Title:  Methods for the identification of inhibitors of 5-aminolevulinate synthase as antibiotics

United States Patent:  6,689,578

Issued:  February 10, 2004

Inventors:  DeZwaan; Todd (Apex, NC); Lo; Sze-Chung (Durham, NC); Montenegro-Chamarro; Maria Victoria (Morrisville, NC); Frank; Sheryl (Durham, NC); Darveaux; Blaise (Hillsborough, NC); Mahanty; Sanjoy K. (Chapel Hill, NC); Heiniger; Ryan (Raleigh, NC); Skalchunes; Amy (Raleigh, NC); Pan; Huaqin (Apex, NC); Tarpey; Rex (Apex, NC); Shuster; Jeffrey (Chapel Hill, NC); Tanzer; Matthew M. (Durham, NC); Hamer; Lisbeth (Durham, NC); Adachi; Kiichi (Durham, NC)

Assignee:  Paradigm Genetics, Inc. (Research Triangle Park, NC)

Appl. No.:  007022

Filed:  December 6, 2001

Abstract

The present inventors have discovered that 5-Aminolevulinate synthase is essential for fungal pathogenicity. Specifically, the inhibition of 5-Aminolevulinate synthase gene expression in fungi results in no signs of successful infection or lesions. Thus, 5-Aminolevulinate synthase can be used as a target for the identification of antibiotics, preferably antifungals. Accordingly, the present invention provides methods for the identification of compounds that inhibit 5-Aminolevulinate synthase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably antifungals.

SUMMARY OF THE INVENTION

Surprisingly, the present inventors have discovered that in vivo disruption of the gene encoding 5-Aminolevulinate synthase in Magnaporthe grisea prevents or inhibits the pathogenicity of the fungus. Thus, the present inventors have discovered that 5-Aminolevulinate synthase is essential for normal rice blast pathogenicity, and can be used as a target for the identification of antibiotics, preferably fungicides. Accordingly, the present invention provides methods for the identification of compounds that inhibit 5-Aminolevulinate synthase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably fungicides.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have discovered that disruption of the ALAS1 gene and/or gene product inhibits the pathogenicity of Magnaporthe grisea. Thus, the inventors are the first to demonstrate that 5-Aminolevulinate synthase is a target for antibiotics, preferably antifungals.

Accordingly, the invention provides methods for identifying compounds that inhibit ALAS1 gene expression or biological activity of its gene product(s). Such methods include ligand binding assays, assays for enzyme activity, cell-based assays, and assays for ALAS1 gene expression. Any compound that is a ligand for 5-Aminolevulinate synthase may have antibiotic activity. For the purposes of the invention, "ligand" refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the invention are useful as antibiotics.

Thus, in one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising:

a) contacting a 5-Aminolevulinate synthase polypeptide with a test compound; and

b) detecting the presence or absence of binding between said test compound and said 5-Aminolevulinate synthase polypeptide;

wherein binding indicates that said test compound is a candidate for an antibiotic.

The 5-Aminolevulinate synthase protein may have the amino acid sequence of a naturally occurring 5-Aminolevulinate synthase found in a fungus, animal, plant, or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence. Preferably the 5-Aminolevulinate synthase is a fungal 5-Aminolevulinate synthase. The cDNA (SEQ ID NO: 1) encoding the M. grisea 5-Aminolevulinate synthase protein, the genomic DNA (SEQ ID NO: 2) encoding the protein, and the polypeptide (SEQ ID NO: 3) can be found herein.

In one aspect, the invention also provides for a polypeptide consisting essentially of SEQ ID NO: 3. For the purposes of the invention, a polypeptide consisting essentially of SEQ ID NO: 3 has at least 80% sequence identity with SEQ ID NO: 3 and catalyses the interconversion of succinyl-CoA and glycine with 5-aminolevulinate, CoA, and CO₂ with at least 10% of the activity of SEQ ID NO: 3. Preferably, the polypeptide consisting essentially of SEQ ID NO: 3 has at least 85% sequence identity with SEQ ID NO: 3, more preferably the sequence identity is at least 90%, most preferably the sequence identity is at least 95% or 97 or 99%, or any integer from 80-100% sequence identity in ascending order. And, preferably, the polypeptide consisting essentially of SEQ ID NO: 3 has at least 25%, at least 50%, at least 75% or at least 90% of the activity of M. grisea 5-Aminolevulinate synthase, or any integer from 60-100% activity in ascending order.

