Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation.

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• G protein-coupled receptors (GPCRs) are crucial for physiological processes and represent a major drug target class.
• Existing methods for studying GPCRs often require complex cell-based assays or purified systems.
• A need exists for robust, adaptable in vitro tools to characterize GPCR-ligand interactions.

Purpose of the Study:

• To develop a generalizable, cell lysate-based in vitro method for detecting activated GPCRs.
• To create a tool, termed IGNiTR (in vitro GPCR split NanoLuc ligand Triggered Reporter), for GPCR and ligand characterization.
• To demonstrate the broad applicability of IGNiTR across different GPCR types and binder formats.

Main Methods:

• Designed a split NanoLuc reporter system reconstituted upon GPCR activation by a ligand.
• Utilized conformation-specific binders (nanobodies, miniG proteins, peptidomimetics) to bridge activated GPCRs and split NanoLuc fragments.
• Validated the IGNiTR assay in a cell lysate format using various Gs- and Gi-coupled GPCRs.

Main Results:

• Successfully reconstituted NanoLuc activity in vitro using IGNiTR across multiple GPCRs and binder types.
• Demonstrated proof-of-concept for GPCR ligand screening using dopamine receptor D1.
• Showcased opioid detection for potential point-of-care applications and GPCR characterization in Nanodiscs.

Conclusions:

• IGNiTR provides a versatile and scalable in vitro platform for GPCR research.
• The cell lysate-based format offers convenience and broad applicability for ligand detection and screening.
• IGNiTR facilitates GPCR characterization and has potential for diagnostic applications.