WO1996020717A1 - Compositions and methods for inhibiting fungal cell wall formation - Google Patents
Compositions and methods for inhibiting fungal cell wall formation Download PDFInfo
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- WO1996020717A1 WO1996020717A1 PCT/US1996/000752 US9600752W WO9620717A1 WO 1996020717 A1 WO1996020717 A1 WO 1996020717A1 US 9600752 W US9600752 W US 9600752W WO 9620717 A1 WO9620717 A1 WO 9620717A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7016—Disaccharides, e.g. lactose, lactulose
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
- A61K36/064—Saccharomycetales, e.g. baker's yeast
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
Definitions
- This invention pertains to antifungal compositions and to methods for using such compositions in the prevention and treatment of fungal infections in plants and animals.
- the invention also encompasses the enzyme that catalyzes the formation of certain fungal cell wall oligosaccharides that are formed by a specific covalent linkage between chitin and glucans.
- Fungi are ubiquitous eukaryotic organisms of varying size and morphology. Fungi typically grow in two basic forms, yeasts and molds. Yeasts are single cell organisms that are usually spherical or ellipsoidal in shape and that generally reproduce by budding. Molds are multicellular organisms that generally form filamentous colonies. Examples of fungi include monomorphic yeasts and yeast-like organisms, including Candida, Cryptococcus, and Saccharomyces; monomorphic molds, such as Aspergillus and Coccidioides; and thermally dimorphic fungi, such as Blastomyces dermatitidis and Histoplasma capsulatum, which grow either in a yeast or a mold phase.
- Fungal cell walls determine the shape of fungal cells and are essential for fungal integrity.
- the cell walls include three types of structural polysaccharides: glucans (polymers of glucose (Glc) containing 0(l-*3) and 0(1 ⁇ 6) linkages), mannans (primarily glycoproteins with attached mannose chains), and chitin.
- glucans polymers of glucose (Glc) containing 0(l-*3) and 0(1 ⁇ 6) linkages
- mannans primarily glycoproteins with attached mannose chains
- chitin is a linear polymer of N-acetylglucosamine (GlcNAc) residues joined by ⁇ ( ⁇ 4) linkages. It is scattered throughout the cell wall, although it is mainly concentrated at the septal region. Because chitin is concentrated at the septal region, it is particularly instrumental in the reproduction of fungi by budding.
- chitin in the dry weight of the cell wall varies according to the fungal species. For example in S. cerevisiae, chitin comprises only 1-2% by weight, whereas in Sclerotium rol it may comprise up to 61 %.
- the architecture of the fungal cell wall is defined by the organization of th cell wall constituents as well as by the constituents themselves.
- Several studies have sugges that different cell wall polysaccharides are covalently linked and particularly that chitin be covalently linked to the glucan component of the yeast cell wall.
- Mol et al. (F.E. Microbiol. Letts. , 41:95-99, 1987) disclose experiments in S. cerevisiae in which treatm of alkali-insoluble glucan with chitinase rendered the material alkali-soluble, suggesting t covalent linkage of glucan to chitin was responsible for its initial alkali insolubility.
- Siets et al. J. Ge/z.
- fungi play an indispensable role in the cyclic transformation of orga matter, such as for example, in food and drug production.
- a broad range of fungi frequent causes of diseases in plants, however.
- An historical example of a fungal plant dise that had extensive adverse effects on man is the potato blight of the nineteenth century wh led to the starvation of over one million people.
- the major fungal animal pathogens in North America are Histoplas capsulatum, Coccidioides immitis, Blastomyces dermatitidis , Cryptococcus neoforma Candida species and Aspergillus species (Medically Important Fungi, Second Edition, Dav H. Larone, Ed. , American Society for Microbiology, Washington, D.C.).
- Figure 1 is an illustration of the elution profile from a Bio-Gel P-2 column of a yeast wall fraction solubilized by digestion with glucanase and chitinase.
- Figure 2 is an illustration of the elution profile from a Bio-Gel P-2 column of compounds I, II, and III (Peaks A, B, and C of Figure 1, respectively) after acid hydrolysis.
- Figure 3a is an illustration of the mass spectrometry determination of the molecular weight of compound I.
- Figure 3b is an illustration of the mass spectrometry determination of the molecular weight of compound II.
- Figure 3c is an illustration of the mass spectrometry determination of the molecular weight of compound HI.
- Figure 4 ⁇ is an illustration of the elution profile from a Bio-Gel P-2 column of compound I after treatment with the 50 ⁇ l of 100 mM citrate-phosphate buffer, pH 5.0.
- Figure 4b is an illustration of the elution profile from a Bio-Gel P-2 column of compound I after treatment with ⁇ -N-acetylglucosaminidase.
- Figure Ac is an illustration of the elution profile from a Bio-Gel P-2 column of compound I after treatment with ⁇ -glucosidase.
- Figure 5a is an illustration of the profile from paper chromatography of the products of a partial acid digestion of compound I.
- Figure 5b is an illustration of the profile from paper chromatography of the products of enzymatic digestion of compound I.
- Figure 6 is an illustration of the HPAEC profile of the products of periodate oxidation of the trisaccharide resulting from N-acetylglucosaminidase digestion of compound I.
- Figure 7 is an illustration of the NMR spectra of compound I, laminaritriitol, and laminaribiitol.
- Figure a is an illustration of the profile from paper chromatography compound II after digestion witii ⁇ -glucosidase.
- Figure 8b is an illustration of the profile from paper chromatography compound III after digestion with ⁇ -glucosidase.
- Figure 9 is a graphic illustration of a scheme for the generation of differ oligosaccharides by chitinase digestion.
- Figure 10 is an illustration of the profile from paper chromatography o diacetylchitobiitol peak.
- Figure 1 ⁇ a is an illustration of the elution profile from a Bio-Gel P-2 colu of material separated from diacetylchitobiitol by paper chromatography.
- Figure lib is an illustration of the elution profile from a Bio-Gel P-2 colu of the material separated from diacetylchitobiitol after treatment with ⁇ - acety lglucosaminidase .
- Figure l ie is an illustration of the elution profile from a Bio-Gel P-2 colu of the material separated from diacetylchitobiitol after treatment with ⁇ -glucosidase.