By "fungal 5-Aminolevulinate synthase" is meant an enzyme that can be found in at least one fungus, and which catalyzes the interconversion of succinyl-CoA and glycine with 5-aminolevulinate, CoA, and CO₂. The 5-Aminolevulinate synthase may be from any of the fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

In one embodiment, the 5-Aminolevulinate synthase is a Magnaporthe 5-Aminolevulinate synthase. Magnaporthe species include, but are not limited to, Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea and Magnaporthe poae and the imperfect states of Magnaporthe in the genus Pyricularia. Preferably, the Magnaporthe 5-Aminolevulinate synthase is from Magnaporthe grisea.

In various embodiments, the 5-Aminolevulinate synthase can be from Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Corn Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and the like.

Fragments of a 5-Aminolevulinate synthase polypeptide may be used in the methods of the invention, preferably if the fragments include an intact or nearly intact epitope that occurs on the biologically active wildtype 5-Aminolevulinate synthase. The fragments comprise at least 10 consecutive amino acids of a 5-Aminolevulinate synthase. Preferably, the fragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, or at least 610 consecutive amino acids residues of a 5-Aminolevulinate synthase. In one embodiment, the fragment is from a Magnaporthe 5-Aminolevulinate synthase. Preferably, the fragment contains an amino acid sequence conserved among fungal 5-Aminolevulinate synthases.

Polypeptides having at least 50% sequence identity with a fungal 5-Aminolevulinate synthase are also useful in the methods of the invention. Preferably, the sequence identity is at least 60%, more preferably the sequence identity is at least 70%, most preferably the sequence identity is at least 80% or 90 or 95 or 99%, or any integer from 60-100% sequence identity in ascending order.

In addition, it is preferred that the polypeptide has at least 10% of the activity of a fungal 5-Aminolevulinate synthase. More preferably, the polypeptide has at least 25%, at least 50%, at least 75% or at least 90% of the activity of a fungal 5-Aminolevulinate synthase. Most preferably, the polypeptide has at least 10%, at least 25%, at least 50%, at least 75% or at least 90% of the activity of the M. grisea 5-Aminolevulinate synthase protein.

Thus, in another embodiment, the invention provides a method for identifying a test compound as a candidate for a fungicide, comprising:

a) contacting a test compound with at least one polypeptide selected from the group consisting of: a polypeptide having at least ten consecutive amino acids of a fungal 5-Aminolevulinate synthase; a polypeptide having at least 50% sequence identity with a fungal 5-Aminolevulinate synthase; and a polypeptide having at least 10% of the activity of a fungal 5-Aminolevulinate synthase; and

b) detecting the presence and/or absence of binding between said test compound and said polypeptide;

wherein binding indicates that said test compound is a candidate for an antibiotic.

Any technique for detecting the binding of a ligand to its target may be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting the binding of a ligand to its target are known in the art, and include, but are not limited to the detection of an immobilized ligand-target complex or the detection of a change in the properties of a target when it is bound to a ligand. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligands are contacted with a 5-Aminolevulinate synthase protein or a fragment or variant thereof, the unbound protein is removed and the bound 5-Aminolevulinate synthase is detected. In a preferred embodiment, bound 5-Aminolevulinate synthase is detected using a labeled binding partner, such as a labeled antibody. In a variation of this assay, 5-Aminolevulinate synthase is labeled prior to contacting the immobilized candidate ligands. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetric methods.

Once a compound is identified as a candidate for an antibiotic, it can be tested for the ability to inhibit 5-Aminolevulinate synthase enzymatic activity. The compounds can be tested using either in vitro or cell based assays. Alternatively, a compound can be tested by applying it directly to a fungus or fungal cell, or expressing it therein, and monitoring the fungus or fungal cell for changes or decreases in growth, development, viability, pathogenicity, or alterations in gene expression. Thus, in one embodiment, the invention provides a method for determining whether a compound identified as an antibiotic candidate by an above method has antifungal activity, further comprising: contacting a fungus or fungal cells with said antifungal candidate and detecting a decrease in the growth, viability, or pathogenicity of said fungus or fungal cells.

