High volume nutrient based yeast two-hybrid assay for the identification of specific protein:protein interacting inhibitors

Abstract

This invention describes how to make and use several effective high volume screens for the discovery of inhibitors of specific protein:protein interactions, using aspects of the yeast two-hybrid system.

We claim: 
     
       1. A method for identifying useful compounds comprising: a) the identification of about 1 to 20 protein::protein interactions of interest, where the protein::protein interactions are indicated X::Y, where protein(s) X and protein(s) Y interact with each other at the cellular level, where the protein(s) X and protein(s) Y may be the same or different proteins,   b) the cloning of one set of proteins, either X or Y, as fusions, or as a set of fusions, to the DNA binding domain of the DNA that codes for a transcription activator protein,   c) the cloning of the other set of proteins, either X or Y, as fusions, or as a set of fusions, to the DNA activation domain of the DNA that codes for a transcription activator protein,   d) the maintaining of the sets of fusions as extrachromosomal elements in cells or the maintaining of the sets of fusions integrated in a yeast chromosome, where the extrachromosomal elements may be plasmids,   e) the introduction of said sets of fusions into several yeast screening strains, said yeast screening strains comprising one or more reporter gene cassette(s), said reporter gene cassette(s) comprising a reporter gene, that is URA3 and comprising a DNA binding site specific to the DNA binding domain of the DNA coding region for said transcription activator protein, and said reporter gene expressed by said yeast screening strains produces a product that converts, 5' Fluoro-orotic acid (FOA), a substance that is non-toxic to the yeast screening strain into a substance that is toxic to the yeast screening strain,   f) growing the yeast in the presence of test compounds, where the test compounds are the compounds to be assayed, and a) growing the yeast in selective medium, where the medium contains material needed for growth; or b) growing the strains, where the strains produce a measurable protein; or c) growing the strains, where the medium contains a non-toxic compound that can be converted by the product of the reporter gene into a substance that is toxic to the yeast screening strain,   g) selecting compounds that inhibit the expression of the reporter gene,   h) measuring said selected compounds that inhibit the expression of the reporter gene in a yeast testing strain, said yeast testing strain being isogenic with said yeast screening strain, and comprising a protein pair, protein A::protein B, where protein A and protein B, are proteins, the same or different from each other, and where protein(s) A and protein(s) B are the same or different from protein(s) X and protein(s) Y, and where protein pair, protein A::protein B are required by said yeast testing strain in order to activate said reporter gene and where said yeast testing strain is either dependent for growth upon the protein produced by said reporter gene, or where said yeast testing strain produces a protein by the reporter gene that is measurable,   i) selecting compounds that inhibit the growth of the screening strain but do not either: inhibit the growth of the testing strain or inhibit the measurable reporter gene.