- Figure 1 Id is an illustration of the elution profile from a Bio-Gel P-2 colu of the material separated from diacetylchitobiitol after treatment with ⁇ - acety lglucosaminidase and ⁇ -glucosidase.
- Figure 12 ⁇ is an illustration of the elution profile from a Bio-Gel P-2 colu of a pentasaccharide which elutes with triacetylchitotriitol.
- Figure 12b is an illustration of the elution profile from a Bio-Gel P-2 colu of the pentasaccharide separated in Figure 12 ⁇ after treatment with ⁇ -N-acety lglucosaminida
- Figure 12c is an illustration of the elution profile from a Bio-Gel P-2 colu of the pentasaccharide separated in Figure 12 ⁇ after treatment with ⁇ -glucosidase.
- Figure 13 ⁇ is an illustration of the elution profile from a Bio-Gel P-2 colu of borotritide-reduced yeast cell walls after endoglucanase treatment and second borotriti reduction.
- Figure 13b is an illustration of the elution profile from a Bio-Gel P-2 colu of borotritide reduced yeast cell walls after endoglucanase treatment.
- Figure 14 ⁇ is an illustration of the elution profile from a Bio-Gel P-2 colu of wild type strain D3C cell walls after treatment with endoglucanase, reduction, a incubation with chitinase.
- Figure 14b is an illustration of the elution profile from a Bio-Gel P-2 column of strain ECY36-3C (chsl chs2: :LEU2) cell walls after treatment with endoglucanase, reduction, and incubation with chitinase.
- Figure 14c is an illustration of the elution profile from a Bio-Gel P-2 column of strain ECY36-3D (chsl call/csd2) cell walls after treatment with endoglucanase, reduction, and incubation with chitinase.
- Figure 14d is an illustration of the elution profile from a Bio-Gel P-2 column of wild type strain ECY36-3D pHV9a cell walls after treatment with endoglucanase, reduction, and incubation with chitinase.
- compositions having antifungal properties in plants and animals are provided. These compositions comprise:
- Another aspect of the invention involves the isolation and identification of the fungal enzyme chitin glucan ⁇ (l ⁇ 4) transferase (CG ⁇ (l ⁇ 4)T). This enzyme catalyzes the formation of a ⁇ (l ⁇ 4) linkage between the terminal reducing GlcNAc residue of chitin and the non-reducing glucose residue of ⁇ (l ⁇ 3) linked glucans.
- the invention also includes methods for the prevention and the treatment of fungal infections in animals and plants.
- prophylactically or therapeutically effective amounts of the antifungal compositions of the invention are administered to the subject plant or animal as a prophylactic measure or for the treatment of an existing fungal disease.
- High-throughput screening methods for identifying CG ⁇ (l ⁇ 4)T inhibitors are also provided.
- the present invention includes several different types of antifungal composit as well as methods, which may include the use of the enzyme CG ⁇ (l ⁇ 4)T, for identif such compositions.
- Preferred antifungal compositions comprise (a) an antifungal effective amount of
- An oligosaccharide is a carbohydrate that is made up of 2-10 monosaccha units, any of which may be the same or different. (Grant &hackh's Chemical Diction 5th Ed. , McGraw Hill Book Co. , (1987)).
- the present inventors have discovered that the wall of fungi, and particularly of S. cerevisiae yeast includes a specific covalent cross-lin between chitin chains and glucans.
- terminal reducing acetylglucosamine residues of chitin are linked j3(l ⁇ 4) to the non-reducing glucose resi of /3(1 ⁇ 3) glucan chains.
- the oligosaccharides formed from N-acetylhexosamine resi linked ⁇ (l-*4) to a hexose are useful in antifungal compositions.
- the hexos either or each constituent of the oligosaccharide is glucose.
- a particular enzyme or class of enzymes catalyzes the forma of a ⁇ (l ⁇ 4) linkage between an N-acetylhexosamine and a hexose and particularly betwee acetylglucosamine and glucose.
- the enzyme (whose identification is described below) ca used in the identification of antifungal agents and in the design of antifungal agents.
- the s moieties that are substrates of the enzyme(s) may be single residues or may be present as of oligosaccharides as in, for example, chitin and glucan.
- This enzyme is found i cerevisiae, and this enzyme or homologues thereof are found in other fungal species includ but not limited to, Candida, Aspergillus, Histoplasma, Cryptococcus, and Coccidio species.
- the enzyme(s) may be identified and isolated biochemically.
- An assay is devised to measure CG ⁇ (l ⁇ 4)T enzymatic activity in a quantitative manner.
- the assay preferably includes a mixture of chitin (Sigma Chemical Co., St. Louis, MO) and in vivo- labeled glucan or laminarin (prepared by growing yeast cells in the presence of [ 3 H] or [ 1 C] glucose and isolating alkali-soluble glucan according to Bowers, B. et al.
- the source of the CG ⁇ (l ⁇ 4)T enzyme is a whole-cell extract of the fungal species studied, such as, for example, S. cerevisiae, produced by bead beating as described for glucan synthase (Kang et al. Proc. Natl. Acad. Sci, USA, 83:5808-5812, 1986).
- CG ⁇ (l ⁇ 4)T is purified using methods that are well known in the art of protein chemistry.
- the whole-cell extract may be fractionated by the sequential application of one or more of the following methods: ion-exchange chromatography, molecular sieve chromatography, and hydrophobic chromatography. These methods may be combined with extraction in detergents and/or mixtures of organic solvents that are known to those of ordinary skill in the art.
- chromatographic fractions and/or extracts are assayed for CG ⁇ (l ⁇ 4)T activity and for total protein content.
- a balance sheet of purification i.e. total activity, total protein, and specific enzymatic activity, is compiled at each step.
- Fractions showing peak CG ⁇ (l-*4)T activity are analyzed in parallel for their polypeptide profiles, using SDS-polyacrylamide gel electrophoresis. In this manner, fractions are obtained containing progressively higher specific activity for CG ⁇ (l ⁇ 4)T and fewer polypeptides.