By decrease in growth, is meant that the antifungal candidate causes at least a 10% decrease in the growth of the fungus or fungal cells, as compared to the growth of the fungus or fungal cells in the absence of the antifungal candidate. By a decrease in viability is meant that at least 20% of the fungal cells, or portion of the fungus contacted with the antifungal candidate are nonviable. Preferably, the growth or viability will be decreased by at least 40%. More preferably, the growth or viability will be decreased by at least 50%, 75% or at least 90% or more. Methods for measuring fungal growth and cell viability are known to those skilled in the art. By decrease in pathogenicity, is meant that the antifungal candidate causes at least a 10% decrease in the disease caused by contact of the fungal pathogen with its host, as compared to the disease caused in the absence of the antifungal candidate. Preferably, the disease will be decreased by at least 40%. More preferably, the disease will be decreased by at least 50%, 75% or at least 90% or more. Methods for measuring fungal disease are well known to those skilled in the art, and include such metrics as lesion formation, lesion size, sporulation, respiratory failure, and/or death.

The ability of a compound to inhibit 5-Aminolevulinate synthase activity can be detected using in vitro enzymatic assays in which the disappearance of a substrate or the appearance of a product is directly or indirectly detected. 5-Aminolevulinate synthase catalyzes the irreversible or reversible reaction succinyl-CoA and glycine=5-aminolevulinate, CoA, and CO₂ (see FIG. 1). Methods for detection of succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂, include spectrophotometry, mass spectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.

Thus, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising:

a) contacting succinyl-CoA and glycine with a 5-Aminolevulinate synthase;

b) contacting succinyl-CoA and glycine with 5-Aminolevulinate synthase and a test compound; and

c) determining the change in concentration for at least one of the following: succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂.

wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising:

a) contacting 5-aminolevulinate, CoA, and CO₂ with a 5-Aminolevulinate synthase;

b) contacting 5-aminolevulinate, CoA, and CO₂ with a 5-Aminolevulinate synthase and a test compound; and

c) determining the change in concentration for at least one of the following: succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂.

wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

Enzymatically active fragments of a fungal 5-Aminolevulinate synthase are also useful in the methods of the invention. For example, an enzymatically active polypeptide comprising at least 100 consecutive amino acid residues of a fungal 5-Aminolevulinate synthase may be used in the methods of the invention. In addition, an enzymatically active polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identity with a fungal 5-Aminolevulinate synthase may be used in the methods of the invention. Most preferably, the polypeptide has at least 50% sequence identity with a fungal 5-Aminolevulinate synthase and at least 10%, 25%, 75% or at least 90% of the activity thereof.

Thus, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising:

a) contacting succinyl-CoA and glycine with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with a 5-Aminolevulinate synthase; a polypeptide having at least 50% sequence identity with a 5-Aminolevulinate synthase and having at least 10% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a 5-Aminolevulinate synthase;

b) contacting succinyl-CoA and glycine with said polypeptide and a test compound; and

c) determining the change in concentration for at least one of the following: succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂ ;

wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a test compound as a candidate for an antibiotic, comprising:

a) contacting 5-aminolevulinate, CoA, and CO₂ with a polypeptide selected from the group consisting of: a polypeptide having at least 50% sequence identity with a 5-Aminolevulinate synthase; a polypeptide having at least 50% sequence identity with a 5-Aminolevulinate synthase and at least 10% of the activity thereof; and a polypeptide comprising at least 100 consecutive amino acids of a 5-Aminolevulinate synthase;

b) contacting 5-aninolevulinate, CoA, and CO₂, with said polypeptide and a test compound; and

c) determining the change in concentration for at least one of the following, succinyl-CoA, glycine, 5-aminolevulinate, CoA, and/or CO₂ ;

wherein a change in concentration for any of the above substances indicates that said test compound is a candidate for an antibiotic.

