title: Identification of protein interaction antagonists using the repressed transactivator two-hybrid system.
abstract: The repressed transactivator (RTA) yeast two-hybrid system was developed to enable genetic identification of interactions with transcriptional activator proteins. We have devised modifications of this system that enable its use in screening for inhibitors of protein interactions from small molecule compound libraries. We show that inhibition of protein interactions can be measured by monitoring growth in selective medium containing 3-aminotriazole (3-AT) and using this assay have identified inhibitors of four independent protein interactions in screens with a 23,000 small molecule compound library. Compounds found to inhibit one of the tested interactions between FKBP12 and the transforming growth factor beta receptor (TGFbeta-R) were validated in vivo and found to inhibit calcineurin-dependent signaling in T cells. One of these compounds was also found to cause elevated basal expression of a TGFbeta-R/SMAD-dependent reporter gene. These results demonstrate the capability of the RTA small molecule screening assay for discovery of potentially novel therapeutic compounds.
authors.affiliation: Interomex Biopharmaceuticals, Vancouver, BC, Canada.
authors.forename: Phalgun B
authors.initials: PB
authors.lastname: Joshi
authors.forename: Martin
authors.initials: M
authors.lastname: Hirst
authors.forename: Tom
authors.initials: T
authors.lastname: Malcolm
authors.forename: Jennifer
authors.initials: J
authors.lastname: Parent
authors.forename: David
authors.initials: D
authors.lastname: Mitchell
authors.forename: Karen
authors.initials: K
authors.lastname: Lund
authors.forename: Ivan
authors.initials: I
authors.lastname: Sadowski
mesh_terms: D004353:Drug Evaluation, Preclinical; D017930:Genes, Reporter; D006801:Humans; D019169:Jurkat Cells; D008024:Ligands; D004364:Pharmaceutical Preparations / Q000378:metabolism; D011485:Protein Binding; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D015398:Signal Transduction; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism; D015534:Trans-Activators / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D008024:Ligands; D004364:Pharmaceutical Preparations; D018125:Receptors, Transforming Growth Factor beta; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D022061:Tacrolimus Binding Protein 1A