ANTIFUNGAL COMPOSITION/TREATMENT

Abstract

An antifungal composition comprising an agent that affects the availability of functional amino acids and either i) another agent that affects the availability of functional amino acids or ii) an aminoglycoside antibiotic.

Claims

1. An antifungal composition comprising an agent that affects the availability of functional amino acids and either i) another agent that affects the availability of functional amino acids or ii) an aminoglycoside antibiotic.

2. An antifungal composition according to claim 1 comprising two different agents that affect the availability of functional amino acids or an agent that affects the availability of functional amino acids and an aminoglycoside antibiotic.

3. (canceled)

4. An antifungal composition according to claim 2, wherein the aminoglycoside antibiotic is selected from the group comprising amikacin, arbekacin, astromicin, bekanamycin, dibekacin, dihydrostreptomycin, framycetin, gentamicin, hygromycin B, isepamicin, kanamycin, neomycin, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin or verdamicin.

5. An antifungal composition of claim 1, wherein the agent that inhibits amino acid availability is an inhibitor of the uptake of one or more amino acids, or an inhibitor of the uptake of substrate(s) required for amino acid synthesis, or an inhibitor of amino acid biosynthesis, or an inhibitor of amino-acyl-tRNA synthetases, or a structural analogue of a proteinogenic amino acid, or an agent that inactivates thiol containing amino acids, or an agent that binds amino acids.

6. The antifungal composition according to claim 5, wherein the inhibitor of the uptake of substrate required for amino acid synthesis is an inhibitor of the uptake of sulphate.

7. The antifungal composition according to claim 6 wherein the inhibitor of the uptake of sulphate is selected from the group comprising alpha-cyanno-4-hydroxycinnamic acid, bicarbonate, chromate, 4,4-diisothio-cyanotostilbene-2,2-disulphonate (DIDS), 4-Acetamido-4-isothiocyano-2,2-disulfonic stilbene (SITS), furosemide, malonate, molybdate, nigericin, oxalate, probenicid, selenate, tetrathionate, thiosulphate, tungstate and vanadate.

8. The antifungal composition according to claim 1 wherein the agent that inhibits amino acid availability may be an inhibitor of the amino acid transport activity of one or more transport proteins.

9. The antifungal composition according to claim 8, wherein the inhibitor of one or more transport proteins is eugenol, zaragozic acid or quinine.

10. The antifungal composition according to claim 5, wherein the inhibitor of amino acid biosynthesis is cyprodinil, mepanipyrim, pyrimethanil or copper.

11. The antifungal composition according to claim 5, wherein the inhibitor of amino-acyl-tRNA synthetases is cispentacin, icofungipen, mupirocin or tavaborole.

12. The antifungal composition according to claim 5, wherein the structural analogue of a proteinogenic amino acid is azetidine, canavanine, 3,4-dihydroxyphenylalanine (DOPA), ethionine, hydroxynorvaline, -N-methylamino-L-alanine (BMAA), thialysine, m-tyrosine, quinine, primaquine, or chloroquine.

13. The antifungal according to claim 5, wherein the agent that inactivates thiol containing amino acids is iodopropynyl butyl carbamate (IPBC), maneb, zineb, mancozeb, thiram or ziram

14. (canceled)

15. A pharmaceutical composition or agricultural composition comprising an antifungal composition according to claim 1 and a pharmaceutically acceptable excipient, diluent, adjuvant or carrier or an agriculturally acceptable support, carrier, filler and/or surfactant, respectively.

16. (canceled)

17. Use of an antifungal composition according to claim 1, for curatively or preventatively controlling or inhibiting growth of phytopathogenic fungi.

18. Use of an antifungal composition according to claim 17, wherein the phytopathogenic fungus or fungus-like organism is of the genera Botrytis (e.g. Botrytis cinerea), Pythium, Phytophthora (e.g. Phytophthora infestans), Fusarium (e,g, Fusarium graminearum), Mycosphaerella (e.g. Mycosphaerella arachidis), Rhizoctonia (e.g. Rhizoctonia solani), Thielavopsis, Sclerotinia, Cylindrocladium, Gibberella, Colletotrichium, Aspergillus (e.g. Aspergillus flavus, Aspergillus fumigatus) or Zymoseptoria (e.g. Zymoseptoria tritici).

19. (canceled)

20. Use of an antifungal composition according to claim 1, for treating or preventing fungal infections in or on the human or animal body; or for treating or preventing fungal growth caused by a non-pathogenic fungus.

21. (canceled)

22. Use of an antifungal composition according to claim 21 wherein the non-pathogenic fungus is a food spoilage organism or is a fungus that grows on synthetic materials or other commercial product.

