WO1999040989A1 - Natural products from terrestrial and marine sources, and drug discovery based thereon - Google Patents

Abstract

The present invention provides methods for isolating directly from terrestrial and marine samples novel natural products. The invention also provides for the use of the aforementioned novel natural products as lead compounds in all manner of drug discovery programs. Additionally, the present invention provides for the use of the novel natural products, and compounds resulting from drug discovery programs based thereon, in agricultural, veterinary, or pharmaceutical formulations or preparations.

Description

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Natural Products From Terrestrial and Marine Sources, and Drug Discovery Based Thereon

Related Applications This application claims the benefit of the filing date of U.S. Provisional Application

No. 60/074,869, filed February 17, 1998.

Government Funding

Work described herein was supported in part with funding from the National Institutes of Health. The United States Government has certain rights in this invention.

Background of the Invention

Cultivation of microorganisms from the environment was assumed until recently to result in the isolation of a good proportion of the resident microorganisms. Phylogenetic analysis of rRNA sequences obtained from direct sampling of environments has shown, however, that this assumption is incorrect (Giovannoni et al. (1990) Nature 345:60-63; Pace et al. (1996) ASM News 62:463-470; Stahl et al. (1985) Appl. Environ. Microbiol. 49:1379- 1384; Suzuki et al. (1997) Appl. Environ. Microbiol. 63:983-989; Ward et al. (1990) Nature 345:63-65). It is now apparent that the subset of microorganisms from any environment that can be cultured using standard techniques represents a minority of the total species present in that environment, indicating that a greater diversity of prokaryotes exists than suggested by these culturing methods (Pace et al. supra; Stahl (1993) ASM News 59:609-613). The realization that the majority of bacteria in an environment are currently nonculturable has revolutionized thinking in microbiology, and has stimulated new approaches to the study of microbes (Woese et al. (1990) Proc. Natl. Acad. Sci. USA 87:4576-4579).

It is estimated that the number of species currently culturable from soil represents 1% or less of the total population (Griffiths et al. (1996) Microbial Ecol. 31:269-280; Torsvik et - 2 - al. (1996) J. Ind. Microbiol. 17:170-178). DNA-DNA reassociation measurements have been used to determine total genetic diversity in a soil sample. The data indicated that greater than 4000 species might be present (Torsvik et al. (1990) Appl. Environ. Microbiol. 56:782-787). This finding indicated at least 200 times more diversity than was observed by examining culturable bacteria from the same sample. Another study based on methods that did not involve culturing suggested 13,000 species in a 100 g soil sample (Torsvik et al. (1994) p.39- 48, In Beyond the Biomass, K. Ritz, j. Dighton and K. E. Giller, Eds., John Wiley and Sons, Chichester). By estimating the total number of cells at 5 x 10¹¹ per gram of soil, this suggested an average of 5 x 10⁷ cells per species assuming even species distribution. Thus, even so-called "rare" species might have fairly large population sizes in the soil.

Summary of the Invention

The present invention, in one aspect, provides methods and protocols for the direct isolation of novel biologically active compounds from samples obtained directly from terrestrial, marine, and aquatic environments. The subject compounds may be the product of de novo organismic biosynthetic pathways, or may be produced by organismic modification of molecules provided ectopically in the given organism's environment. A second aspect of the present invention provides for the use as lead compounds in medicinal chemistry drug discovery programs of the above novel molecules obtained directly from terrestrial, marine, and aquatic samples. Such drug discovery programs may employ classical, combinatorial, or hybrid research methods in their approach to the screening of lead compounds, and the screening and synthesis of compounds based upon those leads. Another aspect of the invention provides pharmaceutical formulations and preparations of the novel molecules obtained directly from the terrestrial, marine, and aquatic samples, or of molecules discovered as a result of drug discovery programs based on the subject novel molecules.

In one practice, the invention provides a method for obtaining novel natural products by, providing a sample, e.g., of soil or water, contacting the sample with a solvent to extract organic components of the sample into the solvent, separating insoluble residue from the solvent, concentrating the organic components, and testing a sample of the organic components in an assay for biological activity. The sample may be terrestrial soil, marine soil, or aquatic water. The sample of soil may be less than 100 kg of soil, less than 10 kg of soil, or less than 1 kg of soil. The sample of water may be less than 1000 kg of water, less than 100 kg of water, or less than 10 kg of water. The solvent may be an organic solvent, or may be supercritical carbon dioxide or supercritical water. The organic components may be purified using methods such as extraction, fractionation, liquid-liquid chromatography, liquid-solid chromatography, affinity chromatography, normal-phase filtration, reverse-phase filtration, ion-exchange filtration, and gel filtration. The assay may be designed to identify antibacterial agents, antifungal agents, cytotoxic drugs, or viral inhibitors. The assay may optionally be selected to identify antitumor agents or cell cycle regulators, or immune stimulants or immunosuppressants. The sample may be treated to lyse cells contained therein. In a related practice, the molecular structure of a component in a sample which exhibits biological activity may be determined. Furthermore, in a drug discovery practice, a new compound having a structure related to the molecular structure of the compound which exhibits biological activity may be prepared and tested in an assay for biological activity.

A compound identified to have biological activity may be formulated as a pharmaceutical preparation. The pharmaceutical preparation may be used to treat a medical condition by administering the preparation to a patient.

In another practice for obtaining novel natural products, the invention provides a method including providing a sample, e.g., of soil or water, separating cells from the sample, contacting the cells with a solvent to extract organic components from the cells, concentrating the organic components, and testing the organic components in an assay for biological activity.

The microorganism responsible for the production of the natural product may be a species of bacteria, fungi, or yeast. In preferred embodiments, the microorganism responsible for the production of the natural product is a species from the Bacteria, such as may be selected from the group consisting of Acetobacter, Actinomyces, Aerobacter, Agrobacterium, Azotobacter, Bacillus, Bacteroides, Bordetella, Brucella, Chlamydia, Clostridium, Corynebacterium, Erysipelothrix, Escherichia, Francisella, Fusobacte ium, Haemophilus, Klebsiella, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Neisseria, Nocardia, Pasteurella, Proteus, Pseudomonas, Rhizobium, Rickettsia, Salmonella, Serratia, Shigella, Spirilla, Spirillum, Staphylococcus, Streptococcus, Streptomyces, Treponema, Vibrio, and Yersinia. - 4 -

Detailed Description of the Invention

I. Overview

Microorganisms have had a major impact on the development of medical science since the discovery that they not only are the cause of infection but also produce organic compounds that can both cure infections and help treat a variety of noninfectious diseases. Traditionally, microbial products discovery has focused on antimicrobial and antitumor activity of soil bacteria and fungi, and, since the original discovery of penicillin in 1929, nearly 50,000 natural products have been discovered from microorganisms (Betina N. 1983. The Chemistry and Biology of Antibiotics. Amsterdam, the Netherlands: Elsevier). Of these, more than 10,000 are reported to have biological activity and more than 100 microbial products are in use today as antibiotics, antitumor agents, and agrochemicals.

Traditional methods of natural product discovery have relied on culturing microbes from the environment and implementing screens to test whether these cultured strains produce metabolites with specific activity profiles (Franco et al. (1991) Crit. Rev. Biotech. 11:193-276). In an attempt to find microbial products having desired activities, those in the art have isolated a number of microorganisms out of naturally occurring soils and conducted extensive research on the products of these microorganisms. For example, the immunosuppressant FK-506 was isolated from a culture of Streptomyces obtained from a soil sample. Likewise, the antibiotic rapamycin was also discovered by culturing another strain of Streptomyces isolated from soil.

In samples from almost any environmental source, including those from extreme environments, one will find a diverse population of microorganisms. However, the set of microorganisms which are isolated by the application of standard culturing techniques to samples from these environments represent only a small fraction of the palette of microorganisms present in a given environment. Thus, the aforementioned culturing steps can dramatically decrease the variety of organisms recovered from a given niche. The classical method of culturing microorganisms therefore gives a skewed picture of the diversity of bacteria and other microorganisms in a natural sample because it essentially ignores organisms that can't be cultured.

In recently described approaches, scientists are bypassing the petri dish and directly characterizing microbial diversity by sub-cloning into culturable organisms genomic DNA from unculturable microbes found in soil. In general, the prior art method provides host cells which have been engineered to express the open reading frames of genomic DNA sub-cloned from a population of heterologous soil microorganisms samples, including genomic DNA from hitherto unaccessed species of organisms. The prior art method takes a functional approach to screening the genomic libraries, requiring that the expression of the cloned genomic DNA recapitulates a biosynthetic pathway from the source organism. In theory, this approach permits access to a wide range of novel natural products by removing much of the biasing produced by direct culturing methods. However, this approach requires that all of the necessary enzymes involved in the biosynthesis of a small molecule be present in the cloned genomic DNA, or be provided upon combination with the gene products of the host organism in a new chimeric pathway. Thus, while a powerful approach, the functional genomic cloning technique can nevertheless miss an appreciable portion of the small molecule diversity in the soil sample.

The Applicant's invention relates to the unexpected discovery that direct extraction of terrestrial and marine soil samples or aquatic water samples can provide hitherto unknown natural products, i.e., small organic, inorganic and hybrid molecules or complexes. Prior to the Applicant's invention, this result would be viewed by one of ordinary skill in the art as unexpected for a number of reasons, e.g., the widely held belief that samples taken directly from the environment do not contain enough material to provide for meaningful biological screening or structure elucidation. The set of natural products isolated directly from the environment may contain greater structural variety than the corresponding set isolated from culturable organisms. Additionally, a wider range of biological activities will accompany the enhanced structural variety contained in the material isolated directly from the environment. Moreover, natural products can be collected from microorganisms which exist only under - 6 - extreme conditions, such as extreme temperature or extreme pH. This feature of the present invention greatly enhances the likelihood that novel natural products can be identified.

In general, the method comprises a step of directly extracting, e.g., into an organic solvent or supercritical carbon dioxide, natural products from a sample taken from a terrestrial, marine, or aquatic environment that may harbor unculturable microorganisms. The resulting extracts can be processed initially to yield fractions defined by characteristics such as polarity, hydrophobicity, acidity, basicity, aggregate charge, molecular weight and the like. The fractions thus obtained are screened for a desired activity in initial assays and fractions that possess the desired activity can be subjected to further analysis. This further analysis can comprise iterative steps of purification and screening. This iterative process leads to a homogeneous sample, or samples, comprising the originally recognized activity. The identity of the active constituent(s) of each of these samples is established, e.g., via the spectroscopic and spectrometric techniques of the art. In this manner, natural products derived from microorganisms which are difficult to culture, or are unculturable by current techniques, are rendered accessible.