- CG ⁇ (l ⁇ 4)T preparation is then sequenced, using the N-terminal Edman degradation reaction. If the preparation contains only a single major polypeptide, the preparation itself is sequenced. If several polypeptides are present, they may be resolved on SDS-polyacrylamide gel electrophoresis and transferred to nylon or other suitable membranes or excised from the gel directly. The individual protein species may then be sequenced separately, using automated microsequencing equipment such as, for example, that available from Applied Biosystems (Foster, City, CA). CG ⁇ (l ⁇ 4)T-related peptide sequences of about 10-20 amino acid residues are obtained.
- DNA fragments of 0.5-3 kb sequenced, and open reading frames are determined.
- the original peptide sequence used to design screening probes, as well deduced amino acid sequences derived from DNA clones, may also be used to des immunogenic peptides for the purpose of producing anti-CG ⁇ (l ⁇ 4)T antibodies.
- Peptides up to 40 residues may be synthesized chemically, and used, in conjunction with appropri carriers and adjuvants, as an immunogen in rabbits or other animals for the production polyclonal and monoclonal antibodies.
- Such antibodies are conveniently made using methods and compositions of Harlow and Lane, Antibodies, A Laboratory Manual, C Spring Harbor Laboratory, 1988.
- Anti-CG ⁇ (l ⁇ 4)T antibodies are used to quantify and/or affinity purify CG ⁇ (l ⁇ 4)T enzyme, as well as to screen cDNA expression libraries described above for purpose of identifying and cloning CG ⁇ (l ⁇ 4)T-related sequences.
- CG ⁇ (l ⁇ 4)T DNA sequence and the amino acid sequence of the CG ⁇ (l ⁇ protein can be used as a basis for large-scale purification of the CG ⁇ (l ⁇ 4)T protein and the design and testing of CG ⁇ (l ⁇ 4)T inhibitors.
- CG ⁇ (l-*4)T polypeptides may be synthesized in large amounts in E. coli, us commercially available vectors and bacterial hosts. An example of a suitable system is Invitrogen XpressTM system (San Diego, CA).
- the sequence "tags" enable the rapid affi purification of the products, after which the "tags” may or may not be proteolytically remo to produce an authentic CG ⁇ (l ⁇ 4)T polypeptide.
- CG ⁇ (l ⁇ 4)T polypeptides may also be modified with a label capable of providing a detectable signal, either directly or indirectly.
- exemplary labels include, but are not limited to, radioisotopes, fluorescent compounds, and the like.
- Labelled CG ⁇ (l-*4)Ts can be used, for example, in assays for inhibitory compounds.
- CG ⁇ (l ⁇ 4)T may be a monomer comprising a single polypeptide, may be a homomultimer, or may be a heteromultimeric molecule comprising different polypeptide chains.
- the polypeptide(s) may be modified by, for example, phosphorylation, sulfation, acylation, glycosylation, or other protein modifications.
- the CG ⁇ (l ⁇ 4)T may be isolated from its natural source or from heterologous organisms or cells, including, but not limited to, bacteria, yeast, insect cells, and mammalian cells, into which the gene or genes encoding CG ⁇ (l-*4)T polypeptide(s) have been incorporated.
- the present invention also contemplates derived proteins, and preferably fungal- derived proteins, with substantial sequence or functional homology to the CG ⁇ (l ⁇ 4)Ts described above.
- Sequence homology describes the relatedness of CG ⁇ (l ⁇ 4)Ts from different sources. Sequences are substantially homologous if at least about 70%, preferably at least about 80%, and most preferably at least about 90% of the two sequences are identical. Functional homology describes the stringency of hybridization conditions under which two sequences effectively or substantially hybridize. "Stringent" hybridization conditions are 0.1X SSC at 5°C.
- CG ⁇ (l-*4)T may be derived from fungal sources such as Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Candida species, and Aspergillus species such as Aspergillus fumigates.
- the CG ⁇ (l ⁇ 4)Ts are derived from other yeast-like organisms such as Candida, and most preferably, C. albicans. Non-S.
- CG ⁇ (l ⁇ 4)Ts may be identified and isolated by methods that are known in the art, such as, for example, antibody cross reactivity, PCR amplification from genomic DNA using degenerate oligonucleotide probes derived from the CG ⁇ (l ⁇ 4)T sequences identified as described above, low-stringency hybridizations using similar S. cerevisiae probes, and finally, functional cloning, in which a cDNA expression library derived from another species is used to transform and to complement an absent or defective CGT function in S. cerevisiae.
- CG ⁇ (l ⁇ 4)T may also be carried out genetically. For example, one could select conditional mutants which are synthetically lethal with chsl mutants or osmotically remedial. Once yeast mutants that lack activity have b isolated, the CG ⁇ (l ⁇ 4)T gene(s) may be identified by transforming the mutant strains yeast genomic DNA libraries or yeast cDNA expression libraries. Transformed clones then screened for re-acquisition of CG ⁇ (l- * 4)T activity. Finally, the DNA clones recovered from the yeast and are analyzed as described above for bacterial cloning syste
- the present invention also encompasses agents that prevent the production the enzyme CG ⁇ (l ⁇ 4)T or that prevent the enzyme from catalyzing the formation of the acetylhexosamine ⁇ (l ⁇ 4) hexose linkage described above.
- the inhibitory agents may comp peptides, oligosaccharides, lipids, derivatives of any of the foregoing, or other small orga molecules.
- the inhibitors comprise modified sugars or oligosaccharides that also highly specific in their inhibitory activity, i.e. inhibit pathogenic fungi without advers affecting animal or plant physiology.
- inhibitors of disaccharide linkages such as for example, allosamidin related analogues (Tet. Letts. , 35:4149, 1994); hydroquinones (Japanese Patent Publication 06-199649); moranoline (PCT Publication No. WO 94/04546); galactosyl-beta-l,3-gly (Scripps Res. Inst.); moenomycin A (Tetrahedron, 50:2029, 1994); di-and tri-saccharides intramolecular NH glycosidic linkages (Carbohyd. Res. , 252: 159, 1994); heteromet maltosides (J. Am. Chem.
- inhibitors of CG ⁇ (l ⁇ 4)T include, but are not limited to, compou comprising a terminal GlcNAc residue in which the carbon at the 1 position of the ring ( 1-carbon) is modified.
- Carbon atom ring numbers are illustrated in Figure 7).