For the in vitro enzymatic assays, 5-Aminolevulinate synthase protein and derivatives thereof may be purified from a fungus or may be recombinantly produced in and purified from an archael, bacterial, fungal, or other eukaryotic cell culture. Preferably these proteins are produced using an E. Coli, yeast, or filamentous fungal expression system. Methods for the purification of 5-Aminolevulinate synthase may be described in Volland and Felix (1984) Eur J Biochem 142: 551-7 (PMID: 6381051). Other methods for the purification of 5-Aminolevulinate synthase proteins and polypeptides are known to those skilled in the art.

As an alternative to in vitro assays, the invention also provides cell based assays. In one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising:

a) measuring the expression of a 5-Aminolevulinate synthase in a cell, cells, tissue, or an organism in the absence of a test compound;

b) contacting said cell, cells, tissue, or organism with said test compound and measuring the expression of said 5-Aminolevulinate synthase in said cell, cells, tissue, or organism; and

c) comparing the expression of 5-Aminolevulinate synthase in steps (a) and (b);

wherein a lower expression in the presence of said test compound indicates that said compound is a candidate for an antibiotic.

Expression of 5-Aminolevulinate synthase can be measured by detecting the ALAS1 primary transcript or mRNA, 5-Aminolevulinate synthase polypeptide, or 5-Aminolevulinate synthase enzymatic activity. Methods for detecting the expression of RNA and proteins are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York, 1995. The method of detection is not critical to the invention. Methods for detecting ALAS1 RNA include, but are not limited to amplification assays such as quantitative reverse transcriptase-PCR, and/or hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using an ALAS1 promoter fused to a reporter gene, DNA assays, and microarray assays.

Methods for detecting protein expression include, but are not limited to, immunodetection methods such as Westem blots, ELISA assays, polyacrylamide gel electrophoresis, mass spectroscopy, and enzymatic assays. Also, any reporter gene system may be used to detect ALAS1 protein expression. For detection using gene reporter systems, a polynucleotide encoding a reporter protein is fused in frame with ALAS1, so as to produce a chimeric polypeptide. Methods for using reporter systems are known to those skilled in the art.

Chemicals, compounds or compositions identified by the above methods as modulators, preferably inhibitors, of ALAS1 expression or activity can then be used to control fungal growth. Diseases such as rusts, mildews, and blights spread rapidly once established. Fungicides are thus routinely applied to growing and stored crops as a preventive measure, generally as foliar sprays or seed dressings. For example, compounds that inhibit fungal growth can be applied to a fungus or expressed in a fungus, in order to prevent fungal growth. Thus, the invention provides a method for inhibiting fungal growth, comprising contacting a fungus with a compound identified by the methods of the invention as having antifungal activity.

Antifungals and antifungal inhibitor candidates identified by the methods of the invention can be used to control the growth of undesired fungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

Examples of undesired fungi include, but are not limited to Powdery Scab (Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot (Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot (Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot (Polyporus squamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus (Armillaria gallica), Root rot (Armillaria luteobubalina), Shoestring Rot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichum musae), Apple-rotting Fungus (Monilinia fructigena), Apple-rotting Fungus (Penicillium expansum), Clubroot Disease (Plasmodiophora brassicae), Potato Blight (Phytophthora infestans), Root pathogen (Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis), Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromyces appendiculatus), Northern Leaf Spot (Cochliobolus carbonum), Milo Disease (Periconia circinata), Southern Corn Blight (Cochliobolus heterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe (Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), Wheat Head Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusarium culmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black Stem Rust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), diseases of animals such as infections of lungs, blood, brain, skin, scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp. Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp., and the like).

Also provided is a method of screening for an antibiotic by determining whether a test compound is active against the gene identified (SEQ ID NO: 1 or SEQ ID NO: 2), its gene product (SEQ ID NO: 3), or the biochemical pathway or pathways it functions on.