In preferred embodiments, the mass of the terrestrial or marine soil samples utilized in the subject method is 100 kg or less. In more preferred embodiments, the mass of the terrestrial or marine soil samples utilized in the subject method is 10 kg or less. In highly preferred embodiments, the mass of the terrestrial or marine soil samples utilized in the subject method is 1 kg or less. In certain embodiments, the mass of the terrestrial or marine soil samples utilized in the subject method is between 10 and 100 kg, preferably between 5 and 50 kg, more preferably between 2 and 20 kg.

In preferred embodiments, the mass of the aquatic water sample utilized in the subject method is 1000 kg or less. In more preferred embodiments, the mass of the aquatic water sample utilized in the subject method is 100 kg or less. In highly preferred embodiments, the mass of the aquatic water samples is 10 kg or less. In certain embodiments, the mass of the - 7 - aquatic water sample utilized in the subject method is between 100 and 1000 kg, preferably between 50 and 500 kg, more preferably between 20 and 200 kg.

In preferred embodiments, the identity of a compound isolated according to the subject invention can be determined from 10 mg or less of a homogeneous, or substantially homogeneous, sample. In more preferred embodiments, the identity of a compound isolated according to the subject invention can be determined from 1 mg or less of a homogeneous, or substantially homogeneous, sample. In highly preferred embodiments, the identity of a compound isolated according to the subject invention can be determined from 100 μg or less of a homogeneous, or substantially homogeneous, sample. In most preferred embodiments, the identity of a compound isolated according to the subject invention can be determined from 10 μg of a homogeneous, or substantially homogeneous, sample.

In one embodiment, the subject method can be used for the discovery of natural products with activity against diseases that afflict mammals, ter alia cancer, immunodeficiency viruses, sensitive and resistant microbial infections, e.g., bacterial and fungal infections, lipid metabolism, inflammation, diabetes and the like. Additional medical conditions which may be amenable to treatment with compounds discovered by methods disclosed herein include conditions characterized by unwanted cell proliferation, aberrant upregulation of enzymic activity, misregulated cell death, and undesirable cell differentiation.

In certain embodiments, such natural products discovered according to the present method can serve as lead compounds in drug discovery programs. Such drug discovery programs predicated on the novel natural products obtained via the present invention may employ the logic and methods of classical medicinal chemistry, computer-aided "rational" drug design, combinatorial or parallel synthesis protocols, combinatorial or parallel assay protocols, or any possible amalgamation of these methods and approaches.

An additional aspect of the present invention is the formulation and use as agricultural, veterinary, or pharmaceutical agents of the novel natural products, or compounds - 8 - resulting from drug discovery programs based on their use as lead compounds, obtained via the methods of the present invention.

It is not expected that every terrestrial, marine, or aquatic sample will yield novel natural products with desirable activity against mammalian diseases; however, even if only a subset of the samples screened provides new compounds with desirable activity profiles, the potential is great for important contributions to the fields of agricultural, veterinary, or pharmaceutical drug discovery.

II. Definitions

As used herein, the term "microorganism" includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism.

The term "prokaryotes" is art recognized and refers to cells which contain no nucleus or other cell organelles. The prokaryotes are generally classified in one of two domains, the

Bacteria and the Archaea. The definitive difference between organisms of the Archaea and

Bacteria domains is based on fundamental differences in the nucleotide base sequence in the 16S ribosomal RNA.

The term "Archaea" refers to a categorization of organisms of the division Mendosicutes, typically found in unusual environments and distinguished from the rest of the procaryotes by several criteria, including the number of ribosomal proteins and the lack of muramic acid in cell walls. On the basis of rRNA analysis, the Archaea consist of two phylogenetically distinct groups: Crenarchaeota and Euryarchaeota. On the basis of their physiology, the Archaea can be organized into three types: methanogens (prokaryotes that produce methane); extreme halophiles (prokaryotes that live at very high concentrations of salt ([NaCl]); and extreme (hyper) thermophiles (prokaryotes that live at very high temperatures). Besides the unifying archaeal features that distinguish them from Bacteria - 9 -

(i.e., no murein in cell wall, ester-linked membrane lipids, etc.), these prokaryotes exhibit unique structural or biochemical attributes which adapt them to their particular habitats. The Crenarchaeota consists mainly of hyperthermophilic sulfur-dependent prokaryotes and the Euryarchaeota contains the methanogens and extreme halophiles.

"Bacteria", or "Eubacteria", refers to a domain of prokaryotic organisms. Bacteria include at least 11 distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions, (a) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) and (b) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic + non-photosynthetic Gram-negative bacteria (includes most "common" Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria ; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (11) Thermotoga and Thermosipho thermophiles.

"Gram-negative bacteria" include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

The term "pathogen" is art recognized and refers generally to any organism which causes a deleterious effect on a selected host under appropriate conditions. Within the scope of this invention the term pathogen is intended to include fungi, bacteria, nematodes, viruses, and insects.

"Gram positive bacteria" include cocci, nonsporulating rods, and sporulating rods. The genera of gram positive bacteria include, for example, Actinomyces, Bacillus, - 10 -

Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

The term "biosynthetic pathway", also referred to as "metabolic pathway", refers to a set of anabolic or catabolic biochemical reactions for converting (transmuting) one chemical species into another. For instance, an antibiotic biosynthetic pathway refers to the set of biochemical reactions which convert primary metabolites to antibiotic intermediates and then to antibiotics.

The term "non-ribosomal synthesis" refers to a biosynthetic step or series of steps other than peptide bond formation in the translation of mRNAs into polypeptides. That is, the term refers to biosynthetic steps other than peptidyl transferase-catalyzed formation of peptide bonds. Likewise, "transformation of a non-proteinaceous compound" refers to the biochemical modification of a compound which is not directly produced by ribosome- mediated formation of peptide bonds

"Ribosomal peptide synthesis", on the other hand, refers to ribosome-mediated formation of peptide bonds in the synthesis of polypeptide; though it does not include post- translational modification of the polypeptide by ribosome-independent reactions.

A "non-proteinaceous compound" refers to a compound which not produced by ribosome-mediated formation of peptide bonds. Thus, the term includes the macrolide class of compounds and the like.

A "small molecule" refers to a compound which is not itself the product of gene transcription or translation (protein, RNA or DNA). Preferably a "small molecule" is a low molecular weight compound, e.g., less than 7500 amu, more preferably less 5000 amu and even more preferably less than 2500 amu. Examples of small molecules include, among the - 11 -

many compounds commonly referred to as "natural products", β-lactam antibiotics, macrolides, steroids, retinoids, polyketides, etc.

"Peptide antibiotics" are classifiable into two groups: (1) those which are synthesized by enzyme systems without the participation of the ribosomal apparatus; and (2) those which require the ribosomally mediated translation of an mRNA to provide the precursor of the antibiotic.

The "non-ribosomal peptide" antibiotics are assembled by large, multifunctional enzymes which activate, modify, polymerize and in some cases cyclize the subunit amino acids, forming polypeptide chains. Other acids, such as aminoadipic acid, diaminobutyric acid, diaminopropionic acid, dihydroxyamino acid, isoserine, dihydroxybenzoic acid, hydroxyiso valeric acid, (4R)-4-[(E)-2-butenyl]-4,N-dimethyl-L-threonine, and omithine can also be incorporated (Katz et al. (1977) Bacteriological Review 41:449-474; Kleinkauf et al. (1987) Annual Review of Microbiology 41:259-289). The products are not encoded by any mRNA, and ribosomes do not directly participate in their synthesis. Peptide antibiotics synthesized non-ribosomally can in turn be grouped according to their general structures into linear, cyclic, lactone, branched cyclopeptide, and depsipeptide categories (Kleinkauf et al. (1990) European Journal of Biochemistry 192:1-15). These different groups of antibiotics are produced by the action of modifying and cyclizing enzymes; the basic scheme of polymerization is common to them all. Non-ribosomally synthesized peptide antibiotics are produced by both bacteria and fungi, and include edeine, linear gramicidin, tyrocidine and gramicidin S from Bacillus brevis, mycobacillin from Bacillus subtilis, polymyxin from Bacillus polymiyxa, etamycin from Streptomyces griseus, echinomycin from Streptomyces echinatus, actinomycin from Streptomyces clavuligerus, enterochelin from Escherichia coil, gamma-(α-L-aminoadipyl)-L-cysteinyl-D-valine (ACV) from Aspergillus nidulans, alamethicine from Trichoderma viride, destruxin from Metarhizium anisolpliae, enniatin from Fusarium oxysporum, and beauvericin from Beauveria bassiana. Extensive functional and structural similarity exists between the prokaryotic and eukaryotic systems, suggesting a common origin for both. The activities of peptide antibiotics are similarly broad, toxic effects of different peptide antibiotics in animals, plants, bacteria, and fungi are known (Hansen - 12 -

(1993) Annual Review of Microbiology 47:535-564; Katz et al. supra; Kleinkauf et al. supra; Kolter et al. (1992) Annual Review of Microbiology 46:141-163).

The "aminoglycosides" and other "carbohydrate-containing" antibiotics refer to organic molecules derived at least part from a saccharide or polysaccharide. For instance, the aminoglycosides are oligosaccharides consisting of an aminocyclohexanol moiety glycosidically linked to other amino sugars. Streptomycin, one of the best studied of the group, is produced by Streptomyces griseus. Streptomycin, and many other aminoglycosides, inhibits protein synthesis in the target organisms.

The "ribosomally synthesized peptide" antibiotics are characterized by the existence of a structural gene for the antibiotic itself, which encodes a precursor that is modified by specific enzymes to create the mature molecule.

The term "terrestrial" refers to any environment which does not lie under a body of salt water, and thus encompasses environments such as freshwater lake and stream beds, freshwater marshes, tundra, beaches, rain forests and the like.

The term "marine" refers to any environment which lies under a body of salt water, and thus encompasses sea floors, saltwater lake beds, saltwater marshes, tidal pools, and the like.

The term "aquatic" refers to water obtained from any naturally occurring water source, including oceans, lakes, ponds, hydrothermal vents, tidal pools, swamps, marshes, and the like.

The term "solvent" as used herein refers to fluids, preferably having a boiling point lower than 200 °C, more preferably lower than 150 °C. Representative solvents include - 13 - supercritical carbon dioxide, supercritical water, and organic solvents such as methanol, ethyl acetate, methylene chloride, hexane, toluene, and the like.

