A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level

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.