounds of that selectively negatively regulate phosphotidylserine synthase expression, polypeptides, peptides, or other molecules that bind to phosphatidylserine synthase may be used to modulate its activity. Some polypeptides comprehended by the present invention are antibody substances (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, and the like) and other binding polypeptides specific for phosphatidylserine synthase. Small molecule chemical compounds specific for phosphatidylserine synthase are also contemplated.

Such modulators which specifically bind to phosphatidylserine synthase can be identified using phosphatidylserine synthase isolated from cell membranes, recombinant phosphatidylserine synthase, phosphatidylserine synthase variants or cells expressing such products. Binding proteins are useful, in turn, for purifying phosphatidylserine synthase, and are useful for detection or quantification of phosphatidylserine synthase in fluid and tissue samples by known immunological procedures. Anti-idiotypic antibodies specific for phosphatidylserine synthase- specific antibody substances are also contemplated.

Biochemical and cellular methods of identifying inhibitors of the enzyme are contemplated by the invention. For example, a biochemical screen is provided that identifies inhibitors based on the diminution of the enzymatic activity of converting serine and CDP-diacylglycerol into phosphatidylserine by a phosphatidylserine synthase polypeptide comprising the amino acid sequence set out in SEQ ID NO:2 or SEQ ID NO: 4. The phosphatidylserine synthase polypeptide may be present in a cell extract, may an isolated polypeptide or may be a polypeptide made by recombinant techniques.

As yet another example, a cellular method of identifying a phosphotidylserine synthase inhibitor is provided that comprises the steps of culturing yeast cells in the presence or absence of a test compound and comparing deathzones of the yeast cells cultured in the presence of the test compound with deathzones of the yeast cells in the absence of the test compound, wherein an increase in the size of, or number of, deathzones in the presence of the test compound indicates that the test compound is a phosphotidylserine synthase inhibitor.

Specificity of an inhibitor identified by methods of the invention for phosphatidylserine synthase versus other proteins may be confirmed by methods that compare the effect of the inhibitor on yeast expressing a wild-type level of phosphatidylserine synthase with the effect of the inhibitor on cells expressing an altered level of the enzyme. In one aspect, if the level of the enzyme in the altered cells is reduced, a specific inhibitor will have a greater effect than on wild-type cells. In another aspect, if the level of the enzyme in the altered cells is increased, a specific inhibitor will have a lesser effect than on wild-type cells. Levels of expression may be altered in cells by introducing a regulatable expression cassette including a nucleic acid sequence set out in SEQ ID NO:1 or SEQ ID NO: 3 into, for example, cells in which the corresponding gene had been knocked out (to generate a lower level of expression in the altered cells) or into wild-type cells (to generate a higher level of expression in the altered cells).

Administration of phosphatidylserine synthase inhibitors to mammalian subjects, especially humans, for the purpose of ameliorating disease states caused by fungi is contemplated by the invention. Fungal infections (mycoses) such as candidiasis, aspergillosis, coccidioidomycosis, blastomycosis, paracoccidioidomycosis, histoplasmosis, cryptococcosis, chromoblastomycosis, sporotrichosis, mucormycosis, and the dermatophytoses can manifest as acute or chronic disease. Pathogenic fungi cause serious, often fatal disease in immunocompromised hosts. Cancer patients undergoing chemotherapy, immunosuppressed individuals, and HIV-infected individuals are susceptible to ■ mycoses caused by Candida, Aspergillus, Pneumocystis carinii, and other fungi.

Amphotericin B, 5-fluorocytosine, and fluconazole are useful therapeutics for fungal infections, but toxicity associated with these drugs causes serious adverse side effects that limit their usefulness. Echinocandins, such as Caspofungin, are cytostatic against Aspergillus species, significantly increasing the opportunity for resistance

development and rendering the therapeutic ineffective in neutropenic patients. .The mortality rate of patients suffering from systemic candidiasis is greater than 50% despite amphotericin B treatment. Therefore, a need exists for agents that inhibit fungal growth in vivo and such products may be used as single agents or in combination with currently approved, conventional anti-fungal compounds. Because the examples herein demonstrate that growing fungi require phosphatidylserine for survival, inhibition of phosphatidylserine synthase may be useful for limiting fungal infections in vivo.

