Figure 2.

Selectivity of the GPR68 PAM 71. Proton concentration-response curves in the absence and presence of increasing concentrations of 1 at human GPR68 (A), 71 at human GPR68 (B), GPR4 (C), and GPR65 (D). Proton-mediated G_s−cAMP production was measured in HEK293T cells transiently transfected with GPR68, GPR4, or GPR65, and GloSensor cAMP plasmids. Results were normalized to the proton activity in the absence of modulator and represented means ± SEM from a minimum of three independent assays, each in quadruplicate. Curves were analyzed in Prism using the built-in four parameter logistic functions. (E) Selectivity of 71 over 48 common GPCRs, ion channels, and transporters in radioligand binding assays. Values represent average binding affinity (pK_i) from a minimum of three independent assays, each in triplicate. Targets with less than 50% inhibition at 10 μM (in quadruplicate) are not shown. BZP for benzodiazepine receptor; DAT for dopamine transporter; DOR for delta (δ) opioid receptor; hERG for human ether-a-go-go-related potassium channel; KOR for kappa (κ) opioid receptor; MOR for mu (μ) opioid receptor; NET for noradrenaline transporter; PBR for peripheral benzodiazepine binding site; SERT for serotonin transporter. (F) Off-target agonist activity of 71 at human GPCRome. 71 was screened across the human GPCRome (318 GPCRs) for off-target agonist activity at 1 μM using the PRESTO-Tango screening platform. Results (fold of basal) represent means of four replicates. Dopamine receptor D₂ with 100 nM quinpirole served as an assay control. The dashed lines indicate an arbitrary cutoff line at 3× fold of basal.