WO1998057176A1 - Protein elongation factor 2 as a target for antifungal and antiparasitic agents - Google Patents
Protein elongation factor 2 as a target for antifungal and antiparasitic agents Download PDFInfo
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- WO1998057176A1 WO1998057176A1 PCT/US1998/011903 US9811903W WO9857176A1 WO 1998057176 A1 WO1998057176 A1 WO 1998057176A1 US 9811903 W US9811903 W US 9811903W WO 9857176 A1 WO9857176 A1 WO 9857176A1
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- compound
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
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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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56961—Plant cells or fungi
Definitions
- PROTEIN ELONGATION FACTOR 2 AS A TARGET
- Elongation factor 2 is an essential protein catalysing ribosomal translocation during protein synthesis in eukaryotic cells. It is highly conserved in all eukaryotes, and has been found to be largely interchangeable in in vitro protein synthesis systems reconstituted from such divergent organisms as human, wheat germ, and fungi. Despite the ubiquitous nature of EF2 in eukaryotic systems and the high degree of amino acid sequence homology between EF2s from various eukaryotic systems, a class of compounds, the sordarins, have now been identified to be selective inhibitors of fungal protein synthesis via a selective interaction with fungal EF2.
- the present invention relates to elongation factor 2 (hereinafter referred to as "EF2") as a target for antifungal and antiparasitic agents.
- EF2 elongation factor 2
- the invention relates to a method for identifying potential antifungal and antiparasitic agents by determining whether a test compound is capable of specifically inhibiting pathogenic protein synthesis via a selective interaction with pathogen EF2.
- the present invention describes the use of mechanism based assays with or without the use of a transformed eukaryotic organism with a heterologous EF2 to facilitate drug discovery.
- the invention relates to a method for treating fungal infections by administering to a host suffering from a fungal or parasitic infection a therapeutically effective amount of a compound that specifically inhibits the pathogen's protein synthesis via EF2.
- This invention provides a method for identifying and evaluating compounds having antifungal and antiparasitic activity comprising: A differential two plate assay containing genetically engineered sordarin sensitive (sSl) and resistant (sRl) strains or naturally selected sordarin resistant strains of yeast.
- the readout of the assay is antimicrobial activity indicated by zones of inhibition which is more apparent against the sordarin sensitive strain relative to the sordarin resistant strain.
- sSl sordarin sensitive
- sRl sordarin resistant
- EFT1 and EFT2 and at least one of these genes is required for survival.
- the co-isogenic strains sSl and sRl were constructed by a series of genetic crosses that result in strains that are disrupted for both the EFT1 and ERG6 genes. The resultant strains are made more permeable due to the ergo disruption (ergosterol deficient), and have either a wild-type or resistant copy of EFT2 as the only source of EF2.
- a known number of these yeast cells are either plated in solid medium or suspended in liquid medium and test compounds or fermentation extracts are applied with the intent of identifying samples which inhibit the growth of these yeast. The cultures are incubated at a specific temperature for a set period of time to allow for the growth of the test organisms (i.e.
- Test samples of interest are those which show a differential effect on the sordarin sensitive strain(s) vs. the resistant strain(s). Those samples which are more potent against the wildtype by definition should be preventing growth via the EF2 target.
- the present invention provides a method for identifying compounds specifically inhibiting pathogenic EF2 function comprising: (a) constructing fungal or protozoan cells dependent upon heterologous EF2 from fungal and parasitic pathogens or from the host species; (b) contacting said cell with a known dilution of a test compound or a natural product extract; and (c) quantitating the minimal inhibitory concentration (MIC) of test compound to completely inhibit growth in liquid or the measurement of an inhibitory zone on a solid substrate.
- Test compounds or fermentation extracts of interest are those which display a differential degree of inhibition (i.e. more inhibitory activity against the wildtype vs. resistant strains of the test organisms). For example a sample which is more effective at inhibiting the growth of a yeast EF2 dependent organism vs. one that is dependent on human EF2.