Specifically contemplated under the invention are phosphatidylserine synthase inhibitor compositions for use in methods for treating a mammal susceptible to or suffering from fungal infections comprising administering phosphatidylserine synthase inhibitor to the mammal in an amount sufficient to inhibit phosphatidylserine synthase activity. It is contemplated that the phosphatidylserine synthase inhibitor may be administered with other conventional anti-fungal agents, including amphotericin B and the structurally related compounds nystatin and pimaricin; 5- fiuorocytosine; azole derivatives such as fluconazole, ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, terconazole, itraconazole and tioconazole; echinocandins, such as anidulafungin, caspofungin, cilofungin, and micafungin; allylamines-thiocarbamates, such as tolnaftate, naftifme and terbinafine; griseofulvin; morpholines, such as amorolfϊne; sordarins; ciclopirox olamine; haloprogin; undecylenic acid; and benzoic acid. [See, e.g., Goodman & Gilman, The Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, NY (1996).] Phosphatidylserine synthase inhibitors may improve the effectiveness of these conventional anti-fungal agents. By reducing the amount of conventional anti- fungal agent needed to exert the desired therapeutic effect, phosphatidylserine synthase inhibitors may allow the drugs to be used at less toxic levels. For example, Davies and Pope, Nature, 273:235-236 (1978) reported that administration of mycolases (enzymes that degrade the fungal cell wall) in conjunction with a normally ineffective dose of anti-fungal drug to Aspergillus-vafected mice provided synergistically effective treatment.

Thus, the invention encompasses the use of phosphatidylserine synthase inhibitor in the preparation of a medicament for the prophylactic or therapeutic treatment of fungal infections, and further contemplates the use of phosphatidylserine

synthase, inhibitor in the preparation of a medicament for co-administration with another anti-fungal agent.

Therapeutic/pharmaceutical compositions contemplated by the invention include phosphatidylserine synthase inhibitor and a physiologically acceptable diluent or carrier and may also include other anti-fungal agents. Dosage amounts indicated would be sufficient to inhibit phosphatidylserine synthase activity. For general dosage considerations see Remington, The Science and Practice of Pharmacy, 19th ed., Mack Publishing Co., Easton, PA (1995). Dosages will vary between about 1 ' μg/kg to 100 mg/kg body weight, and preferably between about 0.1 to about 20 mg phosphatidylserine synthase inhibitor/kg body weight. Therapeutic compositions of the invention may be administered by various routes depending on the infection to be treated, including via subcutaneous, intramuscular, intravenous, intrapulmonary, transdermal, intrathecal, topical, oral, or suppository administration.

Brief Description of the Drawing

Figure 1 shows a sequence alignment for phophatidyl serine syntase polypeptides from S. cerevisiae, C. albicans, A. fumigatus, and consensus.

Detailed Description of the Invention Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples. Examples 1 and 4 describe the isolation of phosphatidylserine synthase genomic DNAs from C. albicans and A. fumigates, respectively. Examples 2 and 3 demonstrate the function of the C. albicans CHOI gene while Example 5 demonstrates the function of ihe A. fumigates CHOI gene. Example 6 addresses determination of the anti-fungal activity of phosphatidylserine synthase inhibitors in vitro. Example 7 addresses determination of the anti-fungal activity of phosphatidylserine synthase inhibitors in vivo in a mouse model, and Examples 8 through 11 similarly address rabbit models of invasive aspergillosis, disseminated candidiasis, Candida ophthalmitis, and Candida endocarditis.

Example 1 Isolation of C. albicans phosphatidylserine synthase genomic DNA ,

In order to clone the C. albicans CHOI gene, the NCBI unfinished eukaryotic genome database was searched with the S. cerevisiae gene [Cummings et at, FEMS Microbiol. Lett, 275:133-138 (2002)] using the BLAST program [Altschul, et al., J MoI Biol, 215:403-10 (1990)]. No homologue was detected at the nucleotide level. However, a single open reading frame was identified, the putative translation of which possessed a basic local alignment search tool expect score of less than 1086. This open reading frame, C. albicans contig 6-2464, contains no introns. The presumed translation possesses 61% identity and 75% similarity with the S. cerevisiae protein (Figure 1).

The gene was cloned by PCR from genomic DNA isolated from C. albicans strain 366 [Ostrander and Gorman, Yeast, 73:871-880 (1997)]. Fungal genomic DNA was isolated from mid-log cells with a BeadBeater, using a variation of standard methods [Hoffman and Winston, Gene, 57:267-272 (1987)]. Cells were broken at 4°C in a mixture of two parts buffer, one part phenol, and one part chloroform. It was found that it was important to allow the cells to cool completely between one-minute BeadBeater pulses. The nucleic acid was precipitated with ethanol and RNA digested with 1 mg DNase-free RNase (Qiagen) in TE for four hours at 37°C. The protein was removed by multiple chloroform extractions. The DNA was precipitated with ethanol and quantified by spectroscopy. The 850 bp gene was amplified by PCR (35 cycles of 95 ⁰ C, I ¹ ; 5O ⁰ C, 2 ¹ ; 72 ⁰ C, 3 ¹ ) using primers, 5'atagaattcatgacagactcatcagctaccgggttctccaagcacc3' (SEQ ID NO: 6)and 5 'atactcgaggattctattttagaatcatctctatggtttagg3 ' (SEQ ID NO: 7).

