DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery

Area of Science:

• Pharmacology and Drug Discovery
• Molecular Biology
• Yeast Genetics

Background:

• G protein-coupled receptors (GPCRs) are a major class of human membrane receptors, with many lacking defined ligands and drugs.
• Existing methods for GPCR ligand discovery are often limited in throughput and scope, particularly for understudied receptors.

Purpose of the Study:

• To develop and validate novel yeast-based technologies for high-throughput GPCR engineering and ligand discovery.
• To identify novel ligand interactions for both well-characterized and understudied GPCRs.

Main Methods:

• Development of Dynamic Cyan Induction by Functional Integrated Receptors (DCyFIR), a CRISPR-integrated yeast system for GPCR and ligand screening.
• Construction and profiling of a yeast library encompassing 30 human GPCRs and their potential Gα coupling combinations.
• Screening of a human metabolite library against GPCRs using DCyFIR to identify novel receptor-ligand interactions.

Main Results:

• DCyFIR accurately recapitulated known GPCR agonism and identified novel interactions for several GPCRs, including ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2.
• Scalable screening identified GPCR-metabolite interactions, notably for pharmacologically dark receptors like GPR4, GPR65, GPR68, and HCAR3.
• Yeast-based findings were validated in mammalian cells, confirming kynurenic acid as an activator of HCAR3.

Conclusions:

• DCyFIR is a powerful and scalable platform for identifying ligand interactions across a wide range of GPCRs, including understudied targets.
• The technology facilitates the discovery of novel therapeutic targets and ligands for GPCRs, advancing drug discovery efforts.