A cell-permeable, activity-based probe for protein and lipid kinases

Area of Science:

• Biochemistry
• Enzymology
• Chemical Biology

Background:

• Protein and lipid kinases are crucial enzymes with dysregulated activity in various diseases.
• Assessing kinase activity within living cells is vital for understanding disease mechanisms.
• Wortmannin is a known inhibitor of protein and lipid kinases.

Purpose of the Study:

• To synthesize and characterize novel wortmannin derivatives as activity-based probes.
• To evaluate the utility of these probes for detecting and isolating kinase activity.
• To explore their potential in cellular imaging and protein analysis.

Main Methods:

• Chemical synthesis of biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin.
• In vitro reactivity assessment with phosphatidylinositol 3-kinase (PI3K) and PI3K-related kinase families.
• Cellular assays to evaluate cell permeability and protein labeling in living cells.

Main Results:

• The synthesized wortmannin derivatives exhibit wortmannin-like reactivity.
• These probes successfully label PI3K and related kinases in cell lysates.
• The probes can distinguish between active and inactive kinase forms, functioning as activity-based probes.
• Biotin-wortmannin facilitates protein isolation, while BODIPY-wortmannin allows for cell-permeable labeling.

Conclusions:

• Novel wortmannin derivatives serve as effective activity-based probes for kinases.
• These probes offer versatile applications in biochemical analysis, protein isolation, and cellular imaging.
• The developed reagents represent valuable tools for studying kinase dysregulation in disease contexts.