23. (canceled)

24. Use of an antifungal composition according to claim 20 wherein the fungal infection is caused by the species of Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis or Stachybotrys.

25. A method of protecting plants from fungal infection comprising applying to the plants and/or seeds thereof and/or to a substrate used for growing said plant an amount of the antifungal composition according to claim 1 to inhibit growth of or kill one or more species of fungi.

26. A method according to claim 25, wherein the species of fungus is a pathogenic fungus.

27. A method for curatively or preventively controlling phytopathogenic fungi of crops and increasing the yield of crops characterised in that an effective and non-phytotoxic amount of an antifungal composition according to claim 1 is applied via seed treatment, foliar application, stem application or drench/drip application (chemigation) to the seed, the plant and/or to the fruit of the plant or to soil and/or to inert substrate, pumice, pyroclastic materials/tuff, synthetic organic substrates and/or to a liquid substrate in which the plant is growing or in which it is desired to grow.

Description

[0055] Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.

[0056] FIG. 1illustrates the synergistic growth inhibition of fungi pathogenic to humans by aminoglycoside antibiotics and sulphate mimetics. FIG. 1A shows growth of C. albicans in YEPD broth supplemented with 200 g ml.sup.1 paromomycin, 10 g ml.sup.1 hygromycin B and/or 25 M chromate. (B) Growth of C. glabrata in YEPD broth supplemented with 400 g ml.sup.1 paromomycin and/or 50 M chromate. (C) Growth of Cryptococcus neoformans in YEPD broth supplemented with 12.5 g ml.sup.1 paromomycin, 0.625 g ml hygromycin B and/or 12.5 M chromate. Data shown are replicates from two independent culturesSEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.

[0057] FIG. 2illustrates the synergistic growth inhibition of other undesirable fungi by aminoglycoside antibiotics and sulphate mimetics. FIG. 1A shows growth of the food spoilage organism Z. bailii after spotting 10-fold serial dilutions of cell suspension on YEPD agar supplemented with 10 g ml.sup.1 paromomycin, 50 M chromate and/or 1 mM molybdate, or to YNB agar with 10 g ml.sup.1 hygromycin B and/or 250 M DIDS. (B) Growth of the plant pathogen R. solani on PDA agar supplemented with 300 g ml.sup.1 paromomycin and 15 mM molybdate. (C) Growth of the plant pathogen Z. tritici in PDB medium supplemented with 0.5 g ml.sup.1 paromomycin, 0.25 g ml.sup.1 hygromycin B and/or 10 M chromate. The data for each condition are representative of at least two independent experiments performed on different days.

[0058] FIG. 3illustrates the synergistic action of an aminoglycoside antibiotic and an agent that inhibits amino acid availability against the fungal plant pathogen Botrylis cinerea. The organism was cultured on Vogel's broth supplemented as indicated with hygromycin and/or quinine. The image was captured after 5 d incubation.

[0059] FIG. 4illustrates the quantitative assessment of the synergistic action of an aminoglycoside antibiotic and an agent that inhibits amino acid availability against B. cinerea. The organism was cultured on Vogel's broth supplemented with 1 g ml.sup.1 hygromycin and/or 1 mM quinine as indicated. Measurements of OD.sub.600 were taken daily up to 5 d and the values shown are means from 8 replicate determinations.

[0060] FIG. 5illustrates the synergistic action of two different agents that inhibit amino acid availability against the fungal plant pathogen B. cinerea. The organism was cultured in Vogel's broth supplemented as indicated with bicarbonate and/or quinine. Data shown are replicates from two independent culturesSEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.

[0061] FIG. 6illustrates the synergistic action of two different agents that inhibit amino acid availability against the fungal plant pathogen R. solani. The organism was cultured in PDB medium supplemented as indicated with thiram and/or copper. Data shown are replicates from two independent culturesSEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.

[0062] FIG. 7illustrates the synergistic action of two more different agents that inhibit amino acid availability against the fungal plant pathogen R. solani. The organism was cultured in PDB medium supplemented as indicated with norvaline and/or selenate. Data shown are replicates from two independent culturesSEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.

[0063] FIG. 8illustrates synergies involving the biocide iodopropynyl-butyl-carbamate (IPBC). Saccharomyces cerevisiae was cultured with sub-inhibitory doses of IPBC (500 ng ml.sup.1) and of the second agents either 10 g ml.sup.1 hygromycin (A) or 7 mM copper (B). Data shown are replicates from two independent culturesSEM where these are larger than the symbol dimensions. The data for each condition are representative of at least two independent experiments performed on different days.

