title: A functionally orthogonal estrogen receptor-based transcription switch specifically induced by a nonsteroid synthetic ligand.
abstract: It is highly desirable to design ligand-dependent transcription regulation systems based on transactivators unresponsive to endogenous ligands but induced by synthetic small molecules unable to activate endogenous receptors. Using molecular modeling and yeast selection, we identified an estrogen receptor ligand binding domain double mutant (L384M, M421G) with decreased affinity to estradiol and enhanced binding to compounds inactive on estrogen receptors. Nonresponsiveness to estrogen was achieved by additionally adding the G521R substitution while introducing an "antagonistic-type" side chain in the compound, as in 4-hydroxytamoxifen. The triple-substituted ligand binding domain is insensitive to physiological concentrations of estradiol and has nanomolar affinity for the ligand. In this binary system, both receptor and ligand are, therefore, reciprocally specific. The mutated variant in the context of a chimeric transcription factor provides tight, ligand-dependent regulation of reporter gene expression.
authors.affiliation: Instituto di Ricerche di Biologia Molecolare P. Angeletti, MRL-Rome, Department of Biochemistry, Via Pontina km 30,600, Pomezia 00040, Italy. paola_gallinari@merck.com
authors.forename: Paola
authors.initials: P
authors.lastname: Gallinari
authors.forename: Armin
authors.initials: A
authors.lastname: Lahm
authors.forename: Uwe
authors.initials: U
authors.lastname: Koch
authors.forename: Chantal
authors.initials: C
authors.lastname: Paolini
authors.forename: Maria Chiara
authors.initials: MC
authors.lastname: Nardi
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Roscilli
authors.forename: Olaf
authors.initials: O
authors.lastname: Kinzel
authors.forename: Daniela
authors.initials: D
authors.lastname: Fattori
authors.forename: Ester
authors.initials: E
authors.lastname: Muraglia
authors.forename: Carlo
authors.initials: C
authors.lastname: Toniatti
authors.forename: Riccardo
authors.initials: R
authors.lastname: Cortese
authors.forename: Raffaele
authors.initials: R
authors.lastname: De Francesco
authors.forename: Gennaro
authors.initials: G
authors.lastname: Ciliberto
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D015195:Drug Design; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D015003:Yeasts / Q000235:genetics
chemical_list: D047628:Estrogen Receptor alpha; D008024:Ligands; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004958:Estradiol