"Purification", as the term is used herein, refers to the separation of unwanted material from a sample. Thus, purification may refer to the separation of cells from soil, the separation of proteins and nucleic acids from small molecules, or the separation of one or more small molecules from other small molecules. A compound which is "pure" contains less than 20% by weight of contaminating material, preferably less than 10%.

The term "variegated population" refers to a population of, e.g., cells, vectors, or the like, including multiple different species. A variegated population of cells preferably includes at least 10², 10³, 10⁴ or 10⁵ different phenotypes in the cell population. Likewise, a variegated population of vectors preferably includes at least 10², 10³, 10⁴ or 10⁵ different vectors.

The term "ED₅₀" means the dose of a drug which produces 50% of its maximum response or effect. Alternatively, the dose which produces a pre-determined response in 50% of test subjects or preparations.

The term "LD₅₀" means the dose of a drug which is lethal in 50% of test subjects.

The term "therapeutic index" refers to the therapeutic index of a drug defined as D₅₀ D₅₀.

The term "ID₅₀" means the dose of a drug which causes 50% of the maximum possible inhibition of a response for the given drug. - 14 -

The term "structure-activity relationship" or "SAR" refers to the way in which altering the molecular structure of drugs alters their interaction with a receptor, enzyme, etc.

The term "agonist" refers to a compound that mimics the action of natural transmitter or, when the natural transmitter is not known, causes changes at the receptor complex in the absence of other receptor ligands.

The term "antagonist" refers to a compound that binds to a receptor site, but does not cause any physiological changes unless another receptor ligand is present.

The term "competitive antagonist" refers to a compound that binds to a receptor site; its effects can be overcome by increased concentration of the agonist.

The term "partial agonist" refers to a compound that binds to a receptor site but does not produce the maximal effect regardless of its concentration.

The term "ligand" refers to a compound that binds at the receptor site.

III. Sources of Terrestrial, Marine, and Aquatic Samples As set out above, the methods of the present invention will allow access to the novel natural products, broadly defined, present in an environment, particularly environments having complex microbial communities. This access is provided by the invention without requiring knowledge of any particular organism or the ability to culture it. The microorganisms from which novel natural products may be isolated include prokaryotic microorganisms, such as eubacteria and archaea, and lower eukaryotic microorganisms such as fungi, some algae and protozoa. The subject methods are based, in part, on the understanding that unculturable microbes can constitute the vast majority of the total - 15 - microbes in any environment, including heavily sampled environments such as soil, marine sediment, or seawater (for a review, see Amann et al. (1995) Microbiol. Rev. 59:143-169).

In one embodiment, novel compounds can be isolated from microorganisms present in soil (land or marine). Soil microbes have been an unparalleled source for natural product discovery based on conventional approaches. Moreover, recent work has revealed that a wide range of previously inaccessible microbes exist in soil samples (Bintrim et al. (1997) PNAS 94:277-282). It is expected, for example, that novel compounds from a range of different bacteria, archaea and other microbes can be isolated from various soil samples, and the majority of those compounds are expected to be from previously unculturable microbes. At the microscopic level, soil is extremely heterogeneous and consists of numerous microenvironments that differ in chemical and physical properties. To access the microbial diversity of soils, microbiologists have long relied on standard microbial cultivation techniques. However, the microbes that were cultivated from soil, in general, reflected neither the abundance nor the phylogenetic diversity in situ. It is estimated that fewer than 1%) of the microbes observed by microscopy are typically recovered by cultivation under standard conditions. Applicant recognizes that the difference between microflora counted by cultivation and that observed by direct microscopy is largely due to the presence in soil of a vast and as yet uncharacterized taxonomic diversity which is not readily accessible by presently available culturing techniques. The instant methods, by utilizing direct isolation techniques independent of microbial cultivation, are well suited for the discovery of novel natural products produced by soil microflora. The subject method may similarly be used to isolate biologically active compounds directly from an aquatic water sample.

In preferred embodiments, terrestrial, marine, and aquatic environments located anywhere on Earth may be sampled via the subject method. Novel compounds may be obtained from a complex microbial sample without an intervening step of culturing cells from the sample. The natural products recovered via the subject method are understood to be relatively unbiased in this respect. Sources, for example, of microbes from which the novel natural products are obtained include, but are not limited to, such environmental samples as may be isolated from terrestrial and marine soil, insect intestines, plant rhizospheres, microbial mats, sulfur springs, ocean and fresh water ecosystems, etc. In certain embodiments, the novel natural products can be obtained from extreme environments, such as - 16 - from samples of arctic or Antarctic ice, water or permafrost sources, samples from environments of extreme pH (acidic or basic), samples from volcanic environments or other high temperature and/or high sulfur environments, samples from soil and the like. Each of the above sample sources are representative of meaningful environments that can be exploited by the subject method as each likely contains a great population of unculturable microorganisms or unculturable combinations of microorganisms. Moreover, many of these environments have not yet been mined heavily for natural products. The samples from which novel natural products may be isolated preferably include at least 100 different microorganisms, more preferably at least 10³, and even more preferably at least 10⁴, 10⁵ and even 10⁶ different microorganisms.

To further illustrate, many invertebrates have been shown to have a diverse collection of microbes associated with their digestive systems. See, for example: Amann et al. (1995) Microbiol. Rev. 59:143-169. The microbes in these environments are phylogenetically diverse and physically accessible. For example, termite intestines contain representatives of the proteobacteria, spirochetes, bacteroides and low G+C Gram-positive groups, as well as members that may represent novel bacterial and archaeal phyla altogether (Ohkuma et al. (1996) Appl. Environ. Microbiol. 62:461-468). The population of gut microbes is often rich in microbes which are unculturable by existing methods; an approach that does not rely on culturing techniques, therefore, should be successful in providing access to the novel natural products produced by these microbes. A terrestrial or marine soil sample or an aquatic water sample comprising a large invertebrate population can be homogenized prior to application of the subject extraction technique; this approach can provide access to natural products derived from microbial parasites or symbionts associated with the invertebrate population.

In another embodiment, in order to assure the isolation of the full range of available novel compounds, the microbial cells of the terrestrial or marine sample must be lysed prior to extraction. To that end, a variety of means are available for lysing organisms. For example, a common method for the mechanical lysis of fungi requires the sample to be alternately vortexed with glass beads and cooled in an ice bath. The resulting extract is recovered by centrifugation after puncturing the bottom of the tube. Similarly, a Mini- Beadbeater™ has been used for lysing bacterial and archaeal species, where cells are ruptured by vigorous shaking with phenol and zirconium beads (see, Hurley, et al. (1987) J Clin - 17 -

Micro. 25:2227-2229). Methods for lysis of soil bacteria have included multiple cycles of freeze-thawing, and passage through a French press, a high-pressure shearing device. One recent method for lysing bacteria calls for the successive application of sonication, microwave heating, and thermal shock (see, Picard, et al. (1992) Applied and Environmental Microbiology. 58:2717-2722). Such methods may also be applied to the lysis of cells isolated from an aquatic environment.

Another common approach for lysis of microorganisms involves enzymes that attack their cell walls. For example, lyticase has proven effective in lysing fungi, while achromopeptidase, mutanolysin, or proteinase K removes cell walls from most Gram-positive microorganisms (see, e.g., Kaneko et al., (1973) Aer. Biol. Chem.. 37:2295-2302; Bollet, et al. (1991) Nucleic Acids Research 19:1955; Siegel et al. (1981) Infection and Immunity 31:808-815).

In an illustrative embodiment, a soil sample can be homogenized or shaken in buffer to disperse soil clumps. The sample is then treated with a mild detergent and/or cation- exchange resin to dissociate microbial cells from soil particles (O'Donnell et al., supra). Microbes are then separated from the soil by differential centrifugation. Final purification of microbes can be by density gradient centrifugation or aqueous two-phase partitioning (O'Donnell et al., supra). Recovery of in excess of 40% - 60% of the total microbial diversity of the initial sample can be achieved using these methods. In this embodiment, these microbial preparations will be the source of novel natural compounds.

IV. Detection Techniques

The ability to detect the presence of novel natural products is central to the practice of the subject invention. In general, assays are carried out to detect organic molecules and the like that are produced as part of an organismic de novo synthesis pathway, or by organismic chemical modification of molecules ectopically provided in the environment. The presence of such molecules can be detected in "test extracts", e.g., which may be direct extracts of the soil sample, conditioned media, cell lystates, cell membranes, or semi-purified or purified fractionation products thereof. The latter may be, as described above, prepared by classical fractionation/purification techniques, including chromatographic separation, or solvent fractionation (e.g., methanol ethanol, acetone, ethyl acetate, tetrahydrofuran (THF), - 18 - acetonitrile, benzene, ether, bicarbonate salts, dichloromethane, chloroform, petroleum ether, hexane, cyclohexane, diethyl ether and the like). Assays that comprise a responder cell, e.g., that test the effect of a soil extract or compound on a whole cell rather than a cell fragment, the extract or compound and the test cell can be combined. In certain embodiments, the assay can directly detect, e.g., by chemical or spectroscopic techniques, a molecular species which is modified, i.e. either elaborated or degraded, by a test organism to yield the biologically active species, e.g., wherein the extract or compound screened comprises a prodrug. In other embodiments, the detection step of the subject method involves characterization of fractionated media cell lysates (the test extract), or application of the test extract to a biochemical or biological detection system. In other embodiments, the invention provides by detecting a phenotypic change in the test organism novel extracts or compounds that potentiate or attenuate a biosynthetic pathway of that organism.

In certain embodiments, analogs related to a known class of compounds will be sought, as for example analogs of alkaloids, aminoglycosides, ansamacrolides, β-lactams

(including penicillins and cephalosporins), carbapenems, terpinoids, prostanoid hormones, sugars, fatty acids, lincosaminides, macrolides, nitrofurans, nucleosides, oligosaccharides, oxazolidinones, peptides and polypeptides, phenazines, polyenes, polyethers, quinolones, tetracyclines, streptogramins, sulfonamides, steroids, terpenoids, vitamins and xanthines. In such embodiments, if an assay is available for directly identifying and/or isolating the natural product, and it is expected that the analogs would behave similarly under those conditions, the detection step of the subject method can be as straight forward as directly detecting analogs of interest in the cell culture media or preparation of the cell. For instance, chromatographic or other biochemical separation of a test extract can be carried out, and the presence or absence of an analog detected, e.g., spectroscopically, in the fraction in which the known compounds would occur under similar conditions. In certain embodiments, such compounds can have a characteristic fluorescence or phosphorescence that can be detected without any need to fractionate the extract.