- the methods of the invention provides a facile and specific assay to screen compounds as potential antifungal and antiparasitic agents. It also allows for the evaluation of test compounds against the EF2 target of obligate pathogens that cannot be cultured in the laboratory.
- EF2 may be cloned from pathogenic organisms for use in growth inhibition assays or purified from these pathogens for use in in vitro binding or translation inhibition assays.
- the EF2 may be from pathogenic fungi of humans, animals or plants such as Candida, Aspergillus spp., Cryptococcus spp., Erysiphe and Puccinia. It may also be from protozoan parasites such as Plasmodium sp., Eimeria sp., Crypto sporidium sp. and Toxoplasma gondii and human and other desired host eukaryotic cells.
- a compound that inhibits EF2 may be one that interferes with the translation of mRNA in target organisms.
- Examples of compounds that inhibit EF2 include diptheria toxin and fusidic acid, however neither of these show any specificity for pathogen over host. Fusidic acid inhibits translation in many organisms by disrupting normal ribosome-EF2 interactions.
- the compound that inhibits EF2 is preferably labeled to allow easy quantitation of the level of interaction between the compound and the enzyme.
- a prefered radiolabel is tritium.
- the test compound may be a synthetic compound, a mixture of synthetic compounds, a crude preparation, a purified preparation or an initial extract of a natural product obtained from plant, microorganism or animal sources.
- sordarin is an antifungal antibiotic isolated from the mould Sordaria araneosa (see GB 1,162,027 and Helvetica Chimica Acta, 1971, 51:119-20).
- Other compounds having the sordarin skeleton have also been reported as antifungal agents.
- Japanese Kokai J62040292 discloses the compound zofimarin isolated from Zofiela marina sp.
- Japanese Kokai J06157582 discloses the compound BE-31405 isolated from Penicillium sp.
- SCH57404 is reported in J. Antibiotics.
- sordaricin the aglycone
- Sordaricin may be obtained from sordarin by acid hydrolysis (Hauser and Sigg, Helvetica Chimica Acta, 1971, 51: 119-20); similarly sordaricin methyl ester is obtained from sordarin methyl ester.
- the total synthesis of sordaricin methyl ester is reported in Kato et al, J. Chem. Soc. Chem. Commun.. 1993, 1002-1004, which also discloses O-methoxymethyl sordaricin methyl ester.
- Rl2 i s CHO R is not (g); Rl is (a) C1-C14 alkyl,
- R5 is (a) R 1 or
- R9 and RIO are independently
- Rl l is (a) OH or
- Rl3 is (a) H
- Rl4 is (a) H
- EF2 inhibitors are useful as antifungal and antiparasitic agents. As such, they may be used in the treatment and prevention of fungal and parasitic diseases in human, animals and plants.
- fungal diseases against which EF2 inhibitors may be used, and their respective causative pathogens include: 1) Erysiphe, Puccinia, Septoria, Botrytis, Phytophthora, Plasmopora and other fungi which cause infections in plants and crops 2) Candida, Aspergillus, Cryptococcus, Fusarium, Penicillium and other fungi which cause fungal infections in man and animals 3) Plasmodium, Eimeria, Toxoplasma, Neospora, Cryptosporidium and other protozoa which infect man and animals.
- the present invention provides a method for the treatment of fungal or parasitic infections comprising administering to a host suffering from a fungal or parasitic infection a therapeutically effective amount of a compound which inhibits EF2 function.
- a therapeutically effective amount may be one that is sufficient to inhibit protein synthesis of the causative fungi or parasite.
- EF2 inhibitors may be administered to a host in need of treatment in a manner similar to that used for other antifungal and antiparasitic agents; for example, they may be administered parenter- ally, orally, topically, or rectally.
