Antifungal Agents From a Streptomycete

RELATED APPLICATIONS

This application claims the benefit of priority to United States Provisional Patent Application serial number 60/965,583, filed August 20, 2007; which is hereby incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with support provided by the National Institutes of Health (grant CA24487) and U.S. Department of Agriculture/Cooperative State Research, Education, and Extension Service (grant 06-CA-l 1330129-042). The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Clinical treatment of human fungal infections has relied mainly on a few types of antifungal agents. Widely used are amphotericin B, flucytosine and nystatin, which are fungicidal and capable of curing fungal infections, but only at the cost of severe side effects to the patient. Also used are fluconazole and other azole agents, which exhibit fewer side effects, but they are only fungistatic. Thus, there exists a need for new types of antifungal agents.

SUMMARY OF THE INVENTION

In studying a Streptomycete associated with the Southern Pine Beetle (SPB), a serious pest on pine trees, a novel antifungal compound was isolated (Figure 1;

"mycangimycin"). It is known that SPB females bore tunnels in pine trees in which they lay their eggs, and upon hatching, the SPB larvae feed on a fungus (Entomocorticium sp.) that the mother has also introduced into the tunnels. The fungal spores are carried in a special structure on the SPB called the mycangium. It has been found that the mycangium of the SPB contains a Streptomycete (herein referred to as SPB074) that produces a potent antifungal agent. Moreover, it has been shown that SPB074 can inhibit the growth of Ophiostoma minus (an example of an 'antagonistic' fungus) and other test fungi, but not Entomocorticium sp. (the 'food fungus' used by the beetles). The spectrum of activity of the isolated antifungal agent is similar to that of amphotericin but approximately three-

times more potent on a weight basis. Remarkably, the isolated antifungal agent is a member of a structural class that has not been reported, either as a natural product or as an antifungal agent.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts the structure of an antifungal agent obtained from a culture of a

Streptomycete (SPB074), which was isolated from the Southern Pine Beetle.

Figure 2 depicts SPB074 (Streptomyces sp.), a mycangial bacteria, in SPB gallery.

Figure 3 depicts (A) SEM micrograph of adult D. frontalis showing the relative placement of the mycangia (arrow), which is used to transport EsA. (B) SEM micrograph from the D. frontalis gallery showing the relative placement of the ac-tinomycetous bacterium (ba), fungus (fu), and beetle larva (Ia). (C) Representative examples of pairwise bioassay challenges illustrating inhibition of the fungal antagonist Om (left), by a D. frontalis symbiotic actinomycete (strain SPB074). In contrast the D. frontalis fungal mutualist EsA is relatively resistant (right), establishing that SPB074 makes a diffusible antifungal agent. (D) The structure of mycangimycin containing a seven-conjugated double bond chain and a fϊve-membered endoperoxide ring.

Figure 4 depicts how relative and absolute (at C3 and C5) stereochemistry was determined.

Figure 5 depicts (A) SEM micrograph of SPB gallery in loblolly pine, showing a nearly complete view of a SPB larva (Ia), and growth of fungal (fu) and actinomycetous (ba) symbionts (top). Micrograph from the same SPB larval gallery, illustrating filamentous bacteria (solid arrows) growing interwoven with the mutualistic fungi (open arrow) (bottom left). Micrograph image of a SPB mycangium, with arrows indicating actinomycete growth within the gland cells surrounding the mycangium in the prothorax (bottom right). Scale bars, 200μm, lOμm, lμm, respectively. (B) Representative examples of pairwise bioassay challenges between the two actinomycete strains (red morphotype = 'StrainR' (top) and white morphotype = 'StrainW (bottom)) and the two fungi Ophiostoma minus (beetle antagonist) or Entomocorticium sp. A (beetle mutualist). The actinomycete is in the middle, while the fungus is inoculated at the edge of the Petri plate. Zones of inhibition (zoi, space between the bacterium and fungus) indicate inhibition of fungus by antibiotic produced by the bacterium (e.g., upper right image). (C) Summary of

160 bioassay challenges, indicating the frequency of different actinomycete strains of each morphotype displaying different degrees of ability to inhibit the two fungi. Black indicates strong inhibition (zoi > 2.5 cm) of the fungus by the bacterium, grey indicates mild inhibition (zoi 0.5-2.5 cm), and light grey indicates little or no inhibition (zoi < 0.5 cm). (D) The susceptibility dose response curves plotting log concentration mycangimycin by fungal density of O. minus and Entomocorticium sp. A, illustrating selective inhibition of the beetle's antagonistic fungus, O. minus, by mycangimycin.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to novel antifungal compounds, and compositions and methods of use thereof. In certain embodiments, the antifungal compounds exhibit broad spectrum antifungal activity. In certain embodiments, the antifungal compounds exhibit broad spectrum antifungal activity against human fungal pathogens.

Certain aspects of the present invention relate to the natural product mycangimycin (as shown Figure 1). As discussed below, the producing organism is a Streptomycete isolated from the mycangium of a Southern Pine Beetle (SPB), a serious pest on pine trees. Streptomyces are a genus of Gram-positive spore-forming bacteria that grow slowly in soil or water as a branching filamentous mycelium similar to that of fungi (Figure 2).

It is known that Southern pine beetles (SPB, Dendroctonus frontalis) engage in a beneficial symbiosis with the fungus Entomocorticium sp. A (EsA), which provides nourishment for their developing larvae. Adult SPBs carry EsA in a specialized storage compartment called a mycangium (Figure 3A); excavate ovipositional galleries within the inner bark and phloem of host pine trees; and inoculate these galleries with EsA. K. D. Klepzig, R. T. Wilkens, AEM 63, 612 (1997); and R. W. Hofstetter, et al. Oecologia 147, 679 (2006). The success of the SPB-EsA mutualism is challenged by an antagonistic fungus, Ophiostoma minus (Om), which can out-compete EsA and thereby disrupt SPB larval development. K. D. Klepzig, R. T. Wilkens, AEM 63, 612 (1997); and R. W. Hofstetter, et al. Oecologia 147, 679 (2006). Remarkably, as described herein, recent experiments indicate that successful maintenance of the SPB-EsA mutualism is likely mediated by an actinomycetous bacterium that produces antibiotics which selectively inhibit Om.