In related embodiments, the sample extract can be assayed by contacting it with a test cell or components thereof. For instance, a test cell, e.g., which can be prokaryotic or eukaryotic, is contacted with an extract or compound from a terrestrial or marine soil sample - 19 - or an aquatic water sample, and the ability of the extract or compound to induce a biological or biochemical response from the target cell is assessed. For instance, the assay can detect cell growth; a phenotypic change in the target cell, for example a change in: the transcriptional or translational rate of a gene; the stability of a protein; the phosphorylation, prenylation, methylation, glycosylation or other post translational modification of a protein, nucleic acid or lipid; the production of 2^nd messengers, such as cAMP, inositol phosphates and the like. Such effects can be measured directly, e.g., by isolating and studying a particular component of the test cell, or indirectly such as by expression of a reporter gene, detection of phenotypic markers, or cytotoxic or cytostatic activity on the test cell. When screening for bioactivity of extracts or compounds, produced by the subject method, intracellular second messenger generation can be measured directly. A variety of intracellular effectors have been identified. For instance, for screens intended to detect inhibitors or potentiators of receptor- or ion channel-regulated events, the level of second messanger production can be detected from downstream signaling proteins, such as adenylyl cyclase, phosphodiesterases, phosphoinositidases, phosphoinositol kinases, and phospholipases, as can the intracellular levels of a variety of ions.

The following examples describe assay formats for natural products that impact receptor or ion channel function; they also provide general guidance, however, for detecting the effects of a test sample on other cellular functions. For instance, in one embodiment, the GTPase enzymatic activity of G proteins can be guaged by evaluating the breakdown of γ32p GTP with techniques that are known in the art (For example, see Signal Transduction: A Practical Approach. G. Milligan, Ed. Oxford University Press, Oxford England). When compounds or extracts are screened for their ability to modulate levels of cAMP, it will be possible to use standard techniques of cAMP detection, including, but not limited to, competitive assays that quantify HjcAMP in the presence of unlabelled cAMP.

Potentiation of certain receptors and ion channels stimulates the activity of phospholipase C which in turn stimulates the breakdown of phosphatidylinositol-4,5,- bisphosphate to 1,4,5-IP3 (which mobilizes intracellular Ca²⁺) and diacylglycerol (DAG) (which activates protein kinase C). Inositol lipids can be extracted and analyzed using standard lipid extraction techniques. DAG can also be measured using thin-layer - 20 - chromatography. Water soluble derivatives of all three inositol lipids (IP1, IP2, IP3) can also be quantified using radiolabeling techniques or HPLC.

DAG can also be produced from phosphatidyl choline. The breakdown of this phospholipid in response to receptor-mediated signaling, as a result of administration of a test compound or extract, can also be measured using a variety of radiolabeling techniques.

The activation by a test compound or extract of phospholipase A2 can easily be quantified using known techniques, including, for example, the generation of arachidonate.

In various cells, specific proteases are induced or activated in each of several arms of divergent signaling pathways. The effects of test extracts or compounds on these pathways may be independently monitored by following their unique activities with substrates specific for each protease.

In cases where modulation of certain receptors and ion channels is desired, it may be appropriate to screen for changes in cellular phosphorylation that result from exposure to test extracts or compounds. Such assay formats may be useful when the receptor pathway of interest is a receptor kinase or phosphatase. For example, immunoblotting (Lyons and Nelson (1984) Proc. Natl. Acad. Sci. USA 81:7426-7430) using anti-phosphotyrosine, anti- phosphoserine or anti-phosphothreonine antibodies. In addition, tests for phosphorylation could be also useful when the targeted receptor itself may not be a kinase, but activates protein kinases or phosphatases that function downstream in a signal transduction pathway. One such cascade is the MAP kinase pathway that appears to mediate both mitogenic differentiation and stress responses in various types of cells. Stimulation of growth factor receptors results in Ras activation followed by the sequential activation of c-Raf, MEK, and p44 and p42 MAP kinases (ERK1 and ERK2). Activated MAP kinase then phosphorylates many key regulatory proteins, including p90RSK and Elk-1; these proteins are phosphorylated upon translocation of MAP kinase to the nucleus. Homologous pathways exist in mammalian and yeast cells. For instance, an essential part of the S. cerevisiae pheromone signaling pathway comprises a protein kinase cascade composed of the products of the STE11, STE7, and FUS3/KSS1 genes (the latter pair are distinct and functionally redundant). Accordingly, phosphorylation and/or activation of members of this kinase cascade can be detected and used to quantify receptor engagement by a test extract or compound. Antibodies specific for phosphotyrosine are available and enable the - 21 - measurement of increases in tyrosine phosphorylation brought about by a test extract or compound.

In yet another embodiment, the signal transduction pathway that is the focus of an assay upregulates expression or otherwise activates an enzyme which is capable of modifying a substrate that may be added to the assay mixture. The effect of the administered test compound or extract on the transduction pathway may be followed with a detectable substrate, in which case loss of the substrate is observed; alternatively, a substrate that produces a easily detected product may be selected. In preferred embodiments, the conversion of substrate to product by the enzyme activated by the test extract or compound produces a detectable change in the optical characteristics of the assay, e.g., the substrate and/or product is chromogenically or fluorogenically active. In an illustrative embodiment, the signal transduction pathway effected by the test extract or compound causes a change in the activity of a proteolytic enzyme, altering the rate at which said enzyme cleaves a substrate peptide (or simply activates the enzyme towards the substrate). The substrate peptide may include a fluorogenic radical, e.g., a fluorescence emitting radical, and an acceptor radical, e.g., a radical which absorbs the fluorescence energy of the fluorogenic radical when the acceptor radical and the fluorogenic radical are held in proximity (see, for example: US Patents 5,527,681, 5,506,115, 5,429,766, 5,424,186, and 5,316,691; and Capobianco et al. (1992) Anal. Biochem. 204:96-102). For example, if the substrate peptide comprises a fluorescence donor group ~ such as 1-aminobenzoic acid (anthranilic acid or ABZ) or aminomethylcoumarin (AMC) ~ and a fluorescence quencher group — such as lucifer yellow, methyl red or nitrobenzo-2-oxo-l,3-diazole (NBD) ~ at a position such that cleavage of the scissile bond results in destruction of the covalent tether between the two fluorophores. The intramolecular fluorescence energy transfer from the fluorescence donor moiety to the acceptor moiety will quench the donor when the two are proximate in space, e.g., when the scissile bond is intact. Upon cleavage of the scissile bond, however, the acceptor moiety is free to diffuse away from the donor group, thereby disrupting fluorescence energy transfer. In this type of assay, activation of the enzyme by the test extract or compound results in proteolysis of the peptide and concomitant destruction of the fluorescence couple. In still other embodiments, the detectable signal can be produced by enzymes or chromogenic probes whose activities are dependent on the concentration of a second - 22 - messenger, e.g., such as calcium, hydrolysis products of inositol phosphate, cAMP, etc. For example, the mobilization of intracellular calcium or the influx of calcium from outside the cell can be measured using standard techniques. The choice of the appropriate calcium indicator, fluorescent, bioluminescent, or Ca²⁺-sensitive microelectrodes, depends on the cell type and the magnitude and time constant of the event under study (Borle (1990) Environ Health Perspect 84:45-56). In an exemplary method of Ca²⁺ detection, cells subjected to a test extract or compound could be loaded, using standard methods, with the Ca²⁺ sensitive fluorescent dye fura-2 or indo-1 and any change in Ca²⁺ concentration quantified spectroscopically. As certain embodiments described above suggest, in addition to directly measuring the effects of test extracts or compounds on secondary messenger production, the signal transduction activity of a receptor or ion channel pathway can be measured by detection of a transcription product, e.g., by detecting receptor/channel-mediated transcriptional activation (or repression) of a gene(s). Detection of the transcription product includes detecting the gene transcript, detecting the product directly (e.g., by immunoassay) or detecting an activity of the protein (e.g., such as an enzymatic activity or chromogenic/fluorogenic activity); each of these methods is generally referred to herein as a means for detecting expression of the indicator gene. The indicator gene may be an unmodified endogenous gene of the host cell, a modified endogenous gene, or a part of a completely heterologous construct, e.g., as part of a reporter gene construct.

In one embodiment, the indicator gene activated by the test extract or compound is an unmodified endogenous gene. For example, the instant method can rely on detecting a change in the transcriptional level of such endogenous genes as the c-fos gene (e.g., in mammalian cells) or the Barl or Fusl genes (e.g., in fungal cells). Many reporter genes and transcriptional regulatory elements have been disclosed and others may be identified or synthesized by methods known to those of skill in the art. Examples of reporter genes include, but are not limited to chloramphenicol acetyl transferase (CAT; Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as β-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984), Proc. Natl. Acad.Sci. 1: 4154-4158; Baldwin et al. (1984), Biochemistry 23: 3663-3667); alkaline - 23 - phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secreted alkaline phosphatase (Cullen and Malim (1992) Methods in Enzvmol. 216:362-368); β-lactamase or GST.

In the case of fungal cells, suitable positively selectable (beneficial) genes include the following: URA3, LYS2, HIS3, LEU2, TRP1; ADE1,2,3,4,5, 7,8; ARGl, 3, 4, 5, 6, 8; MSI, 4, 5; ILV1, 2, 5; THR1, 4; TRP2, 3, 4, 5; LEW, 4; MET2,3,4,8,9, 14, 16,19; URA1, 2,4,5, 10; H0M3,6; ASP3; CHOI; ARO 2, 7; CYS3; OLE1; IN01,2,4; PR01,3. Countless other genes are potential selective markers. The above are involved in well-characterized biosynthetic pathways. The imidazoleglycerol phosphate dehydratase (IGP dehydratase) gene (HIS3) is preferred because it is both quite sensitive and can be selected over a broad range of expression levels. In the simplest case, the cell is auxotrophic for histidine (requires histidine for growth) in the absence of activation. Activation leads to synthesis of the enzyme and the cell becomes prototrophic for histidine (does not require histidine). Thus test extract or compounds may be screened their ability to enable growth in the absence of histidine. Since only a few molecules per cell of IGP dehydratase are required for histidine prototrophy, the assay is very sensitive.

In other embodiments, the reporter gene approach can be used to assay test extracts or compounds for their ability to directly alter the activity of a transcription factor or other DNA associated protein. For instance, the detection step can be used to identify compounds from the test samples which can attenuate or potentiate transcription of a gene by a cellular or viral transcription factor.

