WO1999067418A1 - Beta(1,3)-glucan microfibril assembly assay - Google Patents

Beta(1,3)-glucan microfibril assembly assay Download PDF

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Publication number
WO1999067418A1
WO1999067418A1 PCT/US1999/013429 US9913429W WO9967418A1 WO 1999067418 A1 WO1999067418 A1 WO 1999067418A1 US 9913429 W US9913429 W US 9913429W WO 9967418 A1 WO9967418 A1 WO 9967418A1
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glucan
test solution
solution
agent
renatured
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PCT/US1999/013429
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French (fr)
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Gary R. Ostroff
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The Collaborative Group, Ltd.
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Priority to AU46835/99A priority Critical patent/AU4683599A/en
Publication of WO1999067418A1 publication Critical patent/WO1999067418A1/en

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/12—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar
    • G01N2400/24—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar beta-D-Glucans, i.e. having beta 1,n (n=3,4,6) linkages between saccharide units, e.g. xanthan

Definitions

  • Immunocompromised patients are susceptible to a va ⁇ ety of neoplastic, protozoal, viral, bacterial and fungal diseases; of these, bacte ⁇ al, viral and fungal infections result in the greatest mortality (Bartlett, M. and J. Smith, Clin. Microbiol. Rev. 4:137-149 (1991); Bodey, G. et al, Eur. J. Clin. Microbiol. Infect. Dis. 77:99- 109 (1992); Sternberg, S., Science 266:1632-1634(1994); Cox, G. and J. Perfect, Curr. Opin. Infect. Dis. (5:422-426 (1993); Deepe, G. and W. Bullock, Eur. J. Clin. Microbiol.
  • Candida species especially C. albicans, are the most common (Ainsworth, G., Fungal parasites of vertebrates, in The Fungi, an Advanced Treatise (Ainsworth, G., ed., New York, Academic Press, vol. 3y, 1968; Khardori, N., Eur. J. Clin. Microbiol. Infect Dis. 5:331-351 (1989).
  • Candidemia accounts for 8-10% of all hospital-acquired bloodstream infections and Candida species are the fourth ⁇ riost common cause of nosocomial septicemias. Mortality rates associated with systemic Candida infections are estimated to be as high as 50% of infected patients. Infections caused by other types of fungi (e.g., Aspergillus, Cryptococcus) are also common in immunocompromised patients and result in significant treatment costs and mortality (Meunier, F., Amer. J. Med. 99 (Suppl. ⁇ H):60S-67S (1995)). Although the demand for effective antifungal agents continues to increase, few effective agents are available in the clinic.
  • Aspergillus, Cryptococcus e.g., Aspergillus, Cryptococcus
  • amphotericin B which is a commonly prescribed antifungal drug, interacts with membrane ergosterol (Lyman, C. and T. Walsh, Drugs 44:9-35 (1993)).
  • These drugs have several drawbacks: for example, the development of resistance to azoles has been observed in C. albicans (Bartlett, M. et al, Antimicrobial Agents. Chemo. 55:1859-1861 (1994); Odds, F., Internat. J. Antimicrob. Agents. 6:145-147 (1996)).
  • amphotericin B causes toxic side effects, including renal dysfunction, fever, chills and hypotension.
  • the development of new drugs depends upon the discovery of new therapeutic targets and new assays for assessing the desired biological activity.
  • the present invention is drawn to methods of identifying agents that inhibit folding or association of ⁇ (l,3)-glucan chains.
  • ⁇ (l,3)-glucan chains fold into an unaggregated triple helical conformation (a "single triple helix"), which in turn associates into an aggregated triple helical conformation (a "triple helix aggregate” or “triple helical microfibril”); the methods are accordingly drawn to identifying agents that inhibit either the folding into an single triple helix, or the association into a triple helical microfibril, or both.
  • the methods comprise denaturing a ⁇ (l,3)- glucan test solution that contains a soluble ⁇ (l,3)-glucan composition in an aqueous solution, such as by raising the pH of the solution, by adding dimethyl sulfoxide (DMSO) at concentrations that denature the ⁇ (l,3)-glucan, or by heating the solution; contacting the denatured ⁇ (l,3)-glucan test solution with an agent to be tested; maintaining the denatured ⁇ (l,3)-glucan test solution under renaturing conditions, such as conditions which lower the pH, which remove DMSO, or- which allow cooling of the solution; assessing the conformation of the ⁇ (l,3)-glucan composition in the renatured ⁇ (l,3)-glucan test solution; and comparing the conformation of the ⁇ (l,3)-glucan composition in the renatured ⁇ (l,3)-glucan test solution with the conformation of the ⁇ (l,3)-glucan composition in a ⁇ (l,
  • the agent is an agent that inhibits folding of ⁇ (l,3)-glucan into single triple helix conformation.
  • the invention also provides antifungal agents identified by the methods described above, as well as compositions comprising the agents, and methods of treating fungal infections in vivo or in vitro using the agents or compositions.
  • the methods of the invention provide a simple, convenient means for identifying potential antifungal agents that interfere with ⁇ (l,3)-glucan microfibril assembly.
  • Such agents may interfere with the normal assembly of yeast cell walls; because cell wall assembly is necessary for growth and viability not only of yeast, but of all fungi, agents which affect assembly of yeast cell walls are potential antifungal agents.
  • the methods of the invention facilitate identification and development of novel antifungal agents.
  • Figure 1 is a graphic representation of the ability of Congo red (solid line) to inhibit the assembly of ⁇ (l,3)-glucan triple helical microfibrils, as compared with a solvent control (dotted line).
  • Figure 2 is a graphic representation of the ability of calcofluor (solid line) to inhibit the assembly of ⁇ (l,3)-glucan triple helical microfibrils, as compared with a solvent control (dotted line).
  • Figure 3 is a graphic representation of the ability of congo red, as compared with other agents, to increase survival time in a murine model of Candida infection.
  • DMSO dimethyl sulfoxide
  • the fungal cell wall has a complex composition and structure (Ruiz-Herrera, J. Fungal cell wall Structure, synthesis and assembly, CRC Press, FI, 1992; Wessels, J., New Phytol. 725:397-413 (1993)).
  • the cell wall of human pathogenic fungi contains ⁇ (l,3)-glucan, ⁇ (l,6)-glucan, other cc-linked glucans (including glycogen and ⁇ (l,3)-glucan) mannoproteins, and smaller amounts of peptides, chitin and lipids (Fleet, G.H., in The Yeasts (2 nd edition, Rose, A.H.
  • Cell wall components are either structural, providing mechanical strength to the wall, or cementing, keeping structural components glued together.
  • Structural materials are fibrillar and include chitin, ⁇ -l,3-linked-glucans and cellulose.
  • Cementing materials are amorphous and include ⁇ (l,3)- and ⁇ -linked glucans, chitosan and glycoproteins.
  • Formation of ⁇ (l,3)-glucan fibrils proceeds via the interaction of three ⁇ (l,3)-glucan chains to form a triple helical structure.
  • As the growing ⁇ (l,3)-glucan chains assemble into this triple helical structure they are cross-linked by the enzyme-catalyzed formation of ⁇ (l,6) branch points.
  • the branched triple helical ⁇ (l,3)-glucan chains are incorporated into the cell wall as parallel triple helical microfibrils, thereby forming the basic ⁇ (l,3)-glucan microfibril structure.
  • These ⁇ (l,3)-glucan fibrils are then interwoven and crosslinked with chitin microfibrils and mannoproteins to form the fungal cell wall layer.
  • a potential target of antifungal agents is fungal cell wall biosynthesis and assembly. Interference with cell-wall biosynthesis by inhibitors such as cilofungin ( ⁇ (l,3)-glucan synthesis), nikkomycin (chitin synthesis) and tunicamycin (mannoprotein synthesis), interferes with fungal cell growth (Kurtz, ASM News 64(1):31 (1997)). Inhibition of the non-enzymatic chitin and ⁇ (l,3)-glucan microfibril assembly processes by calcofluor and congo red also interferes with fungal cell growth (Vanni, G. et al, Plant Sci. Letters 31 :9-17 (1983); Ilorza, M. et al, J. Gen. Microbiol.
  • the present invention provides an in vitro method for assessing the ability of an agent to inhibit folding of ⁇ (l,3)-glucan chains into a triple helix, or to inhibit association into a triple helical microfibril.
  • Agents that inhibit ⁇ (l,3)-glucan folding and/or association interfere with ⁇ (l,3)-glucan microfibril formation, and thus prevent the extracellular assembly of the fungal cell wall.
  • Such agents which target the essential, non-enzymatic ⁇ (l,3)-glucan microfibril assembly process, can be useful as antifungal agents.
  • Underivatized aqueous-soluble ⁇ (l,3)-glucan (also referred to as PGG-glucan or BETAFECTLN®) is a polysaccharide composed of glucopyranose units linked in chains via ⁇ (l,3)-glycosidic bonds, with ⁇ (l,3)-linked branches intermittently linked to the main chain via ⁇ (l,6)-glycosidic bonds.
  • Single chains can be isolated (i.e., not substantially interacting with another chain). Three single helix chains can also combine to form a triple helix structure which is held together by interchain hydrogen bonding. Two or more ⁇ (l,3)-glucan triple helices can join together to form a triple helical microfibril.
  • a ⁇ (l,3)-glucan polysaccharide e.g., underivatized, aqueous soluble ⁇ (l,3)-glucan, or other soluble ⁇ (l,3)-glucans
  • a ⁇ (l,3)-glucan polysaccharide e.g., underivatized, aqueous soluble ⁇ (l,3)-glucan, or other soluble ⁇ (l,3)-glucans
  • single disordered chains single helix, single triple helix and triple helical microfibrils.
  • Preparations of the ⁇ (l,3)-glucan can comprise one or more of these forms, depending upon such conditions as pH and temperature.
  • triple helical microfibril refers to a ⁇ (l,3)-glucan conformation in which two or more triple helices are joined together (associated) via non-covalent interactions.
  • the "molecular weight" of a ⁇ (l,3)-glucan composition is the mass average molar mass of the collection of polymer molecules within the composition.
  • the characterization of a collection of polymer molecules in terms of polymer mass average molar mass is well known in the art of polymer science.
  • the "aggregate number" of a ⁇ (l,3)-glucan conformation is the number of single chains which are joined together in that conformation.
  • the aggregate number of a single helix is 1, the aggregate number of a single triple helix is 3, and the aggregate number of a triple helical microfibril is greater than 3.
  • a triple helical microfibril consisting of two triple helices joined together has an aggregate number of 6.
  • the aggregate number of a ⁇ (l,3)-glucan sample under a specified set of conditions can be determined by determining the average molecular weight of the polymer under those conditions.
  • the ⁇ (l,3)-glucan is then denatured, that is, subjected to conditions which separate any aggregates into their component single polymer chains.
  • the average molecular weight of the denatured polymer is then determined.
  • the ratio of the molecular weights of the aggregated and denatured forms of the polymer is the aggregate number.
  • a typical ⁇ (l,3)-glucan composition includes molecules having a range of chain lengths, conformations and molecular weights.
  • the measured aggregate number of a ⁇ (l,3)-glucan composition is the mass average aggregate number across the entire range of ⁇ (l,3)-glucan molecules within the composition. It is to be understood that any reference herein to the aggregate number of a ⁇ (l,3)-glucan composition refers to the mass average aggregate number of the composition under the specified conditions.
  • the aggregate number of a composition indicates which conformation is predominant within the composition. For example, a measured aggregate number of about 6 or more is characteristic of a composition in which the ⁇ (l,3)-glucan is substantially in the triple helical microfibril conformation.
  • the conformation of an aqueous soluble, ⁇ (l,3)-glucan preparation such as PGG-glucan is temperature dependent.
  • an aqueous PGG-glucan solution prepared according to the method disclosed in U.S. Patent No. 5,622,939, incorporated herein by reference in its entirety elutes from a gel permeation chromatography column (GPC, also referred to as size exclusion chromatography) at 25 °C as a single symmetric peak (Fraction A).
  • GPC gel permeation chromatography column
  • Fraction A which elutes first
  • Fraction C which elutes second.
  • Fraction B- which elutes later than Fractions A and C.
  • the molecular weights of fractions A, B and C were determined at 25 °C at both pH 7 and pH 13, at 37°C at pH 7, and at 75°C at pH 7.
  • PGG-glucan is in an unaggregated or single chain conformation.