The sequence of the genomic DNA is set out in SEQ ID NO: 1, while the protein sequence encoded by the genomic DNA is set out both in SEQ ID NOs: 1 and 2.

Example 2

The cloned C. albicans gene encodes a functional phosphatidylserine synthase

The C. albicans CHOI open reading frame was sub-cloned behind a copper- inducible promoter in a low-copy S. cerevisiae vector [Gorman et al, Gene, 48: 13-22 (1986)]. There is a single CUG codon in this gene. In C. albicans, this codon

encodes serine, whereas it will encode leucine in S. cerevisiae [Santos and Tuite, Nucleic Acids Res., 25:1481-1486 (1995)]. The CHOI gene of S. cerevisiae was disrupted by homologous recombination using the flanking regions of the gene containing a selectable marker. The resulting chol strain required 10 mM choline for viability [Hikiji et al, J. Biochem., 704:894-900 (1988)]. The C. albicans plasmid, and the parent plasmid as a control, were then transformed into the chol strain. Transformation was achieved by the lithium procedure [Ostrander and Gorman, Yeast, 13:871-80. (1997)] using 1 μg of linear integrating DNA. Transformants were selected on drop-out media. Quantitative RT-PCR was utilized to demonstrate that the cells, when growth with choline, expressed the C. albicans gene in a copper-dependent manner. Quantification was accomplished by varying either the number of cycles or RNA concentrations. Standard reactions used 22 cycles, 5 pmol of each primer, and 1 μg RNA in 50 μL using the Access System (Promega). Amplicons were separated by agarose electrophoresis, visualized with Sybr ^® Green (Molecular Probes), and quantified using a Storm ^® Fluorescence Imager (Molecular Dynamics). Care was taken to ensure that the amplification was in the linear range for either cycle number or RNA concentration. This showed that the induction plasmid was functioning properly. Further, microsomes were isolated from these cells to demonstrate that the gene encoded a protein with phosphatidylserine synthase activity that increased with copper concentration. Phosphatidylserine synthase enzyme was enriched by isolating microsomes from late exponentially growing cells using variations of established procedures [Yamashita and Nikawa, Biochim Biophys Acta, 4:1-2 (1997)]. The cells were broken with a BeadBeater using a 1 M sorbitol/TE buffer at 4°C and microsomes isolated by centrifugation at 120,000 x g in a TLlOO ultracentrifuge (Beckman). The assay was performed essentially as described [Carman and Bae-Lee, Methods Enzymol, 209:298-305 (1992)] by conversion of labeled serine to an organic product. C. albicans microsomes were incubated at 37°C for ten minutes for a linear conversion. One unit is defined as one nmole of phosphatidylserine produced per minute of reaction.

The results showed that the gene does encode phosphatidylserine synthase activity. Finally, the strain demonstrated copper-dependent growth in the absence of choline showing that the gene is the C. albicans CHOI. No phosphatidylserine

synthase activity with choline or growth without choline was observed with the control strain with any amount of copper.

Example 3 C. albicans phosphatidylserine synthase is essential

A uracil auxotrophic strain of C. albicans was created by homologous recombination disruption of both alleles of URA3 and selection of 5-FOA. The open reading frame from the complete C. albicans CHOI gene was deleted and replaced with the C. albicans URA3 gene. This disruption cassette was then isolated and used to transform the C. albicans uracil auxotroph. Transformation was by integrating

