A GAL4-based yeast three-hybrid system for the identification of small molecule–target protein interactions

We report the development of a yeast strain designed for assaying compound–protein interactions through activation of reporter gene expression. Activation of lacZ expression, driven by the GAL4 promoter, has been demonstrated for precedented compound–protein interactions between FK506 and FK506 binding protein 12 (FKBP12) and also between methotrexate (MTX) and dihydrofolate reductase (DHFR). Reporter gene expression was completely abrogated in a competitive manner by the presence of excess FK506 or MTX, respectively. In addition, a strain expressing a mutated DHFR clone with decreased binding affinity for MTX was not capable of activating reporter gene expression. While strain sensitivity is compound-dependent, the minimum compound concentration necessary to drive reporter gene expression was 20nM for the FK506–FKBP12 interaction. The utility of this strain as a tool for identifying unknown compound-binding proteins has been demonstrated by screening a mouse cDNA library for clones that encode proteins capable of binding MTX. Four library clones of mouse DHFR were identified after screening 5×106 clones. The screen background was low and false positives were easily identified, making this yeast system particularly amenable for use in a screening context for novel compound–protein interactions.