Water-soluble and cell-permeant zwitterionic rhodamine dyes enable quantitative imaging of drug target engagement

Intracellular paired-agent imaging (iPAI) utilizes fluorescent-labeled small molecule therapeutics to measure drug target engagement. Our new water-soluble, cell-permeant fluorophores termed Sulfo-Rh and Sulfo-SiRh show substantially improved optical stability against solvent polarity changes over tetramethylrhodamine (TMR) and silicon-substituted TMR, respectively, while retaining the desirable photophysical properties of the base compounds, including brightness, to facilitate stable biological imaging. Targeted and untargeted versions of an EGFR tyrosine kinase inhibitor labeled with these fluorophores, demonstrated similarity to the parent drug in competitive binding and cytotoxicity assays. Utilizing iPAI to visualize drug target engagement enables quantitative small molecule imaging in living systems.