DNA using electroporation [Ostrander and Gorman, Gene, 148:179-85 (1994)] using a Gene Pulser (BioRad). Most times for electroporation, spheroplasting was required [Ostrander and Gorman, Gene, 148:179-185 (1994)]. Log-phase cells (100 mL) were harvested from rich media and washed into a fresh sorbitol/DTT solution. This was incubated at 37°C for one hour. The cells were spheroplasted using zymolyase, shaking, at 37°C and carefully washed into 1 M sorbitol when spheroplasting reached 75% by microscopic analysis of SDS-treated cells. Cells were added to a tube in which at least 5 μg DNA had been dried. Carrier DNA and 20% PEG were added and the reaction incubated at ambient temperature for thirty minutes. Cells were pelleted and resuspended in fresh 10% rich media, IM sorbitol solution for thirty minutes at 30 ⁰ C. These were plated in selective top agar media and incubated at 30 ⁰ C for one week. Prototrophs were selected and screened for heterozygous disruption of CHOI by genomic DNA PCR. Uracil auxotrophy was then reconstituted by disruption of the URA3 gene within the inactivated chol allele. The disruption cassette was used to re-transform the C. albicans CHOl/cholΔ heterozygote uracil auxotroph. No uracil prototrophs could be recovered, even when selected on media containing 10 mM choline. The C. albicans CHOI gene was cloned into a C. albicans plasmid and co-transformed with the chol Δ disruption cassette into the heterozygous strain. Uracil prototrophs were selected and screened for homozygous disruption of CHOI by genomic DNA PCR. This strongly suggests that the chol gene is essential in C. albicans.

Numerous attempts to select for loss of the plasmid in media containing 10 mM choline proved unsuccessful. Only when the choline level was raised to 100 mM was the plasmid able to be lost. The cells were then completely dependent on choline

for growth demonstrating the essentiality of phosphatidylserine synthase in C. albicans. Successful double disruption of the gene without a covering plasmid was achieved when selected on media containing 100 mM choline.

Various phenotypes of the heterozygous and homozygous cholΔ strains were studied. The growth rate of the heterozygote in different media was unaffected. However, the homozygote, when grown in choline, was found to enter stationary phase growth at a lower cell density and to take longer to recover logarithmic phase growth than either the parental or heterozygous strains. This was only somewhat attenuated by inclusion of the covering CHOI plasmid. Additionally, the homozygous strain exhibited a much higher mortality upon 70°C glycerol storage than the wild type. The percentage of cells demonstrating a hyphal phenotype upon growth at 37°C, in the presence of serum or proline, or at neutral pH (12) was significantly decreased with the homozygote.

The effect of the heterozygous chol Δ deletion on phospholipid composition was minimal. Phospholipid quantification was performed essentially as previously described [Ostrander, et al, J Biol Chem, 276: 25262-72. (2001)]. Cells were labeled for 12 hours (six generations, steady state labeling) or three hours (one generation, synthesis rate labeling) with 50 μCi [ ³² P] Pi (Amersham) in synthetic defined media to the mid-exponential phase of growth. Cells were homogenized with a solution of 10 :5 :4 MeOH, CHCl ₃ , 0.1 N HCl in a BeadBeater without cooling. Lipids were isolated by acid organic extraction (half volumes each Of CHCl ₃ and 0.1 N HCl, 0.5M NaCl), dried by speed vacuum, and resuspended in a minimal volume of CHCI ₃ . Phospholipids were normalized for cpm, separated by thin layer chromatography (65:28:8 CHCl ₃ :MeOH: AcAc) using a 20x20 cm silica gel plate (Whatman) for 2.5 hours, and individual species were identified by co-migration of standards (Sigma). The spots were quantified by phosphoimaging using a Storm ^® Phosphocounter (Molecular Dynamics) and normalized to the total amount of organic soluble 32P. The effect was also minimal when a plasmid containing the wild-type CHOI gene was included in the homozygous disruptant. However, the phospholipid composition was significantly altered in the homozygous disruptant when no CHOI plasmid was present and the strain was maintained on high levels of exogenous choline. Consistent with the absence of a functional phosphatidylserine synthase enzyme, no discernible phosphatidylserine was present. Phosphatidylethanolamine was also much lower than wild-type cells. However, phosphatidylcholine, which is created by

the Kennedy pathway using choline, was proportionately increased in the homozygous disruptant. Phosphatidylinositol was also observed to be increased.

