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

Valaka AP, Nyström H, Håversen L, Benitez-Martin C, Schäfer C, Jang WS, Camponeschi A, Andréasson J, Borén J, Grøtli M

RSC Chem Biol 6 (4) 618-629 [2025-04-02; online 2025-02-20]

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.

Glycoproteomics and MS Proteomics [Service]

PubMed 40026844

DOI 10.1039/d4cb00313f

Crossref 10.1039/d4cb00313f

pmc: PMC11867108
pii: d4cb00313f