  • the ratio of the molecular weights determined at pH 7 and pH 13 is the aggregate number at pH 7 at that temperature.
  • Fraction A had a molecular weight of 238,000 and an aggregate number of 15.0. Upon increasing the temperature to 37 °C, the molecular weight of Fraction A decreased to 164,000 and the aggregate number decreased to 10.3. At 75°C, the molecular weight of this fraction was 52,600 with an aggregate number of 3.3. The temperature dependence of molecular weight and aggregate number was more pronounced for Fraction C. At pH 7.0 and 25 °C, Fraction C had a molecular weight of 71,500 and an aggregate number of 6.0. At 37°C, the molecular weight of Fraction C was 32,000 and the aggregate number was 2.7. At 75 °C, the molecular weight of this fraction was 17,200 and the aggregate number was 1.4. Fraction B showed no temperature dependence of molecular weight. At pH 7 and 25-75 °C, Fraction B had a molecular weight of approximately 15,000 and an aggregate number of approximately 1.
  • the present invention utilizes a soluble ⁇ (l,3)-glucan composition which is substantially in a triple helical microfibril conformation at room temperature (e.g., at about 20-25 °C), which is substantially in a single triple helix conformation under physiological conditions (e.g., at about 37°C), and which is substantially in a single helix conformation at greater than about 75 °C, in assays for assessing the ability of an agent to inhibit folding of ⁇ (l,3)-glucan chains.
  • the soluble ⁇ (l,3)-glucan composition has an aggregate number in the range from greater than 2 to less than 6, and preferably from about 2.5 to about 3.5, under physiological conditions of about 37 °C; and an aggregate number of less than about 2 under conditions of elevated pH and/or temperature.
  • the soluble ⁇ (l,3)-glucan composition is Fraction C, as described above.
  • the soluble ⁇ (l,3)-glucan composition can comprise underivatized or derivatized ⁇ (l,3)-glucan, provided that the ⁇ (l,3)-glucan is capable of folding into a single triple helix conformation and of associating into a triple helical microfibril conformation.
  • the soluble ⁇ (l,3)-glucan composition is underivatized, aqueous soluble, ⁇ (l,3)-glucan.
  • a "soluble ⁇ (l,3)-giucan composition”, as the term is used herein, is a ⁇ (l,3)-glucan composition that dissolves in an aqueous medium at room temperature (about 20-25°C) and neutral pH (from about pH 5.5 to about 7.5) to form a visually clear solution at a concentration up to about 100 mg/mL.
  • a soluble ⁇ (l,3)-glucan composition that is dissolved in an aqueous medium is referred to herein as a "soluble ⁇ (l,3)-glucan solution”.
  • physiological conditions refers to physiological pH, about pH 7, and physiological temperature, about 37°C.
  • physiological conditions the preferred ⁇ (l,3)-glucan composition used in the methods of the invention consists essentially of ⁇ (l ,3)-glucan chains in single triple helix conformation.
  • the soluble ⁇ (l,3)-glucan composition can be prepared from soluble or insoluble glucan particles, from many sources, such as from yeast or fungal sources.
  • the soluble ⁇ (l,3)-glucan composition is derived- from yeasts, as described in U.S. Patent No. 5,622,939; U.S. Serial No. 08/373,251, U.S. Serial No. 08/469,233, U.S. Patent No. 5,322,841, U.S. Serial No. 08/432,303, U.S. Patent No. 5,663,324, U.S. Patent No. 5,633,369 and U.S. Serial No. 08/400,488; the entire teachings are incorporated herein by reference.
  • Glucan particles which are particularly useful as starting materials in the present invention are whole glucan particles as described by Jamas et al. in U.S. Patent Nos. 4,810,646, 4,992,540, 5,082,936, 5,028,703 and 5,622,939, the teachings of each of which are incorporated herein by reference in their entirety.
  • the source of the whole glucan particles can be any yeast, fungus, or alga which contains ⁇ (l,3)-glucans in its cell walls or which secretes ⁇ (l,3)-glucans.
  • yeast strains Saccharomyces cerevisiae R4 NRRL Y-15903 and R4 Ad (ATCC No. 74181).
  • Other strains of yeast which are suitable sources of whole glucan particles include Saccharomyces delbruekii, Saccharomyces rosei, Saccharomyces microellipsodes, Saccharomyces carlsbergensis, Schizosaccharomyces pombe, Kluvveromyces lactis, Kluvveromyces fragilis, Kluvveromyces polysporus, Candida albicans, Candida cloacae, Candida glabrata, Candida tropicalis, Candida utilis, Hansenula wingei, Hansenula ami, Hansenula henricii and Hansenula americana.
  • a soluble ⁇ (l,3)-glucan composition in an aqueous solution is used as a test solution.
  • the soluble ⁇ (l,3)- glucan composition in the test solution is denatured to a random coil, single chain conformation. Any appropriate denaturing method or agent can be used.
  • the soluble ⁇ (l,3)-glucan composition in the solution is denatured by increasing the pH of the solution to greater than about pH 11, to a pH that is between about pH 11 and pH 14 (inclusive), for approximately 1 minute to approximately 1 hour. NaOH/KOH can be used at approximately greater than 0.001 Molar to raise the pH.
  • the soluble ⁇ (l,3)-glucan is denatured by adding dimethyl sulfoxide (DMSO) at a concentration of approximately greater than 30% by volume. At concentrations of less than 5% by volume, DMSO can be used as a solvent; however, at concentrations greater than approximately 30% by volume, DMSO can be used to denature the soluble ⁇ (l,3)-glucan.
  • DMSO dimethyl sulfoxide
  • the soluble ⁇ (l,3)-glucan is denatured by heating the test solution to above about 37 °C. In a preferred embodiment, the test solution is heated to a temperature range of from about 37 to about 135 °C. In a more preferred embodiment, the test solution is heated to a temperature range of from about 90 to about 100°C.
  • the solution is maintained at the higher temperature for an appropriate length of time to result in denaturation of the ⁇ (l,3)- glucan into a random coil, single chain conformation. Generally, the solution is maintained at this temperature for about 10 minutes to about 1 hour.
  • the resultant solution (referred to herein as the "denatured ⁇ (l,3)-glucan test solution") is contacted with an agent to be tested (the "agent of interest” or the "test agent”).
  • the denatured ⁇ (l,3)-glucan test solution that has been contacted with the test agent (referred to herein as the "contacted, denatured ⁇ (l,3)- glucan test solution”) is then maintained under renaturing conditions.
  • “Renaturing conditions” are those conditions that would, in the absence of the agent to be tested, allow folding (renaturation) and reassociation of the ⁇ (l,3)-glucan composition to generate a renatured ⁇ (l,3)-glucan composition. For example, if the soluble ⁇ (l,3)- glucan was denatured by raising the pH of the aqueous solution comprising the ⁇ (l,3)-glucan, then the denatured ⁇ (l,3)-glucan test solution is subjected to renaturing conditions which result in a gradual lowering of the pH.
  • the denatured ⁇ (l,3)-glucan test solution is subjected to renaturing conditions which result in gradual removal of DMSO. If the soluble ⁇ (l,3)-glucan was denatured by heating, the denatured ⁇ (l,3)- glucan test solution is maintained in an appropriate environment (e.g., away from the heat source used for denaturing the ⁇ (l,3)-glucan, at room temperature) so that its temperature can be gradually cooled.
  • an appropriate environment e.g., away from the heat source used for denaturing the ⁇ (l,3)-glucan, at room temperature
  • the endpoint of the renaturing conditions depends on whether the test agent is being assessed for an ability to inhibit formation of single triple helix conformation, or to inhibit formation of triple helical microfibrils.
  • the test agent inhibits folding of ⁇ (l,3)-glucan into a single triple helix conformation
  • the contacted, denatured ⁇ (l,3)-glucan test solution is maintained under renaturing conditions that are sufficient, in the absence of the test agent, to allow the ⁇ (l,3)-glucan to fold into a single triple helix conformation.
  • the contacted, denatured ⁇ (l,3)-glucan test solution is maintained in an environment in which it can cool until its temperature is within a physiological range (e.g., from about 35 to about 50°C). In a preferred embodiment, the denatured ⁇ (l,3)-glucan test solution is allowed to cool until its temperature is from about 37 to about 45 °C. If the ⁇ (l,3)-glucan was denatured by adding DMSO, the contacted, denatured ⁇ (l,3)-glucan test solution is maintained under conditions which lower the amount of DMSO to approximately 15-25% by volume.
  • the amount of DMSO is lowered by approximately 20% by volume. If the ⁇ (l,3)-glucan was denatured by raising the pH, the contacted, denatured ⁇ (l,3)-glucan test solution is maintained under conditions which lower the pH to from greater than about 11 to a pH that is between about pH 9 (inclusive) and pH 11 (inclusive).
  • the contacted, denatured ⁇ (l,3)-glucan test solution is maintained under renaturing conditions that are sufficient, in the absence of the test agent, to allow the ⁇ (l,3)- glucans to associate into triple helical microfibril conformation.
  • the contacted, denatured ⁇ (l,3)-glucan test solution is maintained in an environment in which it can cool until its temperature is less than or equal to room temperature (e.g., from about 2 to 4°C to about 30 °C).
  • the denatured ⁇ (l,3)-glucan test solution is allowed to cool until its temperature is from about 20 to about 25 °C. If the ⁇ (l,3)-glucan was denatured by DMSO, the contacted, denatured ⁇ (l,3)-glucan is maintained in an environment which removes the DMSO until less than approximately 5% by volume is left. If the ⁇ (l,3)-glucan was denatured by raising the pH, the contacted, denatured ⁇ (l,3)-glucan test solution is maintained under conditions which lower the pH from greater than about 11 to approximately a physiologic pH that is between about pH 3 (inclusive) to pH 9 (inclusive). In a preferred embodiment, the pH is lowered to about pH 7.
  • the agent can be assessed for its ability to inhibit folding of ⁇ (l,3)-glucan into a single triple helix conformation; to inhibit association of ⁇ (l,3)- glucans into a triple helical microfibril conformation; or to inhibit both folding and association, by the methods of this invention.
  • denatured ⁇ (l,3)-glucan test solution is subjected to renaturing conditions, the conformation of the ⁇ (l,3)-glucan composition (i.e., the amount of single chain conformation, single triple helix conformation, triple helical microfibril, or a mixture of these conformations) in the renatured ⁇ (l,3)-glucan test solution is then assessed.
  • the assessment of the conformation of the ⁇ (l,3)- glucan can include a determination of the relative amounts of the different conformations.
  • the conformation(s) of the ⁇ (l,3)-glucan composition in the renatured ⁇ (l,3)-glucan test solution can be assessed, for example, by directing the solution through a high performance gel permeation chromatography column, which separates the soluble ⁇ (l,3)-glucan into single chain (slow moving), single triple helix (intermediate moving), and triple helical microfibril (fast moving) forms. Such methods are described below in the Examples.
  • the conformation of the ⁇ (l,3)- glucan composition can also be assessed by using other physical methods, such as by measuring viscosity, polarimetry or light scattering, or by fluorescent polarization.
  • the use of fluorescence polarization is described in detail in U.S. Patent application Serial No. 09/104,560, filed June 25, 1998, entitled "ASSAYS FOR AGENTS WHICH ALTER CELL WALL BIOPOLYMER SYNTHESIS OR ASSEMBLY" by Gary R. Ostroff, the entire teachings of which are incorporated herein by reference.
  • the aggregate number of the ⁇ (l,3)-glucan composition can be determined, as described above.
  • a nonphysical method can be used to measure the amount of random coil, single chain ⁇ (l,3)-glucan, single triple helix ⁇ (l,3)-glucan. or triple helical microfibril ⁇ (l,3)- glucan.
  • the agent that specifically binds to a particular conformation of ⁇ (l,3)-glucan can be labeled, to allow detection of that conformation; alternatively, the agent that specifically binds to a particular conformation of ⁇ (l,3)-glucan can be used in a competition assay, such as an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the renatured ⁇ (l,3)- glucan test solution can be incubated with a monoclonal antibody that specifically binds with single triple helix ⁇ (l,3)-glucan, and the mixture containing residual unbound antibody is then incubated with triple helical ⁇ (l,3)-glucan bound to a solid support, such as an ELISA plate.
  • a solid support such as an ELISA plate.
  • the amount of antibody that binds to the triple helical ⁇ (l,3)-glucan on the solid support can then be determined by reaction with an antibody enzyme conjugate.