The presence of previously unknown actinomycetes within the SPB-EsA mutualisms was established by scanning electron microscopy (SEM) and enrichment culture isolations. SEM revealed unexpected and profuse growth of actinomycetes within the galleries of SPBs, as well as inside the mycangia (Figure 3B and 5). Isolations from 110 beetle individuals yielded 846 colony forming units (CFUs) of actinomycetes, including at least one CFU from each of 92 individuals. Out of 164 actinomycete CFUs selected to be transferred to pure culture, 99 isolates had a red morphotype while 65 isolates had a white morphotype. DNA sequence analyses confirmed the visual morphotype distinction, and within each of the two morphotypes there was complete 16S rDNA sequence identity. The two morphotypes form a monophyletic clade closely related to Streptomyces thermosacchari. The same red morphotype was successfully isolated from 5 of 10 mycangia sampled.

The potential role of the actinomycetes in mediating the SPB fungal community was explored using symbiont pairing bioassays and chemical analyses. The bioassays, which crossed all possible combinations of the two actinomycete morphotypes with EsA and Om, revealed that isolates of the red morphotype produce a diffusible activity that inhibits the beetle's antagonistic fungus, Om, while only slightly affecting the beneficial fungus, EsA (Figures 3C and 5B-C). Extensive chemical and spectral analyses on strains of the red morphotype revealed the antifungal molecule responsible for selective inhibition to be a polyene peroxide, which we named 'mycangimycin'. Mycangimycin (C ₂₀ H ₂₄ O ₄ ), which has not been previously reported, is a linear 20 carbon carboxylic acid with an endoperoxide linking C-3 and C-5 to form a 1,2-dioxolane and a conjugated cis, cis, trans, trans, cis, trans-heptaene spanning C-7 to C-20 (Figures 1 and 3D). Liquid culture antifungal assays using purified mycangimycin showed Om to be almost 20-times more susceptible (minimal inhibitory concentration (MIC) = 1.0 μM) than EsA (MIC = 19.0 μM) (Figure 5D). The discovery of an actinomycete that is localized in the mycangium and galleries, which produces an antibiotic that selectively suppresses the antagonistic fungus Om, indicates that SPBs engage in an additional mutualism with bacteria to regulate the EsA-Om fungal community. Since other bark-beetle species also depend on successfully maintaining beneficial fungi, tripartite beetle-fungus-bacterium mutualisms may be widespread.

In addition, it has also been shown that mycangimycin can inhibit the growth of Saccharomyces cerevisiae and Ceratocystiopsis ranaculosus. The spectrum of activity of

the isolated antifungal agent is similar to that of amphotericin but approximately three- times more potent on a weight basis. Remarkably, the isolated antifungal agent is a member of a structural class that has not been reported, either as a natural product or as an antifungal agent. Interestingly, the results provided herein parallel earlier work on fungus-farming ants, which use actinomycetes to help protect their fungal gardens from pathogens. C. R. Currie, J. A. Scott, R. C. Summerbell, D. Malloch, Nature 398, 701 (1999). Taken together, these findings suggest that the use of antibiotic-producing actinomycetes may be a common method to help maintain beneficial microbes. Indeed, considering the importance of pathogens as a driving force in the evolution of all hosts, the benefit of such associations may extend to helping protect plants and animals from pathogens to which they themselves are susceptible. M. Kaltenpoth, W. Gottler, G. Herzner, E. Strohm, Curr. Biol. 15, 475 (2005); and J. T. Coombs, C. M. M. Franco, AEM 69, 5603 (2003). If, as seems likely, these associations are widespread, targeting them could be an effective strategy for locating novel biologically active natural products.

STREPTOMYCETE CULTURES

One aspect of the invention relates to a culture of Streptomyces cf. thermosacchari SPB074. In certain embodiments, the culture is substantially pure or biologically pure. A "substantially pure" or "biologically pure" culture shall be deemed a culture of a bacteria containing no other bacterial species in quantities sufficient to interfere with replication of the culture. Under the terms of the Budapest Treaty, SPB074 was deposited in the culture collection of the American Type Culture Collection (10801 University Boulevard, Manassas, VA 20110-2209, United States of America) on August 20, 2007, and an accession number (ATCC number PTA-8607) has been assigned. The deposit was tested September 4, 2007 and on that date, the culture was viable.

Another aspect of the invention relates to the use of a cultural filtrate of Streptomycete SPB074. In certain embodiments, such as for example some agricultural applications, it may be preferential to utilize antifungal compounds which have not been purified from the culture filtrate in which they were formed. In other words, one aspect of the invention is the formation and use of a cultural filtrate of a Streptomycete SPB074 culture.

Although the invention is discussed principally with respect to the specific strain, it is well known in the art that the properties of microorganisms can be varied naturally and artificially. Thus, all strains derived from SPB074 including varieties and mutants, whether obtained by natural selection, produced by the action of mutating agents, such as ionizing radiation or ultraviolet irradiation, or by the action of chemical mutagens, such as nitrosoguanidine, are contemplated to be within the scope of this invention.