In still other embodiments, the assay is provided in the form of a cell-free system, e.g., a cell-lysate or purified or semi-purified protein or nucleic acid preparation. The samples obtained from the terrestrial or marine soil samples or aquatic water samples can be tested for such activities as attenuating or potentiating such pairwise complexes (the "target complex") as involve protein-protein interactions, protein-nucleic acid interactions, protein- carbohydrate interactions, protein-hormone interactions, nucleic acid-nucleic acid interactions, and the like. The assay can detect the gain or loss of the target complexes, e.g., by endogenous or heterologous activities associated with one or both molecules of the complex. - 24 -

Assays which are performed in cell-free systems, such as may be conducted with purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target when contacted with a test extract or compound. Moreover, the issues of cellular toxicity and/or bioavailability of the test extract or compound are obviated in the in vitro system, the assay instead being focused primarily on the effect of the test extract or compound on the selected molecular target(s). These effects may be manifest in an alteration of binding affinity between molecules or changes in enzymatic properties (if applicable) of given target. Detection and quantification of the pairwise complexes provides a means for determining the efficacy of the test extract or compound at inhibiting (or potentiating) the formation of complexes. The efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test extract or compound. Moreover, a control assay can also be performed to provide a baseline for comparison. For instance, in the control, conditioned media lacking test extracts or compounds can be added to the assay system.

The modulation of the target complex may be detected by a variety of techniques. For instance, modulation in the formation of complexes can be quantified using, for example, labeled proteins (e.g. radiolabeled, fluorescently labeled, or enzymatically labeled) or the like, by immunoassay, or by chromatographic means.

Additionally, the effect of a test extract or compound on a target complex can be determined by use of an interaction trap assay (see, for example: U.S. Patent No: 5,283,317; PCT publication WO94/10300; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). The interaction trap assay relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins, one of which comprises the DNA-binding domain of a transcriptional activator fused to one of the proteins of the target complex. The second fusion protein comprises a transcriptional activation domain (e.g. able to initiate RNA polymerase transcription) fused to the other protein of the target complex. When the two protein interact, the two domains of the transcriptional activator protein are brought into sufficient proximity as to cause transcription of a reporter gene. Thus, test extracts or compounds which are able to inhibit or potentiate interaction of - 25 - the fusion proteins will result in modulation of the expression of the reporter gene. Versions of the interaction trap assay also exist for detecting protein-nucleic acid and nucleic acid- nucleic acid interactions and may be readily adapted for use in the subject method.

In still other embodiments, a purified or semi-purified enzyme can be used to assay the test extracts or compounds. The ability of a test extract or compound to inhibit or potentiate the activity of the enzyme can be conveniently detected by following the rate of conversion of a enzymatic substrate.

In further embodiments, the detection step can be designed to detect a phenotypic change in the cell line of the assay that is induced by test extracts or compounds. For instance, the assay can detect the ability of a test extract or compound to reverse antibiotic resistance in an otherwise resistant cell line.

The subject screening methods can be carried out in a differential format, e.g., by comparing the efficacy of a test extract or compound in a detection assay derived from mammalian components with its efficacy in an assay derived from, e.g., fungal or bacterial components. Thus, bactericidal or fungicidal activity can be the basis for selection.

A test extract need not contain unduly high amounts of the novel compounds for the method to be successful. Isolation of the novel compounds from the terrestrial or marine soil samples or aquatic water samples may not be optimal, or the absolute quantities of the novel compounds in the sample may be low. The ability to detect the effects of the extracts or compounds will often not require maximal levels of the active agents when the bioassay is sensitive to small amounts of natural products. Thus initial access to small quantities of novel compounds need not be a limitation to the success of the subject method.

Finally, as indicated above, the test extract or compound may be derived from, for example, conditioned media or cell lysates obtained directly, or after some degree of processing, from the terrestrial or marine soil sample or the aquatic water sample. For example, cells may be separated from the soil sample, lysed, and then extracted; the extract will then serve as a source of useful pharmaceuticals.

The following scenarios, though not intended to be limiting in any manner, provide further guidance. - 26 -

A) High-throughput robotic screening of subject extracts or compounds.

The high throughput processing and analysis of large libraries of test extracts or compounds may be automated, e.g., using automated/robotic systems. This automation can include, for instance, such activities as: 1) arraying and storage of libraries of extracts or compounds; and 2) screening subject extracts and compounds in biological and biochemical assays. The details of the specific methods utilized will vary from one embodiment to the next, but can be readily implemented by those skilled in the art.

High throughput assays: The subject extracts or compounds may be tested for activity in high throughput biochemical or biological assays adapted for automated readouts. For instance, the method can employ established procedures for robotic antimicrobial testing. In general, such assays are performed in multi-well plates (96 or 384) or by placing small aliquots of test extracts or solutions of homogeneous compounds onto plates seeded with a bacterial lawn, fungal lawn or the like. The goal is to develop an automated method that is sensitive and rapid. In addition to antimicrobial assays, as described above, the test extracts or compounds can be tested in biochemical assays, such as competitive binding assays or enzyme activity assays, as well as whole cell assays, e.g., which detect changes in phenotype dependent on addition of test extracts or compounds. To increase throughput, it may be desirable to test pools of extracts from more than one soil or water sample in certain instances.

B) Screen the libraries of subject extracts or compounds for activity against invertebrate pests and pathogens, using nematodes as the model.

In one embodiment, the subject method can be used to provide test extracts and compounds which are tested for nematicidal activity. For instance, aliquots of a test extract or of a solution of a homogeneous test compound can be added to culture of C. elegans, e.g., an easily culturable nematode. If the aliquots of the test extract or of the solution of a homogeneous test compound comprise a nematicidal compound, then no growth will be seen. Test extracts or compounds that are active in this assay will be retested in a variety of insect bioassays to establish a spectrum of insecticidal activity. - 27

V. Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically effective amount of one or more of the compounds described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. Pharmaceutical preparations may be used for testing or therapeutic purposes. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam.

The phrase "therapeutically effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect by inhibiting an intracellular signaling pathway in at least a sub-population of cells in an animal and thereby blocking the biological consequences of that pathway in the treated cells, at a reasonable benefit/risk ratio applicable to any medical treatment.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject antibacterial agent from one organ, or portion of the body, to another organ, or portion - 28 - of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non- toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present natural products may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term

"pharmaceutically acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical

Salts", J. Pharm. Sci. 66:1-19)

The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non- toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, - 29 - propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, topical

(including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be - 30 - combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium - 31 - stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract or, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, - 32 - ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and - 33 - polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the antibacterial in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel. Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compositions may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like - 34 - into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral and topical administrations are preferred.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, infracapsular, intraorbital, intracardiac, infradermal, intraperitoneal, franstracheal, - 35 - subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular antibacterial employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the - 36 - pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day.

If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically effective amount of one or more of the subject antibacterials, as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intravectally, for example, as a pessary, cream or foam.

The antibiotic compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other antibiotics.

The term "treatment" is intended to encompass also prophylaxis, therapy and cure. - 37 -

The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with other antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy, thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered. The addition of the active compound of the invention to animal feed is preferably accomplished by preparing an appropriate feed premix containing the active compound in an effective amount and incorporating the premix into the complete ration.

Alternatively, an intermediate concentrate or feed supplement containing the active ingredient can be blended into the feed. The way in which such feed premixes and complete rations can be prepared and administered are described in reference books (such as "Applied

Animal Nutrition", W.H. Freedman and CO., San Francisco, U.S.A., 1969 or "Livestock

Feeds and Feeding" O and B books, Corvallis, Ore., U.S.A., 1977).

The compounds covered in this invention may be administered alone or in combination with other pharmaceutically active agents or in combination with a pharmaceutically acceptable carrier of dilutent. The compounds of the invention may be administered intravenously, intramuscularly, intraperitoneally, subcutaneously, topically, orally, or by other acceptable means. The compounds may be used to treat mammals (i.e., humans, livestock, and domestic animals), birds, reptiles, and any other organism which can tolerate the compounds, and also to inhibit growth in cell culture. The compounds can also be used for effects related to their predominant activity such as for increasing the weight gain of livestock.

VI. Exemplary Uses

There are a wide range of uses for the natural products which can be identified by the subject method. Secondary metabolites produced by microorganisms, such as fungi, reflect a - 38 - wide variety of chemical structures affecting numerous biological activities in different classes of organisms, including both prokaryotes (bacteria) and eukaryotes (animals, plants, and insects). Antibiotics constitute the largest group of known bioactive secondary metabolites, acting on such diverse processes as cell wall synthesis, DNA replication, and protein synthesis. In addition to their use as antibiotics, secondary metabolites are being successfully developed and used in agriculture as pesticides, herbicides, and anti-parasitic compounds, and in treating non-infectious human diseases as inhibitors of enzymes.

To further illustrate, in animal therapies, the present method may be used to provide, e.g., angiogenesis inhibitors, insecticidal agents, antibacterial agents, antifimgal agents, antiprotazoan agents, antiinflammatory drugs, antiparasitic agents, antitumor agents, cell cycle regulators, cytotoxic drugs, immune stimulants, immunosuppressants, ion channel blockers, fibrinolytic agents, free radical scavengers, prostaglandins and precursors, vasodilators, hypolipidemic agents, viral inhibitors (including reverse transcriptase and protease inhibitors), and modulators of microtubule dynamics, receptor-ligand interactions and enzyme activity (inhibitors or activators), the subject method can also provide biological activity molecules for use in agricultural applications, such as antibiotics, antifeedants, bactericides, enzymes with antibiosis activities (lysozymes, chitinases, glucanases, cellulases), fungicides, herbicides, pesticides (e.g., antihelminthics, insecticides, acaricides, anticoccidials, antitreponemals, and antitrichomonals), ion channel blockers and promoters, miticides, nematicides, pheromones, siderophores, viricides and the like. The subject method can also produce compounds which have applications in the food industry, such as may be useful as enzymes, fatty acids, flavorings, gums, novel carbohydrates, peptides, pigments and dyes, sweeteners, and vitamins. Still other industrial applications include compounds and/or extracts useful in bioremediation (e.g., degradation of pesticides, toxic waste, oil, grease), as biotech enzymes (restriction enzymes, new reporter genes, antibiotic resistance markers), as industrial enzymes (amylases, proteases, lipases, phosphatases), or as new sources of polysaccharides (lubricants, thickeners). The test extracts of the subject method can provide natural products having such activities can be assessed using methods standard in the art (see, e.g., Franco et al. (1991) Crit. Rev, in Biotech. 11:193-276, and references therein). The subject method, therefore, can further involve the use of, inter alia, biochemical assays, cell or tissue culture assays, and animal model systems. Several exemplary embodiments of these assays are described below. - 39 -

Antibiotic and Antiviral Activities

In one aspect, the method of the present invention can be used to discover extracts or compounds which display some antibiotic activity, e.g., antibacterial, antifungal and/or antiviral. Historically, discovery of antibiotics occurred through evaluation of fermentation broths for anti-bacterial or anti-fungal activity. For instance, many proteobacteria produce β- lactam antibiotics. This has been documented in Chromobacterium, Pseudomonas, Agrobacterium, Serratia, and Erwinia (de Lorenzo et al. (1984) TIBS 9: 266). Additionally, production of metabolites having antifungal activity, such as phenazines and phloroglucinols, have been documented in Pseudomonas (see, for example, Buysens et al. (1996) Appl. Environ. Microbiol. 62:865-871). Myxobacteria have emerged as major producers of novel biologically active compounds (Reichenbach et al., 1993, In Third International Conference on the Biotechnology of Microbial Products: Novel Pharmacological and Agrobiological Activities. Developments in Industrial Microbiology Series Volume 33. V. P. Gullo, J. C. Hunter-Cevera, R. Cooper, and R. K. Johnson, Eds. Society for Industrial Microbiology). Therefore, the extracts of the present invention should be an excellent and abundant source of compounds (e.g., new metabolites) having antibiotic activities.