  • the control ⁇ (l,3)-glucan solution is an aqueous solution comprising the soluble ⁇ (l,3)-glucan composition, and that has been subjected to the same denaturing and renaturing conditions as the test solution, but in the absence of the test agent, or in the presence of a control agent (e.g., DMSO, water, or a compound that is known not to interfere with ⁇ (l,3)-glucan microfibril assembly) in lieu of the test agent.
  • a control agent e.g., DMSO, water, or a compound that is known not to interfere with ⁇ (l,3)-glucan microfibril assembly
  • the presence of a greater amount of random chain ⁇ (l,3)-glucan in the renatured ⁇ (l,3)-glucan test solution than in the ⁇ (l,3)-glucan control solution; and/or the presence of a lesser amount of single triple helix ⁇ (l,3)-glucan in the renatured test solution than in the ⁇ (l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit ⁇ (l,3)-glucan folding into a single triple helix formation.
  • a difference between the test solution and the control solution that is greater than or equal to two indicates that the test agent inhibits folding of ⁇ (l,3)-glucan into single triple helix conformation, and/or inhibits association of ⁇ (l,3)-glucan into triple helical microfibril formation.
  • test agent is identified by the methods described above as an agent that inhibits ⁇ (l,3)-glucan folding
  • further experiments can be conducted to assess the agent's potential as an antifungal agent. For example, experiments can be performed to determine whether the agent inhibits fungal cell growth, as described in Example 3, or to determine whether any fungal cell death caused by the agent is due to inhibition of ⁇ (l,3)-glucan microfibril assembly, as described in Example 4.
  • the agent can also be tested for in vivo efficacy, as described in Example 5.
  • Agents identified by the methods described herein can be used as antifungal agents that target the essential, non-enzymatic ⁇ (l,3)-glucan microfibril assembly process, thereby avoiding the problems associated with targeting enzymatic activities.
  • compositions comprise a therapeutically (or prophylactically) effective amount of an agent identified by the methods described above, and a pharmaceutically acceptable carrier or excipient.
  • the carrier and composition can be sterile.
  • the formulation should suit the mode of administration.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof.
  • the pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
  • compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • composition is administered by injection
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, can be employed.
  • Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • the agent may be incorporated into a cosmetic formulation.
  • sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.
  • a pressurized volatile, normally gaseous propellant e.g., pressurized air.
  • Agents described herein can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- e hylamino ethanol, histidine, procaine, etc.
  • the amount of agents which will be effective in the treatment of a particular disorder or condition will depend on the nature of the infection, disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the infection, disease or disorder, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use of sale for human administration.
  • the pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g., separately, sequentially or concurrently), or the like.
  • the pack or kit may also include means for reminding the patient to take the therapy.
  • the pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages.
  • the agents can be separated, mixed together in any combination, present in a single vial or tablet.
  • Agents assembled in a blister pack or other dispensing means is preferred.
  • unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each agent and administered in FDA approved dosages in standard time courses.
  • the agents identified by the methods of the invention as inhibiting folding of ⁇ (l,3)-glucan into single triple helix conformation, and or association of ⁇ (l,3)- glucan into triple helical microfibril formation, as well as the compositions described above, can be used either in vitro or in vivo to kill fungi and/or treat fungal infection.
  • the agent is generally administered to an animal, a human, or a location of fungal contamination or growth (e.g., an environmental location) in an amount sufficient to inhibit and or eliminate fungal infection or growth (a "therapeutically effective amount” or an "effective amount”).
  • the mode of administration of the agent (or composition), in the case of in vivo administration, can be oral, enteral, parenteral, intravenous, subcutaneous, intraperitoneal, intramuscular, topical or intranasal.
  • the agent (or composition) is administered by a means that allows contact of the agent (or composition) with the fungal growth.
  • the form in which the composition will be administered e.g., powder, tablet, capsule, solution, emulsion
  • the quantity of the agent or composition to be administered in vivo will be determined on an individual basis, and will be based at least in part on consideration of the severity of infection or injury in the patient, the patient's condition or overall health, the patient's weight and gender. In general, a single dose will preferably contain approximately 0.01-100 mg per kilogram of body weight, and preferably about 1 mg/kg.
  • the quantity of the agent or composition to be administered in vitro will also be determined on a case-by-case basis, and will be based at least in part on consideration of the type and extent of the fungal contamination or growth.
  • agents or compositions of the present invention can be administered to an individual or applied to an in vitro fungal source as necessary to treat the fungal infection or contamination.
  • An individual skilled in the medical arts will be able to determine the length of time during which the agent or composition is administered and the dosage.
  • a PGG-Glucan composition prepared as described in U.S. Patent No. 5,622,939 was concentrated to approximately 20 mg/mL.
  • An aliquot of the concentrated sample was fractionated at 25 °C on a preparative scale gel permeation chromatography column (5 cm TSK HW55F resin column) using 0.15 M sodium chloride as the mobile phase.
  • the composition eluted as a single symmetrical band.
  • a second aliquot of the concentrated sample was fractionated on a preparative GPC column maintained in a hot room at 37 °C.
  • the PGG-glucan eluted from the column in two distinct fractions.
  • a third aliquot of the concentrated sample was fractionated on a preparative GPC column maintained in a hot room at 75 °C. These fractions were collected and designated Fraction A and Fraction C, and Fraction B, respectively.
  • EXAMPLE 2 DEVELOPMENT OF AN 77V VITRO ⁇ (l,3)-GLUCAN MICROFIBRIL ASSAY
  • An assay was developed to assess the ability of agents to inhibit folding of ⁇ (l,3)-glucan into unaggregated triple helical structures, or into aggregated triple helical structures (e.g., triple helical microfibrils).
  • a 1 mg/ml solution of neutral soluble glucan e.g., Fraction C as described above, or BETAFECTIN® or PGG-glucan
  • water is heated to 100° C for 30 minutes to denature the triple- helical bundles into a single chain random-coil conformation.
  • a 100 ⁇ l volume of the hot, denatured neutral soluble glucan solution is mixed with 1.0 ⁇ l of a solvent control (water or DMSO), a positive control (congo red, 1 mg/ml; or calcofluor 1 mg/ml), or a test compound?
  • a solvent control water or DMSO
  • a positive control congo red, 1 mg/ml; or calcofluor 1 mg/ml
  • the solutions are then cooled for approximately 1 hour to room temperature to allow the glucan to refold/reassociate into native triple- helical bundles. If a test agent interferes with the refolding/reassociation reaction, then the glucan remains in the denatured, single-chain form.
  • the molecular size of the assembled product i.e., the refolded/reassociated glucan
  • GPC gel permeation chromatography
  • % inhibition [1- (THB/SC)test/(THB/SC)control] x 100, where THB is the peak height of triple helical microfibril bundles (which are fast migrating), and SC is the peak height of single chain (slow migrating).
  • IC 50 determinations for active test compounds can be calculated by regression analysis of a 3-point, % inhibition dilution curve of each test compound.
  • ⁇ (l,3)-glucan microfibril inhibitors and structurally- related compounds were evaluated for their ability to inhibit ⁇ (l,3)-glucan microfibril assembly, using this assay.
  • Known ⁇ (l,3)-glucan microfibril inhibitors such as congo red and calcofluor, inhibited the assembly of neutral soluble glucan relative to a solvent control, as shown in Figures 1 and 2.
  • EXAMPLE 3 ASSESSMENT OF FUNGAL CELL GROWTH INHIBITION
  • Compounds identified by the assay described above as inhibiting ⁇ (l,3)- glucan microfibril assembly can be tested for their ability to inhibit the growth and protoplast regeneration of the human pathogenic fungus C. albicans using standard agar diffusion assays. Co ⁇ elation between ⁇ (l,3)-glucan and microfibril assembly inhibition with fungal cell killing was demonstrated using such an assay.
  • a 96-well microtiter plate agar diffusion assay was conducted as follows: a 2.5 ⁇ l volume of solvent control (water or DMSO), positive control (cilofungin, 10 mg/ml; nikkomycin Z, 10 mg/ml; amphotericin B. 10 mg/ml; fluconazole, 10 mg/ml), or a test compound (congo red, calcofluor, or structurally similar microfibril-inactive compounds as described above, at 10 mg/ml) was added to individual wells of a 96-well microtiter plate.
  • solvent control water or DMSO
  • positive control cilofungin, 10 mg/ml
  • nikkomycin Z 10 mg/ml
  • amphotericin B 10 mg/ml
  • fluconazole 10 mg/ml
  • test compound congo red, calcofluor, or structurally similar microfibril-inactive compounds as described above, at 10 mg/ml
  • YPDA molten yeast extract-peptone-dextrose agar
  • Pathogenic C. albicans such as ATCC deposit 20402 cells grown to mid-log phase in YPD liquid medium at 30 °C were washed twice with sterile water by centrifugation at 3,000 x g for 10 minutes, adjusted to 5 x 10 6 cells/ml, then mixed with YPD soft agar (YPDSA; 0.4% agar) medium and 30 ⁇ l of YPDSA containing approximately 1,500 cells, and added to each well.
  • EXAMPLE 4 DEMONSTRATION THAT CELL DEATH IS DUE TO INHIBITION OF TARGET The effect of microfibril inhibitors on protoplast cell-wall regeneration were assessed to verify that the cause of cell death was due to inhibition of ⁇ (l,3)-glucan microfibril assembly. A 96-well microtiter plate protoplast regeneration agar diffusion assay was used.
  • a 2.5 ⁇ l volume of solvent control water or DMSO
  • positive control cilofungin, 10 mg/ml; nikkomycin Z, 10 mg/ml; amphotericin B, 10 mg/ml; fluconazole, 10 mg/ml
  • a test compound congo red, calcofluor, or structurally similar micro fibril-inactive compounds, at 10 mg/ml
  • YPDA molten yeast- extract-peptone-dextrose-KCl agar
  • YPDA 1.5% agar, 0.6 M KCl
  • Protoplast formation was followed by microscopic observation, and when complete, protoplasts were washed three times with sterile 0.6M KCl by centrifugation at 500 x g for 10 minutes. Protoplasts were then adjusted to 5 x 10 6 cells/ml, mixed with YPDK soft agar (YPDSAK; 0.4% agar, 0.6 M KCl) medium and 30 ⁇ l of YPDSAK containing approximately 15,000 protoplasts was added to each well. Plates were incubated at 30 °C and scored for growth and protoplast regeneration by visual and microscopic video imaging at 24 and 48 hours.
  • YPDK soft agar YPDSAK
  • control protoplasts After incubation for 24 hours at 30 °C, control protoplasts regenerated cell wall and reinitiated cell division to an 8- 16 cell microcolony stage as visualized by light microscopy. If a fungicidal compound was present, such as positive control amphotericin B, the protoplasts died and there were no observable cells present at 24 hours. If a fungistatic compound was present, such as positive control fluconazole, 1-2 regenerated cell microc ⁇ lonies were observed. If a cell- wall active compound was present, such as positive controls cilofungin or nikkomycin Z, live, single cell, non-regenerated protoplasts were seen.
  • mice Male ICR mice (weight approximately 20 g) were intraperitoneally administered 1.25 x 10 7 cfu of C. albicans (ATCC deposit number 10231) was intraperitoneally administered. Administration of this C. albicans dose has been previously shown to be lethal for 90-100% of placebo-treated mice within 10 days of infection (data not shown). One hour after mice were infected with C.
  • mice were administered various doses of test compound (congo red), or control compound (DMSO or amphotericin B at 10 mg/kg) in a 0.1 ml volume via intraperitoneal injection at +1 hour (single dose schedule) or at +1 hour, +1 day, +2 day, -r3 day, +4 day (multi- dose schedule), and survival monitored twice daily for a 10-day period.
  • results shown in Figure 3, indicated that congo red increased survival time, relative to a vehicle control, at a dose of 3 mg/kg.

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Abstract

Methods of identifying agents that inhibit folding of β(1,3)-glucan chains, comprising denaturing a β(1,3)-glucan test sollution; contacting the denatured β(1,3)-glucan test solution with an agent to be tested; maintaining the denatured β(1,3)-glucan test solution under renaturing conditions; assessing the conformation of the β(1,3)-glucan composition in the renatured β(1,3)-glucan test solution; and comparing the conformation of the β(1,3)-glucan composition in the renatured β(1,3)-glucan test solution with the conformation of the β(1,3)-glucan composition in a β(1,3)-glucan control solution, are disclosed, as are agents identified by the methods, compositions comprising the agents, and methods of treating a fungal infection or a fungal growth by administering the agents or compositions.