The production of the compound shown in Figure 1 may be carried out by cultivating SPB074 in a suitable nutrient medium under conditions described herein until a substantial amount of antifungal activity is detected in the fermentation broth, harvesting by extracting the active components from the mycelial growth with a suitable solvent, concentrating the solution containing the desired component, and subjecting the concentrated material to chromatographic separation to isolate the compound from other metabolites also present in the cultivation medium.

Broadly, the sources of carbon include glucose, fructose, mannose, maltose, galactose, mannitol and glycerol, other sugars and sugar alcohols, starches and other carbohydrates, or carbohydrate derivatives, such as dextran, cerelose, as well as complex nutrients, such as oat flour, com meal, millet, corn and the like. The exact quantity of the carbon source which is utilized in the medium will depend, in part, upon the other ingredients in the medium, but it is usually found that an amount of carbohydrate between about 0.5 and 15 percent by weight of the medium is satisfactory. These carbon sources can be used individually or several such carbon sources may be combined in the same medium. Certain carbon sources are preferred as hereinafter set forth.

The sources of nitrogen include amino acids, such as glycine, arginine, threonine, methionine and the like, ammonium salt, as well as complex sources, such as yeast hydrolysates, yeast autolysates, yeast cells, tomato paste, soybean meal, casein hydrolysates, yeast extract, corn steep liquors, distillers solubles, cottonseed meal, meat extract, and the like. The various sources of nitrogen can be used alone or in combination in amounts ranging from about 0.05 to 5 percent by weight of the medium.

Among the nutrient inorganic salts that may be incorporated in the culture media are the customary salts capable of yielding sodium, potassium, magnesium, calcium, phosphate, sulfate, chloride, carbonate, and like ions. Also included are trace metals, such as cobalt, manganese, iron, molybdenum, zinc, cadmium, and the like.

Representative suitable solid and liquid production media, as well as representative seed medium, may be found in the Tables 1 and 2 of U.S. Patent 5,712,109, hereby incorporated by reference in its entirety. These, however, are merely illustrative of the wide variety of media which may be employed and are not intended to be limiting. One aspect of the invention relates to a culture comprising SPB074 bacteria. In certain embodiments, the present invention relates to the aforementioned culture, wherein said culture is biologically pure. In certain embodiments, the present invention relates to the aforementioned culture, wherein said culture is capable of producing a compound of formula I, II or III, in recoverable amounts. In certain embodiments, the present invention relates to the aforementioned culture, wherein said culture is capable of producing a compound of formula I, II or III, in recoverable amounts.

ANTIFUNGAL ASSAYS

The usefulness of the compound as an antifungal agent, especially as an antimycotic agent, may be demonstrated with the compound in a broth microdilution assay for the determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) against fungi. The compound is found to be effective in the assay against a panel of fungi selected for their resistance/susceptibility to known compounds, animal virulence, source and clinical importance, at concentrations less than an established antifungal agent, amphotericin B. For example, in a microbroth dilution assay, microorganisms may be selected by streaking a yeast culture on Sabouraud dextrose agar (SDA) and incubating for 24-48 hours at 35° C to 37° C. Three to five characteristic colonies may be selected and transferred to a fresh plate and incubated under similar conditions. From the regrowth, 3 to 5 colonies may be selected and suspended in 10 milliliters of YM broth (Difco) and incubated for 4 hours at 35° C to 37° C shaking at 225 rpm. The 4 hour broth cultures may be adjusted optically to 86% transmission resulting in a concentration of 1-5 x 10 ⁶ cfu/mL which may be further diluted 1 : 100 in YNBD (yeast nitrogen base with 1 % dextrose) to obtain a concentration of 1-5 x 10 ⁴ cfu/mL for use as inocula.

The test compound may be dissolved at in 10% DMSO and diluted 2 times into the first well to achieve a concentration of the test compound in 5% DMSO in the first well.

Compounds may be subsequently serially diluted 2 times and cell suspension may be added to each well resulting in an additional 2 times dilution of compound. 75 μL of said solution

may be delivered to each well in column 1 of a 96-well, U-bottomed plate. The compounds in column 1 may then be serially diluted two-fold to yield concentrations from 128 μg/mL to 0.03 μg/mL.

Amphotericin B, or a different control compound, may be prepared as a stock solution of 256 μg/mL in 10% DMSO and 75 μL of said solution may be delivered to column 1 of a 96-well, U-bottomed plate. The compounds in column 1 may then be serially diluted two-fold to yield concentrations from 128 μg/mL to 0.03 μg/mL.

The plates containing the diluted compounds may then be inoculated with 75 μL/well of the appropriate microorganism and incubated for 48 hours at 35° C to 37° C with MIC (minimum inhibitory concentration) determinations made out after 24 hours of incubation (except Cryptococcus strains which are read at 48 hours). Growth and sterility controls for each organism and sterility checks for the compounds may also be carried out.

After recording MICs at 24 hours, the microtiter plates may be shaken gently to resuspend the cells. A 1.5 μL sample may be transferred from each well of the 96-well plate to a single reservoir inoculum plate containing SDA. The inoculated SDA and corresponding microtiter plates may be incubated for 24 hours at 35° C to 37° C. For Cryptococcus neoformans, SDA plates may be inoculated at 48 hours after recording MICs and incubated 48 hours before reading the MFC. MFC is the lowest concentration of compound at which either no growth or growth of < 4 colonies occurs. No MFC values should be indicated for Aspergillus furnigatus, and the like, since colony counts are unreliable with filamentous species. Instead, a Minimum Effective Concentration (MEC) may be reported. The MEC is defined as the lowest concentration of drug which effects a severe morphological change in the cells. The MEC is scored macroscopically by direct observation of the plate wells after 24 hours and reflects microscopic alterations in cell morphology (see: Kurtz et al, AAC 1994 38: 1480-1489).

Another aspect of the invention relates to inhibiting the growth of filamentous fungi. Such use may be illustrated in the following tests with Aspergillus flavus, Fusarium oxysporum, Ustilago zeae and the like.