- suitable modifications at this position include, but are not limited to, (CH 2 ) n - wherein n is an integer from 1 to 12, C r C ⁇ 2 alkyl, unsubstituted aryl, aryl substitu preferably with C,-C 10 alkyl or alkenyl, or allosamiz ⁇ line (Takehaski et al. , Tet. Letts. , 32:5123, 1991).
- hexose and preferably glucose molecules in which the carbon at the 4 position of the ring (the 4-carbon) is modified to prevent formation of the (31 ⁇ 4) GlcNAc linkage see Figure 7
- Suitable modifications of glucose include, but are not limited to, (CH 2 ) n -OH, wherein n is an integer from 1 to 12,), C,-C ⁇ 2 alkyl, unsubstituted aryl, aryl subtituted preferably with C,-C, 0 alkyl or alkenyl, or allosamizoline (Takehaski et al., Tet. Letts. , 32:5123, 1991). Either constituent of the oligosaccharide can be further modified at the other carbons.
- Rational design of oligosaccharide-based inhibitors is based on an extensive enzymological analysis of CG ⁇ (l ⁇ 4)T activity. That is, the relative activity of different
- GlcNAc polymers (chitin precursors) of different lengths, as well as for different configurations and lengths of (
- Both GlcNAc- and glucose-based inhibitory compounds may comprise one or more sugar units. These compounds may also contain other modifications to enhance their efficacy, including those that cause the compound to be retained in the periplasmic space of the target organism. Preferably, inhibitors will be freely taken up across the fungal cell wall but will not cross the plasma membrane. The only limitation is that the modified compounds retain their capacity to bind CG ⁇ (l-*4)T and to inhibit its enzyme activity.
- Antifungal compositions are prepared from the N-acetylhexosamine residue ⁇ (l-*4) hexose oligosaccharides; CG ⁇ (l ⁇ 4)T inhibitors, including, but not limited to. modified N-acetylglucosamine or modified glucose; or any combination thereof as an active agent in a biologically acceptable carrier.
- Suitable biologically acceptable carriers include, but are not limited to, phosphate-buffered saline, saline, deionized water, or the like. Preferred biologically acceptable carriers are physiologically or pharmacologically acceptable carriers.
- the antifungal compositions include an antifungal effective amount of active agent.
- Antifungal effect amounts are those quantities of the antifungal agents of the present invention that afford prophylactic protection against fungal infections in plants and animals, and which result in amelioration or cure of an existing fungal infection in plants or animals.
- This antifungal effective amount will depend upon the fungus, the agent, and the host. amount can be determined by experimentation known in the art, such as by establishi matrix of dosages and frequencies and comparing a group of experimental units or subjec each point in the matrix.
- the antifungal compositions could act, for example, via inhibition transglycosidation, by a competitive or non-competitive mechanism. These compositions c also inhibit the reaction by mass action end product inhibition.
- compositions could inhibit the cleavage of the chitin-glucan linkage that may normally o during growth or expansion of the cell wall.
- the antifungal active agents or compositions can be formed into dosage forms, such as for example, creams, ointments, lotions, powders, liquids, tablets, caps suppositories, sprays, or the like.
- the dosage unit form may contain an antifungal effective amount of active ag
- the dosage unit form may include less than such an amount if multiple do unit forms or multiple dosages are to be used to administer a total dosage of the active ag
- Dosage unit forms can include, in addition, one or more excipient(s), diluen disintegrant(s), lubricant(s), plasticizer(s), colorant(s), dosage vehicle(s), absorp enhancer(s), stabilizer(s), bactericide(s), or the like.
- the antifungal agents and compositions of the present invention are useful preventing or treating fungal infections in plants and animals.
- Fungal infection preven methods incorporate a prophylactically effective amount of an antifungal agent or composit
- a prophylactically effective amount is an amount effective to prevent fungal infection and depend upon the fungus, the agent, and the host. These amounts can be determ experimentally by methods known in the art and as described above.
- Fungal infec treatment methods incorporate a therapeutically effective amount of an antifungal agen composition.
- a therapeutically effective amount is an amount sufficient to stabilize o ameliorate a fungal infection. Preferably, this amount will yield a reduction to less than 1 of the amount of fungus present at initiation of treatment.
- prophylactically and/or therapeutically effective amounts can administered in one administration or over repeated administrations.
- Therape administration can be followed by prophylactic admimstration, once the initial fungal infection has been resolved.
- the antifungal agents and compositions can be applied to plants topically or non- topically, i.e., systemically. Topical application is preferably by spraying onto the plant. Systemic admimstration is preferably by application to the soil and subsequent absorption by the roots of the plant.
- the antifungal composition that includes an N- Acetylhexosamine ( ⁇ - * 4) hexose oligosaccharide can be administered in an amount that effectively saturates the fungus and its environment, thereby inhibiting the CG ⁇ (l ⁇ 4)T enzyme.
- the antifungal agents and compositions can be administered to animals topically or systemically.
- Systemic administration with respect to animals include both oral and parental routes. Parental routes include, without limitation, subcutaneous, intramuscular, intraperitoneal, intraduodenal, and intravenous administration.
- Antifungal compounds may be identified using screening methods including, but not limited to, high-throughput screening methods that are based on a modified CG ⁇ (l ⁇ 4)T assay.
- a screen for compounds that differentially affect the viability of chsl versus chs3 imttant yeast strains would be expected to detect inhibitors of CG ⁇ (l ⁇ 4)T.
- Another screening method involves growing yeast cells in an osmotic remedial medium in the present of potential inhibitors, followed by an assay for alkali-soluble versus alkali-insoluble glucan. This ratio would increase upon inhibition of CG(1 ⁇ 4)T.
- compounds are screened for their ability to bind to CG ⁇ (l ⁇ 4)T polypeptides purified as above.
- Assays involve screening test inhibitory compounds from large libraries of synthetic or natural compounds. Synthetic compound libraries are commercially available from, for example, Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical library is available from Aldrich Chemical Company, Inc. (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from, for example, Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and bioche means.