Example 4 Isolation of A. fumigatus phosphatidylserine synthase genomic DNA

In order to clone the A. fumigatus. CHOI gene, the NCBI unfinished eukaryotic genome database was searched with the S. cerevisiae gene. No homologue was detected at the nucleotide level. However, two open reading frames, separated by 59 base pairs, were identified. Although a sequencing error may have occurred within this non-homologous sequence, it is assumed that the interruption is intronic (see below). The putative translation of these reading frames together possessed a basic local alignment search tool expect score of less than 1050. The translation possesses 55% identity and 69% similarity with the S. cerevisiae protein and 59% identity and 69% similarity with the C. albicans protein (Figure 1). The gene was cloned by PCR from genomic DNA isolated from A. fumigatus strain ND 158 as described above in Example 1. The gene was amplified by PCR as described above using the primers set out in that Example (SEQ ID NOS: 6 and 7). The sequence of the genomic DNA is set out in SEQ ID NO: 3, while the protein sequence encoded by the genomic DNA is set out in both SEQ ID NOs: 3 and 4. . The beginning of the open reading frame of the Aspergillus CHOI gene is not apparent from the genomic sequence. No significant homology with the N-terminal regions of either the predicted S. cerevisiae or C. albicans protein sequences is found with any possible translation up to a kilobase upstream of the two homologous open reading frames identified. Therefore, the entire region, including more than a kilobase upstream of identified open reading frames, was cloned into a copper- inducible promoter in a low-copy S. cerevisiae vector [Gorman et al, Gene, 48:13-22 (1986)]. This plasmid was transformed into the chol strain of S. cerevisiae, but no complementation was observed.

In order to test that the two identified open reading frames could encode a functional phosphatidylserine synthase, these were sub-cloned, fused together, and fused to the 5' region of the S. cerevisiae gene where the homology stopped. This chimeric gene was cloned into the CUPl promoter vector used previously for the C. albicans gene. The S. cerevisiae chol Δ strain was transformed with this plasmid. Quantitative RT-PCR was utilized to demonstrate that the cells, when grown with

choline, expressed the S. cerevisiaelA. fumigatus fusion gene in a copper-dependent manner. This showed that the induction plasmid was functioning properly. Further, microsomes were isolated from these cells to demonstrate that the gene encoded a protein with phosphatidylserine synthase activity that increased with copper concentration. This shows that the A. fumigatus exons do encode phosphatidylserine synthase activity when coupled to the S. cerevisiae N-terminal region. Finally, the strain demonstrated copper-dependent growth in the absence of choline. No phosphatidylserine synthase enzymatic activity or growth without copper could be ' detected from the exons themselves without the S. cerevisiae N-terminal region. As the N-terminal region is not well conserved between species, it is inferred that the A. fumigatus exons encode the catalytic domains of the enzyme and, therefore, that the exons are indeed part of the A. fumigatus CHOI gene.

Example 5 A. fumigatus phosphatidylserine synthase is essential

A uracil auxotrophic strain of A. fumigatus was created by homologous recombination disruption of PYR4 and selection of 5-FOA. The open reading frame from the complete A. fumigatus CHOI gene was deleted and replaced with the A. fumigatus PYR4 gene. However, no transformants could be isolated.. A plasmid containing the entire A. fumigatus CHOI gene was used for co-transformation with the disruption cassette. A. fumigatus were transformed by electroporation essentially as described [Weidner, et al., Curr Genet., 33:378-85 (1998)]. Uracil prototrophs were selected and screened for disruption of CHOI by genomic DNA PCR. After more than a hundred generationsj this covering plasmid was still found in the disrupted strain. Therefore, it is strongly suggested that A. fumigatus CHOI is essential. Incidentally, co-transformation of a plasmid containing the S. cerevisiae first quarter gene fused to the second and third exons of the A. fumigatus gene also afforded selection of a CHOI strain. This demonstrated that the inter-exon region of the gene is indeed intronic. No amount of choline was able to cause loss of the CHOI plasmid from the

CHOI Δ strain. However, labeling cells with choline demonstrated that A. fumigatus possesses a Kennedy pathway, synthesizing phosphatidylcholine from exogenous choline. Therefore, phosphatidylcholine cannot compensate for loss of phosphatidylserine in A. fumigatus.

Only a single phenotypic difference was observed in the CHOI disruption strain containing the covering plasmid compared with the wild type parent. The strain was completely refractory to storage at -70 ⁰ C. Several different conditions were attempted, but no recovery was from frozen storage was ever observed. Mortality of the strain on culture plates was also comparatively high. However, no significant difference in growth rate, nutrient requirements, phospholipid composition, or phospholipid synthesis rates was observed between the disruptant and the parental strain. The only difference was a minor decrease in phosphatidylserine synthesis levels in the disruption strain.

