Design and application of a fluorescent probe for imaging of endogenous Bruton's tyrosine kinase with preserved enzymatic activity
Journal article, 2025

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.

Agammaglobulinemia

Protein Tyrosine Kinase

Structure Activity Relationship

Author

Anna P. Valaka

University of Gothenburg

Hampus Nyström

University of Gothenburg

Liliana Håversen

Sahlgrenska University Hospital

Carlos Benitez-Martin

University of Gothenburg

Clara Schäfer

University of Gothenburg

Woo Suk Jang

University of Gothenburg

Alessandro Camponeschi

University of Gothenburg

Joakim Andreasson

Chalmers, Chemistry and Chemical Engineering, Chemistry and Biochemistry

Jan Borén

Sahlgrenska University Hospital

Morten Grötli

University of Gothenburg

RSC Chemical Biology

26330679 (eISSN)

Vol. In Press

Breaking the Resolution Limit in Two-Photon Microscopy Using Molecular Photoswitches

Swedish Research Council (VR) (2021-05311), 2022-01-01 -- 2025-12-31.

Subject Categories (SSIF 2025)

Molecular Biology

Cell and Molecular Biology

DOI

10.1039/d4cb00313f

More information

Latest update

3/21/2025