A functionally orthogonal estrogen receptor-based transcription switch specifically induced by a nonsteroid synthetic ligand

Paola Gallinari, Armin Lahm, Uwe Koch, Chantal Paolini, Maria Chiara Nardi, Giuseppe Roscilli, Olaf Kinzel, Daniela Fattori, Ester Muraglia, Carlo Toniatti, Riccardo Cortese, Raffaele De Francesco, Gennaro Ciliberto

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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.

Original languageEnglish
Pages (from-to)883-893
Number of pages11
JournalChemistry and Biology
Volume12
Issue number8
DOIs
Publication statusPublished - Aug 2005

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ASJC Scopus subject areas

  • Organic Chemistry

Cite this

Gallinari, P., Lahm, A., Koch, U., Paolini, C., Nardi, M. C., Roscilli, G., Kinzel, O., Fattori, D., Muraglia, E., Toniatti, C., Cortese, R., De Francesco, R., & Ciliberto, G. (2005). A functionally orthogonal estrogen receptor-based transcription switch specifically induced by a nonsteroid synthetic ligand. Chemistry and Biology, 12(8), 883-893. https://doi.org/10.1016/j.chembiol.2005.05.018