[0064] FIG. 9illustrates the absence of synergy against non-target cells. (A) E. coli growing in LB broth supplemented without () or with 1 g ml.sup.1 paromomycin (), 15 M chromate (.circle-solid.), or both agents (.square-solid.). (B) Human cells were incubated in DMEM broth supplemented with 1 mg ml.sup.1 paromomycin and/or 10 M chromate and relative viability was estimated from tetrazolium reduction activity. Data are representative of more than one independent experiment performed on different days.

EXAMPLES

Materials and Methods

Fungal Strains and Maintenance

[0065] The pathogenic, food spoilage or laboratory fungi tested in this study were the yeasts Candida albicans, Candida glabrata, Zygosaccharomyces bailii, Saccharomyces cerevisiae and the filamentous fungi Botrytis cinerea, Rhizoctonia solani and Z. tritici, all from culture collections at the University of Nottingham. The yeasts were routinely grown and maintained on YEPD agar: 1% (w/v) yeast extract (Oxoid), 2% (w/v) peptone (Oxoid), 2% (w/v) glucose, 2% (w/v) bacteriological agar (Sigma-Aldrich). R. solani and Z. tritici were routinely maintained and grown on potato dextrose agar (PDA; Oxoid). B. cinerea was routinely maintained on malt extract agar (MEA): 2% (w/v) malt extract (Fluka Analytical), 0.6 (w/v) % peptone, 1.6% (w/v) bacteriological agar.

Chemicals

[0066] All chemicals were from Sigma-Aldrich except hygromycin B (Panreac Applichem). Stock solutions were prepared in distilled water except for DIDS (in 0.1 M KHCO.sub.3), quinine (in 70% ethanol) and IPBC or thiram (in DMSO). The final concentrations of KHCO.sub.3, ethanol or DMSO in control and test media containing DIDS, quinine, IPBC or thiram were matched. All stock solutions were filter-sterilized before additions to media.

Growth Inhibition Assays in Broth

[0067] Candida spp. or S. cerevisiae were inoculated from YEPD plates to YEPD broth (composition as above, minus agar) and cultured overnight at 30 C., 120 rev min.sup.1. Overnight cultures were diluted to OD.sub.6000.5 and cultured for a further 4 h in fresh YEPD before dilution of these experimental cultures to OD.sub.6000.01 or 0.1 in the same medium. Aliquots (300 l) of the diluted culture plus any chemical supplements (see above), as specified, were transferred to 48-well plates (Greiner Bio-One). OD.sub.600 was monitored continuously in a BioTek Powerwave-XS microplate spectrophotometer, with shaking at 30 C. The bacterium Escherichia coli was tested in a similar way but with growth in LB broth and at 37 C. Spores of R. solani or Z. tritici were inoculated from PDA plates to potato dextrose broth (PDB; Fluka) (20,000 spores ml.sup.1). Aliquots (150 l) of the diluted culture plus any chemical supplements, as specified, were transferred to 96-well plates (Greiner Bio-one) and cultured over 8 d at 24 C., 120 rev min-1. OD.sub.600 was monitored every day in a BioTek EL800 microplate spectrophotometer. Spore suspensions from Botrytis cinerea on MEA were prepared in 0.1% (v/v) tween-80. Aliquots of spore suspensions were diluted in Vogel's broth (http://www.fgse.net/methods/vogels.html) to give a final density of 10.sup.4 spores ml.sup.1. Aliquots (300 l) of the diluted suspension plus any chemical supplements (see above), as specified, were incubated as above. Human TE671 cells were cultured in DMEM supplemented with 10% foetal bovine serum, L-glutamine (2 mM), penicillin (100 U ml.sup.1), streptomycin (100 g ml.sup.1) in 25 cm.sup.2 cell culture flasks, 36.5 C., 5% oxygen. Cells were detached with trypsin/EDTA and washed in 10 ml DMEM. Then 100 ul of cell suspension (in DMEM without antibiotics) were dispensed to 5000 cells/well in a 96-well plate. After 24 h, paromomycin or chromate were added as specified. After a further 24 h, 10 l of CCK-8 reagent (Sigma) were added to each well. After 4 h incubation, formazan production was determined at 450 nm using a BioTek EL800 microplate spectrophotometer.

