title: A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level.
abstract: Late stage failures of candidate drug molecules are frequently caused by off-target effects or inefficient target engagement in vivo. In order to address these fundamental challenges in drug discovery, we developed a modular probe strategy based on bioorthogonal chemistry that enables the attachment of multiple reporters to the same probe in cell extracts and live cells. In a systematic evaluation, we identified the inverse electron demand Diels-Alder reaction between trans-cyclooctene labeled probe molecules and tetrazine-tagged reporters to be the most efficient bioorthogonal reaction for this strategy. Bioorthogonal biotinylation of the probe allows the identification of drug targets in a chemoproteomics competition binding assay using quantitative mass spectrometry. Attachment of a fluorescent reporter enables monitoring of spatial localization of probes as well as drug-target colocalization studies. Finally, direct target occupancy of unlabeled drugs can be determined at single cell resolution by competitive binding with fluorescently labeled probe molecules. The feasibility of the modular probe strategy is demonstrated with noncovalent PARP inhibitors.
authors.affiliation: Cellzome GmbH, a GlaxoSmithKline Company , Meyerhofstrasse 1, D- 69117 Heidelberg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Rutkowska
authors.affiliation: Cellzome GmbH, a GlaxoSmithKline Company , Meyerhofstrasse 1, D- 69117 Heidelberg, Germany.
authors.forename: Douglas W
authors.initials: DW
authors.lastname: Thomson
authors.affiliation: Cellzome GmbH, a GlaxoSmithKline Company , Meyerhofstrasse 1, D- 69117 Heidelberg, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Vappiani
authors.affiliation: Cellzome GmbH, a GlaxoSmithKline Company , Meyerhofstrasse 1, D- 69117 Heidelberg, Germany.
authors.forename: Thilo
authors.initials: T
authors.lastname: Werner
authors.affiliation: Cellzome GmbH, a GlaxoSmithKline Company , Meyerhofstrasse 1, D- 69117 Heidelberg, Germany.
authors.forename: Katrin M
authors.initials: KM
authors.lastname: Mueller
authors.affiliation: Cellzome GmbH, a GlaxoSmithKline Company , Meyerhofstrasse 1, D- 69117 Heidelberg, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Dittus
authors.affiliation: Cellzome GmbH, a GlaxoSmithKline Company , Meyerhofstrasse 1, D- 69117 Heidelberg, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Krause
authors.affiliation: Cellzome GmbH, a GlaxoSmithKline Company , Meyerhofstrasse 1, D- 69117 Heidelberg, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Muelbaier
authors.affiliation: Cellzome GmbH, a GlaxoSmithKline Company , Meyerhofstrasse 1, D- 69117 Heidelberg, Germany.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Bergamini
authors.affiliation: Cellzome GmbH, a GlaxoSmithKline Company , Meyerhofstrasse 1, D- 69117 Heidelberg, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Bantscheff
mesh_terms: D006367:HeLa Cells; D006801:Humans; D015335:Molecular Probes / Q000737:chemistry*; D059010:Single-Cell Analysis
chemical_list: D015335:Molecular Probes