Inocula for filamentous fungi may be prepared by scraping the surface of stock plates maintained on potato dextrose agar with a moistened sterile dacron swab. The spores and mycelia may then be suspended in 10 milliliters of sterile potato dextrose broth and adjusted to 70 percent transmission at 660 nm.

The samples to be tested for production of antifungal agent may be applied directly to the agar plates as methanol solutions. When the sample to be tested is crude broth, it may be centrifuged prior to application. The assay plates may then be incubated at either 28° C or 37° C for 24 hours. Following incubation, the inhibition zones may be measured and appearance noted. In this way one can show that a compound is effectively inhibiting growth of a fungal organism.

ANTIFUNGAL AGENTS AND PHARMACEUTAL COMPOSITIONS THEREOF

One aspect of the invention relates to a pure and isolated compound of formula I: I wherein, R is hydrogen, alkyl, acyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; X is -O-, -S-, -N(hydrogen)-, -N(alkyl)-, -N(aralkyl)-, -N(heteroaralkyl)- or -CH ₂ -; A is alkylene which is unsubstituted or monosubstituted, disubstituted or trisubstituted by halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, aryl, heteroaryl, trifiuoromethyl or cyano; and the stereochemical configuration at any stereocenter of a compound of formula I is R, S, or a mixture of these configurations.

In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen or alkyl. In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen, methyl, ethyl, n-propyl, or i-propyl. In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen or methyl. In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen. In certain embodiments, the present invention relates to the aforementioned compound, wherein X is -O-. In certain embodiments, the present invention relates to the aforementioned compound, wherein X is -S-. In certain embodiments, the present invention relates to the aforementioned compound, wherein X is -N(hydrogen)-, -N(alkyl)-, -N(aralkyl)- or-N(heteroaralkyl)-. In certain embodiments, the present invention relates to the aforementioned compound, wherein X is -CH ₂ -.

In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen or alkyl; and X is -O- or -N(hydrogen)-. In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen; and X is -O-. In certain embodiments, the present invention relates to the aforementioned compound, wherein A is C _2-25 alkylene. In certain embodiments, the present invention relates to the aforementioned compound, wherein A is Cio _-2 o alkylene. In certain embodiments, the present invention relates to the aforementioned compound, wherein A is Ci ₃ -i ₅ alkylene. In certain embodiments, the present invention relates to the aforementioned compound, wherein A is Ci ₄ alkylene.

In certain embodiments, the present invention relates to the aforementioned compound, wherein A is C _4-25 polyenylene. In certain embodiments, the present invention relates to the aforementioned compound, wherein A is Cio _-2 o polyenylene. In certain embodiments, the present invention relates to the aforementioned compound, wherein A is Cj _3-I5 polyenylene. In certain embodiments, the present invention relates to the aforementioned compound, wherein A is Ci ₄ polyenylene.

Another aspect of the invention relates to a pure and isolated compound of formula II:

II wherein, R is hydrogen, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl; and =.=-= is independently for each occurrence a single bond, cis-double bond, or trans-double bond.

In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen or alkyl. In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen, methyl, ethyl, n-propyl, or i-propyl. In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen or methyl. In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen.

In certain embodiments, the present invention relates to the aforementioned compound, wherein =-=--: is a cis-double bond or trans-double bond. Another aspect of the invention relates to a pure and isolated compound of formula III:

III wherein, R is hydrogen, alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl.

In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen or alkyl. In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen, methyl, ethyl, n-propyl, or i-propyl. In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen or methyl. In certain embodiments, the present invention relates to the aforementioned compound, wherein R is hydrogen. In view of the activity, the compound of the present invention, either singly or as a mixture, is adaptable to being utilized in various applications of antifungal compositions. In such case, one or more compounds of formula I, II, or III, or a pharmaceutically acceptable salt thereof, may be admixed with a biologically inert carrier, generally with the aid of a surface active dispersing agent, the nature of which would vary depending on whether the use is for the control of pathogens infecting man or animals, or for control of fungi in agriculture, such as in soil or plant parts, or for the control of fungi in inanimate objects.

In compositions for medical applications, the compound may be admixed with a pharmaceutically acceptable carrier, the nature of which will depend on whether the composition is to be topical, parenteral or oral. If said application is to be topical, the drug may be formulated in conventional creams and ointments, such as white petrolatum, anhydrous lanolin, cetyl alcohol, cold cream, glyceryl monostearate, rose water and the like.

For parenteral applications, the compounds may be formulated in conventional parenteral solutions, such as 0.85 percent sodium chloride or 5 percent dextrose in water, or other pharmaceutically acceptable compositions.

Compositions for oral administration may be prepared by mixing the component drugs with any of the usual pharmaceutical media, including for liquid preparations, liquid carriers, such as water, glycols, oils, alcohols, and the like; and for solid preparations, such as capsules and tablets, solid carriers, such as starches, sugars, kaolin, ethyl cellulose,

surface active dispersing agents, generally with lubricants, such as calcium stearate, together with binders, disintegrating agents and the like. Water is the preferred liquid carrier for the compound of the invention.

These compositions are then administered in amounts sufficient to obtain the desired antifungal effect. For medical applications, the method comprises administering to a subject in need of treatment a therapeutically effective antifungal amount of the compounds. The appropriate dose will vary depending on age, severity, body weight and other conditions. For topical application, the compositions are applied directly to the area where control is desired. For internal administration, the composition may be applied by injection or may be administered orally.

For non-medical application, the product of the present invention, either alone or as a mixture, may be employed in compositions in an inert carrier which included finely divided dry or liquid diluents, extenders, fillers, conditioners and excipients, including various clays, diatomaceous earth, talc, and the like or water and various organic liquids, such as lower alkanols, such as ethanol and isopropanol.

