Pathhunter assay

The PathHunter™ assay from the DiscoverX company (DiscoveRx, Birmingham, United Kingdom) was used to measure the recruitment of β-arrestin2 to melatonin receptors as previously discribed⁵⁷ . Briefly, HEK293 parental cells stably expressing a fusion protein of β-arrestin2 and the larger N-terminal deletion mutant of β-gal were transiently transfected by X-tremeGENE 9 with different quantitates of expression vectors for Flag-MT₁, HA-MT₂, myc-GPR61, myc-GPR62 or myc-GPR135 fused at their C-terminal part with the small enzyme fragment ProLink tag (PK2). On the next day, cells were spread to poly-L-lysine (Sigma)-coated 98-well plate. Two days after transfection, the cells were treated with or without 1 µM melatonin for 2–3 hours at 37 °C. β-arrestin2 recruitment resulted in the complementation of the two enzyme fragments and the formation of an active β-gal enzyme. Luminescence signals were determined after 60 min incubation at room temperature.

The measurement of β -arrestin-2 recruitment to activated-receptors was performed utilizing the PathHunter assay (DiscoverX, Birmingham, UK) as described previously^{60 (link)}. HEK293 cells stably expressing the EA-tagged β-arrestin-2 fusion protein (provided by DiscoverX) were transiently transfected with the ProLink(ARMS2-PK2)-tagged dopamine D_2S or D_2L receptors, respectively, using Mirus TransIT-293 (Mobitech, Göttingen, Germany) transfection reagent. 24 h after transfection, cells were detached using Versene (Invitrogen) and 5,000 cells per well were seeded into white, clear bottom 384-well plates (Greiner Bio-One) and maintained at 37 °C, 5% CO₂ for 24 h in assay medium. After incubation with different concentrations of test compounds (from 10⁻¹² to 10⁻⁵ M final concentration) in duplicates for 5 h, the detection mix was added and incubation was continued for further 60 min. Chemiluminescence was determined on a CLARIOstar microplate reader. Resulting responses were normalized to the maximum effect obtained with quinpirole (100%) and the basal response (vehicle, 0%). Dose–response curves were calculated by nonlinear regression using the algorithms of Prism 6.0.

A Pathhunter assay [35 (link)] kit was obtained from DiscoverX, and enzyme fragment complementation assay was carried out. U2OS cells transfected with HER3:HER2 were used for the assay. After cells were attached to the wells, they were incubated with compound 18 and controls at different concentration in the presence of neuregulin-1 (NRG-1) at 0.3 µM concentration. After 24 h of incubation, reagents were added, and luminescence was measured and compared with a control without any compound. A plot of luminescence versus concentration was obtained to measure the dimerization of HER3:HER2 and its inhibition by compound 18.