Proximity-assisted screening of class A/B hybrid GPCRs

Class A G protein-coupled receptors (GPCRs) are key media-tors in numerous signaling pathways and important drug targets for several diseases. A major shortcoming in GPCR ligand screening is the detection limit for weak binding mole-cules, which is especially critical for poorly druggable GPCRs. Here we present a proximity-based screening system for class A GPCRs, which adopts the natural two-step activation mech-anism of class B GPCRs. In this approach, class A/B chimeras with the extracellular domain of the class B receptor CRF1R, grafted to the transmembrane domain of target class A recep-tors are stimulated with hybrid ligands. The latter contain a high-affinity peptide derived from CRF, which recruits the hybrid ligands to the engineered target GPCR, dramatically increasing the local concentration of the test substances. We exemplified this method for the neurotensin receptor 1 (NTR1) and endothelin receptor B (ETB), two important class A GPCR drug targets for pulmonary arterial hypertension or psychological disorders and neurodegenerative diseases. We observed >20x activity enhancement by the directed proximi-ty approach, enabling the detection of weakly activating se-quences that would have otherwise remained undetected. Our approach allows to probe GPCR activation in the membrane of living cells and may be especially useful for GPCRs for which it has been difficult to generate small drug-like molecules.