Activity At the Serotonin 5-HT2B Receptor

Activity at the 5-HT_2B receptor was examined as previously described (Luethi et al., 2018). In summary, HEK 293 cells expressing the human 5-HT_2B receptor were seeded at a density of 50,000 cells per well in 96-well poly-D-lysine-coated plates overnight at 37°C. The cells were then incubated in growth medium consisting of high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Zug, Switzerland), 10% fetal calf serum (non-dialyzed, heat-inactivated), 250 mg/l Geneticin and 10 ml/l PenStrep (Gibco), overnight at 37°C. After removal of the growth medium via snap inversion, calcium indicator Fluo-4-solution (100 µl) was injected into each well (Molecular Probes, Eugene, OR, USA) and incubated for 45 min at 31°C. Afterwards, the Fluo-4 solution was removed (snap inversion) and a further 100 µl of the Fluo-4 solution was added and incubated (45 min, 31°C). Thereafter, using the EMBLA cell washer, the cells were washed just before testing with HBSS and 20 mM HEPES and exposed to 100 µl of assay buffer. The well plate was positioned in the FLIPR and while online, 25 µl of the test compounds diluted in assay buffer were added. Concentration-response curves were calculated using nonlinear regression, and EC₅₀ values were obtained. The maximal activity at the receptors was calculated relative to 5-HT activity which was defined as 100%. Since setting up a stable and reliable binding assay for the 5-HT_2B receptor has been proven to be difficult in the past, we did not try to include binding data for this receptor in our investigation. However, because the 5-HT_2B receptor activity is important for determining the potential cardiotoxicity of a derivative, we have included this data to estimate whether any substances have the potential to induced endocardial fibrosis.