- the dosage to be administered will vary according to the particular compound used, the infectious organism involved, the particular host, the severity of the disease, physical condition of the host, and the selected route of administration; the appropriate dosage can be readily determined by a person skilled in the art.
- the dosage may range from 0.01 mg/kg to 500 mg/kg.
- the dosage may range from 0.01 mg/kg to 100 mg/kg.
- the compositions of the present invention comprises an
- compositions for human and veterninary usage, or in the form of feed composition.
- composition is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions of one or more of the ingredients.
- the composition of the present invention thus includes a composition when made by admixing an EF2 inhibitor and inert carrier.
- compositions of the present invention comprise an EF2 inhibitor as an active ingredient, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients.
- the compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administrations, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered.
- the pharmaceutical compositions may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.
- an EF2 inhibitor can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
- the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous).
- any of the usual pharmaceutical media may be employed.
- oral liquid preparations such as suspensions, elixirs and solutions
- water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like may be used; or in the case of oral solid preparations such as powders, capsules and tablets, carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be included.
- carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be included.
- tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed.
- tablets may be coated by standard aqueous or nonaqueous techniques.
- compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.
- Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients.
- compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
- a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients.
- Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
- each tablet contains from about 1 mg to about 500 mg of the active ingredient and each cachet or capsule contains from about 1 to about 500 mg of the active ingredient.
- compositions of the present invention suitable for parenteral administration may be prepared as solutions or suspensions of these active compounds in water suitably mixed with a surfactant such as hydroxypropylcellulose.
- Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
- the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.
- Suitable topical formulations include transdermal devices, aerosols, creams, ointments, lotions, dusting powders, and the like. These formulations may be prepared via conventional methods containing the active ingredient. To illustrate, a cream or ointment is prepared by mixing sufficient quantities of hydrophilic material and water, containing from about 5-10% by weight of the compound, in sufficient quantities to produce a cream or ointment having the desired consistency.
- compositions suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories.
- Suitable carriers include cocoa butter and other materials commonly used in the art, and the suppositories may be conveniently formed by admixture of the combination with the softened or melted carrier(s) followed by chilling and shaping moulds.
- the pharmaceutical formulations described above may include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and. substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
- compositions containing a compound of formula I may also be prepared in powder or liquid concentrate form .
- conventional water soluble excipients such as lactose or sucrose, may be incorporated in the powders to improve their physical properties.
- suitable powders of this invention comprise 50 to 100% w/w, and preferably 60 to 80% w/w of the combination and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients.
- These powders may either be added to animal feedstuffs, for example by way of an intermediate premix, or diluted in animal drinking water.
- Liquid concentrates of this invention suitably contain a water-soluble compound combination and may optionally include a veterinarily acceptable water miscible solvent, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol.
- the liquid concentrates may be administered to the drinking water of animals, particularly poultry.
- EXAMPLES The following non-limiting examples are provided to illustrate the invention.
- the assays may be run in 96 well or other appropriate sized plates or in the appropriate liquid medium.
- yeast strain YEFD 12h/pURA3-EFT 1 that is deleted for both chromosomal copies of genes which encode EF2, has been obtained from the laboratory of James Bodley (Phan et al., Journal of Biological Chemistry (1993). 268:8665-8668). This strain also contains an episomal copy of a gene encoding EF2, and is essential for
- Yeast strains expressing EF2 genes from pathogens of interest may be constructed by (1) transforming YEFD12h/pURA3- EFT1 with yeast expression plasmids that contain heterologous EF2 encoding genes, and (2) evicting the plasmid containing the native EF2 gene from the cell. This may be used in the growth inhibition assay
- a competitive assay can be performed involving the displacement of a radiolabeled compound with specificity for pathogen EF2 such as H-Compound I binding to Saccharomyces cerevisiae EF2 in crude S30 extracts.
- pathogen EF2 such as H-Compound I binding to Saccharomyces cerevisiae EF2 in crude S30 extracts.