- a preferred screening method includes the steps of (a) contacting a CG ⁇ (l enzyme as described above with an N-acetylhexosamine and a hexose in the presence o antifungal candidate to form a test mixture; (b) contacting the enzyme with the same acetylhexoseamine and hexose in the absence of the candidates to form a control mixtur (c) detecting any formation of a ⁇ (l ⁇ 4) linkage between the N-acetylhexoseamine and hexose in the test mixture and in the control mixture; (d) comparing the efficienc formation of that linkage in the test mixture and in the control mixture; and (e) selectin an antifungal compound the test candidate compound that causes a decrease in the effici of formation of the linkage in the test mixture relative to the efficiency of formation of linkage in the control mixture.
- test compound Once a particular test compound has been identified in a high-throughput scr its inhibitory activity is then confirmed by measuring its effect on CG ⁇ (l ⁇ 4)T activity standard (i.e. low-throughput) assay. Finally, the compound is tested for two properties: the ability to inhibit fungal growth; and (2) a lack of effect on animal and/or plant c
- Fungal growth is measured by any method well-known in the art, for example, optical de of a liquid culture or colony formation on agar.
- the potential toxic ity of an agent mammalian cells is measured by monitoring its effect in a typical mammalian cell culture, s as, for example, L-cells.
- Enzymes and Reagents ⁇ -N-Acetylglucosaminidases from jack beans or from Diploco pneumoniae(Oxfo ⁇ d Glycosystems, Inc.-Rosedale, NY). ⁇ -N-Acetylglucosaminidase from beef kidney and ⁇ -glucosidase (Boehri Mannheim-Indianapolis, IN). ⁇ (l ⁇ 3) endoglucanase.
- ECY36-3C (MATa chsl-23 chs .LEUl trpl-1 ura3-51 leul-1) - CHsl- and Chs2-deficient.
- ECY36-3D pHV9A - contains plasmid pHV9A which carries the CAL1/CSD2 gene that restores Chs3 activity.
- Strains D3C and ECY36-3D were grown in YEPD (1 % yeast extract, 2% peptone, 2% glucose), and strains ECY36-3C and ECY36-3D pHV9A were grown in minimal medium (2% glucose, 0.7% Difco yeast nitrogen base without amino acids) plus nutritional requirements. In all cases growth was at 30°C.
- the extract was aspirated from the glass beads, and the aspirated extract was washed several times with small portions of Tris buffer. Cell walls were sedimented by centrifugation at 4,000 x g for 10 minutes, the pellets were washed five times with Tris buffer. The washed cell walls were suspe in the same buffer to a final volume of 495 ml. ⁇ (l ⁇ 3) endoglucanase (Zymolyase 100 T) (4.3 ml of a 7.5 mg/ml solutio 50 mM sodium phosphate, pH 6.3) was added. The suspension was incubated at 37° C shaking, and the absorbance at 660 nm was monitored. After about one hour, the absorb had decreased to about 5% of the original value.
- the suspension was centrifuged for 10 minutes at 16,000 g.
- the pellet washed twice with Tris buffer and twice with 1 % SDS.
- the suspension was placed f minutes in a boiling water bath in the second SDS washing.
- the pellet was then washed t more times with water and was suspended in water to a concentration of 10 mg chiti (about one ml in the preparation described.)
- the ratio of total N-acetylglucosamine to glucose at this step was 1:0.7.
- 600 ⁇ l of the resultant suspension was treated with 600 ⁇ l of NaB 3 H 4 (6 in dimethylformamide at room temperature for 5 hours.
- the reaction was terminated by addition of 300 ⁇ l of 1 M acetic acid, and the insoluble material was washed by repe centrifugation, followed by suspension in 50 mM potassium phosphate at pH 6.3. washed, reduced chitin was digested overnight at 30 °C with 600 ⁇ l (184 mU) of marcescens chitinase. Any insoluble residue was removed by centrifugation.
- the supernatant fluid was used directly for Bio-Gel P-2 chromatography. compounds isolated from the eluate of the Bio-Gel column were again reduced with an ex of unlabeled sodium borohydride and repurified by Bio-Gel chromatography prior to fur analysis.
- HPAEC Dionex high performance amino exch chromatography
- PAD2 pulsed amperometric detector M PAD2
- pellicular anion-exchange columns PA-1 or MA-1, 4 x 250 mm.
- the Di eluent degas module was used to sparge and pressurize the eluents with the helium set at p.s.i.
- the flow rate was maintained at 0.8 ml/min.
- the applied pulse potential was 0.0 and detector sensitivity was set at 300 nA.
- the system was used at ambient temperat Samples were applied via a Dionex microinjection valve with a 50- ⁇ l loop. Areas unde curve were recorded and integrated with a Spectra-Physics integrator.
- the eluent contained 100-500 mM NaOH.
- Mass spectrometry Chemical iodization mass spectra were obtained with a Finigan 1015D spectrometer, using ammonia as the reactive gas.
- Optical rotations were measured at 25° C with a Perkin Elmer Model 241 MC automatic polarimeter. All reactions were monitored by thin-layer chromatography on pre- coated slides of silica gel G F254 (Analtech). Detection was effected by charring with 5% sulfuric acid in ethanol or, when applicable, with UV light. Preparative chromatography was performed by gradient elution from columns of Silica Gel 60 (Merck, No. 9385). Reacti requiring anhydrous conditions were performed under dry nitrogen using common laborat glassware and reagents and solvents were handled with gas-tight syringes.
- 1,2,3,4-Tetra-O-acetyl- ⁇ -D-glucopyranose (0.7 g, 2 mmol) was dissolved in nitromethane (30 ml), and 4A molecular sieve (1 gram) was added. After cooling to 30 the reaction mixture was stirred for 30 minutes, and sym-collidine (0.28 ml, 2 mmol) silver triflate (0.54 gram , 2. 1 mmol) were added .
- F inal bromo-2-deoxy-2-N-phtalimido-3,4,6- tri-O-acetyl- ⁇ , ⁇ -D-glucopyranose (1 gram, 2 m dissolved in nitromethane (5 ml) was added dropwise.
