Reading dynamic kinase activity in living cells for high-throughput screening

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Protein kinases are vital signaling molecules and emerging drug targets.
• Assaying kinase activity in living cells is crucial for drug discovery and genomics.
• Existing methods may not capture dynamic kinase activity effectively.

Purpose of the Study:

• To develop a general method for high-throughput screening of dynamic kinase activities in living cells.
• To showcase the application of this method for assaying protein kinase A (PKA) and the cyclic adenosine monophosphate (cAMP)/PKA pathway.
• To demonstrate the utility of this method for identifying modulators of cellular signaling pathways.

Main Methods:

• Utilized ratiometric fluorescent sensors for real-time monitoring of kinase activity.
• Applied the method to measure intracellular protein kinase A (PKA) activity.
• Screened a library of clinical compounds to identify pathway modulators.

Main Results:

• Successfully established a general method for high-throughput dynamic kinase activity assays.
• Demonstrated the ability to monitor the cAMP/PKA pathway, which is downstream of G-protein coupled receptors (GPCRs).
• Identified novel modulators of the GPCR/cAMP/PKA pathway from a collection of clinical compounds.

Conclusions:

• Ratiometric fluorescent sensors provide a powerful tool for high-throughput screening of dynamic kinase activities.
• This method facilitates drug discovery by enabling the identification of modulators for critical cellular signaling pathways.
• The developed screening approach is effective for identifying compounds that modulate the GPCR/cAMP/PKA pathway in living cells.