Anti-bacterial activities can be identified using a number of standard assays known in the art. For example, a culture of bacteria, such as a bacterial lawn, can be contacted with an extract or compound derived from a terrestrial or marine soil sample or aquatic water sample, e.g., filter paper discs doped with the extract, and the areas of lysis characterized. In other embodiments, the extracts are added to a liquid culture of a target organism, and the inhibition of bacterial cell growth can be determined, e.g., by turbidimetric readings. In addition to detecting general effects on bacterial growth and viability, the screening methods of the invention can involve assays for effects on bacteria-specific structures, enzymes, or processes.

A large number of antifungal compounds have been identified using classic approaches, e.g., evaluating samples in primary tests directly against a range of filamentous fungi and yeasts, e.g., Candida albicans, grown in agar plates, or in some cases, directly against phytopathogenic infestations (Bastide et al. (1986) Mircen J. Appl. Microbiol.

Biotechnol. 2:453; and Haruo, (1987) Gendai Kagaku Zokan 9:16). Such asssays can be - 40 - readily adapted for use in a detection step of the subject method. Several examples of fungi- specific targets include chitin and glucan synthases (Selitrennikoff et al., (1983) Antimicrob. Agents Chemother. 23:757; Kirsch et al., (1986) J. Antibiot. 39:1620; and Denisot et al., (1990) 9th Int. Symp. Future Trends in Chemother.. Geneva, Mar. 26 to 28, page 47), and cutinases (Roller et al, (1990) J. Antibiot. 43:734; Umezawa et al. (1980) J. Antibiot. 33:1594).

To further illustrate, compounds which modulate sterol biosynthesis have valuable pharmacological properties. In particular, they can have a pronounced antifungal activity, e.g., such as ketoconazole and terbinafine. These compounds can accordingly be used as medicaments, especially for the control or prevention of topical or systemic infections which are caused by pathogenic fungi in mammals.

Ergosterol is the principal membrane sterol of fungi. It is structurally similar to its animal counterpart, cholesterol, except that ergosterol has a methyl group and two double bonds not present in cholesterol. In yeast, ergosterol affects membrane fluidity and permeability and plays an essential role in the yeast cell cycle. Yeast cells can take up cholesterol and decrease their requirement for ergosterol to very low levels, but cholesterol alone cannot completely substitute for ergosterol (Gaber et al. (1989) Mol. Cell. Biol. 9:3447- 3456). Though the biosynthesis of ergosterol in fungi involves steps distinct from cholesterol biosynthesis in animals, sterol biosynthesis in different organisms shares many common steps. At least one cytochrome P450 is implicated in sterol biosynthesis. The term "cytochrome P450" is a trivial name for a class of cytochromes that includes a number of heme proteins exhibiting a characteristic absorption maximum at 450 nm when combined with CO in the reduced state ('P' denotes pigment; hence, the name). These cytochromes occur in most animal tissues, plants and microorganisms and catalyze the monooxygenation of a wide variety of hydrophobic substances, including lipophilic endogenous compounds and xenobiotics; these enzymes serve as oxygenating catalysts in the presence of one or more electron transfer proteins or redox enzymes.

In certain embodiments, the test extracts or compounds are screened for sterol biosynthesis inhibitors that may be of potential use as fungicides or antihypercholesterolemic agents via the induction of lanosterol 14-α-demethylase, an enzyme in the biosynthetic pathway of ergosterol and cholesterol. Test extracts or compounds that inhibit ergosterol - 41 -

biosynthesis in this system induce lanosterol 14-α-demethylase activity in the culture. In one screening test, test extracts or compounds are incubated in a culture of a Saccharomyces cerevisiae strain sensitive to ergosterol biosynthesis and containing a gene fusion of a lanosterol 14-α-demethylase clone with a gene for bacterial β-galactosidase. After incubation of the culture, an increase in lancsterol 14-α-demethylase activity is determined indirectly by measuring β-galactosidase activity. The culture media contains a chromogenic substrate of β- galactosidase such as ort zø-nitrophenyl-β-D-galactopyranoside or 5-bromo-4-chloro-3- indoyl-β-D-galactopyranoside, so that active samples are identified by the production of a visibly colored product. For comparison purposes, screening tests may employ a lanosterol 14-α-demethylase inhibitor such as dinaconazole as a positive control.

Anti-viral extracts and compounds can be identified by screening for inhibitors of virus-specific enzymes, such as retroviral reverse transcriptases. Other virus-specific processes, such as viral uncoating, viral receptor binding, and cell fusion (e.g., syncytium formation caused by HIV) can also be targeted in the screening methods of the invention. The antiviral properties of the test extracts or compounds may be determined in an assay which exploits the unique properties of the virus. For instance, the influenza virus is a negative strand virus with a segmented genome. The synthesis of viral mRNA is accomplished by a virally encoded transcription complex. Influenza virus is unique in that it requires capped and methylated palmers which are obtained from host cell RNA polymerase H transcripts to initiate mRNA synthesis. An in vitro influenza transcription assay was established to detect agents that may be present in natural product extracts that are capable of inhibiting the transcription apparatus of the influenza virus.

U.S. Patent 5,624,928 describes an exemplary assay for detecting inhibitors of the transcription apparatus of the influenza virus which are required to initiate viral mRNA (messenger RNA) synthesis. Briefly, to each well of a 96-well microtiter plate is added a stock mix of the virus, the test extract or compound, labeled nucleotides, and water. Ten microliters of primer (alfalfa mosaic virus (ALMV) RNA at 0.015 mu g/ml) is also added to the wells. The plates are gently mixed on a shaker for 30 seconds and then incubated for 60 minutes in a 31 °C. water bath. At the end of this period, the plates are removed from the water bath, placed on a bed of ice and the reaction stopped with (i) sterile saturated sodium pyrophosphate solution containing 0.5 mg/ml RNase-free tRNA and (ii) ice-cold 40% TCA, - 42 - and the plates allowed to stand on ice for 15 minutes. The samples are then collected, using a cell harvester, washed twice with 5% TCA, then twice with 95% ethanol and then transferred to sealing bags. The incorporation of the labeled nucleotides into a reverse transcript of the ALMV RNA is detected.

Anti-Tumor Activities

To identify anti-tumor activities, cultured tumor cell lines or cultured tumors can be contacted with subject extracts or compounds and their effects on cell growth and viability monitored. Another approach involves screening for extracts or compounds that induce differentiation of tumor cells, e.g., that cause these cells to lose their tumorigenicity (Franco et al., (1991) Crit. Rev, in Biotech. 11:193-276). An in vitro disease-oriented screening program can utilize a large panel of human tumor cell lines grown initially in vitro and assessed for cytotoxicity by the MTT assay (Carmichael et al. (1987) Cancer Res 47:936-42) and subsequently the sulforhodamine B protein assay (Skehan et al. (1991) Eur J Cancer 27:1162-8). The aim of this screen is to select test extracts or compounds exhibiting selective activity against distinct histological tumor types.

Enzymes can also be used as targets for identifying anti-tumor activities. Enzymes that have been successfully employed as targets in the search for anti-tumor agents include protein tyrosine kinases, which are components of signal transduction pathways regulated by a number of oncogenes, phosphatidylinositol kinase, spermidine synthase, and topoisomerases. As the differences between tumor and non-tumor cells become more apparent, tumor cell-specific targets can be used in the screens in order to identify extracts or compounds that are not toxic to the patient.

Extracts or compounds that exhibit anti-tumor activities in biochemical and cell culture assays can be tested further in appropriate animal model systems.

Immunosuppressive Activities

Immunosuppressive activities can be identified using a number of standard methods in the art, including the mixed lymphocyte reaction, which measures lymphocyte proliferation (Goto et al., (1982) J. Antibiot. 35:1286), and screens for macrophage activation (Tanida et - 43 - al. (1989) J. Antibiot. 42:1619). Inhibitors of T cell activation can be identified by growing cultured T cells in the presence of the candidate extract or compound, crosslinking with activating agents, such as antibodies to CD3 and CD4 surface molecules and a secondary antibody, which normally activate T cells, and determining the level of T cell activation. T cell activation can be quantified by, e.g., a bioassay in which IL-2 production is measured by applying the T cell culture supernatant to CTLL-20 cells, which require IL-2 to live (Sleckman et al., (1987) Nature 328:351).

The cellular immune response involves a very complex set of interactions between antigens, T cells, B cells, macrophages, and numerous factors, such as cytokines, which are released by the cells during the course of the interactions. In one embodiment, the test extracts or compounds can be tested for their effect on T cell activation. While specificity of the T cell response is determined by antigen-specific binding to the T cell antigen receptor (TCR), binding to at least one secondary receptor is also necessary for activation. One such secondary receptor is CD28 which, upon stimulation, induces the activity of nuclear proteins which can increase the production of interleukin-2 and possibly other cytokines by binding to an enhancer region associated with the cytokine genes. Immunosuppressive drugs which act by suppression of the CD28 pathway may have a number of advantages over drugs which act through other mechanisms. Thus, according to the present invention, screening assays for immunosuppressive compositions can comprise exposing cultured T cells to test extract or compound, where the T cells produce an observable signal as a result of normal CD28 stimulation. The T cells are cultured under conditions which will, in the absence of effective CD28 stimulation, produce the observable signal, generally requiring the presence of substances which result in stimulation of both CD28 and the T cell receptor (TCR). The assay can thus identify test extracts or compounds that at least partially suppress the stimulation of CD28, thus resulting in a decrease in the observable signal.