Compositions for injection, a preferred route of delivery, may be prepared in unit dosage form in ampules, or in multidose containers. The injectable compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain various formulating agents Alternatively, the active ingredient may be in powder (lyophillized or non-lyophillized) form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water. In injectable compositions, the carrier is typically comprised of sterile water, saline or another injectable liquid, e.g., peanut oil for intramuscular injections. Also, various buffering agents, preservatives and the like can be included. Topical applications may be formulated in carriers, such as hydrophobic or hydrophilic bases to form ointments, creams, lotions, in aqueous, oleaginous or alcoholic liquids to form paints or in dry diluents to form powders.

Oral compositions may take such forms as tablets, capsules, oral suspensions and oral solutions. The oral compositions may utilize carriers, such as conventional formulating agents, and may include sustained release properties as well as rapid delivery forms.

The dosage to be administered depends to a large extent upon the condition and size of the subject being treated, the route and frequency of administration, the sensitivity of the

pathogen to the particular compound selected, the virulence of the infection and other factors. Such matters, however, are left to the routine discretion of the physician according to principles of treatment well known in the antibacterial arts. Another factor influencing the precise dosage regimen, apart from the nature of the infection and peculiar identity of the individual being treated, is the molecular weight of the compound.

The compositions for human delivery per unit dosage, whether liquid or solid, may contain from about 0.01% to as high as about 99% of active material, the preferred range being from about 10-60%. The composition will generally contain from about 15 mg to about 2.5 g of the active ingredient; however, in general, it is preferable to employ dosage amounts in the range of from about 250 mg to 1000 mg. In parenteral administration, the unit dosage will typically include the pure compound in sterile water solution or in the form of a soluble powder intended for solution, which can be adjusted to neutral pH and isotonic.

METHODS OF USE

In certain embodiments, the compounds of the invention are useful against organisms causing systemic human pathogenic mycotic infections, such as, for example, Candida albicans, Candida tropicalis, Candida guillermondii, Candida glabrata, Aspergillus fumigatus, Candida pseudotropicalis, Saccharomyces cerevisiae, and Aspergillus flavus. The compounds of the invention are also useful against organisms causing superficial fungal infections, such as Trichoderma sp. and Candida sp. These properties may be effectively utilized by administering compositions containing an antifungal amount of the compound to an area, object or subject, on or in which fungi are to be controlled. Thus, compositions containing an antifungally effective amount of the compound and their use for the control of fungi are aspects of the present invention. An especially preferred aspect of the present invention are compositions in a pharmaceutically acceptable carrier (see above) and their use for the control of mycotic infections by administering a therapeutically effective amount of one or both of the compounds.

One aspect of the invention relates to a method for treating or preventing a fungal infection in subject, comprising administering to the subject a therapeutically effective amount of a compound of formula I, II or III, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

Another aspect of the invention relates to a method for treating or preventing a fungal infection in a patient, comprising administering to the human a therapeutically

effective amount of a compound of formula I, II or III, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the patient is a human. Another aspect of the invention relates to a method for preventing or treating an agricultural fungal infection, comprising administering to the site where growth is to be treated an amount of the compound or composition of a compound of formula I, II or III sufficient to exert an antifungal activity.

Another aspect of the invention relates to a method for preventing or treating an agricultural fungal infection, comprising : (a) growing the SPB074 bacteria in a nutrient medium and filtering the resulting mixture, thereby forming a cultural filtrate; (b) administering to the site where growth is to be treated an amount of the cultural filtrate of step (a) in order to exert an antifungal activity.

In certain embodiments, the present invention relates to any of the aforementioned methods, wherein fungal infection is caused by a fungus of a genus selected from the group consisting of Candida, Aspergillus, Cryptococcus, Mucor, Histoplasma, Blastomyces, Coccidioides, Paracoccidioides, Trichophyton, Epidermophyton, Microsporum, Malassezia, Pseudallescheria, Sporothrix, Rhinosporidium, Fonsecaea, wangiella, Phialophora, Exophiala, Cladosporium, Alternaria, Aureobasidium, Chaetomium, Curvularia, Drechslera, Mycocentrospora, Phoma, Hendersonula, Scytalidium, Corynespora, Leptosphaeria, Madurella, Neotestudina, Sedosporium, Pyrenochaeta, Geotrichum, Trichosporon, Chrysosporium, Coprinus, Schizophyllum, Pneumocystis, Conidiobolus, Basidiobolus, Paecilomyces, Penicilliun, Acremonium, Fusarium, Scopulariopsis, Saccharomyces, Cephalosporium, Loboa, Rhizopus, Rhizomucor and Absidia. In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the method of administration of the antifungal compound is selected from the group consisting of oral and parenteral, e.g., i.v. infusion, i.v. bolus and i.m. injection.

In certain embodiments, the present invention relates to any of the aforementioned methods, wherein the administration is by intravenous administration, intramuscular administration or subcutaneous administration.