Example 6 Anti-fungal Activity of Phosphatidylserine Synthase Inhibitors In Vitro

Phosphatidylserine synthase inhibitors are tested for inhibition of fungal growth in vitro. The two fungi C. albicans and A. fumigatus are serious pathogens for immunocompromised patients

The anti-fungal activity of phosphatidylserine synthase inhibitors are evaluated in an agar diffusion assay, in a broth assay according to National Committee on Clinical Laboratory Standards, and in a cell wall inhibition assay according to Selitrennikoff, Antimicrob. Agents Chemother., 23:757-765 (1983). In the agar diffusion assay, approximately 1 x 10 cells/mL of C. albicans

(ATCC no. 90028) inoculated into 1.5% agar (RPMI 1640 media buffered with 2-(N- morpholino)propanesulfόnic acid (MOPS), pH 7.0. A disk containing 50 μg of the sample (A: recombinant phosphatidylserine synthase, B: buffer control, C: control protein, D: a bacterial lysate with phosphatidylserine synthase activity, or a known anti-fungal agent) was placed on the agar, and the zone of growth inhibition was measured.

In the broth assay, 50 μg/mL of the sample (A: phosphatidylserine synthase inhibitor, B: buffer control, C: control protein, D: a bacterial lysate with phosphatidylserine synthase activity, or a known anti-fungal agent) was added with a certain concentration of the test fungal organism to RPMI 1640 media buffered with MOPS, pH 7.0. The samples were incubated at 35°C, with shaking at 120 rpm, for 48 .hours, and then growth was evaluated by measuring the turbidity of the suspension. The approximate concentrations of the test fungi were as follows: 2.5 x 10 ⁴ cells/mL of C. albicans (ATCC No. 90028); 5 x 10 ⁴ cells/mL of C. albicans-polyene resistant

(ATCC no. 38247); 1 x 10 ⁴ cells/mL of A. fumigatus (ATCC No. 16424); 1 x 10 ⁴ cells/mL of Neurospora crassa (ATCC No. 18889); and 1 x 10 ⁴ cells/mL of S. cerevisiae (ATCC No. 26108).

Example 7

Several animal models have been developed for testing efficacy of anti-fungal compounds [see Louie et ah, Infect. Immun., 62: 2761-2772, 1994; Kinsman et al, Antimicrobial Agents and Chemotherapy, 37:1243-1246, 1993 ; Nakajima et ah, Antimicrobial Agents and Chemotherapy 3.9:1517-1521., 1995; Tonetti et al, Eur. J. Immunol, 25:1559-1565 (1995); Denning and Stevens, Antimicrob. Agents

Chemother., 35:1329-1333 (1991); see also Stevens, J. Mycol. Med, 6(Suppl I)-J-IO (1996)]. Briefly, the animal host is infected with the fungi, varying doses of phosphatidylserine synthase inhibitor are administered to the animals, and their survival is measured over time. The experiments are performed using phosphatidylserine synthase inhibitor as the sole therapeutic agent, or with a combination of conventional anti-fungal agents such as amphotericin B and fluconazole to determine if the phosphatidylserine synthase inhibitor improves the efficacy of such compounds. Specifically, acute systemic candidiasis is achieved in mice by intraperitoneal or intravenous challenge of 10 x 10 ⁶ CFU Candida albicans. The therapeutic agents are administered before or at 1 to 5 hours after challenge, and the number of survivors is determined after five days. In addition, the mice can be sacrificed and fungal load can be determined in specific organs such as brain, kidney, lung, liver and spleen. Alternatively, the mice are challenged with lower doses of fungi, e.g., Aspergillus (8-10 x 10 ⁶ CFU) or Candida (1 x 10 ⁶ CFU), in which case survival can be measured at more distant time points, e.g., 45 days. The long term fungicidal/fungistatic activity of phosphatidylserine synthase inhibitor alone or with another anti-fungal drug may be evaluated by continuing therapy for a week or more, e.g., 11 days, and following the animals over several weeks, e.g., 18 days to one month. Effective anti-fungal agents enhance the long term survival of animals and reduce fungal load in blood and organs.

Example 8

Activity of Phosphatidylserine Synthase Inhibitors hi Vivo in a Rabbit Model of Invasive Aspergillosis The efficacy of phosphatidylserine synthase inhibitor, alone or in combination with other conventional anti-fungal agents, is assessed in an imimmosuppressed rabbit model of invasive aspergillosis which has been used for over ten years to evaluate a variety of anti-fungal therapies. See, e.g., Andriole et al., Clin. Infect. Dis., 14(Suppl. 1):S 134-S 138 (1992). The study is conducted generally according to Patterson et al., . Antimicrob. Agents Chemother., 37:2307-2310 (1993) or George et al., J. Infect. Dis., 168:692-698 (1993). Briefly, on day one the rabbits are given cyclophosphamide (200 mg) intravenously to render them leukopenic, followed by triamcinolone acetonide (10 mg) subcutaneously each day for the duration of the experiment. On day two, 24 hours after immunosuppression, the animals are challenged intravenously with about 10 ⁶ (lethal challenge) or about 10 ⁵ (sublethal challenge) A. fumigatus conidia. Anti-fungal therapy (phosphatidylserine synthase inhibitor alone, or in combination with other conventional anti-fungal agents, e.g., amphotericin B, fluconazole, or 5-fluorocytosine) is initiated at 24 hours after challenge or 48 hours before challenge (for prophylaxis) and is continued for 5 to 6 days or until death. Exemplary doses of conventional anti-fungal agents are 1.5 or 0.5 mg/kg/day intravenous amphotericin B, 60 or 120 mg/kg/day oral fluconazole and 100 mg/kg/day oral 5-fluorocytosine. Control rabbits are not treated with any anti-fungal agent.