T cells used in the screening assays of the present invention can be obtained from T cell lines which have been modified to incorporate a CD28 enhancer region in a reading frame with a reporter gene so that exposure of the cells to conditions selected to induce the

CD28 receptor will result in expression of the reporter gene. The T cell lines may be derived by modifying previously established human or mouse T cell lines and hybridomas, where the - 44 - starting cell lines and hybridomas are capable of expressing certain cytokine gene(s), as discussed below.

The CD28 enhancer region may be derived from the 5' flanking region of a cytokine gene, where the cytokine gene selected should be one which is normally expressed in the cell line being modified. The enhancer region will include at least that portion of the 5' flanking region which is bound by the CD28 nuclear protein which is produced as a result of stimulation of the CD28 receptor, as described below. Suitable enhancer regions may be obtained from such genes as the IL-2 gene, the GM-CSF gene, the IL-3 gene, the G-CSF gene, or the γ-IFN gene. Extracts or compounds of the present invention found to possess immunosuppressive activity in the cell culture assays can be further tested in animal model systems. An extract containing a candidate compound, or a purified or semi-purified fraction thereof, is administered to an immunocompetent animal, for example, a mouse which has a non-MHC matched skin graft, and the effect of the compound on, e.g., T cell or macrophage activation is determined by monitoring the immune response of the mouse.

As mentioned above, preferable screening assays are designed to identify biological activities directed specifically against the target cell, e.g., an infectious pathogen or a tumor cell, and not cells of the host organism, in order to decrease the likelihood of toxicity problems. Especially in cases where the potential therapeutic biological activity is directed against a process or structure which may be similar in the target cell and the host, it is critical to determine the relationship between the effectiveness and the toxicity of the treatment. This can be determined by standard methods using both cell culture assays and animal model systems (The Pharmacological Basis of Therapeutics. Goodman and Gilman, Eds. MacMillan Publishing, New York, 1980, pp. 28-39, and 1602-1614).

Lipid Biosynthesis

The subject method can also be used to identify extracts or compounds that effect lipid biosynthesis. To illustrate, surface-exposed unusual lipids containing phthiocerol and phenolphthiocerol are found only in the cell wall of slow-growing pathogenic mycobacteria and are thought to play important roles in host-pathogen interaction. The enzymology and - 45 - molecular genetics of biosynthesis of phthiocerol and phenolphthiocerol are unknown; though it has been postulated that a set of multifunctional enzymes are involved in their synthesis, and that these genes are clustered on the bacterial genome (Azad et al. (1997) J Biol Chem 272: 16741-5).

Modulators of Extracellular Factors

In one embodiment, the subject extracts or compounds can be assayed for their ability to alter the bioactivity of an extracellular protein, lipid, carbohydrate or the like. For instance, extracts or compounds may be sought that inhibit the action of blood coagulation factors, thrombolytic factors, or enzymes aberrantly upregulated in diseases states, such as superoxide dismutase or the like.

Ligands for Cell Surface Receptors

In another embodiment, the subject extracts and compounds can be used to discover ligands for cell surface receptor proteins or ion channels, e.g. proteins which interact with an extracellular molecule (i.e. hormone, growth factor, peptide, ion) to modulate a signal in the cell. Exemplary receptors include: a receptor tyrosine kinase, e.g. an EPH receptor; an ion channel; a cytokine receptor; an multisubunit immune recognition receptor; a chemokine receptor; a growth factor receptor; or a G-protein coupled receptor, such as a chemoattractant peptide receptor, a neuropeptide receptor, a light receptor, a neurofransmitter receptor, or a polypeptide hormone receptor. In addition, the subject method is amenable to providing and identifying ligands for an orphan receptor, i.e., a receptor with no known ligand, regardless of the class of receptors to which it belongs.

In certain embodiments, the receptor targeted by the subject extracts or compounds is a G protein coupled receptor, such as αl A-adrenergic receptor, αlB-adrenergic receptor, α2- adrenergic receptor, α2B-adrenergic receptor, βl-adrenergic receptor, β2-adrenergic receptor, β3-adrenergic receptor, ml acetylcholine receptor (AChR), m2 AChR, m3 AChR, m4 AChR, m5 AChR, Dl dopamine receptor, D2 dopamine receptor, D3 dopamine receptor, D4 dopamine receptor, D5 dopamine receptor, Al adenosine receptor, A2b adenosine receptor, 5-HTla receptor, 5-HTlb receptor, 5HTl-like receptor, 5-HTld receptor, 5HTld-like - 46 - receptor, 5HTld beta receptor, substance K (neurokinin A) receptor, fMLP receptor, fMLP- like receptor, angiotensin II type 1 receptor, endothelin ETA receptor, endothelin ETB receptor, thrombin receptor, growth hormone-releasing hormone (GHRH) receptor, vasoactive intestinal peptide receptor, oxytocin receptor, somatostatin SSTR1 and SSTR2, SSTR3, cannabinoid receptor, follicle stimulating hormone (FSH) receptor, leutropin (LH HCG) receptor, thyroid stimulating hormone (TSH) receptor, thromboxane A2 receptor, platelet-activating factor (PAF) receptor, C5a anaphylatoxin receptor, Interleukin 8 (IL-8) IL- 8RA, IL-8RB, Delta Opioid receptor, Kappa Opioid receptor, mip-1/RANTES receptor, Rhodopsin, Red opsin, Green opsin, Blue opsin, metabotropic glutamate mGluRl-6, histamine H2 receptor, ATP receptor, neuropeptide Y receptor, amyloid protein precursor receptor, insulin-like growth factor II receptor, bradykinin receptor, gonadotropin-releasing hormone receptor, cholecystokinin receptor, melanocyte stimulating hormone receptor receptor, antidiuretic hormone receptor, glucagon receptor, and adrenocorticotropic hormone II receptor. In other embodiments, the targeted receptor is a receptor tyrosine kinase, e.g., an EPH receptor such as eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyrol, tyro4, tyroδ, tyroό, tyroll, cek4, cek5, cekό, cek7, cek8, cek9, ceklO, bsk, rtkl, rtk2, rtk3, mykl, myk2, ehkl, ehk2,pagliaccio, htk, erk and nuk receptors.

The modulation of cell surface proteins can also include effecting the bioactivity of the adherin proteins, e.g., cadherins, integrins, lectins and the like.

In certain embodiments the subject extracts and compounds can effect the production of second messengers and thereby cause changes in ligand engagement by the receptor. A "second messenger" is defined as an intermediate compound whose concentration, either intercellularly or within the surrounding cell membrane, is raised or lowered as a consequence of the activity of an effector protein. Some examples of second messengers include cyclic adenosine monophosphate (cAMP), phosphotidyl inositols (PI), such as inositol triphosphate (IP3), diacylglycerol (DAG), calcium (Ca²⁺) and arachidonic acid derivatives. In preferred embodiments, changes in GTP hydrolysis, calcium mobilization, or phospholipid hydrolysis resulting from exposure to the test extracts or compounds can be effected and detected. - 47 -

Modulators of Intracellular Signaling

Still another class of molecules which can be obtained and identified in the method of the present invention are those which modulate intracellular signalling, e.g., by inhibiting or potentiating protein-protein (intermolecular or intramolecular interactions), protein-DNA, protein-lipid or protein-2nd messenger interactions, inhibiting or potentiating intracellular enzymes, or inhibiting or potentiating ion channel passivity, and the like. As described above, the subject extracts or compounds can be sampled with purified or semi-purified components, lysates, whole cells or any other convenient way of contacting the products of the invention with the intended target in a manner which permits generation of a detectable signal. That signal may be, for instance, a change in the a cell's phenotype, rate of proliferation or survival, transcription of a reporter gene, changes in 2^nd messenger levels, a change in an enzyme's activity towards a detectable substrate (or one which produces a detectable product), a change in the amount or characteristics of protein complexes or the localization of a protein, e.g., within various cellular compartments. To further illustrate, the detection step of the instant assays can be derived to identify extracts or compounds of the subject invention that, for illustration, modulate a protein kinase (e.g., serine/threonine kinase, tyrosine kinase), a protein phosphatase (e.g., serine/theronine phosphatase, tyrosine phosphatase), interactions mediated by SH2 domains (e.g., with phosphotyrosine residues), interactions mediated by SH3 domains, interactions mediated by leucine zipper domains, phosphatidyl inositol kinases, adenyl cyclases, interactions involving G proteins (e.g., with a G protein coupled receptor, between the α subunit with β/γ dimer, or downstream signal transduction proteins), phospholipases, phosphodiesterases, interactions between DNA binding proteins and DNA, and ion flux through ion channels. The interactions can occur between components of the same cell compartment, as between two intracellular proteins, or different compartments, such as between a cell surface receptor and an intracellular signal transduction protein.

Additional Natural Products - 48 -

In one embodiment, the invention can be used to identify novel biologically active polyketides. Polyketides are naturally occurring compounds, most often produced by microorganisms such as fungi and the filamentous bacteria (the actinomycetes). The polyketides comprise a vast array of natural products with structures varying from simple aromatic compounds like 6-methylsalicylic acid (6-MSA) to the gigantic polycyclic ether maitotoxin, whose molecular weight of 3422 Da makes it one of the largest known secondary metabolites. Apart from microorganisms, polyketides are also isolated from a wide range of marine organisms (for example, brevitoxin) and higher plants (flavonoids). Many other metabolites contain polyketide-derived moieties as part of a larger structure from another biosynthetic origin, for example, the unusual amino acids, such as 4-[2-butenyl]-4-methyl-L- threonine (Bmt) found in cyclosporin, and algal peptide toxins and monoterpenoids such as tetrahydrocannabinol.

In addition to their wide occurrence and structural diversity, polyketides display a huge range of biological activities. These activities include antibiotics (for example tetracyclines), anti-cancer agents (daunomycin and dynemycin A), antifungals (griseofulvin and strobilurins), antiparasitics (avermectin and monensin), immunosuppressive agents (FK506 and rapamycin), and cholesterol-lowering agents (lovastatin and squalestatins). Thus they have long been of interest to scientists from many disciplines, including natural product chemists, microbiologists and pharmacologists. Many of the most challenging synthetic targets currently being pursued by organic chemists are polyketides.

