384 well microplate

Investigation of α_2AAR and α_2BAR stimulated β-arrestin-2 recruitment was performed applying an assay which is based on fragment complementation of β-galactosidase (PathHunter assay, DiscoverX, Birmingham, UK) as described (79 (link)). In detail, HEK293T cells stably expressing the enzyme acceptor (EA) tagged β-arrestin-2 were cotransfected with human α_2AAR or α_2BAR each fused to the ProLink-ARMS2-PKS2 fragment for enzyme complementation and GRK2 (cDNA Resource Center) at equal amounts and subsequently transferred into 384 well microplates (Greiner) after 1 day. After incubation for further 24 hours cells were incubated with test compounds for 60 min (α_2AAR) or 90 min (α_2BAR), arrestin recruitment was stopped by adding detection regent and the resulting chemoluminescence was monitored with a Clariostar plate microreader. Data were normalized relative to buffer (0%) and the maximum effect of norepinephrine (100%). Three to nine repeats for α_2AAR (three to six for α_2BAR) in duplicate were measured.

HEK293T-ACE2-GFP cells were seeded in white, solid bottom 384-well microplates (Greiner BioOne) at 6,000 cells/well in 15 μl medium and incubated at 37°C with 5% CO₂ overnight (~16 h). Compounds were titrated 1:3 in DMSO and dispensed via pintool at 23 nl/well to assay plates. Cells were incubated for 1 h at 37°C 5% CO₂ before 15 μl/well of media was added. The plates were then incubated at 37°C for 48h at 37°C 5% CO₂. After incubation, 30 μl/well of ATPLite (PerkinElmer) was added to assay plates and incubated for 15 min at room temperature. The luminescence signal was measured using a Viewlux plate reader (PerkinElmer). Data were normalized with wells containing cells as 100%, and wells containing media only as 0%.

Determination of G protein–mediated signaling by human α_2AAR, murine α_2AAR, and human α_2BAR was performed applying an IP accumulation assay (IP-One HTRF, Cisbio, Codolet, France) according to the manufacturer’s protocol and in analogy to previously described protocols (77 (link), 78 (link)). In brief, HEK 293T cells were cotransfected with the cDNA for a receptor and the hybrid G protein Gα_qi (Gα_q protein with the last five amino acids at the C terminus replaced by the corresponding sequence of Gα_i) (gift from The J. David Gladstone Institutes, San Francisco, CA), respectively, in a ratio of 1:2. After 1 day, cells were transferred into 384-well microplates (Greiner) and incubated for further 24 hours. On the day of the experiment, cells were incubated with test compounds for 90 min (α_2AAR) or 120 min (α_2BAR), and accumulation of second messenger was stopped by adding detection reagents (IP1-d2 conjugate and Anti-IP1cryptate TB conjugate). After 60 min, TR-FRET was monitored with a Clariostar plate reader. FRET-signals were normalized to buffer (0%) and the maximum effect of norepinephrine (100%). Three to nine (murine α_2AAR, α_2BAR) or 4 to 11 repeats (human α_2AAR), respectively, were performed for each test compound all done in duplicate.

FRET assay experiments were carried out using a CLARIOstar^® microplate reader (BMG Labtech, DE) following the protocol for measuring a three-way protein-protein interaction using FRET from Martin et al (2008)^{32 (link)} and Kopp et al (2018)²² for apo protein. In brief, a mixture containing 30 μM of each fluorescent protein (CFP-IRE1 LD and YFP-BiP), and increasing concentrations of C_H1 were made into a volume of 160 μL in buffer E. Next, 50 μL was transferred to a 384 well microplate (Greiner Bio-one, AT). The protocol was adapted for nucleotide experiments to include a 30 min incubation at RT with 1mM nucleotide in the reaction mixture. Calculation of Ki was based on the protocol from Martin et al (2008)^{32 (link)}. The data were plotted using percentage inhibition as a function of the C_H1 concentration and fitted to an exponential curve (two phase association), from which 50% dissociation value or IC₅₀ (equivalent to half-life) for each phase were measured in GraphPad prism 8. These values were then used to calculate the Ki for each phase.