CellEKT: A Robust Chemical Proteomics Workflow to Profile Cellular Target Engagement of Kinase Inhibitors

Area of Science:

• Biochemistry
• Chemical Biology
• Proteomics

Background:

• The human genome has 518 protein kinases, crucial targets for drug discovery in oncology and autoimmune diseases.
• Developing selective kinase inhibitors is challenging due to the conserved ATP-binding pocket targeted by most drugs.
• Efficient methods are needed to assess cellular target engagement (TE) across the kinome for drug candidate prioritization.

Purpose of the Study:

• To present CellEKT (Cellular Endogenous Kinase Targeting), an optimized chemical proteomics platform.
• To investigate cellular TE of endogenously expressed kinases using novel probes.
• To enable characterization and prioritization of kinase-inhibiting drug candidates.

Main Methods:

• Developed and optimized the CellEKT platform using sulfonyl fluoride-based probes (XO44, ALX005, ALX011).
• Applied CellEKT to determine the kinome interaction landscape of covalent and noncovalent drugs.
• Quantified drug-kinome interactions as IC50 values across over 300 kinases.
• Validated CellEKT findings using phosphoproteomics and NanoBRET assays.

Main Results:

• The CellEKT platform successfully mapped the interaction landscape of drugs across >300 kinases.
• Determined IC50 values for drug-kinome interactions, providing quantitative target engagement data.
• Linked target engagement profiles to kinase substrate space, offering insights into drug mechanisms.
• Validated the platform's robustness and accuracy through orthogonal methods.

Conclusions:

• CellEKT is a robust platform for investigating cellular target engagement of endogenous kinases.
• The platform aids in decrypting drug actions and guiding the discovery and development of selective kinase inhibitors.
• CellEKT facilitates the characterization and prioritization of kinase-inhibiting drug candidates for therapeutic development.