Despite their enormous structural variety, all of the polyketides are related by their common biosynthetic origins (O'Hagan et al. (1995) Nat. Prod. Rep.. 12:1). They are derived from highly functionalized carbon chains whose assembly are controlled by multifunctional enzyme complexes called polyketide synthases. Like the closely related fatty acid synthases, polyketide synthases catalyse a repetitious sequence of decarboxylative condensation reactions between simple acyl thioesters and malonate. Each condensation is followed by a cycle of modifying reactions: ketoreduction, dehydration and enoyl reduction.

Such extracts and compounds can be screened in, for example, the assays described above for identifying antibiotic agents, e.g., by biological, biochemical or chemical means. Another class of small molecule natural products which can be obtained by the subject method are the macrocyclic lactones. This group of compounds shares the presence of a large - 49 - lactone ring with various ring substituents. They can be further classified into subgroups, depending on the ring size and other characteristics. The macrolides, for example, contain 12-, 14-, 16-, or 17-membered lactone rings glycosidically linked to one or more aminosugars and/or deoxysugars. They are inhibitors of protein synthesis, and are particularly effective against gram-positive bacteria. Erythromycin A, a well-studied macrolide produced by Saccharopolyspora erythraea, consists of a 14-membered lactone ring linked to two deoxy sugars.

Still another class of molecules which can be provided by the subject method are quinones and derivatives thereof. Quinones can be broadly classified into subgroups according to the number of aromatic rings present, i.e., benzoquinones, napthoquinones, etc. A well studied group is the tetracyclines, which contain a naphthacene ring with different substituents. Tetracyclines are protein synthesis inhibitors and are effective against both gram-positive and gram-negative bacteria, as well as rickettsias, mycoplasma, and spirochetes. The aromatic rings in the tetracyclines are derived from polyketide molecules. Derivatives of several other types of small molecule products are also likely to be identified by the subject method. One of these is the antibiotic 2-hexyl-5-propylresorcinol which is produced by certain strains of Pseudomonas. It was first isolated from the Pseudomonas strain B-9004 (Kanda et al. (1975) J. Antibiot. 28:935-942) and is a dialkyl- substituted derivative of 1,3-dihydroxybenzene. It has been shown to have antipathogenic activity against Gram-positive bacteria (in particular Clavibacter sp.), mycobacteria, and fungi. Another class are the methoxyacrylates, such as strobilurin B. Strobilurin B is produced by Basidiomycetes and has a broad spectrum of fungicidal activity (Anke et al. (1977) Journal of Antibiotics (Tokyo) 30:806-810). In particular, strobilurin B is produced by the fungus Bolinia lutea. Strobilurin B appears to have antifungal activity as a result of its ability to inhibit cytochrome-b dependent electron transport thereby inhibiting respiration (Becker et al. (1981) FEBS Letters 132:329-333.

Nematicidal Agents

In another aspect, the subject methods are useful for the identification of extracts or compounds which can be used to control pests and, in particular, plant pests. Specifically, the subject method can be used to identify new toxins useful for the control of nematodes. - 50 -

Certain extracts and compounds of the subject invention can also be used to control coleopteran pests, including corn rootworm.

Control of nematodes, or coleopterans, using such extracts or compounds can be accomplished by a variety of methods known to those skilled in the art. These methods include, for example, the application of the extract or compound to the pests or their niche.

Exemplary assays formats for detecting nematicidal agents include: a. The Split-pot test: this test detects anti-nematode agents having a repellent or antifeedant effect on the nematodes and/or a nematicidal effect. A 'split-pot', i.e. a pot divided into two sections by a fine mesh material (see Alphey et al (1988) Revue Nematol. 11:399-404), can be used. Each side is filled with soil. Test extracts or compounds are added to the soil on the side in which a seedling (Petunia) has been planted. To the other side a population of nematodes, e.g., adult Xiphinema diversicaudatum, are added. After a certain period of time, the two halves of the pot are separated and the nematodes extracted from the soil in each half. Root galls are recorded on plants from the treated sides (antifeedant action). The numbers of live and dead nematodes from each half are also counted (nematotoxic effect). b. Mini-pot test: This test identifies the nematicidal effect of a test extract or compound in soil and its effect on nematode feeding behavior. Briefly, seedlings (Petunia) are planted in soil. The test extracts, along with a population of nematodes, is added to the soil. Some time later, the nematodes are extracted and the number of galls induced by nematode feeding on the roots are determined.

Identification of Compounds Responsible for the Biological Activities

The biological activity can be further characterized by purifying the compound(s) responsible for the activity of a subject extract using standard methods, such as liquid-liquid, liquid-solid, or affinity chromatography with normal phase, reverse-phase, ion-exchange, and gel filtration techniques being implemented as needed (Box, (1991) in Discovery and Isolation of Microbial Products. Verall, M. S., Ed., Ellis Horwood, Chichester, 1985; Franco et al. (1991) Crit. Rev, in Biotech. 11:193-276). The purification process can be monitored by co-fractionation of the biological activity, using any of the screening assays described - 51 - above. Once purified, the structure of the compound can be determined using standard methods, including nuclear magnetic resonance spectroscopy, mass spectrometry, and X-ray crystallography.

VII. 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. Example 1

Composted soil (1 kg) was extracted with organic solvent, counter-extracted with hexane, concentrated by evaporation and redissolved in ethyl acetate. The extract had an antibacterial and anti-yeast activity with an minimum inhibitory concentration (MIC) at which growth is completely suppressed of 300 μg/mL. A similar result was obtained from two additional resamplings of the same source at different times.

Example 2

A marine sample (1 kg) rich in microorganisms was extracted with an organic solvent. The extract was evaporated and the solid residue redissolved in a small volume of DMSO. The extract was then tested for antimicrobial activity using Staphylococcus aureus and yeast, Saccharomyces cerevisiae. The extract showed good activity against both microorganisms. The extraction was repeated from the same area on dates separated by 1 to 2 months. The four extracts had comparable activities. Minimal inhibitory concentration (MIC) of the crude extract was 100 μg/mL.

Example 3

Marine sediment was sampled from Nahant bay on 15 separate occasions in different locations and at different times of the year, from June through November. The composition of the community of microorganisms at different locations and different seasons varies - 52 - significantly. Each sample (1 - 2 kg) was extracted with ethyl acetate as described above, and the extracts were tested for antimicrobial activity against S. aureus. The minimal inhibitory activity as determined by the microtiter plate broth microdilution method varied among extracts between 0.1 and 2.5 mg/ml. This survey demonstrates that the method described herein can be used to extract antimicrobial agents from randomly selected natural sources with a high probability of success.

All of the references and publications cited herein are hereby incorporated by reference.

- 53 -

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. Such equivalents are intended to be encompassed by the following claims.

Claims

- 54 -

1. A method for obtaining novel natural products, comprising: providing a sample of soil; contacting the sample of soil with a solvent to extract components of the soil into the solvent; separating solid residue from the solvent and the components dissolved therein; concentrating the solvent and components dissolved therein to give cocentrated material; and testing a sample of the concentrated material in an assay for biological activity.

2. The method of claim 1, wherein the sample of soil is terrestrial soil.

3. The method of claim 1 , wherein the sample of soil is marine soil.

4. The method of claim 1 , wherein the sample of soil consists of less than 100 kg of soil.

5. The method of claim 1 , wherein the sample of soil consists of less than 10 kg of soil.

6. The method of claim 1, wherein the sample of soil consists of less than 1 kg of soil.

7. The method of claim 1 , wherein the solvent is an organic solvent.

8. The method of claim 1, wherein the solvent is selected from the group consisting of supercritical carbon dioxide and supercritical water.

9. The method of claim 1 , further comprising the step of purifying the concentrated material. - 55 -

10. The method of claim 9, wherein the step of purifying the concentrated material comprises at least one method selected from the group consisting of extraction, fractionation, liquid- liquid chromatography, liquid-solid chromatography, affinity chromatography, normal- phase filtration, reverse-phase filtration, ion-exchange filtration, and gel filtration.

11. The method of claim 1, wherein the assay is selected to identify a component of the concentrated material selected from the group consisting of antibacterial agents, antifungal agents, cytotoxic drugs, and viral inhibitors.

12. The method of claim 1, wherein the assay is selected to identify a component of the concentrated material selected from the group consisting of antitumor agents and cell cycle regulators.

13. The method of claim 1, wherein the assay is selected to identify a component of the concentrated material selected from the group consisting of immune stimulants and immunosuppressants.

14. The method of claim 1, further comprising the step of treating the sample of soil to lyse cells in the sample of soil.

15. A method for identifying a biologically active compound, comprising: the method of claim 1; and determining the molecular structure of a component of a sample of soil which exhibits biological activity.

16. A component identified by the method of claim 15. - 56 -

17. A method for identifying a drug candidate, comprising: the method of claim 15; and preparing a new compound having a structure related to the molecular structure of the component which exhibits biological activity; and testing the new compound in an assay for biological activity.

18. A compound identified by the method of claim 17.

19. A method for manufacturing a pharmaceutical preparation, comprising: the method of claim 15; and formulating the component as a pharmaceutical preparation suitable for administration to a patient.

20. A method for manufacturing a pharmaceutical preparation, comprising: the method of claim 17; and formulating the compound as a pharmaceutical preparation suitable for administration to a patient.

21. A pharmaceutical preparation, comprising a compound of claim 16 formulated for administration to a patient.

22. A pharmaceutical preparation comprising a compound of claim 18 formulated for administration to a patient.

23. A method for treating a medical condition, comprising: - 57 - the method of claim 19; and administering the pharmaceutical preparation to a patient.

24. A method for treating a medical condition, comprising: the method of claim 20; and administering the pharmaceutical preparation to a patient.

25. A method for treating a medical condition, comprising administering to a patient a pharmaceutical preparation of claim 21.

26. A method for treating a medical condition, comprising administering to a patient a pharmaceutical preparation of claim 22.

27. A method for obtaining novel natural products, comprising: providing a sample of soil; separating cells from the soil; contacting the cells with a solvent to extract components; concentrating the solvents and components dissolved therein; and testing a sample of the concentrated components in an assay for biological activity.

28. The method of claim 27, further comprising the step of treating the cells to lyse the cells.

29. A method for obtaining novel natural products, comprising: providing a sample of aquatic water; contacting the sample of aquatic water with a solvent to extract components of - 58 - the water into the solvent; separating the water from the solvent; concentrating the solvent and components dissolved therein; and testing a sample of the concentrated components in an assay for biological activity.