Description

BETA(1,3)-GLUCAN MICROFIBRIL ASSEMBLY ASSAY
RELATED APPLICATIONS
This application claims pπoπty to U.S. Application No. 09/324,814, filed June 2, 1999, which is a continuation of U.S. Application No. 09/104,914, filed June 25, 1998, the contents of which are incorporated by reference in their entirety herein.
BACKGROUND OF THE INVENTION
Immunocompromised patients are susceptible to a vaπety of neoplastic, protozoal, viral, bacterial and fungal diseases; of these, bacteπal, viral and fungal infections result in the greatest mortality (Bartlett, M. and J. Smith, Clin. Microbiol. Rev. 4:137-149 (1991); Bodey, G. et al, Eur. J. Clin. Microbiol. Infect. Dis. 77:99- 109 (1992); Sternberg, S., Science 266:1632-1634(1994); Cox, G. and J. Perfect, Curr. Opin. Infect. Dis. (5:422-426 (1993); Deepe, G. and W. Bullock, Eur. J. Clin. Microbiol. Infect. Dis. 9:377-380 (1990); Fox, J.L., ASM News 59:515-518 (1993); Kujath, P., Mycoses 55:225-228 (1992); Pfaller, M. and R. Wenzel, Eur. J. Clin. Microbiol. Infect. Dis. 11:281-291 (1992); and Samonis, G. and D. Bafaloukos, In vivo (5: 183-194 (1992)). Duπng the last three decades there has been a dramatic increase in the frequency of fungal infections, especially disseminated systemic mycoses in immunodeficient hosts (Odds, F., Antimicrob. Chemother 57:463-471 (1993); Rmgel, S., Mycopath. 109:15-81 (1990); Walsh, T. et al.. Diagn. Microbiol. Infect. Dis. 75:37-40 (1990); Nouza, M., Infection. 20:113-117 (1992); Rhodes, J. et al.. J. Med. Vet. Mvc. 30:51-51 (1992); Saral, R., Rev. Infect. Dis. 75:487-492 (1991); Levitz, S., Clin. Infect.Dis 14:31-42 (1992); Polak, A. and P. Hartman, Progress in Drug Research. Basel Birkhauser Verlag 57: 181-269 (1991); Matthewson, H.S.. Resp Care 35 987-989 (1990V. Hoepπch, P . Prog Drug Res 44-81-121 (1995)) Fungal infections m immunocompromised patients are mamly the result of opportunistic infections by normally harmless, asymptomatic commensals, which can be pathogenic under certain conditions (Odds, F , Antimicrob. Chemother. 57:463-471 (1993); Rhodes, J. et al, J. Med. Vet. Myc. 50:51-57 (1992); Saral, R., Rev. Infect. Dis. 75:487-492 (1991)). Species of Cryptococcus, Candida, Coccidioides, Histoplasma, Blastomyces, Sporothrix and Aspergillus, as well as other opportunistic fungi, are important causative agents; of these, Candida species, especially C. albicans, are the most common (Ainsworth, G., Fungal parasites of vertebrates, in The Fungi, an Advanced Treatise (Ainsworth, G., ed., New York, Academic Press, vol. 3y, 1968; Khardori, N., Eur. J. Clin. Microbiol. Infect Dis. 5:331-351 (1989). Candidemia accounts for 8-10% of all hospital-acquired bloodstream infections and Candida species are the fourth ^riost common cause of nosocomial septicemias. Mortality rates associated with systemic Candida infections are estimated to be as high as 50% of infected patients. Infections caused by other types of fungi (e.g., Aspergillus, Cryptococcus) are also common in immunocompromised patients and result in significant treatment costs and mortality (Meunier, F., Amer. J. Med. 99 (Suppl. <H):60S-67S (1995)). Although the demand for effective antifungal agents continues to increase, few effective agents are available in the clinic. Available drugs for the treatment of mycotic infections include azoles and polyenes, both of which inhibit sterol biosynthesis. The polyene amphotericin B, which is a commonly prescribed antifungal drug, interacts with membrane ergosterol (Lyman, C. and T. Walsh, Drugs 44:9-35 (1993)). These drugs, however, have several drawbacks: for example, the development of resistance to azoles has been observed in C. albicans (Bartlett, M. et al, Antimicrobial Agents. Chemo. 55:1859-1861 (1994); Odds, F., Internat. J. Antimicrob. Agents. 6:145-147 (1996)). Further, amphotericin B causes toxic side effects, including renal dysfunction, fever, chills and hypotension. The development of new drugs depends upon the discovery of new therapeutic targets and new assays for assessing the desired biological activity.
SUMMARY OF THE INVENTION
The present invention is drawn to methods of identifying agents that inhibit folding or association of β(l,3)-glucan chains. β(l,3)-glucan chains fold into an unaggregated triple helical conformation (a "single triple helix"), which in turn associates into an aggregated triple helical conformation (a "triple helix aggregate" or "triple helical microfibril"); the methods are accordingly drawn to identifying agents that inhibit either the folding into an single triple helix, or the association into a triple helical microfibril, or both. The methods comprise denaturing a β(l,3)- glucan test solution that contains a soluble β(l,3)-glucan composition in an aqueous solution, such as by raising the pH of the solution, by adding dimethyl sulfoxide (DMSO) at concentrations that denature the β(l,3)-glucan, or by heating the solution; contacting the denatured β(l,3)-glucan test solution with an agent to be tested; maintaining the denatured β(l,3)-glucan test solution under renaturing conditions, such as conditions which lower the pH, which remove DMSO, or- which allow cooling of the solution; assessing the conformation of the β(l,3)-glucan composition in the renatured β(l,3)-glucan test solution; and comparing the conformation of the β(l,3)-glucan composition in the renatured β(l,3)-glucan test solution with the conformation of the β(l,3)-glucan composition in a β(l,3)-glucan control solution. If a greater amount of random chain β(l,3)-glucan is present in the renatured β(l,3)-glucan test solution than in the β(l,3)-glucan control solution, or if a lesser amount of single triple helix β(l,3)-glucan is present in the renatured test solution than in the β(l,3)-glucan control solution, then the agent is an agent that inhibits folding of β(l,3)-glucan into single triple helix conformation. If a lesser amount of triple helical microfibril β(l,3)-glucan in the renatured test solution than in the β(l,3)-glucan control solution, then the agent inhibits β(l,3)-glucan association into a triple helical microfibril conformation. The invention also provides antifungal agents identified by the methods described above, as well as compositions comprising the agents, and methods of treating fungal infections in vivo or in vitro using the agents or compositions. The methods of the invention provide a simple, convenient means for identifying potential antifungal agents that interfere with β(l,3)-glucan microfibril assembly. Such agents may interfere with the normal assembly of yeast cell walls; because cell wall assembly is necessary for growth and viability not only of yeast, but of all fungi, agents which affect assembly of yeast cell walls are potential antifungal agents. Thus, the methods of the invention facilitate identification and development of novel antifungal agents. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graphic representation of the ability of Congo red (solid line) to inhibit the assembly of β(l,3)-glucan triple helical microfibrils, as compared with a solvent control (dotted line). Figure 2 is a graphic representation of the ability of calcofluor (solid line) to inhibit the assembly of β(l,3)-glucan triple helical microfibrils, as compared with a solvent control (dotted line).
Figure 3 is a graphic representation of the ability of congo red, as compared with other agents, to increase survival time in a murine model of Candida infection. Diamonds, amphotericin B; squares, congo red; triangles, dimethyl sulfoxide (DMSO).
The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings.
DETAILED DESCRIPTION OF THE INVENTION
THE FUNGAL CELL WALL
The fungal cell wall has a complex composition and structure (Ruiz-Herrera, J. Fungal cell wall Structure, synthesis and assembly, CRC Press, FI, 1992; Wessels, J., New Phytol. 725:397-413 (1993)). In general, the cell wall of human pathogenic fungi contains β(l,3)-glucan, β(l,6)-glucan, other cc-linked glucans (including glycogen and α(l,3)-glucan) mannoproteins, and smaller amounts of peptides, chitin and lipids (Fleet, G.H., in The Yeasts (2nd edition, Rose, A.H. and Harrison, J.S., eds) 4:199-277, Academic Press, London (1991)). Cell wall components are either structural, providing mechanical strength to the wall, or cementing, keeping structural components glued together. Structural materials are fibrillar and include chitin, β-l,3-linked-glucans and cellulose. Cementing materials are amorphous and include β(l,3)- and α-linked glucans, chitosan and glycoproteins.
Normal cell-wall biosynthesis and assembly is essential for growth and viability of fungi, as shown using cell-wall-acting drugs, as well as by analysis of mutants defective in key cell wall biosynthesis and assembly steps (Scott, W., Ann. Rev. Microbiol. 50:85-104 (1972)). Cell wall assembly is a complex, incompletely understood process (Klis, F., Yeast 70:851-569 (1994)). β(l,3)-glucan polymers of the cell wall are vectorially synthesized through the plasma membrane by a transmembrane multi-subunit enzyme complex into the periplasmic space in a random coil conformation; subsequently they undergo an ordered assembly process to form microfibrils. Formation of β(l,3)-glucan fibrils proceeds via the interaction of three β(l,3)-glucan chains to form a triple helical structure. As the growing β(l,3)-glucan chains assemble into this triple helical structure they are cross-linked by the enzyme-catalyzed formation of β(l,6) branch points. The branched triple helical β(l,3)-glucan chains are incorporated into the cell wall as parallel triple helical microfibrils, thereby forming the basic β(l,3)-glucan microfibril structure. These β(l,3)-glucan fibrils are then interwoven and crosslinked with chitin microfibrils and mannoproteins to form the fungal cell wall layer.
A potential target of antifungal agents is fungal cell wall biosynthesis and assembly. Interference with cell-wall biosynthesis by inhibitors such as cilofungin (β(l,3)-glucan synthesis), nikkomycin (chitin synthesis) and tunicamycin (mannoprotein synthesis), interferes with fungal cell growth (Kurtz, ASM News 64(1):31 (1997)). Inhibition of the non-enzymatic chitin and β(l,3)-glucan microfibril assembly processes by calcofluor and congo red also interferes with fungal cell growth (Vanni, G. et al, Plant Sci. Letters 31 :9-17 (1983); Ilorza, M. et al, J. Gen. Microbiol. 729:1577-1582 (1983)). Assays to identify agents that inhibit β(l,6)-glucan synthesis and cell wall assembly are described in detail in U.S. Patent application Serial No. 09/104,873, , filed on June 25, 1998, entitled "β(l,6)- GLUCAN SYNTHESIS AND CELL WALL ASSEMBLY ASSAY" by Gary R. Ostroff, the entire teachings of which are incorporated herein by reference. Assays to identify agents that inhibit chitin microfibril assembly are described in detail in U.S. Patent application Serial No. 09/104,315, filed on June 25, 1998, entitled "CHITIN MICROFIBRIL ASSEMBLY ASSAY" by Gary R. Ostroff, the entire teachings of which are incorporated herein by reference. Targeting the enzymatic processes involved in cell wall biosynthesis and assembly has proven to be difficult. From genome sequencing of S. cerevisiae, it is known that the fungal genome includes redundant genes for many critical enzymatic activities. The resulting isozymes enable the fungal cell to synthesize a cell wall even if a single isoenzyme is inhibited or a particular gene is genetically inactivated. This redundancy complicates the drug discovery process and has resulted in significant difficulties in developing new cell wall biosynthesis targeting antifungal drug candidates.
The present invention provides an in vitro method for assessing the ability of an agent to inhibit folding of β(l,3)-glucan chains into a triple helix, or to inhibit association into a triple helical microfibril. Agents that inhibit β(l,3)-glucan folding and/or association interfere with β(l,3)-glucan microfibril formation, and thus prevent the extracellular assembly of the fungal cell wall. Such agents, which target the essential, non-enzymatic β(l,3)-glucan microfibril assembly process, can be useful as antifungal agents.
