Objective: GPR52 is a constitutively active, orphan GPCR which is highly expressed in the striatum and has been proposed as a target for schizophrenia. No endogenous ligands for GPR52 have been identified and very few tool ligands reported. Here, we describe the characterisation of the first known radioligand for GPR52, [3H]-HTL45725.

Methods: [3H]-HTL45725 binding assays: 25µg human or rat GPR52 expressing membranes were incubated for 2h at room temperature with [3H]-HTL45725 and competing ligands in binding buffer (20mM HEPES + 10mM MgCl2 + 0.1% BSA). Membranes were harvested by rapid filtration using TomTec harvester onto 0.1% PEI coated GF/B plates and washed 3x with cold PBS. For kinetic experiments, membranes were added at indicated time points. Non-specific binding was determined by 10µM HTL’892.

cAMP assays: Frozen GPR52 expressing cells were seeded 4000 cells per well on proxiplates containing test compound and incubated for 30min at 37oC in stimulation buffer (Cisbio) containing 500µM IBMX. cAMP was detected using HiRange cAMP kit (Cisbio) according to the manufacturer’s instructions.

Results: [3H]-HTL45725 was shown to bind specifically to both human and rat GPR52 with pKDs of 7.0 ± 0.2 and 7.1 ± 0.4 respectively, with a kon of 8.52 ± 0.23 x105 M-1 min-1 and koff of 0.14 ± 0.02 min-1 at the human receptor. No specific binding was detected in untransfected HEKs. A panel of GPR52 ligands showed full displacement of [3H]-HTL45725, with excellent correlation between pKis and pEC50s from functional cAMP assays.

Conclusion: Here we have characterised [3H]-HTL45725, a novel radioligand for GPR52. GPR52 is a target of interest for CNS disorders but lacks many ligands for pharmacological evaluation. The identification of a high affinity, specific radioligand for GPR52 that allows pharmacological characterisation of compounds provides an excellent tool to further probe this orphan receptor.