Design and application of a fluorescent probe for imaging of endogenous Bruton's tyrosine kinase with preserved enzymatic activity†

Fluorophore integration into proteins within living cells is essential for exploring proteins in their natural environment. Bruton's tyrosine kinase (BTK), is a validated oncology target and is crucial for B cell proliferation and activation. Developing BTK-labelling probes is key to understand BTK's dynamic signalling pathway. In this work, we aimed to develop a novel fluorescent labelling probe for endogenous BTK imaging while preserving its enzymatic activity. Evobrutinib, a second-generation BTK inhibitor with high selectivity, was chosen as the scaffold. We designed two probes, Evo-1 and Evo-2, with a BODIPY fluorescent group, guided by molecular modelling. The synthesis was achieved using optimised Suzuki–Miyaura cross-coupling and amide coupling reactions. Biochemical assays confirmed covalent binding to Cys481 of BTK while preserving its enzymatic activity. Labelling of endogenous BTK with Evo-2 with reduced off-target effects in Ramos cells was validated in cellular assays. The dynamic signalling pathway of BTK in its native environment was investigated by confocal microscopy with Evo-2. This methodology is a valuable asset in the chemical biology toolbox for studying protein dynamics and interactions in real time without interfering with the protein activity.