β(l,3)-GLUCAN STRUCTURE, FOLDING, ASSOCIATION AND PREPARATION The methods of the invention are based on observations of the characteristics of underivatized, aqueous soluble β(l,3)-glucan preparations at different temperatures. Underivatized aqueous-soluble β(l,3)-glucan (also referred to as PGG-glucan or BETAFECTLN®) is a polysaccharide composed of glucopyranose units linked in chains via β(l,3)-glycosidic bonds, with β(l,3)-linked branches intermittently linked to the main chain via β(l,6)-glycosidic bonds. Single chains can be isolated (i.e., not substantially interacting with another chain). Three single helix chains can also combine to form a triple helix structure which is held together by interchain hydrogen bonding. Two or more β(l,3)-glucan triple helices can join together to form a triple helical microfibril. A β(l,3)-glucan polysaccharide (e.g., underivatized, aqueous soluble β(l,3)-glucan, or other soluble β(l,3)-glucans) can exist in at least four distinct conformations: single disordered chains, single helix, single triple helix and triple helical microfibrils. Preparations of the β(l,3)-glucan can comprise one or more of these forms, depending upon such conditions as pH and temperature. The term "single triple helix", as used herein, refers to a β(l,3)-glucan conformation wherein three single chains are joined together to form a triple helix structure. In this conformation, there is no higher ordering of these triple helices, that is, there is no substantial aggregation of triple helices. The term "triple helical microfibril", as used herein, refers to a β(l,3)-glucan conformation in which two or more triple helices are joined together (associated) via non-covalent interactions. The "molecular weight" of a β(l,3)-glucan composition, as the term is used herein, is the mass average molar mass of the collection of polymer molecules within the composition. The characterization of a collection of polymer molecules in terms of polymer mass average molar mass is well known in the art of polymer science. The "aggregate number" of a β(l,3)-glucan conformation is the number of single chains which are joined together in that conformation. The aggregate number of a single helix is 1, the aggregate number of a single triple helix is 3, and the aggregate number of a triple helical microfibril is greater than 3. For example, a triple helical microfibril consisting of two triple helices joined together has an aggregate number of 6. The aggregate number of a β(l,3)-glucan sample under a specified set of conditions can be determined by determining the average molecular weight of the polymer under those conditions. The β(l,3)-glucan is then denatured, that is, subjected to conditions which separate any aggregates into their component single polymer chains. The average molecular weight of the denatured polymer is then determined. The ratio of the molecular weights of the aggregated and denatured forms of the polymer is the aggregate number. A typical β(l,3)-glucan composition includes molecules having a range of chain lengths, conformations and molecular weights. Thus, the measured aggregate number of a β(l,3)-glucan composition is the mass average aggregate number across the entire range of β(l,3)-glucan molecules within the composition. It is to be understood that any reference herein to the aggregate number of a β(l,3)-glucan composition refers to the mass average aggregate number of the composition under the specified conditions. The aggregate number of a composition indicates which conformation is predominant within the composition. For example, a measured aggregate number of about 6 or more is characteristic of a composition in which the β(l,3)-glucan is substantially in the triple helical microfibril conformation. The conformation of an aqueous soluble, β(l,3)-glucan preparation such as PGG-glucan is temperature dependent. For example, as described in Example 1 , an aqueous PGG-glucan solution prepared according to the method disclosed in U.S. Patent No. 5,622,939, incorporated herein by reference in its entirety, elutes from a gel permeation chromatography column (GPC, also referred to as size exclusion chromatography) at 25 °C as a single symmetric peak (Fraction A). When the elution is conducted at 37 °C, however, two distinct peaks are observed, denoted Fraction A, which elutes first, and Fraction C, which elutes second. When the elution is conducted at 75 °C, only one peak is observed, denoted Fraction B-, which elutes later than Fractions A and C.
The molecular weights of fractions A, B and C were determined at 25 °C at both pH 7 and pH 13, at 37°C at pH 7, and at 75°C at pH 7. At pH 13, PGG-glucan is in an unaggregated or single chain conformation. Thus, at a given temperature the ratio of the molecular weights determined at pH 7 and pH 13 is the aggregate number at pH 7 at that temperature.
At pH 7 and 25 °C, Fraction A had a molecular weight of 238,000 and an aggregate number of 15.0. Upon increasing the temperature to 37 °C, the molecular weight of Fraction A decreased to 164,000 and the aggregate number decreased to 10.3. At 75°C, the molecular weight of this fraction was 52,600 with an aggregate number of 3.3. The temperature dependence of molecular weight and aggregate number was more pronounced for Fraction C. At pH 7.0 and 25 °C, Fraction C had a molecular weight of 71,500 and an aggregate number of 6.0. At 37°C, the molecular weight of Fraction C was 32,000 and the aggregate number was 2.7. At 75 °C, the molecular weight of this fraction was 17,200 and the aggregate number was 1.4. Fraction B showed no temperature dependence of molecular weight. At pH 7 and 25-75 °C, Fraction B had a molecular weight of approximately 15,000 and an aggregate number of approximately 1.
The results indicated that at 25 °C and pH 7, both Fraction A and Fraction C exist predominantly in a triple helical microfibril conformation. When the temperature is increased to 37 °C, Fraction A remains predominantly in a triple helical microfibril conformation, while Fraction C is primarily in a single triple helix conformation. At 75 °C. Fraction A remains predominantly in a single triple helix conformation, while Fraction C is primarily in a single chain random coil conformation. Fraction B exists primarily in a single chain conformation, remaining primarily as a single chain at 25-75 °C.
Additional information regarding Fractionation of PGG-glucan is provided in U.S. Patent application Serial No. 08/902,586, filed July 28, 1997, entitled
"CONFORMATIONS OF PGG-GLUCAN", the teachings of which are incorporated herein by reference in their entirety.
METHODS OF THE INVENTION
The present invention utilizes a soluble β(l,3)-glucan composition which is substantially in a triple helical microfibril conformation at room temperature (e.g., at about 20-25 °C), which is substantially in a single triple helix conformation under physiological conditions (e.g., at about 37°C), and which is substantially in a single helix conformation at greater than about 75 °C, in assays for assessing the ability of an agent to inhibit folding of β(l,3)-glucan chains. In a preferred embodiment, the soluble β(l,3)-glucan composition has an aggregate number in the range from greater than 2 to less than 6, and preferably from about 2.5 to about 3.5, under physiological conditions of about 37 °C; and an aggregate number of less than about 2 under conditions of elevated pH and/or temperature. In a particularly preferred embodiment, the soluble β(l,3)-glucan composition is Fraction C, as described above. The soluble β(l,3)-glucan composition can comprise underivatized or derivatized β(l,3)-glucan, provided that the β(l,3)-glucan is capable of folding into a single triple helix conformation and of associating into a triple helical microfibril conformation. In a preferred embodiment, the soluble β(l,3)-glucan composition is underivatized, aqueous soluble, β(l,3)-glucan. A "soluble β(l,3)-giucan composition", as the term is used herein, is a β(l,3)-glucan composition that dissolves in an aqueous medium at room temperature (about 20-25°C) and neutral pH (from about pH 5.5 to about 7.5) to form a visually clear solution at a concentration up to about 100 mg/mL. An "aqueous medium", as the term is used herein, refers to water or a water-rich phase, particularly physiologically acceptable aqueous phases, including phosphate-buffered saline, saline and dextrose solutions. A soluble β(l,3)-glucan composition that is dissolved in an aqueous medium is referred to herein as a "soluble β(l,3)-glucan solution". The term "physiological conditions", as used herein, refers to physiological pH, about pH 7, and physiological temperature, about 37°C. Under physiological conditions, the preferred β(l,3)-glucan composition used in the methods of the invention consists essentially of β(l ,3)-glucan chains in single triple helix conformation.
The soluble β(l,3)-glucan composition can be prepared from soluble or insoluble glucan particles, from many sources, such as from yeast or fungal sources. In a preferred embodiment, the soluble β(l,3)-glucan composition is derived- from yeasts, as described in U.S. Patent No. 5,622,939; U.S. Serial No. 08/373,251, U.S. Serial No. 08/469,233, U.S. Patent No. 5,322,841, U.S. Serial No. 08/432,303, U.S. Patent No. 5,663,324, U.S. Patent No. 5,633,369 and U.S. Serial No. 08/400,488; the entire teachings are incorporated herein by reference. A general procedure for the preparation of insoluble yeast glucans is provided by Manners et al, Biol. J. 135 : 19-30 (1973). Glucan particles which are particularly useful as starting materials in the present invention are whole glucan particles as described by Jamas et al. in U.S. Patent Nos. 4,810,646, 4,992,540, 5,082,936, 5,028,703 and 5,622,939, the teachings of each of which are incorporated herein by reference in their entirety. The source of the whole glucan particles can be any yeast, fungus, or alga which contains β(l,3)-glucans in its cell walls or which secretes β(l,3)-glucans. Particularly useful are whole glucan particles obtained from the yeast strains Saccharomyces cerevisiae R4 (NRRL Y-15903) and R4 Ad (ATCC No. 74181). Other strains of yeast which are suitable sources of whole glucan particles include Saccharomyces delbruekii, Saccharomyces rosei, Saccharomyces microellipsodes, Saccharomyces carlsbergensis, Schizosaccharomyces pombe, Kluvveromyces lactis, Kluvveromyces fragilis, Kluvveromyces polysporus, Candida albicans, Candida cloacae, Candida glabrata, Candida tropicalis, Candida utilis, Hansenula wingei, Hansenula ami, Hansenula henricii and Hansenula americana.
In the methods of the invention, a soluble β(l,3)-glucan composition in an aqueous solution, as described above, is used as a test solution. The soluble β(l,3)- glucan composition in the test solution is denatured to a random coil, single chain conformation. Any appropriate denaturing method or agent can be used. In one embodiment, the soluble β(l,3)-glucan composition in the solution is denatured by increasing the pH of the solution to greater than about pH 11, to a pH that is between about pH 11 and pH 14 (inclusive), for approximately 1 minute to approximately 1 hour. NaOH/KOH can be used at approximately greater than 0.001 Molar to raise the pH. In another embodiment, the soluble β(l,3)-glucan is denatured by adding dimethyl sulfoxide (DMSO) at a concentration of approximately greater than 30% by volume. At concentrations of less than 5% by volume, DMSO can be used as a solvent; however, at concentrations greater than approximately 30% by volume, DMSO can be used to denature the soluble β(l,3)-glucan. In a third embodiment, the soluble β(l,3)-glucan is denatured by heating the test solution to above about 37 °C. In a preferred embodiment, the test solution is heated to a temperature range of from about 37 to about 135 °C. In a more preferred embodiment, the test solution is heated to a temperature range of from about 90 to about 100°C. If the soluble β(l,3)-glucan is denatured by heating, the solution is maintained at the higher temperature for an appropriate length of time to result in denaturation of the β(l,3)- glucan into a random coil, single chain conformation. Generally, the solution is maintained at this temperature for about 10 minutes to about 1 hour.
Once the soluble β(l,3)-glucan composition in the β(l,3)-glucan test solution is denatured, the resultant solution (referred to herein as the "denatured β(l,3)- glucan test solution") is contacted with an agent to be tested (the "agent of interest" or the "test agent"). The denatured β(l,3)-glucan test solution that has been contacted with the test agent (referred to herein as the "contacted, denatured β(l,3)- glucan test solution") is then maintained under renaturing conditions. "Renaturing conditions" are those conditions that would, in the absence of the agent to be tested, allow folding (renaturation) and reassociation of the β(l,3)-glucan composition to generate a renatured β(l,3)-glucan composition. For example, if the soluble β(l,3)- glucan was denatured by raising the pH of the aqueous solution comprising the β(l,3)-glucan, then the denatured β(l,3)-glucan test solution is subjected to renaturing conditions which result in a gradual lowering of the pH. If the soluble β(l,3)-glucan was denatured by adding DMSO, then the denatured β(l,3)-glucan test solution is subjected to renaturing conditions which result in gradual removal of DMSO. If the soluble β(l,3)-glucan was denatured by heating, the denatured β(l,3)- glucan test solution is maintained in an appropriate environment (e.g., away from the heat source used for denaturing the β(l,3)-glucan, at room temperature) so that its temperature can be gradually cooled.