In one particular embodiment, the method is performed by providing an organism having a first form of the gene corresponding to either SEQ ID NO: 1 or SEQ ID NO: 2, either a normal form, a mutant form, a homologue, or a heterologous ALAS1 gene that performs a similar function as ALAS1. The first form of ALAS1 may or may not confer a growth conditional phenotype, i.e., a 5-aminolevulinate requiring phenotype, and/or a hypersensitivity or hyposensitivity phenotype on the organism having that altered form. In one particular embodiment a mutant form contains a transposon insertion. A comparison organism having a second form of an ALAS1, different from the first form of the gene is also provided, and the two organisms are separately contacted with a test compound. The growth of the two organisms in the presence of the test compound is then compared.

Thus, in one embodiment, the invention provides a method for identifying a test compound as a candidate for an antibiotic, comprising:

a) providing cells having one form of a 5-Aminolevulinate synthase gene, and providing comparison cells having a different form of a 5-Aminolevulinate synthase gene; and

b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and said comparison cells in the presence of the test compound,

wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

It is recognized in the art that the optional determination of the growth of said first organism and said comparison second organism in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different genes. It is also recognized that any combination of two different forms of an ALAS1 gene, including normal genes, mutant genes, homologues, and functional homologucs may be used in this method. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like. In a preferred embodiment the organism is Magnaporthe grisea.

Conditional lethal mutants may identify particular biochemical and/or genetic pathways given that at least one identified target gene is present in that pathway. Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics as inhibitors of the substrates, products and enzymes of the pathway. Pathways known in the art may be found at the Kyoto Encyclopedia of Genes and Genomes and in standard biochemistry texts (Lehninger, A., D. Nelson, et al. (1993) Principles of Biochemistry. New York, Worth Publishers).

Thus, in one embodiment, the invention provides a method for screening for test compounds acting against the biochemical and/or genetic pathway or pathways in which ALAS1 functions, comprising:

a) providing cells having one form of a gene in the heme biochemical and/or genetic pathway and providing comparison cells having a different form of said gene;

b) contacting said cells and said comparison cells with a test compound; and

c) determining the growth of said cells and said comparison cells in the presence of said test compound;

wherein a difference in growth between said cells and said comparison cells in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

The use of multi-well plates for screening is a format that readily accommodates multiple different assays to characterize various compounds, concentrations of compounds, and fungal strains in varying combinations and formats. Certain testing parameters for the screening method can significantly affect the identification of growth inhibitors, and thus can be manipulated to optimize screening efficiency and/or reliability. Notable among these factors are variable sensitivities of different mutants, increasing hypersensitivity with increasingly less permissive conditions, an apparent increase in hypersensitivity with increasing compound concentration, and other factors known to those in the art.

Conditional lethal mutants may identify particular biochemical and/or genetic pathways given that at least one identified target gene is present in that pathway. Knowledge of these pathways allows for the screening of test compounds as candidates for antibiotics. Pathways known in the art may be found at the Kyoto Encyclopedia of Genes and Genomes and in standard biochemistry texts (Lehninger, A., D. Nelson, et al. (1993) Principles of Biochemistry. New York, Worth Publishers).

Thus, in one embodiment, the invention provides a method for screening for test compounds acting against the biochemical and/or genetic pathway or pathways in which ALAS1 functions, comprising:

(a) providing paired growth media comprising a first medium and a second medium, wherein said second medium contains a higher level of 5-aminolevulinate than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism;

wherein a difference in growth of the organism between said first and said second media indicates that said test compound is a candidate for an antibiotic.

It is recognized in the art that determination of the growth of said organism in the paired media in the absence of any test compounds may be performed to control for any inherent differences in growth as a result of the different media. Growth and/or proliferation of an organism is measured by methods well known in the art such as optical density measurements, and the like. In a preferred embodiment, the organism is Magnaporthe grisea.

Claim 1 of 31 Claims

What is claimed is:

1. A method for identifying a test compound as a candidate for an antibiotic, comprising:

a) contacting a 5-Aminolevulinate synthase polypeptide with a test compound; and

b) detecting the presence or absence of binding between said test compound and said 5-Aminolevulinate synthase polypeptide;

wherein binding indicates that said test compound is a candidate for an antibiotic.

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