Methods for the identification of inhibitors of homocitrate synthase as antibiotics

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

• a) contacting acetyl-CoA and H2O and 2-oxoglutarate with a homocitrate synthase;
• b) contacting acetyl-CoA and H2O and 2-oxoglutarate with a homocitrate synthase and said test compound;
• and
• c) determining the change in concentration for at least one of the following 2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate, acetyl-CoA, CoA, and/or H2O;
• 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-hydroxybutane-1,2,4-tricarboxylate and CoA with a homocitrate synthase;
• b) contacting 2-hydroxybutane-1,2,4-tricarboxylate and CoA with a homocitrate synthase and said test compound;
• and
• c) determining the change in concentration for at least one of the following:
• 2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate, acetyl-CoA, CoA, and/or H2O;
• 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 acetyl-CoA and H2O and 2-oxoglutarate with a polypeptide selected from the group consisting of:
• a polypeptide having at least 50% sequence identity with a homocitrate synthase, a polypeptide having at least 50% sequence identity with a homocitrate synthase and having at least 10% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a homocitrate synthase
• b) contacting acetyl-CoA and H2O and 2-oxoglutarate with said polypeptide and said test compound;
• and
• c) determining the change in concentration for at least one of the following:
• 2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate, acetyl-CoA, CoA, and/or H2O;
• 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-hydroxybutane-1,2,4tricarboxylate and CoA with a polypeptide selected from the group consisting of:
• a polypeptide having at least 50% sequence identity with a homocitrate synthase, a polypeptide having at least 50% sequence identity with a homocitrate synthase and at least 10% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a homocitrate synthase
• b) contacting 2-hydroxybutane-1,2,4-tricarboxylate and CoA, with said polypeptide and said test compound;
• and
• c) determining the change in concentration for at least one of the following:
• 2-hydroxybutane-1,2,4-tricarboxylate, 2-oxoglutarate, acetyl-CoA, CoA, and/or H2O;
• 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 homocitrate synthase in a cell, cells, tissue, or an organism in the absence of said compound;
• b) contacting said cell cells, tissue, or organism with said test compound and measuring the expression of said homocitrate synthase in said fungus or fungal cell;
• c) comparing the expression of homocitrate 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.

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 lysine biochemical and/or genetic pathway and providing comparison cells having a different form of said gene,
• b) contacting said cells and comparison cells with a said test compound,
• c) determining the growth of said cells and 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 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 paired growth media;
• comprising a first medium and a second medium, wherein said second medium contains a higher level of lysine than said first medium;
• (b) contacting an organism with said test compound;
• (c) inoculating said first and 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 second media indicates that said test compound is a candidate for an antibiotic.