Abstract

Class A G protein-coupled receptors (GPCRs) are key mediators 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 molecules, 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 mechanism 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 receptors are stimulated with hybrid ligands. These ligands 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 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 >20× activity enhancement by the directed proximity approach, enabling the detection of weakly activating sequences 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.

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