The endpoint of the renaturing conditions depends on whether the test agent is being assessed for an ability to inhibit formation of single triple helix conformation, or to inhibit formation of triple helical microfibrils. To assess whether the test agent inhibits folding of β(l,3)-glucan into a single triple helix conformation, the contacted, denatured β(l,3)-glucan test solution is maintained under renaturing conditions that are sufficient, in the absence of the test agent, to allow the β(l,3)-glucan to fold into a single triple helix conformation. For example, if the β(l,3)-glucan was denatured by heating, the contacted, denatured β(l,3)- glucan test solution is maintained in an environment in which it can cool until its temperature is within a physiological range (e.g., from about 35 to about 50°C). In a preferred embodiment, the denatured β(l,3)-glucan test solution is allowed to cool until its temperature is from about 37 to about 45 °C. If the β(l,3)-glucan was denatured by adding DMSO, the contacted, denatured β(l,3)-glucan test solution is maintained under conditions which lower the amount of DMSO to approximately 15-25% by volume. In a preferred embodiment, the amount of DMSO is lowered by approximately 20% by volume. If the β(l,3)-glucan was denatured by raising the pH, the contacted, denatured β(l,3)-glucan test solution is maintained under conditions which lower the pH to from greater than about 11 to a pH that is between about pH 9 (inclusive) and pH 11 (inclusive). To assess whether the test agent inhibits association of β(l,3)-glucan into a triple helical microfibril conformation, the contacted, denatured β(l,3)-glucan test solution is maintained under renaturing conditions that are sufficient, in the absence of the test agent, to allow the β(l,3)- glucans to associate into triple helical microfibril conformation. For example, if the β(l,3)-glucan was denatured by heating, the contacted, denatured β(l,3)-glucan test solution is maintained in an environment in which it can cool until its temperature is less than or equal to room temperature (e.g., from about 2 to 4°C to about 30 °C). In a preferred embodiment, the denatured β(l,3)-glucan test solution is allowed to cool until its temperature is from about 20 to about 25 °C. If the β(l,3)-glucan was denatured by DMSO, the contacted, denatured β(l,3)-glucan is maintained in an environment which removes the DMSO until less than approximately 5% by volume is left. If the β(l,3)-glucan was denatured by raising the pH, the contacted, denatured β(l,3)-glucan test solution is maintained under conditions which lower the pH from greater than about 11 to approximately a physiologic pH that is between about pH 3 (inclusive) to pH 9 (inclusive). In a preferred embodiment, the pH is lowered to about pH 7. The agent can be assessed for its ability to inhibit folding of β(l,3)-glucan into a single triple helix conformation; to inhibit association of β(l,3)- glucans into a triple helical microfibril conformation; or to inhibit both folding and association, by the methods of this invention. After the contacted, denatured β(l,3)-glucan test solution is subjected to renaturing conditions, the conformation of the β(l,3)-glucan composition (i.e., the amount of single chain conformation, single triple helix conformation, triple helical microfibril, or a mixture of these conformations) in the renatured β(l,3)-glucan test solution is then assessed. Generally, a mixture of different conformations of β(l,3)- glucan are present; therefore, the assessment of the conformation of the β(l,3)- glucan can include a determination of the relative amounts of the different conformations. The conformation(s) of the β(l,3)-glucan composition in the renatured β(l,3)-glucan test solution can be assessed, for example, by directing the solution through a high performance gel permeation chromatography column, which separates the soluble β(l,3)-glucan into single chain (slow moving), single triple helix (intermediate moving), and triple helical microfibril (fast moving) forms. Such methods are described below in the Examples. The conformation of the β(l,3)- glucan composition can also be assessed by using other physical methods, such as by measuring viscosity, polarimetry or light scattering, or by fluorescent polarization. The use of fluorescence polarization is described in detail in U.S. Patent application Serial No. 09/104,560, filed June 25, 1998, entitled "ASSAYS FOR AGENTS WHICH ALTER CELL WALL BIOPOLYMER SYNTHESIS OR ASSEMBLY" by Gary R. Ostroff, the entire teachings of which are incorporated herein by reference. Alternatively, the aggregate number of the β(l,3)-glucan composition can be determined, as described above. In another embodiment, a nonphysical method can be used to measure the amount of random coil, single chain β(l,3)-glucan, single triple helix β(l,3)-glucan. or triple helical microfibril β(l,3)- glucan. For example, in a preferred embodiment, an agent that specifically binds to a particular conformation of β(l,3)-glucan (e.g., a monoclonal antibody that specifically binds to random coil β(l,3)-glucan, to single chain β(l,3)-glucan, to single triple helix β(l,3)-glucan, or to triple helical microfibril β(l,3)-glucan; or a monoclonal antibody that specifically binds to single triple helix β(l,3)-glucan and to triple helical microfibril β(l,3)-glucan, but not to single chain β(l,3)-glucan; or a receptor or other binding agent for a particular conformation of β(l,3)-glucan) can be used to identify the presence of that conformation of β(l,3)-glucan as described in U.S. Serial No. 08/637,934, U.S. Serial No. 08/664,173, U.S. Serial No. 08/797,696, U.S. Serial No. 08/990,155 and U.S. Serial No. 08/990,125; the entire teachings of these are incorporated herein by reference. The agent that specifically binds to a particular conformation of β(l,3)-glucan can be labeled, to allow detection of that conformation; alternatively, the agent that specifically binds to a particular conformation of β(l,3)-glucan can be used in a competition assay, such as an enzyme-linked immunosorbent assay (ELISA). For example, the renatured β(l,3)- glucan test solution can be incubated with a monoclonal antibody that specifically binds with single triple helix β(l,3)-glucan, and the mixture containing residual unbound antibody is then incubated with triple helical β(l,3)-glucan bound to a solid support, such as an ELISA plate. The amount of antibody that binds to the triple helical β(l,3)-glucan on the solid support can then be determined by reaction with an antibody enzyme conjugate. If an agent inhibits folding of β(l,3)-glucan into triple helix conformation, there would be no competition for the monoclonal antibody, and a colored, secondary antibody-enzyme product would appear in the ELISA reaction. Once the conformation (or the relative amounts of different conformations) of the β(l,3)-glucan in the renatured β(l,3)-glucan test solution is assessed, it is compared with the conformation (or the relative amounts of different conformations) of β(l,3)-glucan in a control β(l,3)-glucan solution. The control β(l,3)-glucan solution is an aqueous solution comprising the soluble β(l,3)-glucan composition, and that has been subjected to the same denaturing and renaturing conditions as the test solution, but in the absence of the test agent, or in the presence of a control agent (e.g., DMSO, water, or a compound that is known not to interfere with β(l,3)-glucan microfibril assembly) in lieu of the test agent. The presence of a greater amount of random chain β(l,3)-glucan in the renatured β(l,3)-glucan test solution than in the β(l,3)-glucan control solution; and/or the presence of a lesser amount of single triple helix β(l,3)-glucan in the renatured test solution than in the β(l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit β(l,3)-glucan folding into a single triple helix formation. The presence of a greater amount of single chain β(l,3)-glucan in the renatured β(l,3)-glucan test solution than in the β(l,3)-glucan control solution; and/or the presence of a lesser amount of single triple helix β(l,3)- glucan in the renatured β(l,3)-glucan test solution than in the β(l,3)-glucan control solution; and or the presence of a lesser amount of triple helical microfibril β(l,3)- glucan in the renatured test solution than in the β(l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit β(l,3)-glucan association into a triple helical microfibril formation.
If the quantitative average aggregate number is used to assess the conformation of the β(l,3)-glucan, a difference between the test solution and the control solution that is greater than or equal to two indicates that the test agent inhibits folding of β(l,3)-glucan into single triple helix conformation, and/or inhibits association of β(l,3)-glucan into triple helical microfibril formation.
If a test agent is identified by the methods described above as an agent that inhibits β(l,3)-glucan folding, further experiments can be conducted to assess the agent's potential as an antifungal agent. For example, experiments can be performed to determine whether the agent inhibits fungal cell growth, as described in Example 3, or to determine whether any fungal cell death caused by the agent is due to inhibition of β(l,3)-glucan microfibril assembly, as described in Example 4. The agent can also be tested for in vivo efficacy, as described in Example 5. Agents identified by the methods described herein can be used as antifungal agents that target the essential, non-enzymatic β(l,3)-glucan microfibril assembly process, thereby avoiding the problems associated with targeting enzymatic activities.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically (or prophylactically) effective amount of an agent identified by the methods described above, and a pharmaceutically acceptable carrier or excipient. The carrier and composition can be sterile. The formulation should suit the mode of administration. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc., as well as combinations thereof. The pharmaceutical preparations can, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrollidone, sodium saccharine, cellulose, magnesium carbonate, etc.
The composition can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, compositions for intravenous administration typically are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. For topical application, nonsprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, can be employed. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. The agent may be incorporated into a cosmetic formulation. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., pressurized air.
Agents described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- e hylamino ethanol, histidine, procaine, etc.
The amount of agents which will be effective in the treatment of a particular disorder or condition will depend on the nature of the infection, disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the infection, disease or disorder, and should be decided according to the judgment of a practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use of sale for human administration. The pack or kit can be labeled with information regarding mode of administration, sequence of drug administration (e.g., separately, sequentially or concurrently), or the like. The pack or kit may also include means for reminding the patient to take the therapy. The pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages. In particular, the agents can be separated, mixed together in any combination, present in a single vial or tablet. Agents assembled in a blister pack or other dispensing means is preferred. For the purpose of this invention, unit dosage is intended to mean a dosage that is dependent on the individual pharmacodynamics of each agent and administered in FDA approved dosages in standard time courses.
The agents identified by the methods of the invention as inhibiting folding of β(l,3)-glucan into single triple helix conformation, and or association of β(l,3)- glucan into triple helical microfibril formation, as well as the compositions described above, can be used either in vitro or in vivo to kill fungi and/or treat fungal infection. The agent is generally administered to an animal, a human, or a location of fungal contamination or growth (e.g., an environmental location) in an amount sufficient to inhibit and or eliminate fungal infection or growth (a "therapeutically effective amount" or an "effective amount"). The mode of administration of the agent (or composition), in the case of in vivo administration, can be oral, enteral, parenteral, intravenous, subcutaneous, intraperitoneal, intramuscular, topical or intranasal. For in vitro administration, the agent (or composition) is administered by a means that allows contact of the agent (or composition) with the fungal growth. The form in which the composition will be administered (e.g., powder, tablet, capsule, solution, emulsion) will depend on whether it is used in vivo or in vitro, as well as (in the case of in vivo administration) the route by which it is administered. The quantity of the agent or composition to be administered in vivo will be determined on an individual basis, and will be based at least in part on consideration of the severity of infection or injury in the patient, the patient's condition or overall health, the patient's weight and gender. In general, a single dose will preferably contain approximately 0.01-100 mg per kilogram of body weight, and preferably about 1 mg/kg. The quantity of the agent or composition to be administered in vitro will also be determined on a case-by-case basis, and will be based at least in part on consideration of the type and extent of the fungal contamination or growth.
In general, the agents or compositions of the present invention can be administered to an individual or applied to an in vitro fungal source as necessary to treat the fungal infection or contamination. An individual skilled in the medical arts will be able to determine the length of time during which the agent or composition is administered and the dosage.
The following Examples are offered for the purpose of illustrating the present invention and are not to be construed to limit the scope of this invention. The teachings of all references cited are hereby incorporated herein by reference.
EXAMPLES
EXAMPLE 1 TEMPERATURE DEPENDENCE OF PGG-GLUCAN
CONFORMATION, FRACTIONATION OF PGG-GLUCAN AND CHARACTERIZATION OF FRACTIONS
FIRST FRACTIONATION OF PGG-GLUCAN AT 25°C, 37°C AND 75°C A PGG-Glucan composition prepared as described in U.S. Patent No. 5,622,939 was concentrated to approximately 20 mg/mL. An aliquot of the concentrated sample was fractionated at 25 °C on a preparative scale gel permeation chromatography column (5 cm TSK HW55F resin column) using 0.15 M sodium chloride as the mobile phase. The composition eluted as a single symmetrical band. A second aliquot of the concentrated sample was fractionated on a preparative GPC column maintained in a hot room at 37 °C. The PGG-glucan eluted from the column in two distinct fractions. A third aliquot of the concentrated sample was fractionated on a preparative GPC column maintained in a hot room at 75 °C. These fractions were collected and designated Fraction A and Fraction C, and Fraction B, respectively.
CHARACTERIZATION OF FRACTIONS A, B AND C The molecular weights of each fraction at pH 7 and 25 °C, 37°C and 75°C, and at pH 13 and 25 °C are presented in Table 1. For each fraction, molecular weight decreases with increasing temperature. The aggregate numbers of each fraction at pH 7 and 25 °C, 37°C and 75 °C are shown in Table 2. At 25 °C, fractions A and C have an aggregate number greater than 6, indicating that each fraction is predominantly in a triple helical microfibril conformation at this temperature; Fraction B has an aggregate number of approximately 1 , indicating that it is in the single chain conformation at this temperature. At 37 °C, Fraction A remains predominantly in a triple helical microfibril conformation, Fraction C is predominantly in a single triple helix conformation. At 75 °C, however, Fraction A remains predominantly in a single triple helix conformation, while Fractions B and C are predominantly in a single chain random-coil conformation. These data are summarized in Tables 1 and 2, below.