Growth Inhibition Assays on Solid Medium

[0068] For qualitative growth-inhibition assays with yeasts on solid medium, experimental cultures prepared as described above were adjusted to OD.sub.6002.0, 0.2, 0.02, 0.002, 0.0002 and the dilution series spotted (4 l) on to either YEPD agar (above) or yeast nitrogen base (YNB) agar: 0.69% (w/v) yeast nitrogen base without amino acids (Formedium; Norfolk, UK) supplemented with 2% (w/v) D-glucose, 0.06 mg ml.sup.1 leucine, 0.02 mg ml.sup.1 histidine, 0.02 mg ml.sup.1 uracil and 2% (w/v) bacteriological agar (Sigma-Aldrich). Images were captured after 2 d (YEPD) or 3 d (YNB) growth at 30 C. For growth-inhibition assays on solid medium with R. solani, circular sections of 0.5 cm diameter were excised from cultures on PDA and transferred to the centre of fresh plates. Images were captured after 3 d growth at 28 C and the total mycelial area determined using ImageJ and GIMP2 software. Where specified, chemical supplements were included in the solid media.

Results

[0069] The effects of different combinations of aminoglycosides with inhibitors of amino acid availability were tested in key fungi of interest. Agents were supplied at doses which, individually, were just sub-inhibitory to the fungi. Accordingly, inclusion of either 25 M chromate, 200 g ml.sup.1 paromomycin or 10 g ml.sup.1 hygromycin in the growth medium had no discernible inhibitory effect on growth of the yeast Candida albicans (FIG. 1A). However, growth of this human pathogen was slowed when Cr was combined with aminoglycoside, especially hygromycin which gave 90% growth inhibition in combination with Cr. Checkerboard analysis (not shown) indicated that these combinations decreased the MICs of the individual agents by 16-fold. Synergistic growth inhibition was also evident with another pathogenic Candida species, C. glabrata, although the effect did not become apparent until mid-exponential growth (6 h) in this case (FIG. 1B). There was also synergistic growth inhibition of the human pathogen Cryptococcus neoformans (FIG. 1C). The food spoilage yeast Zygosaccharomyces bailii was hyper-sensitive to combinations of aminoglycoside and inhibitors of sulphate transport (which limit availability of sulphur-containing amino acids) (FIG. 2A): most dilutions of Z. bailii cell suspensions showed little growth on agar supplemented with two agents (paromomycin or hygromycin combined with Cr, Mo or DIDS), supplied at concentrations where neither agent alone had a significant effect on growth. Higher concentrations of molybdate than chromate were required to achieve this effect, reflecting the fact that molybdate is the less toxic of these two agents. Synergistic growth inhibition of the fungal plant pathogen Rhizoctonia solani was also evident (FIG. 2B). Outward growth of R. solani from a central, point inoculum on agar was compromised by incorporation of paromomycin and molybdate into the medium, supplied at concentrations where neither agent alone had a marked growth-inhibitory effect. Combinations of Cr with different aminoglycosides also produced strong, synergistic inhibition of growth of another fungal plant pathogen, Zymoseptoria tritici (FIG. 2C).

[0070] The antimalarial drug quinine has recently been shown to mimic the amino acid tryptophan and cause tryptophan starvation. In combination with hygromycin, quinine produced synergistic growth inhibition of the fungal plant pathogen Botrylis cinerea. For example, growth of B. cinerea was slowed at 5 g ml.sup.1 hygromycin in the absence of quinine, but was strongly inhibited at 0.5 g ml.sup.1 hygromycin in the presence of 1 mM quinine (FIG. 3). At appropriate sub-inhibitory doses of these agents, growth of B. cinerea was inhibited 90% when they were combined compared to growth with either agent alone (FIG. 4).

[0071] Other effective combinations did not include an aminoglycoside. Combinations of two different agents that inhibit amino acid availability also demonstrated synergistic growth inhibition. Thus, quinine combined with a sulphate transport inhibitor like bicarbonate produced synergistic growth inhibition of B. cinerea (FIG. 5). Similarly, combinations of thiram and copper (FIG. 6) or of norvaline and selenate (FIG. 7), produced striking synergistic inhibition of R. solani growth, at concentrations where none of these agents had an inhibitory effect individually. The biocide iodopropynyl-butyl-carbamate (IPBC) was tested against the yeast model S. cerevisiae, where it produced synergistic inhibition in combinations with hygromycin or copper (FIG. 8).

[0072] The invention did not affect alternative (non-fungal) cell types that were tested. Paromomycin and Cr were supplied to the bacterium E. coli at concentrations that, individually, were just sub-inhibitory to growth. The combination had no further inhibitory effect (FIG. 9A). Similarly, combination of paromomycin and Cr had no effect on mammalian cells that was not already present when these agents were supplied individually (FIG. 9B). The results indicate that compositions of the invention act on fungi with some specificity.