- binding competition can also be performed with purified EF2 in the presence of washed ribosomes
- H-Compound I Specific binding of H-Compound I is found with Saccharomyces S30 extracts and requires the presence of ribosomes as well as EF2. The binding is displaceable by unlabelled L-793,422, sordarin and analogs. No binding is seen with mammalian cell or wheat germ S30 extracts.
- the specificity of H-L793,422 for yeast resides in the EF2 molecule since substitution of yeast ribosomes with either rat or wheat germ has no effect on binding, while substitution of yeast EF2 with rat or wheat germ EF2 abolishes binding.
- Buffer A 50 mM Tris-HCl PH 7.5,150 mM NaCl,10 mM MgCl 2 ,1 mM EDTA
- Buffer B 50 mMTris-HCl PH 7.5,10 mM MgCl 2, 1 mM EDTA GTP- ⁇ -s (Sigma)
- Yeast S-30 prepared as below.
- 3H-Compound I (20 mCi/mg, 8000mCi/mmol; 0.004mg/ml)
- Saccharomyces S30 extract A Saccharomyces cerevisiae strain containing wild-type EF2 is inoculated into medium containing in g/1: lOg Bacto Yeast Extract, 20g Bacto Peptone, 20 g dextrose and 60mg adenine and incubated with shaking at 30°C until mid to late logarithmic phase (A600-2). The cells are harvested and washed twice with water. Pellets may be stored at -70°C indefinitely prior to disruption. For breakage, cells are resuspended in 2 vol of buffer containing 50 mM Tris-Cl pH 7.4, 10% (wt/vol) glycerol, 2mM MgCl 2 ,
- This assay involves the same procedure as disclosed above substituting 0.4 A260 units salt-washed S. cerevisiae ribosomes and 1 pmol purified EF2 for the 10 ⁇ g S30 in the above assay.
- the ribosomes and EF2 are both prepared by published methods (L. Skogerson,
- this assay may be used to either identify the component binding drug, or to examine competition by unknown agents. Results
- Saccharomyces EF2 0.65 pmol purified Saccharomyces EF2 plus 4 pmol purified Saccharomyces ribosomes binds approximately 0.6 pmol labelled compound, with similar displacement (IC50 3ng/ml) by active analogs.
- Replacement of Saccharomyces ribosomes by those of either rat liver or wheat germ does not reduce binding.
- Ribosomes, EF1, EF2 and EF3 are purified from Saccharomyces cerevisiae, and the assay is performed, as described in (L. Skogerson, Methods in Enzymology, Vol LX,p676-685). Sordarin, its analogs and any unknowns may be titrated in this assay and an IC 50 value determined for inhibition.
- Ribosomes and EF2 from other eukaryotic systems may be purified as described in the literature. When EF2 from rat liver (prepared as described by J. F. Collins, F. Raeburn and E.S. Maxwell. J. Biol.
- An assay has been developed to identify antifungal compounds with sordarin like activities using S. cerevisiae as as a surrogate organism. It consists of a two plate differential zone assay using sordarin sensitive (sSl) and resistant strains (sRl) that contain an ergo deletion, which increases membrane permeability and facilitates the uptake of various substances. In this screen, active compounds show a clear zone on the sensitive strain plate and no zone for the resistant strain plate.
- Methods Approximately 1 x 10 ⁇ 6° cells per ml are added to growth medium containing 2% agar. Medium and cells are mixed, poured into plates, and allowed to solidify. Test compounds or fermentation extracts are applied with the intent of identifying samples which inhibit the growth of these yeast. The cultures are incubated at 30 C for 16-24 hours. A similar assay can also be run in a high-throughput microtiter format by inoculating cells into liquid growth medium containing test compounds or fermentation extracts. Active compounds can be identified by assaying for growth inhibition, which can be determined by measuring the optical density of the individual cultures.
- sordarin gives a clear zone of 20mm with the sensitive strain and no zone with the resistant strain.