DEFINITIONS

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language, such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not

necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non- limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

The term "alkyl" is art-recognized, and includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has about 30 or fewer carbon atoms in its backbone (e.g., Ci-C _3O for straight chain, C ₃ -C ₃₀ for branched chain), and alternatively, about 20 or fewer. Likewise, cycloalkyls have from about 3 to about 10 carbon atoms in their ring structure, and alternatively about 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, "lower alkyl" refers to an alkyl group, as defined above, but having from one to ten carbons, alternatively from one to

about six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

The term "alkylene" is art-recognized, and as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound, which may be aliphatic or alicyclic, or a combination thereof, and which may be saturated, partially unsaturated, or fully unsaturated. Examples of linear saturated Ci-ioalkylene groups include, but are not limited to, -(CH ₂ ) _n - where n is an integer from 1 to 10, for example, - CH ₂ - (methylene), -CH ₂ CH ₂ - (ethylene), -CH ₂ CH ₂ CH ₂ - (propylene), -CH ₂ CH ₂ CH ₂ CH ₂ - (butylene), -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ - (pentylene) and -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ - (hexylene). Examples of branched saturated Ci-ioalkylene groups include, but are not limited to, -CH(CH ₃ )-, -CH(CH ₃ )CH ₂ -, -CH(CH ₃ )CH ₂ CH ₂ -, -CH(CH ₃ )CH ₂ CH ₂ CH ₂ -, - CH ₂ CH(CH ₃ )CH ₂ -, -CH ₂ CH(CH ₃ )CH ₂ CH ₂ -, -CH(CH ₂ CH ₃ )-, -CH(CH ₂ CH ₃ )CH ₂ -, and - CH ₂ CH(CH ₂ CH ₃ )CH ₂ -. Examples of linear partially unsaturated Ci-ioalkylene groups include, but are not limited to,-CH=CH- (vinylene), -CH=CH-CH ₂ -, -CH=CH-CH ₂ -CH ₂ -, - CH=CH-CH ₂ -CH ₂ -CH ₂ -, -CH=CH-CH=CH-, -CH=CH-CH=CH-CH ₂ -, -CH=CH-CH=CH- CH ₂ -CH ₂ -, -CH=CH-CH ₂ -CH=CH-, and -CH=CH-CH ₂ -CH ₂ -CH=CH-. Examples of branched partially unsaturated Ci-ioalkylene groups include, but are not limited to, -C(CH ₃ )=CH-, -C(CHa)=CH-CH ₂ -, and -CH=CH-CH(CH ₃ )-. Examples of alicyclic saturated Ci-ioalkylene groups include, but are not limited to, cyclopentylene (e.g., cyclopent-l,3-ylene), and cyclohexylene (e.g., cyclohex-l,4-ylene). Examples of alicyclic partially unsaturated Ci.ioalkylene groups include, but are not limited to, cyclopentenylene (e.g., 4-cyclopenten-l,3-ylene), and cyclohexenylene (e.g., 2-cyclohexen-l,4-ylene, 3- cyclohexen-l,2-ylene, and 2,5-cyclohexadien-l,4-ylene). The term "polyenylene" as used herein is a type of alkylene which contains one or more sequences of alternating double bonds (cis or trans) and single carbon-carbon bonds. For example, -CH=CH-CH=CH-CH=CH-.

The term "aralkyl" is art-recognized, and includes alkyl groups substituted with an aryl group (e.g., an aromatic or heteroaromatic group). The terms "alkenyl" and "alkynyl" are art-recognized, and include unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

The term "heteroatom" is art-recognized, and includes an atom of any element other than carbon or hydrogen. Illustrative heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur and selenium, and alternatively oxygen, nitrogen or sulfur.

The term "aryl" is art-recognized, and includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, naphthalene, anthracene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "heteroaryl" or "heteroaromatics." The aromatic ring may be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF ₃ , -CN, or the like. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms ortho, meta and para are art-recognized and apply to 1,2-, 1,3- and 1,4- disubstituted benzenes, respectively. For example, the names 1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

The terms "heterocyclyl" and "heterocyclic group" are art-recognized, and include 3- to about 10-membered ring structures, such as 3- to about 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles may also be polycycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams, such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring may be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,

hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF ₃ , -CN, or the like.

The terms "polycyclyl" and "polycyclic group" are art-recognized, and include structures with two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms, e.g., three or more atoms are common to both rings, are termed "bridged" rings. Each of the rings of the polycycle may be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF ₃ , -CN, or the like.

The term "carbocycle" is art recognized and includes an aromatic or non-aromatic ring in which each atom of the ring is carbon. The flowing art-recognized terms have the following meanings: "nitro" means -NO ₂ ; the term "halogen" designates -F, -Cl, -Br or -I; the term "sulfhydryl" means -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" means -SO ₂ ^" .

The terms "amine" and "amino" are art-recognized and include both unsubstituted and substituted amines, e.g., a moiety that may be represented by the general formulas:

wherein R50, R51 and R52 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH ₂ ) _m -R61, or R50 and R51, taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R61 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In certain embodiments, only one of R50 or R51 may be a carbonyl, e.g., R50, R51 and the nitrogen together do not form an imide. In other embodiments, R50 and R51 (and optionally R52) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH ₂ ) _m -R61. Thus, the term "alkylamine" includes an amine group, as defined

above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R50 and R51 is an alkyl group.

The term "acylamino" is art-recognized and includes a moiety that may be represented by the general formula:

O

-N- -R54 R50 wherein R50 is as defined above, and R54 represents a hydrogen, an alkyl, an alkenyl or -(CH ₂ ) _m -R61, where m and R61 are as defined above.

The term "amido" is art recognized as an amino-substituted carbonyl and includes a moiety that may be represented by the general formula:

wherein R50 and R51 are as defined above. Certain embodiments of the amide in the present invention will not include imides which may be unstable.

The term "alkylthio" is art recognized and includes an alkyl group, as defined above, having a sulfur radical attached thereto. In certain embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH ₂ ) _m -R61, wherein m and R61 are defined above. Representative alkylthio groups include methylthio, ethyl thio, and the like.