Table 1. Average Molecular Weight Values of Fractions A, B and C at 25 °C and 37°C
Figure imgf000022_0001
Table 2. Aggregate Number of Fractions A and C at 25° C and 37° C
Figure imgf000023_0001
EXAMPLE 2 DEVELOPMENT OF AN 77V VITRO β(l,3)-GLUCAN MICROFIBRIL ASSAY An assay was developed to assess the ability of agents to inhibit folding of β(l,3)-glucan into unaggregated triple helical structures, or into aggregated triple helical structures (e.g., triple helical microfibrils). In the assay, a 1 mg/ml solution of neutral soluble glucan (e.g., Fraction C as described above, or BETAFECTIN® or PGG-glucan) in water is heated to 100° C for 30 minutes to denature the triple- helical bundles into a single chain random-coil conformation. A 100 μl volume of the hot, denatured neutral soluble glucan solution is mixed with 1.0 μl of a solvent control (water or DMSO), a positive control (congo red, 1 mg/ml; or calcofluor 1 mg/ml), or a test compound? The solutions are then cooled for approximately 1 hour to room temperature to allow the glucan to refold/reassociate into native triple- helical bundles. If a test agent interferes with the refolding/reassociation reaction, then the glucan remains in the denatured, single-chain form. The molecular size of the assembled product (i.e., the refolded/reassociated glucan), is analyzed using a single column, high performance gel permeation chromatography (GPC) system (column, Shodex KB-803; solvent, 0.9% saline; flow rate, 1 ml min). Data is electronically captured using a Perkin-Elmer/Nelson Access Chrom system. From manual analysis of the DRI chromatograms, the percent inhibition of refolding/reassociating by the test compound is determined as, % inhibition = [1- (THB/SC)test/(THB/SC)control] x 100, where THB is the peak height of triple helical microfibril bundles (which are fast migrating), and SC is the peak height of single chain (slow migrating). IC50 determinations for active test compounds can be calculated by regression analysis of a 3-point, % inhibition dilution curve of each test compound.
A number of known β(l,3)-glucan microfibril inhibitors and structurally- related compounds were evaluated for their ability to inhibit β(l,3)-glucan microfibril assembly, using this assay. Known β(l,3)-glucan microfibril inhibitors, such as congo red and calcofluor, inhibited the assembly of neutral soluble glucan relative to a solvent control, as shown in Figures 1 and 2. Structurally-related compounds, acid red 97 and brilliant yellow, were inactive (data not shown). ~In addition, inhibitors of β(l,3)-glucan- and chitin-biosynthesis, cilofungin and nikkomycin Z were also inactive (data not shown), demonstrating specificity of the β(l,3)-glucan microfibril assembly assay. These results (summarized in Table 3, below) demonstrated the feasibility of the in vitro assay to screen for inhibitors of β(l,3)-glucan microfibril assembly. The measurement of physical size differences can also be performed by batch-type light scattering (Wyatt miniDawn), capillary type viscometer, or polarimetric techniques. The measurement of conformational differences can also be performed by competitive ELISA assays.
EXAMPLE 3 ASSESSMENT OF FUNGAL CELL GROWTH INHIBITION Compounds identified by the assay described above as inhibiting β(l,3)- glucan microfibril assembly can be tested for their ability to inhibit the growth and protoplast regeneration of the human pathogenic fungus C. albicans using standard agar diffusion assays. Coπelation between β(l,3)-glucan and microfibril assembly inhibition with fungal cell killing was demonstrated using such an assay. A 96-well microtiter plate agar diffusion assay, was conducted as follows: a 2.5 μl volume of solvent control (water or DMSO), positive control (cilofungin, 10 mg/ml; nikkomycin Z, 10 mg/ml; amphotericin B. 10 mg/ml; fluconazole, 10 mg/ml), or a test compound (congo red, calcofluor, or structurally similar microfibril-inactive compounds as described above, at 10 mg/ml) was added to individual wells of a 96-well microtiter plate. A 75 μl volume of molten yeast extract-peptone-dextrose agar (YPDA; 1.5% agar) medium cooled to 50°C was added to each well and allowed to solidify at room temperature. Pathogenic C. albicans (such as ATCC deposit 20402) cells grown to mid-log phase in YPD liquid medium at 30 °C were washed twice with sterile water by centrifugation at 3,000 x g for 10 minutes, adjusted to 5 x 106 cells/ml, then mixed with YPD soft agar (YPDSA; 0.4% agar) medium and 30 μl of YPDSA containing approximately 1,500 cells, and added to each well. Plates were incubated at 30 °C and scored for growth by visual and microscopic video imaging at approximately 20 and 48 hours. Results, shown in Table 3 below, demonstrated that inhibition of β(l,3)-glucan microfibril assembly resulted in C. albicans cell death, validating use of microfibril assembly as an antifungal target.
EXAMPLE 4 DEMONSTRATION THAT CELL DEATH IS DUE TO INHIBITION OF TARGET The effect of microfibril inhibitors on protoplast cell-wall regeneration were assessed to verify that the cause of cell death was due to inhibition of β(l,3)-glucan microfibril assembly. A 96-well microtiter plate protoplast regeneration agar diffusion assay was used. A 2.5 μl volume of solvent control (water or DMSO), positive control (cilofungin, 10 mg/ml; nikkomycin Z, 10 mg/ml; amphotericin B, 10 mg/ml; fluconazole, 10 mg/ml) or a test compound (congo red, calcofluor, or structurally similar micro fibril-inactive compounds, at 10 mg/ml) were added to individual wells of a 96-well microtiter plate. Then a 75 μl volume of molten yeast- extract-peptone-dextrose-KCl agar (YPDA; 1.5% agar, 0.6 M KCl) medium cooled to 50°C will be added to each well and allowed to solidify at room temperature. Pathogenic C. albicans (such as ATCC deposit 20402) cells grown to mid-log phase of growth in YPD liquid medium at 30°C were washed twice with sterile water by centrifugation at 3,000 x g for 10 minutes and adjusted to an optical density at 600 ran of approximately 2. Washed cells in protoplast buffer (40 mM NA2HPO , 5 mM EDTA, 5 mM β-mercaptoethanol, 0.6 M KCl, pH = 7.2) were resuspended in sterile lyticase (0.75 mg/ml in protoplast buffer, Sigma) followed by incubation at 37°C. Protoplast formation was followed by microscopic observation, and when complete, protoplasts were washed three times with sterile 0.6M KCl by centrifugation at 500 x g for 10 minutes. Protoplasts were then adjusted to 5 x 106 cells/ml, mixed with YPDK soft agar (YPDSAK; 0.4% agar, 0.6 M KCl) medium and 30 μl of YPDSAK containing approximately 15,000 protoplasts was added to each well. Plates were incubated at 30 °C and scored for growth and protoplast regeneration by visual and microscopic video imaging at 24 and 48 hours. After incubation for 24 hours at 30 °C, control protoplasts regenerated cell wall and reinitiated cell division to an 8- 16 cell microcolony stage as visualized by light microscopy. If a fungicidal compound was present, such as positive control amphotericin B, the protoplasts died and there were no observable cells present at 24 hours. If a fungistatic compound was present, such as positive control fluconazole, 1-2 regenerated cell microcόlonies were observed. If a cell- wall active compound was present, such as positive controls cilofungin or nikkomycin Z, live, single cell, non-regenerated protoplasts were seen. Congo red and calcofluor were similar to cell-wall polymer synthesis inhibitors, preventing the regeneration of the fungal cell wall, but not killing the protoplasts. In contrast, the structurally similar, microfibril-inactive compounds had no effect on protoplast regeneration or viability. These results are also summarized in Table 3, below. The results demonstrate that β(l,3)-glucan microfibril inhibitors have a direct cell-wall effect, and that lethal disruption of the fungal cell wall can be accomplished through inhibition of either cell-wall polymer biosynthesis or β(l,3)- glucan microfibril assembly.
Table 3. Summary of Results for Anti-Micro fibril Agents and Control Cell- wall Active Compounds
Figure imgf000027_0001
EXAMPLE 5 CORRELATION BETWEEN IN VITRO INHIBITION AND IN VIVO EFFICACY
To assess the β(l,3)-glucan microfibril assembly inhibitors for antifungal activity in vivo, a murine model of acute Candidiasis was used. Male ICR mice (weight approximately 20 g) were intraperitoneally administered 1.25 x 107 cfu of C. albicans (ATCC deposit number 10231) was intraperitoneally administered. Administration of this C. albicans dose has been previously shown to be lethal for 90-100% of placebo-treated mice within 10 days of infection (data not shown). One hour after mice were infected with C. albicans, groups of 10 mice were administered various doses of test compound (congo red), or control compound (DMSO or amphotericin B at 10 mg/kg) in a 0.1 ml volume via intraperitoneal injection at +1 hour (single dose schedule) or at +1 hour, +1 day, +2 day, -r3 day, +4 day (multi- dose schedule), and survival monitored twice daily for a 10-day period. Results, shown in Figure 3, indicated that congo red increased survival time, relative to a vehicle control, at a dose of 3 mg/kg.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

CLAIMSWhat is claimed is:
1. A method of assessing an agent of interest for an ability to inhibit folding or association of ╬▓(l,3)-glucan, comprising contacting a ╬▓(l,3)-glucan test solution comprising a denatured, soluble ╬▓(l,3)-glucan composition in an aqueous solution with the agent of interest, maintaining the solution under renaturing conditions, and assessing the conformation of the soluble ╬▓(l,3)- glucan composition in the solution, wherein the presence of a greater amount of random chain ╬▓(l,3)-glucan, a lesser amount of single triple helix ╬▓(l,3)- glucan, or a lesser amount of triple helical microfibrils, in the solution as compared with the amount in a denatured, soluble ╬▓(l,3)-glucan composition in aqueous solution maintained under the renaturing conditions in the absence of the agent of interest, is indicative of the ability of the test agent to inhibit folding or association of ╬▓(l,3)-glucan.
2. The method of Claim 1, wherein the soluble ╬▓(l,3)-glucan composition comprises aqueous soluble ╬▓(l,3)-glucan.
3. A method of assessing an agent of interest for the ability to inhibit folding or association of ╬▓(l,3)-glucan, comprising the steps of: a) providing a ╬▓(l,3)-glucan test solution comprising a soluble ╬▓(l,3)- glucan composition in an aqueous solution; b) denaturing the soluble ╬▓(l,3)-glucan composition in the test solution, thereby forming a denatured ╬▓(l,3)-glucan test solution; c) contacting the denatured ╬▓(l,3)-glucan test solution with the agent of interest, thereby forming a contacted, denatured ╬▓(l,3)-glucan test solution; d) maintaining the contacted, denatured ╬▓(l,3)-glucan test solution under renaturing conditions, thereby forming a renatured ╬▓(l,3)- ucan test solution; e) assessing the conformation of the ╬▓(l,3)-glucan composition in the renatured ╬▓(l ,3)-glucan test solution; and f) comparing the conformation of the ╬▓(l,3)-glucan composition in the renatured ╬▓(l,3)-glucan test solution with the conformation of the ╬▓(l ,3)-glucan composition in a ╬▓(l ,3)-glucan control solution, wherein the presence of a greater amount of random chain ╬▓(l,3)-glucan in the renatured ╬▓(l,3)-glucan test solution than in the ╬▓(l,3)-glucan control solution; the presence of a lesser amount of single triple helix ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution; or the presence of a lesser amount of triple helical microfibril ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit ╬▓(l,3)-glucan folding or association.
4. The method of Claim 3, wherein the soluble ╬▓(l ,3)-glucan composition comprises aqueous-soluble ╬▓(l,3)-glucan.
5. The method of Claim 3, wherein the soluble ╬▓(l,3)-glucan composition comprises Fraction C of aqueous-soluble ╬▓(l,3)-glucan.
