title: Displacement of protein-bound aptamers with small molecules screened by fluorescence polarization.
abstract: Small molecule inhibitors of proteins are invaluable tools in research and as starting points for drug development. However, their screening can be tedious, as most screening methods have to be tailored to the corresponding drug target. Here, we describe a detailed protocol for a modular and generally applicable assay for the identification of small organic compounds that displace an aptamer complexed to its target protein. The method relies on fluorescence-labeled aptamers and the increase of fluorescence polarization upon their binding to the target protein. The assay has high Z'-factors, making it compatible with high-throughput screening. It allows easy automation, making fluorescence readout the time-limiting step. As aptamers can be generated for virtually any protein target, the assay allows identification of small molecule inhibitors for targets or individual protein domains for which no functional screen is available. We provide the step-by-step protocol to screen for antagonists of the cytohesin class of small guanosine exchange factors.
authors.affiliation: Program Unit Chemical Biology & Medicinal Chemistry, LIMES Institute, c/o Kekulé Institut für Organische Chemie und Biochemie, University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Hafner
authors.forename: Elena
authors.initials: E
authors.lastname: Vianini
authors.forename: Barbara
authors.initials: B
authors.lastname: Albertoni
authors.forename: Laura
authors.initials: L
authors.lastname: Marchetti
authors.forename: Imke
authors.initials: I
authors.lastname: Grüne
authors.forename: Christian
authors.initials: C
authors.lastname: Gloeckner
authors.forename: Michael
authors.initials: M
authors.lastname: Famulok
mesh_terms: D052157:Aptamers, Nucleotide / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005454:Fluorescence Polarization / Q000379:methods*; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D052157:Aptamers, Nucleotide; D020662:Guanine Nucleotide Exchange Factors