The term "carbonyl" is art recognized and includes such moieties as may be represented by the general formulas:

wherein X50 is a bond or represents an oxygen or a sulfur, and R55 represents a hydrogen, an alkyl, an alkenyl, -(CH ₂ ) _m -R61or a pharmaceutically acceptable salt, R56 represents a

hydrogen, an alkyl, an alkenyl or -(CH ₂ ) _m -R61, where m and R61 are defined above. Where X50 is an oxygen and R55 or R56 is not hydrogen, the formula represents an "ester". Where X50 is an oxygen, and R55 is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R55 is a hydrogen, the formula represents a "carboxylic acid". Where X50 is an oxygen, and R56 is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiocarbonyl" group. Where X50 is a sulfur and R55 or R56 is not hydrogen, the formula represents a "thioester." Where X50 is a sulfur and R55 is hydrogen, the formula represents a "thiocarboxylic acid." Where X50 is a sulfur and R56 is hydrogen, the formula represents a "thioformate." On the other hand, where X50 is a bond, and R55 is not hydrogen, the above formula represents a "ketone" group. Where X50 is a bond, and R55 is hydrogen, the above formula represents an "aldehyde" group.

The terms "alkoxyl" or "alkoxy" are art recognized and include an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O-(CH ₂ ) _m -R61, where m and R61 are described above.

The term "oxime" is an art recognized moiety that may be represented by the general formula:

An "oximate anion" is a deprotonated oxime. Examples of useful oximes readily forming oximate anions include, but are not limited to, salicylaldoxime, 2-pyridinealdoxime, 2- hydroxy-5-nonylacetophenone oxime, l-cetyl-3-(2-oximopropyl)imidazolium chloride, oxime methacrylate, hexadecyltrimethylammonium anti-pyruvaldehyde 1 -oximate, anti- pyruvaldehyde 1-oxime (monoisonitrosoacetone), O-(2,3,4,5,6- pentafluorobenzyl)hydroxylamine hydrochloride, l,l'-trimethylene bis(4- hydroxyiminomethyl) pyridinium dichloride (trimedoxime), 4-amino-4-methyl-2-pentanone oxime, and the like.

The term "hydroxamic acid" is an art recognized moiety that may be represented by the general formula:

A "hydroxamate anion" is a deprotonated hydroxamic acid. The term "sulfonate" is art recognized and includes a moiety that may be represented by the general formula:

O

— OR57

O in which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term "sulfate" is art recognized and includes a moiety that may be represented by the general formula:

in which R57 is as defined above.

The term "sulfonamido" is art recognized and includes a moiety that may be represented by the general formula:

O N S OR56 R50 O in which R50 and R56 are as defined above.

The term "sulfamoyl" is art-recognized and includes a moiety that may be represented by the general formula:

in which R50 and R51 are as defined above.

The term "sulfonyl" is art recognized and includes a moiety that may be represented by the general formula:

in which R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

The term "sulfoxido" is art recognized and includes a moiety that may be represented by the general formula:

in which R58 is defined above.

Analogous substitutions may be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls. The definition of each expression, e.g. alkyl, m, n, and the like, when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.

The term "selenoalkyl" is art-recognized and refers to an alkyl group having a substituted seleno group attached thereto. Exemplary "selenoethers" which may be substituted on the alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and - Se-(CH2)m-R61, m and R61 being defined above.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations.

Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. In addition, polymers of the present invention may also be optically active. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (d)-isomers, (I)- isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent, such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which

does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, or other reaction.

The term "substituted" is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms, such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

For purposes of the invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles,

materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

EXEMPLIFICATION

The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Southern Pine Beetle Symbiosis. SPB reproductive adults bore holes through a tree's outer bark to create ovipositional galleries within the inner bark and phloem. T. D. Paine, K.F. Raffa, T.C. Harrington, Annu. Rev. Entomol. 42, 179 (1997); and D. Six, K.D Klepzig, Symbiosis 37, 207 (2004). These galleries serve as the nursery for larvae. During the process of gallery construction, female SPBs inoculate the phloem and xylem of the tree with their mutualistic fungus Entomocorticium sp. A. The fungus colonizes the uninfected phloem within the newly constructed galleries and serves as an important food source for the developing larvae. Additional fungal symbionts occur within this insect-fungal community. Ophiostoma minus has a context-dependent relationship with the SPB: it can out-compete the SPB 's beneficial fungus for uncolonized substrate and thereby disrupt SPB larval development, while it can also assist attacking adults in killing healthy trees. O. minus engages in its own mutualism with tarsonemid mites, which ride from tree to tree on the exoskeleton of SPBs. The mites feed on O. minus and in exchange the fungus is vectored to new host trees (S2). Thus the SPB system includes at least five symbionts: one mutualistic pair - SPB and its beneficial fungus - a second mutualistic pair - O. minus and the tarsonemid mites - with a net antagonistic relationship to the first pair, and the host tree in the middle. The interactions between actinomycetes and Entomocorticium sp. A. and O. minus were focused on because the ecological roles of these fungal symbionts have been previously established. This allowed testing of the a priori prediction that if SPBs engage in a mutualistic association with actinomycetes, paralleling the fungus-growing ant system, the bacterial symbionts should inhibit O. minus, the fungus antagonistic to the beetles' larvae, but not Entomocorticium sp. A, the fungus that benefits SPBs. Because the ecological role of C. ranaculosus is not clearly resolved, making a priori prediction was not possible.

Southern Pine Beetle (SPB) Collection. Sections of loblolly pine (Pinus taeda L.) logs were collected from SPB infestations located within the Homochitto National Forest, near Meadville, MS, USA. Logs were placed in emergence containers in a climate controlled insectary at the USDA Forest Service, Southern Research Station laboratories in Pineville, LA, U.S.A. As beetles emerged they fell into sterile refrigerated collection cups. Only live, moving beetles were used in this study.