At autopsy or death, semi-quantitative fungal cultures and histopathologic examination are conducted on the liver, spleen, kidneys, lungs and brain. Cultures of the heart, urine and blood may also be performed. Blood samples are obtained at intervals and assayed for white blood cell counts and circulating Aspergillus carbohydrate antigen using an ELISA assay. The effect of treatment with the test drug is evaluated on three endpoints: reduction in mortality rate, reduction in number of Aspergillus organisms cultured from target organs (fungal burden), and reduction in level of circulating Aspergillus antigen. Effective anti-fungal agents reduce mortality and/or fungal load.

Alternatively, pulmonary aspergillosis may be evaluated in this model generally according to Chilvers et al., Mycopathologia, 108:163-71 (1989), in which

" the immunosuppressed rabbits are challenged with intratracheal instillation of

Aspergillus fumigatus conidia, followed by bronchoalveolar lavage on days 1, 2, 4, 7 and 10 following challenge; fungal culture, chitin assay, white cell counts and histopathology are performed on the lavage fluids to determine infective load within the lung. Effective fungal agents reduce the infective load or inflammation within the lung.

Example 9 Activity of Phosphatidylserine Synthase Inhibitors In Vivo in a Rabbit Model of Disseminated Candidiasis

The efficacy of phosphatidylserine synthase inhibitor, alone or in combination with other conventional anti-fungal agents, is assessed in a rabbit model of disseminated candidiasis generally according to Rouse et al., Antimicrob. Agents Chemother., 36:56-58 (1992). New Zealand white rabbits are infected systemically with about 3 x 10 ⁶ Candida albicans blastospores. Anti-fungal therapy is initiated 48 hours after challenge with Candida (or before challenge for prophylaxis) and is continued for, e.g., four days. Surviving animals are sacrificed, and fungal cultures are performed on the aortic valve with attached vegetation, lung, kidney and spleen. Fungal cultures and histopathological examination may also be performed on these and other organs, such as liver, brain, and heart. Urine and blood cultures may also be done. The effect of the anti-fungal therapy on mortality and circulating or tissue fungal burden is determined.

Bayer et al., Antimicrob. Agents Chemother., 19:179-184 (1981), in which rabbits are inoculated intraperitoneally with about 5 x 10 ⁸ CFU Candida albicans. A saline peritoneal aspirate is obtained and cultured from each animal four days after intraperitoneal inoculation, and animals with a positive fungal cμlture aspirate are randomly assigned to control or treatment groups. Anti-fungal treatment is begun . seven days after challenge. The eyes of all rabbits are evaluated using indirect ophthalmoscopy, as disseminated candidiasis may result in Candida endophthalmitis. Animals are sacrificed at 7, 11 and 14 days after initiation of therapy and their abdomens inspected for evidence of peritonitis and intraabdominal abscess formation. Eyes are examined for macroscopic lesions. Tissue samples from peritoneal abscesses, all other visible abscesses, kidneys, livers, spleens and ocular structures are weighed, homogenized in brain heart infusion broth, serially diluted and cultured to

determine the CFU per gram of tissue. Renal and peritoneal abscesses are also .fixed in 10% neutral formaldehyde and examined for histopathology. Sections are stained with periodic acid-Schiff reagent to determine the fungal burden and, fungal morphology. Effect of the test drugs on improving survival and reducing fungal burden is evaluated.