6. A method of assessing an agent of interest for the ability to inhibit folding or association of ╬▓(l,3)-glucan, comprising the steps of: a) providing a ╬▓(l,3)-glucan test solution comprising a soluble ╬▓(l,3)- glucan composition in an aqueous solution; b) denaturing the soluble ╬▓(l,3)-glucan composition in the test solution by raising the pH of the ╬▓(l,3)-glucan test solution, thereby forming a denatured ╬▓(l,3)-glucan test solution; c) contacting the denatured ╬▓(l,3)-glucan test solution with the agent of interest, thereby forming a contacted, denatured ╬▓(l,3)-glucan test solution; d) maintaining the contacted, denatured ╬▓(l,3)-glucan test solution under renaturing conditions that lower the pH of the contacted, denatured ╬▓(l,3)-glucan test solution, thereby forming a renatured ╬▓(l,3)-glucan test solution; e) assessing the conformation of the ╬▓( 1 ,3)-glucan composition in the renatured ╬▓(l,3)-glucan test solution; and f) comparing the conformation of the ╬▓(l,3)-glucan composition in the renatured ╬▓(l,3)-glucan test solution with the conformation of the ╬▓(l,3)-glucan composition in a ╬▓(l,3)-glucan control solution, wherein the presence of a greater amount of random chain ╬▓(l,3)-glucan in the renatured ╬▓(l,3)-glucan test solution than in the ╬▓(l,3)-glucan control solution; the presence of a lesser amount of single triple helix ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution; or the presence of a lesser amount of triple helical microfibril ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit ╬▓(l,3)-glucan folding.
7. The method of Claim 6, wherein the soluble ╬▓(l,3)-glucan composition comprises aqueous-soluble ╬▓(l,3)-glucan.
8. The method of Claim 6, wherein the soluble ╬▓(l,3)-glucan composition comprises Fraction C of aqueous-soluble ╬▓(l,3)-glucan.
9. The method of Claim 6, wherein the pH is raised in step (b) to greater than about 11.
10. The method of Claim 9, wherein the pH is lowered in step (d) to from greater than about 11 to a pH that is between about pH 9 and pH 11 , inclusive.
11. The method of Claim 10, wherein the presence of a greater amount of random chain ╬▓(l,3)-glucan in the renatured ╬▓(l,3)-glucan test solution than in the ╬▓(l,3)-glucan control solution, or the presence of a lesser amount of single triple helix ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit ╬▓(l,3)-glucan folding into single triple helix conformation.
12. The method of Claim 9, wherein the pH is lowered in step (d) to a pH that is between about pH 3 and pH 9, inclusive.
13. The method of Claim 12, wherein the pH is lowered in step (d) to about 7.
14. The method of Claim 12, wherein the presence of a lesser amount of triple helical microfibril ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit ╬▓(l,3)-glucan association into triple helical microfibril conformation.
15. A method of assessing an agent of interest for the ability to inhibit folding of ╬▓(l,3)-glucan, comprising the steps of: a) providing a ╬▓(l,3)-glucan test solution comprising a soluble ╬▓(l,3)- glucan composition in an aqueous solution; b) denaturing the soluble ╬▓(l,3)-glucan composition in the test solution by heating the ╬▓(l,3)-glucan test solution, thereby forming a denatured ╬▓(l,3)-glucan test solution; c) contacting the denatured ╬▓(l,3)-glucan test solution with the agent of interest, thereby forming a contacted, denatured ╬▓(l,3)-glucan test solution; d) maintaining the contacted, denatured ╬▓(l,3)-glucan test solution under renaturing conditions that allow the contacted, denatured ╬▓(l,3)-glucan test solution to cool, thereby forming a renatured ╬▓(l,3)-glucan test solution; e) assessing the conformation of the ╬▓(l,3)-glucan composition in the renatured ╬▓(l,3)-glucan test solution; and f) comparing the conformation of the ╬▓(l,3)-glucan composition in the renatured ╬▓(l,3)-glucan test solution with the conformation of the ╬▓(l,3)-glucan composition in a ╬▓(l,3)-glucan control solution, wherein the presence of a greater amount of random chain ╬▓(l ,3)-glucan in the renatured ╬▓(l,3)-glucan test solution than in the ╬▓(l,3)-glucan control solution; the presence of a lesser amount of single triple helix ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution; or the presence of a lesser amount of triple helical microfibril ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit ╬▓(l,3)-glucan folding.
16. The method of Claim 15, wherein the soluble ╬▓(l,3)-glucan composition comprises aqueous-soluble ╬▓(l,3)-glucan.
17. The method of Claim 15, wherein the soluble ╬▓(l,3)-glucan composition comprises Fraction C of aqueous-soluble ╬▓(l,3)-glucan.
18. The method of Claim 15, wherein the ╬▓(l ,3)-glucan test solution is heated to at least about 37┬░C.
19. The method of Claim 18, wherein the ╬▓(l ,3)-glucan test solution is heated to a temperature in the range of about 37┬░ to about 135 ┬░C.
20. The method of Claim 19, wherein the ╬▓(l,3)-glucan test solution is heated to a temperature in the range of about 90 ┬░C to about 100┬░C.
21. The method of Claim 18, wherein the contacted, denatured ╬▓(l,3)-glucan test solution in step (d) is cooled to a temperature in the range of about 35 ┬░C to 50┬░C.
22. The method of Claim 21 , wherein the contacted, denatured ╬▓(l ,3)-glucan test solution in step (d) is cooled to a temperature in the range of about 37 ┬░C to about 45 ┬░C.
23. The method of Claim 21 , wherein the presence of a greater amount of random chain ╬▓(l,3)-glucan in the renatured ╬▓(l,3)-glucan test solution than in the ╬▓(l ,3)-glucan control solution, or the presence of a lesser amount of single triple helix ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution, is indicative of the ability of the agent of interest to inhibit the folding of ╬▓(l,3)-glucan into single triple helix conformation.
24. The method of Claim 18, wherein the contacted, denatured ╬▓(l,3)-glucan test solution in step (d) is cooled to a temperature in the range of about 2┬░C to about 30┬░C.
25. The method of Claim 24, wherein the contacted, denatured ╬▓(l,3)-glucan test solution in step (d) is cooled to a temperature in the range of about 20 ┬░C to about 25 ┬░C.
26. The method of Claim 24, wherein the presence of a lesser amount of triple helical microfibril ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit ╬▓(l,3)-glucan folding into triple helical microfibril conformation.
27. A method of assessing an agent of interest for the ability to inhibit folding or association of ╬▓(l,3)-glucan, comprising the steps of: a) providing a ╬▓(l,3)-glucan test solution comprising a soluble ╬▓(l,3)- glucan composition in an aqueous solution; b) denaturing the soluble ╬▓(l,3)-glucan composition in the test solution by adding dimethyl sulfoxide to the ╬▓(l,3)-glucan test solution, thereby forming a denatured ╬▓(l,3)-glucan test solution; c) contacting the denatured ╬▓(l ,3)-glucan test solution with the agent of interest, thereby forming a contacted, denatured ╬▓(l,3)-glucan test solution; d) maintaining the contacted, denatured ╬▓(l,3)-glucan test solution under renaturing conditions that remove dimethyl sulfoxide from the contacted, denatured ╬▓(l,3)-glucan test solution, thereby forming a renatured ╬▓(l,3)-glucan test solution; e) assessing the conformation of the ╬▓(l ,3)-glucan composition in the renatured ╬▓(l,3)-glucan test solution; and f) comparing the conformation of the ╬▓(l ,3)-glucan composition in the renatured ╬▓(l,3)-glucan test solution with the conformation of the ╬▓(l,3)-glucan composition in a ╬▓(l,3)-glucan control solution, wherein the presence of a greater amount of random chain ╬▓(l,3)-glucan in the renatured ╬▓(l,3)-glucan test solution than in the ╬▓(l,3)-glucan control solution; the presence of a lesser amount of single triple helix ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution; or the presence of a lesser amount of triple helical microfibril ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit ╬▓(l,3)-glucan folding.
28. The method of Claim 27, wherein the soluble ╬▓(l ,3)-glucan composition comprises aqueous-soluble ╬▓(l,3)-glucan.
29. The method of Claim 27, wherein the soluble ╬▓(l ,3)-glucan composition comprises Fraction C of aqueous-soluble ╬▓(l,3)-glucan.
30. The method of Claim 27, wherein dimethyl sulfoxide is added in step (b) to a concentration of greater than about 30% by volume.
31. The method of Claim 30, wherein the dimethyl sulfoxide is removed in step (d) to result in a concentration of dimethyl sulfoxide that is approximately 15-25% by volume.
32. The method of Claim 31 , wherein the dimethyl sulfoxide is removed in step (d) to result in a concentration of dimethyl sulfoxide that is approximately 20% by volume.
33. The method of Claim 31 , wherein the presence of a greater amount of random chain ╬▓(l,3)-glucan in the renatured ╬▓(l,3)-glucan test solution than in the ╬▓(l,3)-glucan control solution, or the presence of a lesser amount of single triple helix ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit ╬▓(l,3)-glucan folding into single triple helix conformation.
34. The method of Claim 30, wherein the dimethyl sulfoxide is removed in step (d) to result in a concentration of dimethyl sulfoxide that is less than approximately 5% by volume.
35. The method of Claim 34, wherein the presence of a lesser amount of triple helical microfibril ╬▓(l,3)-glucan in the renatured test solution than in the ╬▓(l,3)-glucan control solution, is indicative of the ability of the test agent to inhibit ╬▓(l,3)-glucan association into triple helical microfibril conformation.
36. An agent, having the ability to inhibit folding of ╬▓(l ,3)-glucan, identifiable by the method of Claim 1.
37. An agent, having the ability to inhibit folding of ╬▓(l,3)-glucan, identifiable by the method of Claim 3.
38. An agent, having the ability to inhibit folding of ╬▓(l,3)-glucan, identifiable by the method of Claim 6.
39. An agent, having the ability to inhibit folding of ╬▓(l,3)-glucan, identifiable by the method of Claim 15.
40. An agent, having the ability to inhibit folding of ╬▓(l,3)-glucan, identifiable by the method of Claim 27.
41. A pharmaceutical composition comprising an agent of Claim 36.
42. A pharmaceutical composition comprising an agent of Claim 37.
43. A pharmaceutical composition comprising an agent of Claim 38.
44. A pharmaceutical composition comprising an agent of Claim 39.
45. A pharmaceutical composition comprising an agent of Claim 40.
46. A method of treating a fungal infection in an individual, comprising administering to the individual an agent having the ability to inhibit folding of ╬▓-(l,3)-glucan, identifiable by the method of Claim 1, in a therapeutically effective amount.
47. A method of treating a fungal growth, comprising administering to the growth an agent having the ability to inhibit folding of ╬▓(l,3)-glucan, identifiable by the method of Claim 1, in an effective amount.
48. A method of assessing an agent of interest for antifungal activity, comprising assessing the ability of the agent to inhibit folding of ╬▓(l,3)-glucan, wherein if the agent of interest inhibits folding of ╬▓(l,3)-glucan, it is an agent that has antifungal activity.]
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008001368A3 (en) * 2006-06-27 2009-04-30 Hadasit Med Res Service Use of stilbene derivatives for treatment and prevention of aquatic mold infections

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5622939A (en) * 1992-08-21 1997-04-22 Alpha-Beta Technology, Inc. Glucan preparation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5622939A (en) * 1992-08-21 1997-04-22 Alpha-Beta Technology, Inc. Glucan preparation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
G L VANNINI, F POLI, A DONINI, S PANCALDI: "Effects of Congo Red on Wall Synthesis and Morphogenesis in Saccharomyces Cerevisiae", PLANT SCIENCE LETTERS, vol. 31, 1983, pages 9 - 17, XP002121692 *
KOPECKA, MARIE; KREGER, D. R.: "Assembly of microfibrils in vivo and in vitro from (1 .fwdarw. 3)-.beta.-D-glucan synthesized by protoplasts of Saccharomyces cerevisiae", ARCHIVES OF MICROBIOLOGY, vol. 143, no. 4, 1986, pages 387 - 395, XP002121691 *
M V ELORZA, H RICO, R SENTANDREU: "Caclofluor White Alters the Assembly of Chitin Fibrils in Saccharomyces cerevisiae and Candida albicans cells", JOURNAL OF GENERAL MICROBIOLOGY, vol. 129, 1983, pages 1577 - 1582, XP002121693 *

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WO2008001368A3 (en) * 2006-06-27 2009-04-30 Hadasit Med Res Service Use of stilbene derivatives for treatment and prevention of aquatic mold infections
US8377934B2 (en) 2006-06-27 2013-02-19 State Of Israel, Ministry Of Agriculture & Rural Development, Agricultural Research Organization Use of stilbene derivatives for treatment and prevention of aquatic mold infections

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