title: A mammalian genetic system to screen for small molecules capable of disrupting protein-protein interactions.
abstract: A mammalian two-hybrid system was developed for high-throughput screening of compounds that disrupt specific protein-protein interactions. The existing mammalian systems are unsatisfactory for drug screening due to nonregulated expression of interacting proteins. To construct a tightly regulated system, the tetracycline repressor was fused with the inhibitory KRAB domain as a suppressor. The binding of the suppressor to the tet operator entirely blocked expression of two interacting proteins. When both the inducer doxycycline and drugs were added to the culture, the reporter gene was either activated by interaction of the paired proteins with ineffective drugs or remained silent due to disruption of the protein interactions by the effective drugs. We demonstrate that interactions of the type I receptor for TGFbeta with FKBP12 and the epidermal growth factor receptor (EGFR) with p85 are effectively disrupted by FK506 and EGFR kinase inhibitor AG1478, respectively. The power of this system for drug screening was further demonstrated by rapid identification of inhibitors from a druglike library for the receptor kinases.
authors.affiliation: Health Sector, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
authors.forename: Hui-Fen
authors.initials: HF
authors.lastname: Zhao
authors.forename: Taira
authors.initials: T
authors.lastname: Kiyota
authors.forename: Shafinaz
authors.initials: S
authors.lastname: Chowdhury
authors.forename: Enrico
authors.initials: E
authors.lastname: Purisima
authors.forename: Denis
authors.initials: D
authors.lastname: Banville
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Konishi
authors.forename: Shi-Hsiang
authors.initials: SH
authors.lastname: Shen
mesh_terms: D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D005818:Genetic Engineering; D006801:Humans; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology*
chemical_list: D004268:DNA-Binding Proteins; D011506:Proteins; D018125:Receptors, Transforming Growth Factor beta; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D013752:Tetracycline; D004318:Doxycycline