Example 10

Activity of Phosphatidylserine Synthase Inhibitors In Vivo in a Rabbit Model of Fungal Endophthalmitis The efficacy of phosphatidylserine synthase inhibitor, alone or in combination with other conventional anti-fungal agents, is assessed in a rabbit model of Candida endophthalmitis, generally according to Park et al., Antimicrob. Agents Chemother., 39:958-963 (1995). Briefly, New Zealand albino rabbits, 2 to 2.5 kg, are infected with an intravitreal inoculation of about 1,000 CFU of Candida albicans. Endophthalmitis is confirmed 5 days after inoculation by indirect ophthalmoscopy, and is defined as moderate to severe vitreous haze with partial or complete obscuration of greater than 50% of the retinal and choroidal vasculature. The vitreous turbidity is graded on a scale, and the fundus appearance may be graded and documented by fundus photography. The rabbits are then randomized to the following treatment conditions: phosphatidylserine synthase inhibitor alone for 2 to 4 weeks, a combination of phosphatidylserine synthase inhibitor and another conventional anti-fungal agent (e.g., amphotericin B, fluconazole or 5-fluorocytosine) for 2 to 4 weeks, or no treatment (control). Exemplary doses of conventional antifungal agents are 80 mg/kg/day of oral fluconazole and 100 mg/kg every 12 hours of oral 5-fluorocytosine.

The treatment effect is assessed at 2 and 4 weeks after therapy by indirect ophthalmoscopy, quantitative fungal culture, and histopathology. For quantitative fungal culture, the eyes are dissected and weighed, and a weighed fraction of each sample is homogenized and cultured on brucella agar-5% horse blood plates for 48 hours at 35°C in 5 to 10% CO ₂ . The homogenized sample may also be diluted 10- or 100-fold with sterile saline before plating. The colonies are counted and the total CFU in the eye calculated on the basis of the growth yielded from the measured fractions of sample. Treatment effect is assessed in terms of a reduction in the total intraocular fungal burden. For histopathology, representative eyes are removed, fixed

" in formalin, embedded in plastic, and sliced into 5 μm sections. The sections are stained with hematoxylin-eosin or Gomori's methenamine silver stain and examined by light microscopy for inflammation, fibrous organization and fungal elements. The effect of the anti-fungal agents on reducing mortality, reducing fungal load, or reducing the inflammation associated with fungal infection, is evaluated.

Alternatively, a rabbit model of Aspergillus endophthalmitis may be used generally according to Jain et al., Doc. Ophthalmol, 69:227-235 (1988). Briefly, New Zealand white rabbits are inoculated in one eye with about forty spores of Aspergillus fumigatus. Their contralateral (control) eyes receive a similar but sterile inoculum. After treatment with the test drug (phosphatidylserine synthase inhibitor alone, or phosphatidylserine synthase inhibitor in combination with another agent), the rabbits' eyes may be evaluated for clinical appearance, electroretinogram waveforms, indirect ophthalmoscopy, quantitative fungal culture, and histopathology. Clinically evident endophthalmitis typically develops within three to seven days after inoculation.

Example 11

Activity of Phosphatidylserine Synthase Inhibitors hi Vivo in a Rabbit Model of Fungal Endocarditis The efficacy of phosphatidylserine synthase inhibitor, alone or in combination with other conventional anti-fungal agents, is assessed in a rabbit model of Candida endocarditis generally according to Witt and Bayer, Antimicrob. Agents Chemother., 35:2481-2485 (1991). See also Longman et al., Rev. Infect. Dis., 12(Suppl. 3):S294- 298 (1990). Sterile thrombotic endocarditis is produced in New Zealand white rabbits by transaortic valvular placement of a sterile polyethylene catheter (internal diameter, 0.86 mm), which remained in place for the duration of the study, Infective endocarditis is then established 48 hours after catheterization by intravenous injection of about 2 x 10 ⁷ C. albicans blastospores. Alternatively, C. parapsilosis may be used. Anti-fungal therapy (phosphatidylserine synthase inhibitor or phosphatidylserine synthase inhibitor in combination with another conventional anti-fungal agent) is initiated either 24 hours before or 24 to 60 hours after fungal challenge. Therapy is continued daily for 9 or 12 days. Exemplary doses of conventional anti-fungal agents are 1 mg/kg/day intravenous amphotericin B, 50 mg/kg/day or 100 mg/kg/day intravenous or intraperitoneal fluconazole. Control rabbits are given no anti-fungal

agent. At sacrifice, hearts are removed and the position of the indwelling catheter verified. Cardiac vegetations from each animal are removed, pooled, weighed and homogenized in 1 mL of sterile saline. The homogenate is serially diluted and quantitatively cultured on yeast potassium dextrose agar at 35°C for 48 hours. Culture-negative vegetations are considered to contain less than 2 loglO CFU/grarn on the basis of average vegetation weight.

Numerous modifications and variations of the above-described invention are expected to occur to those of skill in the art. Accordingly, only such limitations as appear in the appended claims should be placed thereon.