De novo design of selective kinase modulators

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Signaling

Background:

• Protein kinases regulate cellular processes but are difficult to modulate due to conserved structures.
• Targeting kinase activity precisely is crucial for understanding and manipulating cell signaling pathways.

Purpose of the Study:

• To design genetically encoded miniproteins for precise activation or inhibition of focal adhesion kinase (FAK).
• To establish a versatile platform for selective, genetically encoded kinase control.

Main Methods:

• De novo design of genetically encoded miniproteins targeting the FAK kinase domain.
• Biochemical characterization of miniprotein binders for FAK activity modulation.
• In vitro and in-cell validation of miniprotein efficacy and specificity.

Main Results:

• 96 miniprotein binders were designed, with 33 modulating FAK activity.
• Four potent modulators were identified: two inhibitors with low-nanomolar IC50 values and two activators.
• Designed modulators demonstrated conserved inhibitory and activating effects in vitro and in living cells.
• FAK inhibitors were successfully redesigned to inhibit Src kinase, demonstrating platform versatility.

Conclusions:

• Designed conformational binders can directly tune FAK signaling in living cells.
• This approach provides a versatile platform for selective and genetically encoded kinase control.
• The study offers a starting point for discovering novel kinase modulatory sites.