Abstract
Description
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP50307999A JP2002511933A (en) | 1997-06-10 | 1998-06-09 | Protein elongation factor 2 as a target for antifungal and antiparasitic drugs |
CA002291653A CA2291653A1 (en) | 1997-06-10 | 1998-06-09 | Protein elongation factor 2 as a target for antifungal and antiparasitic agents |
AU80627/98A AU741017B2 (en) | 1997-06-10 | 1998-06-09 | Protein elongation factor 2 as a target for antifungal and antiparasitic agents |
EP98928944A EP0995120A1 (en) | 1997-06-10 | 1998-06-09 | Protein elongation factor 2 as a target for antifungal and antiparasitic agents |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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US4927197P | 1997-06-10 | 1997-06-10 | |
US60/049,271 | 1997-06-10 | ||
GBGB9714331.7A GB9714331D0 (en) | 1997-07-07 | 1997-07-07 | Protein elongation factor 2 as a target for antifungal and antiparasitic agents |
GB9714331.7 | 1997-07-07 |
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WO1998057176A1 true WO1998057176A1 (en) | 1998-12-17 |
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PCT/US1998/011903 WO1998057176A1 (en) | 1997-06-10 | 1998-06-09 | Protein elongation factor 2 as a target for antifungal and antiparasitic agents |
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EP (1) | EP0995120A1 (en) |
JP (1) | JP2002511933A (en) |
AU (1) | AU741017B2 (en) |
CA (1) | CA2291653A1 (en) |
WO (1) | WO1998057176A1 (en) |
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US7885665B2 (en) | 2003-09-26 | 2011-02-08 | Siemens Enterprise Communications, Inc. | System and method for failsafe presence monitoring |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992003455A1 (en) * | 1990-08-16 | 1992-03-05 | Isis Pharmaceutics, Inc. | INHIBITION OF $i(CANDIDA) |
US5403713A (en) * | 1988-11-14 | 1995-04-04 | Brigham & Women's Hospital | Antibodies specific for ELAM-1 and the use thereof |
US5641627A (en) * | 1993-10-25 | 1997-06-24 | Ribogene, Inc. | Methods for screening for antimycotics |
-
1998
- 1998-06-09 WO PCT/US1998/011903 patent/WO1998057176A1/en not_active Application Discontinuation
- 1998-06-09 CA CA002291653A patent/CA2291653A1/en not_active Abandoned
- 1998-06-09 EP EP98928944A patent/EP0995120A1/en not_active Withdrawn
- 1998-06-09 JP JP50307999A patent/JP2002511933A/en active Pending
- 1998-06-09 AU AU80627/98A patent/AU741017B2/en not_active Ceased
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5403713A (en) * | 1988-11-14 | 1995-04-04 | Brigham & Women's Hospital | Antibodies specific for ELAM-1 and the use thereof |
WO1992003455A1 (en) * | 1990-08-16 | 1992-03-05 | Isis Pharmaceutics, Inc. | INHIBITION OF $i(CANDIDA) |
US5641627A (en) * | 1993-10-25 | 1997-06-24 | Ribogene, Inc. | Methods for screening for antimycotics |
Non-Patent Citations (1)
Title |
---|
JONES D. E., ET AL.: "MOLECULAR CLONING AND CHARACTERIZATION OF A CRYPTOSPORIDIUM PARVUM ELONGATION FACTOR-2 GENE.", MOLECULAR AND BIOCHEMICAL PARASITOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 71., 1 February 1995 (1995-02-01), NL, pages 143 - 147., XP002914520, ISSN: 0166-6851, DOI: 10.1016/0166-6851(95)00051-2 * |
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EP0995120A1 (en) | 2000-04-26 |
CA2291653A1 (en) | 1998-12-17 |
JP2002511933A (en) | 2002-04-16 |
AU741017B2 (en) | 2001-11-22 |
AU8062798A (en) | 1998-12-30 |
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