- the reaction mixture was kept under reflux (65° C) for 2 hours, at which point starting material was consumed as monitored by TLC (ethyl acetate/ethanol/water, 8:4:2). After cooling to room temperature, acetic anhydride (0.5 gram, 0.46 ml, 4.9 mmol) was added, and the mixture was stirred for 20 minutes. When the starting material was no longer detected by TLC (propanol/ethylacetate/ water 2: 1: 1), the reaction mixture was concentrated and purified on a Bio-Gel P-2 extra-fine, 2 x 90 cm column to yield 6-O-(2- acetamido-2-deoxy- ⁇ -D-glucopyranosyl)- ⁇ , ⁇ -D-glucopyranose .
- yeast cell walls w digested with a ⁇ (l-3) endoglucanase (zymolyase), with the expectation of forming s glucose oligosaccharide stubs attached to the chitin.
- the insoluble fraction was reduced with sodium borotritide, to label the reducing e of the stubs.
- This treatment also reduced and labeled GlcNAc residues at the reducing en chitin chains not bound to glucan.
- the labeled material was then digested with S.
- marcesc exo-chitinase an enzyme that sequentially cleaves diacetylchitobiose residues from chi starting from the non-reducing end (See, Roberts et & ⁇ . , Anal. Biochem. , 127:402-412, 198
- the chitinase-solubilized fraction from 6 mg of glucanase-resistant insolu residue was applied to an extra-fine Bio-Gel P-2 column (2 x 90 cm) and was eluted with
- 2-8 indicate the positions of the following standards: 2, triacetylchitotriitol (or laminarihexaitol); 3, laminaripentaitol; 4, diacetylchitobiitol (laminaritetraitol); 5, laminaritriitol; 6, GlcNAc-ol (or laminaribiitol); 7, glucitol; 8, Glc ([ 14 C]) glucose was added as internal standard).
- Peak A which corresponds to a reduced glucose pentasaccharide standard.
- Compound I (Peak A - Figure 1) was treated with N-acetyl- ⁇ -glucosamini and ⁇ -glucosidase to determine the manner and position at which the GlcNAc was attac Aliquots ( ⁇ 130 pmol, 130,000 cpm) of compound I were evaporated to dryness and redissolved in 50 ⁇ l of 100 mM citrate-phosphate buffer, pH 5.0 ( Figure 4a); in the s buffer plus 5 ⁇ l (135 mU) of ⁇ -N-acetylglucosaminidase from jack beans ( Figure 4b); a 60 ⁇ l of 0.1M acetate buffer pH 4.5, containing 0.1 mg of sweet almond ⁇ -glucosidase (Fi 4c).
- Results are illustrated in Figure 4. The positions of peaks A, B and C are indicated.
- Results are illustrated in Figure 5b.
- the tentative structure of compound I and of the hydrolysis products are shown, where an open square stands for GlcNAc, an open circle for Glc and a filled circle for glucitol.
- Trisaccharide Periodate Oxidation A portion of compound I (60 nmol) was digested with jack bean ⁇ acetylglucosaminidase and was subjected to Bio-Gel P-2 chromatography essentially described above. The recovered trisaccharide (50 ⁇ l) was oxidized with 700 nmol of sodi metaperiodate for 70 hours at 4°C in the dark. Ethyleneglycol (1 % , 23 ⁇ l) was added. A 2 hours at room temperature, 40 ⁇ l of 0.1 NaOH and 50 ⁇ l of sodium borohydride in 0.01 NaOH were added. Incubation was continued for 3 additional hours.
- the sample evaporated to dryness under nitrogen, dissolved in 100 ⁇ l of 2 M trifluoracetic acid, heated at 100°C for 2 hours. After evaporation to dryness, the residue was dissolved in ⁇ l water and a 50- ⁇ l portion was subjected to HPAEC on a PA-1 column with 0.2 M Na as solvent.
- Laminaribiitol and laminaritriitol (50 nmol of each) were subjected to the sa treatment and chromatographed.
- GlcNAc( ⁇ l-6)Glc was synthesized. This compound eliminated the 1 ⁇ 6 linkage as a possibility for the chitin/N-acetylglucosamine linkage because the synthetic compound was decomposed by beef kidney ⁇ -N-acetylglucosaminidase whereas compound I was resistant (data not shown). The possibility that GlcNAc was attached to Glc by a 1-2, 1-3 or 1-4 linkage still remained. Since the amount of material available was insufficient for methylation analysis, NMR spectroscopy was employed. "C-NMR Spectrum Approximately 1 ⁇ mol of compound I was evaporated to dryness several times with D 2 O, and then was dissolved in 600 ⁇ l of D 2 O.
- C"-2 and C"-6 were eliminated as participants in the bond, because their chemical shifts, 73.55 and 73.35 ppm for C"-2 and 60.65 and 60.86 ppm for C"-6, are the same for compound I and for reduced laminaritriose. If the glycosidic linkage were at position C"-3, one of the signa the region 75.41-75.97 ppm would move to lower field in the spectrum of compoun because this is the area in which carbons C-5, C"-5 and C"-3 are located. This shift di occur.
- a portion (5000 cpm) of compound II was evaporated to dryness and disso in 30 ⁇ l of acetate buffer at pH 4.5, containing 10 ⁇ g of sweet almond ⁇ -glucosidase. 16 hours of incubation at 37° C, the sample was analyzed by paper chromatography.
- ⁇ -Glucosidase Digestion A portion (10 pmol, 10,000 cpm) of compound III was evaporated to dry and dissolved in 30 ⁇ l of acetate buffer at pH 4.5, containing 10 ⁇ g of sweet almon glucosidase. After 16 hours of incubation at 37° C, the sample was analyzed by pa chromatography. Standards: Glc; 2, laminaribiitol; 3, sophoritol; 4, cellobiitol; gentiobiitol.
- Figure 9 illustrates a scheme for the generation of different oligosaccharides chitinase digestion.
- Chitinase is able to cut between a GlcNAc and a Glc residue, if linkage between the two sugars is ⁇ (l ⁇ 4). Therefore, compounds I and II would be deri from chitin chains with an odd or even number of GlcNAc residues, respectively, b attached to a reduced laminaritriose.
- Compound III would result from hydrol of an even-numbered chain linked to laminaribiitol.
- the slow-moving labeled band was excised and eluted with water.
- the standards were: laminaritriitol; 2, laminaribiitol; 3, diacetylchitobiitol; 4, glucitol.
