A FRET-Based Biosensor for the Src N-Terminal Regulatory Element.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Intrinsically disordered regions (IDRs) in signaling proteins act as crucial regulatory elements.
• IDRs are sensitive to environmental factors, ligand binding, and post-translational modifications, influencing downstream signaling.
• The dynamic nature of IDRs poses challenges for traditional drug screening methods relying on fixed structures.

Purpose of the Study:

• To design and validate a genetically encodable biosensor for the N-terminal regulatory element of c-Src kinase.
• To enable high-throughput screening for compounds modulating c-Src kinase activity.
• To overcome limitations of classical drug screening for intrinsically disordered proteins.

Main Methods:

• Development of a Förster Resonance Energy Transfer (FRET)-based biosensor using fluorescent proteins.
• Incorporation of the SH4, unique, and SH3 domains of c-Src kinase into the biosensor construct.
• Inclusion of an engineered proteolytic site for an internal control.
• High-throughput screening of a compound library (1669 compounds).
• Confirmation of hits using Nuclear Magnetic Resonance (NMR) spectroscopy.

Main Results:

• Demonstrated FRET variations in the biosensor upon ligand binding, indicating conformational changes.
• Successfully screened a library of 1669 compounds.
• Identified seven confirmed hit compounds modulating the c-Src regulatory element.

Conclusions:

• The developed biosensor is effective for studying c-Src kinase regulation.
• This FRET-based biosensor facilitates high-throughput screening for modulators of intrinsically disordered protein regions.
• The identified hits represent potential leads for therapeutic development targeting c-Src kinase.