Scanning electron microscopy (SEM). To visualize potential actinomycetes associated with SPBs, 50 adult female SPB beetles were used, and 20 beetle galleries were removed with sterile razor blades. All specimens were fixed in half-strength Karnovsky's fixative. O. Pechanova, W.D. Stone, W. Monroe, T. E. Nebeker, K.D. Klepzig, C. Yuceer, Insect MoL Biol. 17, 261 (2008). Specimens were prepared for SEM by following previously published methods. MJ Karnovsky, J. Cell. Biol. 27, 137A (1965). All the specimens were examined with a JSM-6500F scanning electron microscope (JEOL).

Strain Isolation. SPB074 was isolated from the mycangia of the southern pine beetle, Dendroctonus frontalis Zimmermann. Isolations were carried out by dissecting the mycangia from a live beetle and placing that structure in the center of a chitin plate (see below). Once growth was observed the isolate was allowed to grow for about 2 weeks before being transferred and streaked onto a new chitin plate. After about 1 week of growth a single colony was picked under a dissecting microscope and streaked onto yeast malt extract agar (YMEA; see below).

Strain Identification. Once confident of culture purity the DNA of the strain was extracted. Mycelium was collected from plates and suspended in 250 μL of cetyltrimethylammonium bromide buffer (CTAB; see Hillis DM, Moritz C & Mable BK (1996) Molecular Systematics, 2nd edition. Sunderland, MA: Sinauer Associates, Inc.) in 1.5mL microcentrifuge tubes. Cells were broken manually (grinding) and by cycles of freezing and thawing (20 min at -80 ⁰ C followed by 10 min at 70 ⁰ C; repeated 3X). One volume of chloroformrisoamyl alcohol (24:1) was added, tubes were vortexed briefly, and then centrifuged for 15 min at 10 00Og. Precipitation was carried out by removing the aqueous layer and adding it to 1 volume of 100% isopropanol. Samples were incubated at -80 ⁰ C for 20 min and centrifuged for 15 min at 10 000g. After centrifugation, the DNA pellet was washed twice with 70% ethanol and air dried. DNA was resuspended in 50 μL of ddH ₂ O aliquots were diluted (1:1000) for PCR amplification. Universal eubacterial primers (Lane 1991) for 16S ribosomal genes were used to amplify the SSU gene. The

amplification reactions were standardized for a total final volume of 20 μL. Cycle parameters for PCR followed the protocol: initial denaturation at 95°C for 4 min, 35 cycles of 94 ⁰ C for 45 s, 51°C for 50 s, 72°C for 2 min, a final extension at 72°C for 4 min, and hold at 4°C.

Using multiple phylogenetic analysis programs (ARB, PAUP, Mr. Bayes) it was determined that the strain is most closely related to Streptomyces thermosacchari. The two strains differed by 8 of 1471 base pairs of the 16S rRNA gene.

Table 1: Chitin Media

Strain Growth. Once growth is confirmed (fuzzy white colonies in ~1 week) a colony should be transferred to YMEA medium (see below). On this medium the strain should be pink to red in color and produce white spores.

Table 2: YMEA medium

Bioassays. Bioassay challenges were conducted between actinomycete isolates and the main fungi associated with the SPB system (Entomocorticium sp. A and Ophiostoma minus). Forty strains of each of the two morphotypes of actinomycetes found associated with SPB were used in the bioassays. Challenges between bacterial strains and each of the

fungi were conducted on yeast malt extract agar and followed previously published methods (S5). Bioassays were run for six weeks or until fungal growth ceased.

Structural determination of mycangimycin. ¹ H, ¹³ C, and 2D NMR data were collected on a Varian Inova 600 MHz spectrometer. UV spectra were acquired in an Amersham Biosciences Ultraspec 5300 Pro spectrometer. A high-resolution mass spectrum was obtained using Waters Q-Tof Ultima with chemical ionization source. The structure of mycangimycin was determined by NMR, UV, and mass spectral data. The production of mycangimycin was confirmed in six of six isolates of the red morphotypes and by none of the white morphtypes (n=3). Culturing of Bacterial Strains. Ophiostoma minus and Entomocorticium sp. A strains were cultivated in potato dextrose medium (Difco). The cell suspensions were transferred to the wells in 96-well plates and various concentrations of pure mycangimycin were applied. The plates were incubated for 24 h and alamarBlue (Soretec Ltd.) was added. The fluorescence was measured with excitation at 540 nm and emission at 590 nm by Wallac Vector 2 plate reader after 6 h.

Vouchers, Sequences and Phylogenetic Analysis. Approximately 1445 base pairs of 16S rDNA were sequenced from 33 strains of actinomycetes isolated from SPB (red=20 and white=13). Sequence analysis revealed 100% (1445/1445) sequence identity within morphotypes and 99.5% (1438/1445) between isolates of red and white morphotypes. The evolutionary relationship among the actinomycetes isolated from the SPB system was elucidated by phylogenetic analyses {Scott et al. unpublished data). Maximum parsimony (MP) and maximum likelihood analyses (ML) were performed in PAUP* 4.0 using heuristic searches (stepwise addition and tree bisection-reconnection branch swapping). Characters were all treated as unordered, and gaps as missing data. Bootstrap support for MP was calculated for internal branches after 1000 pseudoreplicates. The model of sequence evolution was estimated using ModelTest 3.7.1, indicating the general time reversible model (GTR+F+G) under a gamma distribution for the among-site rate variation (base frequencies = (A:0.2137 C:0.2452 G:0.3445 T:0.1996); rate matrix = (AC:0.5306 AG: 1.8143 AT:0.9797 CG:0.7552 CT:4.3220 GT: 1.0000); shape parameter for gamma distribution=0.5063; number of estimated parameters for discrete gamma approximation^ 0; proportion of invariant sites=0.3256). These analyses revealed that the

actinomycetes associated with SPB form a monophyletic clade, most closely related to Streptomyces thermosacchari.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications cited herein are hereby incorporated by reference.