TY - JOUR
T1 - CellEKT
T2 - A Robust Chemical Proteomics Workflow to Profile Cellular Target Engagement of Kinase Inhibitors
AU - Rüegger, Joel
AU - Gagestein, Berend
AU - Janssen, Antonius P. A.
AU - Valeanu, Alexandra
AU - Mori, Alger Lazo
AU - van der Peet, Marielle
AU - Boutkan, Michael S.
AU - Florea, Bogdan I.
AU - Henneman, Alex A.
AU - Hochstrasser, Remo
AU - Wang, Haiyan
AU - Westwood, Paul
AU - Topp, Andreas
AU - Gomez Barila, Patricia M.
AU - Medema, Jan Paul
AU - Jimenez, Connie R.
AU - Woersdoerfer, Bigna
AU - Kirchner, Stephan
AU - Zhang, Jitao David
AU - Grether, Uwe
AU - Rufer, Arne C.
AU - van der Stelt, Mario
N1 - Publisher Copyright:
© 2025 THE AUTHORS.
PY - 2025/6/1
Y1 - 2025/6/1
N2 - The human genome encodes 518 protein kinases that are pivotal for drug discovery in various therapeutic areas, such as cancer and autoimmune disorders. The majority of kinase inhibitors target the conserved ATP-binding pocket, making it difficult to develop selective inhibitors. To characterize and prioritize kinase-inhibiting drug candidates, efficient methods are desired to determine target engagement (TE) across the cellular kinome. In this study, we present CellEKT (Cellular Endogenous Kinase Targeting), an optimized and robust chemical proteomics platform for investigating cellular TE of endogenously expressed kinases using the sulfonyl fluoride-based probe XO44 and two new probes ALX005 and ALX011. The optimized workflow enabled the determination of the kinome interaction landscape of covalent and noncovalent drugs across over 300 kinases, expressed as IC50, which were validated using distinct platforms like phosphoproteomics and NanoBRET. With CellEKT, TE profiles were linked to their substrate space. CellEKT has the ability to decrypt drug actions and to guide the discovery and development of drugs.
AB - The human genome encodes 518 protein kinases that are pivotal for drug discovery in various therapeutic areas, such as cancer and autoimmune disorders. The majority of kinase inhibitors target the conserved ATP-binding pocket, making it difficult to develop selective inhibitors. To characterize and prioritize kinase-inhibiting drug candidates, efficient methods are desired to determine target engagement (TE) across the cellular kinome. In this study, we present CellEKT (Cellular Endogenous Kinase Targeting), an optimized and robust chemical proteomics platform for investigating cellular TE of endogenously expressed kinases using the sulfonyl fluoride-based probe XO44 and two new probes ALX005 and ALX011. The optimized workflow enabled the determination of the kinome interaction landscape of covalent and noncovalent drugs across over 300 kinases, expressed as IC50, which were validated using distinct platforms like phosphoproteomics and NanoBRET. With CellEKT, TE profiles were linked to their substrate space. CellEKT has the ability to decrypt drug actions and to guide the discovery and development of drugs.
KW - CellEKT
KW - activity-based protein profiling
KW - broad-spectrum kinase probes
KW - cellular target engagement
KW - chemical proteomics
KW - endogenous kinome profiling
UR - https://www.scopus.com/pages/publications/105010569180
U2 - 10.1016/j.mcpro.2025.100961
DO - 10.1016/j.mcpro.2025.100961
M3 - Article
C2 - 40187492
SN - 1535-9476
VL - 24
SP - 100961
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
IS - 6
M1 - 100961
ER -
