Methods for the identification of inhibitors of 3-isopropylmalate dehydratase as antibiotics

A method for identifying a test compound as a candidate for an antibiotic, 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 3-Isopropylmalate dehydratase;
• a polypeptide having at least 50% sequence identity with a fungal 3-Isopropylmalate dehydratase;
• and a polypeptide having at least 10% of the activity of a fungal 3-Isopropylmalate dehydratase;
• 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.

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

• a) contacting 2-Isopropylmalate and H2O with a 3-Isopropylmalate dehydratase;
• b) contacting 2-Isopropylmalate and H2O with 3-Isopropylmalate dehydratase and a test compound;
• and
• c) determining the change in concentration for at least one of the following:
• 2-Isopropylmalate, H2O, and/or 3-Isopropylmalate,
• wherein a change in concentration for any of the above substances between steps (a) and
• (b) indicates that said test compound is a candidate for an antibiotic.

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

• a) contacting 3-Isopropylmalate with a 3-Isopropylmalate dehydratase;
• b) contacting 3-Isopropylmalate with a 3-Isopropylmalate dehydratase and a test compound;
• and
• c) determining the change in concentration for at least one of the following:
• 2-Isopropylmalate, H2O, and/or 3-Isopropylmalate,
• wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

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

• a) contacting 2-Isopropylmalate and H2O with a polypeptide selected from the group consisting of:
• a polypeptide having at least 50% sequence identity with 3-Isopropylmalate dehydratase;
• a polypeptide having at least 50% sequence identity with a 3-Isopropylmalate dehydratase and having at least 10% of the activity thereof;
• and a polypeptide comprising at least 100 consecutive amino acids of a 3-Isopropylmalate dehydratase;
• b) contacting 2-Isopropylmalate and H2O with said polypeptide and a test compound;
• and
• c) determining the change in concentration for at least one of the following:
• 2-Isopropylmalate, H2O, and/or 3-Isopropylmalate,
• wherein a change in concentration for any of the above substances between steps (a) and (b) indicates that said test compound is a candidate for an antibiotic.

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

• a) contacting 3-Isopropylmalate with a polypeptide selected from the group consisting of:
• a polypeptide having at least 50% sequence identity with a 3-Isopropylmalate dehydratase;
• a polypeptide having at least 50% sequence identity with a 3-Isopropylmalate dehydratase and at least 10% of the activity thereof;
• and a polypeptide comprising at least 100 consecutive amino acids of a 3-Isopropylmalate dehydratase;
• b) contacting 3-Isopropylmalate, with said polypeptide and a test compound;
• and
• c) determining the change in concentration for at least one of the following:
• 2-Isopropylmalate, H2O, and/or 3-Isopropylmalate,
• wherein a change in concentration for any of the above substances between steps (a) and
• (b) indicates that said test compound is a candidate for an antibiotic.

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

• a) measuring the expression of a 3-Isopropylmalate dehydratase 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 3-Isopropylmalate dehydratase in said cell, cells, tissue, or organism;
• and
• c) comparing the expression of 3-Isopropylmalate dehydratase in steps (a) and (b),
• wherein a lower expression in the presence of said test compound indicates that said test compound is a candidate for an antibiotic.

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

• a) providing cells having one form of a 3-Isopropylmalate dehydratase gene, and providing comparison cells having a different form of a 3-Isopropylmalate dehydratase gene;
• and
• b) contacting said cells and said comparison cells with a test compound and determining the growth of said cells and 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 compound indicates that said compound is a candidate for an antibiotic.

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

• a) providing cells having one form of a gene in the L-leucine 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 said test compound,
• 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.

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

• (a) providing paired growth media;
• comprising a first medium and a second medium, wherein said second medium contains a higher level of L-leucine 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.