- the slow moving radioactive material was eluted with water from paper, concentrated and treated with ⁇ -N-acetylglucosaminidase followed by Bio-Gel P-2 chromatography as described above. Results are illustrated in Figure 12b.
- the pentasaccharide was also incubated with ⁇ -glucosidase followed by Bio-Gel P-2 chromatography as described above. Results are illustrated in Figure 12c.
- the standards were: 1, triacetychitobiitol or laminarihexaitol; 2, laminaripentaitol; 3, diacetychitobiitol or laminaritetraitol; 4, laminaritriitol; 5, compound III or laminaribiitol; 6, glucose.
- This substance was resistant to ⁇ -glucosidase, but was digested by ⁇ -N- acetylglucosaminidase, with concomitant displacement to the laminaritetraitol position in the P-2 column.
- the substance had the expected properties of compound VI (GlcNAc- ⁇ -Glc- ⁇ - Glc- ⁇ -Glc- ⁇ -Glc-ol).
- glucose oligosaccharides attached to chitin preexisted as such in the intact cell wall before glucanase digestion, rather than being part of a larger chain. If the glucose oligosaccharides did pre-exist, they would be labeled i borotritide reduction were performed before, rather than after treatment with gluca Therefore, walls were reduced twice, before and after ⁇ (l ⁇ 3) endoglucanase treatment were chromatographed on a Bio-Gel P-2 column. Results are illustrated in Figure 13a.
- Chsl , Chs2 and Chs3 Three different chitin synthetases (Chsl , Chs2 and Chs3) participate in diffe aspects of chitin synthesis in yeast (Shaw et al. , J. Cell Biol , 114: 111-123, 1991; Cabi al., J. Cell. Biol , 108: 1665-1672, 1989). Mutants in each of the three synthetases available.
- Wild type strain D3C cell walls were treated with endoglucanase, redu incubated with chitinase, and chromatographed on Bio-Gel P-2 columns as described ab Results are illustrated in Figure 14 ⁇ .
- the standards were: 1 , void volume; triacetylchitotriitol; 3, diacetylchitobiitol; 4, diacetylchitobiose; 5, laminaritriitol; laminaribiitol; 7, GlcNAc; 8, Glc.
- Results are illustrated in Figures 14b, c, and d, respectively.
- Tritium-labeled Void Volume Peak The material solubilized by glucanase and chitinase digestion was fractionated on P-2 columns. A fairly large amount of radioactivity emerged at the void volume (Figure 1). This material was rechromotographed on Sephacryl S-200 and Sephacryl S-300 columns. Results indicated that the material was heterogeneous and of high molecular weight, in the 200,000-300,000 dalton range. NMR spectra were similar to those of pustulan, a ⁇ (l ⁇ 6)- linked glucan, although other components appeared to be present. Acid hydrolysis released glucose and some mannose.
- the void volume material was barely detectable in the Chs3 mutant ( Figure 14c) and was restored by the C i/CSD2plasmid ( Figure I4d), which indicated that it was originally bound to chitin whose synthesis depends on the presence of Chs3.
- the void volume labeled material was also somewhat reduced in the chsl -chsl mutant ( Figure 14b) as well as in the wild-type fraction resulting from cell walls reduced with borotritide before glucanase digestion ( Figure 13b).
- Example 1 illustrates that the oligosaccharides containing GlcNAc linked ⁇ (l ⁇ 4) to glucose were not solubilized until cell walls were digested with both ⁇ -glucanase and chitinase. This indicates that the oligosaccharides originate in the linkage region of glucan and chitin. The presence of both N-acetylglucosamine and glucose in some of the compounds confirmed this. The short glucose chains were originally part of the glucan. because they are protected from reduction when the polysaccharide is intact.
- the structure of compound I corresponds to an original oligosaccharide (before reduction) containing one N-acetylglucosaminyl group linked in ⁇ (l ⁇ 4) to laminaritriose.
- Compound I and the other five compounds studied can be arranged in two homologous series, one containing 2, 3, or 4 ⁇ (l-3)-linked glucose units and the other with the same units plus an N-acetylglucosaminyl group at the non-reducing end.
- the different lengths of the glucose moieties was due to some variability in the position of the ⁇ (l ⁇ 3) linkage hydrolyzed by the zymolyase preparation.
- the sum of reduced diacetylchitobiose and triacetylchitobiose is equivalen the number of free chitin chains.
- the sum of oligosaccharides should be equivalent to number of glucan-linked chains. This analysis suggests that between 40 and 50% of the c chains are engaged in linkage with glucan.
- the chitin to glucan ratio in the cell wall is a 1: 10 in strain D3C.
- the effect of small amounts of chitin on the solubilization in hot al of about 70% of the glucan Mol et al., F.E.M.S. Microbiol. Lett.
- Chs3 is enzyme responsible for the formation of the chitin that is incorporated into the chitin- ⁇ (l linked-hexose oligosaccharide. This is consistent because it is known that Chs3 is invol in the synthesis of 80-90% of the cell wall chitin, including that present in a ring at the of an emerging bud and that dispersed throughout the wall (Shaw et al. , J. Cell Bi 114: 111-123, 1991; Bulawa et al., P.N.A.S., USA, 87:7424-7428, 1990).
- chitin glucan bond may be formed in periplasmic space by transglycosylation from a newly-formed chitin chain (Cabib et Microbiol. Sci. , 5:370-375, 1988). According to this hypothesis, a portion of the chitin c would be released in the reaction.
- An alternative mechanism is possible if chitin chains g from reducing end, as does the O-antigen of Gram-negative bacteria (Robbins et al., Scie 158: 1536-1542, 1967). In that case, the GlcNAc residue at the reducing end would re activated during synthesis, and the whole nascent chain could be transferred directly to gluc Example 2 - An Enzymatic Assay for Chitin-Glucan Linkage
- chitin (10 mg/ml in PBS), 50 ⁇ l of 3 H-glucan (prepared as in Example 1) (10 4 cpm/mg, 10 mg/ml in PBS), and 10 ⁇ l of CG ⁇ (l ⁇ 4)T source are mixed.
- the mixture is incubated at 30°C for 1 hour.
- 5 ⁇ l of a solution of zymolyase 100T (7.5 mg/ml, prepared as in Example 1) are added, and the incubation is continued for 1 hour at 37°C.
- 200 ⁇ l of ice-cold 20% trichloroacetic acid is added, and the resultant mixture is incubated on ice for 5 minutes.
- the reaction mixture is filtered through Whatman GF/C filters. The radioactivity associated with the filters is then quantified by liquid scintillation counting. Controls are prepared either omitting the enzyme source or with 2 mg unlabelled glucan.
- a dose-dependent increase in acid-precipitable radioactivity indicates the formation of a chitin-glucan linkage.
- Example 2 The assay described in Example 2 is adapted for high-throughput screening as follows:
- a known source of the enzyme is used, such that 50% of the 3 H-glucan (i.e. 2500 cpm) is converted from an acid-soluble to acid-insoluble form during the reaction.
- Reaction mixtures are formed in 96-well microliter dishes according to the procedure of Example 2, with the addition of 15 ⁇ l of a solution containing test inhibitory compounds. After incubation at 30 °C for 1 hour, zymolyase 100T is added according to the procedure of Example 2 and incubation is continued for an additional hour.
- each well is transferred to a sheet of Whatman 3MM filter paper.
- the paper is immersed in ice-cold 10% trichloroacetic acid for 10 minutes and is then washed in 5% trichloroacetic acid. Areas of the paper corresponding to each well are excised and counted. A reduction in the number of cpm detected in a given well indicates a candidate inhibitory compound.
- a candidate antifungal agent is dissolved in a biologically acceptable solvent such as saline.
- Serial 10-fold dilutions of the agent are prepared in yeast growth medium. 10 ml aliquots of each dilution are inoculated with 10 4 yeast cells, followed by incubatio 30°C. At hourly intervals, the growth of the cultures is ascertained by measuring absorbance at 600 nm.
- An effective antifungal agent is one that suppresses the growth of yeast cells >90% at concentrations that are practical for agricultural or medicinal applications.
- An antifungal formulation for agricultural use is prepared by mixing acetylglucosamine-3(l ⁇ 4)-glucose with deionized water.
- the resultant composition is spra on a fungally infected plant.
- An antifungal formulation suitable for animal use is prepared by mixing acetylglucosamine ⁇ (l ⁇ 4)glucose with saline.
- the resultant solution is administe systemically to a mammal suffering from a fungal infection.
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AU49644/96A AU702725B2 (en) | 1995-01-04 | 1996-01-02 | Compositions and methods for inhibiting fungal cell wall formation |
EP96906177A EP0796101A4 (en) | 1995-01-04 | 1996-01-02 | Compositions and methods for inhibiting fungal cell wall formation |
JP8521293A JPH10512247A (en) | 1995-01-04 | 1996-01-02 | Compositions and methods for inhibiting fungal cell wall formation |
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US5030619A (en) * | 1990-04-16 | 1991-07-09 | Miles Inc. | Synergistic fungicidal composition |
US5413991A (en) * | 1991-01-31 | 1995-05-09 | Ajinomoto Co., Inc. | Allosamidin compounds |
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US5326561A (en) * | 1992-12-15 | 1994-07-05 | Cornell Research Foundation, Inc. | Antifungal synergistic combination of enzyme fungicide and non-enzymatic fungicide and use thereof |
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1996
- 1996-01-02 EP EP96906177A patent/EP0796101A4/en not_active Withdrawn
- 1996-01-02 AU AU49644/96A patent/AU702725B2/en not_active Ceased
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- 1996-01-02 JP JP8521293A patent/JPH10512247A/en active Pending
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US5030619A (en) * | 1990-04-16 | 1991-07-09 | Miles Inc. | Synergistic fungicidal composition |
US5413991A (en) * | 1991-01-31 | 1995-05-09 | Ajinomoto Co., Inc. | Allosamidin compounds |
Non-Patent Citations (5)
Title |
---|
CABIB E, ET AL.: "Chitin Synthase 1, an Auxiliary Enzyme for Chitin Synthesis in Saccharomyce s Cerevisiae", THE JOURNAL OF CELL BIOLOGY : JCB, THE ROCKEFELLER UNIVERSITY PRESS, US, vol. 108, 1 May 1989 (1989-05-01), US, pages 1665 - 1672, XP002969748, ISSN: 0021-9525, DOI: 10.1083/jcb.108.5.1665 * |
GAUGHRAN J P, ET AL.: "Nikkomycin Z is a Specific Inhibitor of Saccharomyces Cerevisiae Chitin Syn thase Isozyme Chs3 in Vitro and in Vivo", JOURNAL OF BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 176, no. 18, 1 September 1994 (1994-09-01), US, pages 5857 - 5860, XP002971208, ISSN: 0021-9193 * |
HEUER M, ET AL.: "STRUCTURAL ANALOGUES OF THE ANTIBIOTIC MOENOMYCIN A WITH A D-GLUCOSE-DERIVED UNIT F", TETRAHEDRON, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 50, no. 07, 1 January 1994 (1994-01-01), AMSTERDAM, NL, pages 2029 - 2046, XP001061722, ISSN: 0040-4020, DOI: 10.1016/S0040-4020(01)85066-3 * |
See also references of EP0796101A4 * |
SHAW J A, ET AL.: "The Function of Chitin Synthase 2 and 3 in the Saccharomyces Cerevisiae Cel l Cycle", THE JOURNAL OF CELL BIOLOGY : JCB, THE ROCKEFELLER UNIVERSITY PRESS, US, vol. 114, no. 1, 1 July 1991 (1991-07-01), US, pages 111 - 123, XP002969749, ISSN: 0021-9525, DOI: 10.1083/jcb.114.1.111 * |
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CN110387364A (en) * | 2019-08-05 | 2019-10-29 | 河北农业大学 | A kind of recombinant chitinase and its relevant biological material and application |
CN110387364B (en) * | 2019-08-05 | 2023-09-01 | 河北农业大学 | Recombinant chitinase and related biological material and application thereof |
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JPH10512247A (en) | 1998-11-24 |
CA2209663A1 (en) | 1996-07-11 |
EP0796101A1 (en) | 1997-09-24 |
AU702725B2 (en) | 1999-03-04 |
AU4964496A (en) | 1996-07-24 |
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EP2167674B1 (en) | Nutritional compositions |
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