Cryptates

The fluorescent binding assay employs the native MUR fused at the N-terminus to a SNAP-tag® enzyme and expressed on HEK293 cells. SNAP-tag-mu-opioid is then covalently labeled with terbium cryptate (Lumi4®-Tb), a long lifetime FRET donor. An analog of the potent opioid antagonist naltrexone that contains the d2 dye (red-naltrexone) is used as the fluorescence energy transfer acceptor. Upon ligand binding, a FRET process occurs between the Lumi4-Tb donor (emission at 620 nm) in SNAP-Lumi4-Tb-mu-opioid receptor and the red-naltrexone acceptor (emission at 665 nm). The fluorescence emission from the acceptor is detected in a time resolved manner (TR-FRET). For HTRF assay (Cisbio Bioassays, Bedford, MA), Tag-lite® mu opioid cells suspended in culture medium were dispensed in white 384-well low-volume microplates (Greiner Bio-one Greiner Bio-One North America, Monroe, NC) at 3700 cells/10 µL/well Tag-lite m opioid labeled cells with 60 nM of Tag-lite opioid receptors red-naltrexone, and 5 µL of the wsMUR-TM with 11 further 1∶2 serial dilutions from the µM to the nM range. All samples were mixed with final volume 20 µl and incubated at RT for 2 h. After incubation, HTRF signals were measured using a plate reader (BMG, Cary, NC) after excitation at 337 nm at both 620 and 665 nm emission, HTRF signal was calculated as a two-wavelength signal ratio: [intensity (665 nm)/intensity (620 nm)]. IC₅₀ determination and statistical analysis IC₅₀ values for wsMUR-TM were determined by fitting the dose–responses curves using the Prism program (GraphPad Software, San Diego, CA).

cAMP accumulation in CHO-K1 cells expressing wild-type eLH/CGR or the eLH/CGR mutants was measured using cAMP Dynamic 2 competitive immunoassay kits [10 (link)]. The transfected cells were seeded in a 384-well plate (10,000 cells per well). The standard samples were prepared to cover an average cAMP concentration of 0.17–712 nM (final concentration of cAMP per well). We added MIX to the cell dilution buffer to prevent cAMP degradation. To each well, 5 μL of compound medium buffer containing rec-eCG mutants was added. The plate was sealed and incubated for cell stimulation at room temperature for 30 min. The samples were incubated with the detection reagents, cAMP-d2 and anti-cAMP-cryptate (diluted five-fold in lysis buffer, 5 μL/well), for 1 h at RT. cAMP was detected by measuring the decrease in homogeneous time-resolved fluorescence (HTRF) energy transfer (665 nm/620 nm) using an Artemis K-101 HTRF microplate reader (Kyoritsu Radio, Tokyo, Japan). The specific signal-Delta F (energy transfer) is inversely proportional to the concentration of cAMP in the standard or the sample. The results were calculated on the basis of the 665 nm/620 nm ratio, and expressed as Delta F% (cAMP inhibition), according to the following equation: (Delta F% = (standard or sample ratio-mock transfection) × 100/mock transfection). The cAMP concentrations for the Delta F% values were calculated in nM using the GraphPad Prism software (GraphPad, Inc., La Jolla, CA, USA).

The inhibition of SARS-CoV-2 NSP3 Mac1 was assessed by the displacement of an ADP-ribose-conjugated biotin peptide from His₆-tagged protein using an HTRF-technology-based screening assay, which was performed as previously described [36 (link)]. Compound library screens (including the MIDAS and FDA-approved screening set and the curated BioAscent hit compound library) were performed at a compound concentration of 25 µM in duplicate measurements, while for hit confirmation, IC₅₀ curves were acquired with a top compound concentration of 125 µM (MIDAS and FDA-approved hit compounds) or 187 µM (BioAscent hit compounds), followed by an 8-point 1:1 dilution series in duplicate measurements. The compounds were dispensed into ProxiPlate-384 Plus (PerkinElmer, Waltham, MA, US) assay plates using an Echo 525 liquid handler (Labcyte, San Jose, CA, US). Binding assays were conducted in a final volume of 16 μL with 12.5 nM of SARS-CoV-2 NSP3 Mac1, 400 nM of peptide ARTK(Bio)QTARK(Aoa-RADP)S, 1:20,000 Anti-His₆-Eu³⁺ cryptate (HTRF donor, PerkinElmer), and 1:125 Streptavidin-XL665 (HTRF acceptor, PerkinElmer) in assay buffer (25 mM of HEPES pH 7.0, 20 mM of NaCl, 0.05% bovine serum albumin and 0.05% Tween-20). Assay reagents were dispensed into plates using a Multidrop combi (Thermo Scientific, Waltham, MA, US). Macrodomain protein and peptide were first dispensed and incubated for 30 min at room temperature. This was followed by the addition of the HTRF reagents and incubation at room temperature for 1 h. Fluorescence was measured using a PHERAstar microplate reader (BMG) using the HTRF module with dual emission protocol (A = excitation of 320 nm, emission of 665 nm, and B = excitation of 320 nm, emission of 620 nm). Raw data were processed to give an HTRF ratio (channel A/B × 10,000), which was used to generate IC₅₀ curves. The IC₅₀ values were determined by nonlinear regression using GraphPad Prism v.9 (GraphPad Software, San Diego, CA, USA). Of note is that we judged—based on our experience in medicinal chemistry and FRET-based assays, as well as references in the literature (e.g., Baell and Walters, 2014 [41 (link)])—the screening compounds for the presence of chemical features known to cause assay interference and promiscuous binding behaviour. Compounds were excluded from the hit validation processes without further biophysical testing or computational predictions when certain motifs were identified. However, where stated as “showed assay effects at higher concentrations”, the compounds did not show structural features suspicious for assay interference per se and were tested in the HTRF-based assay. At higher compound concentrations of the titration, we observed a decrease or were even unable to determine Mac1 inhibition values, indicating that these compounds had unspecific assay effects unrelated to true Mac1 inhibition.

In vitro activity of human RSK CTKD was evaluated in the HTRF KinEASE assay (Cisbio Bioassays). For activity evaluation, RSK CTKD proteins (2 μM) were either untreated or pre-activated by ERK2 (0.1 μM) (SignalChem) in reaction buffer containing 200 μM ATP and 10 mM MgCl₂ in kinase buffer (50 mM HEPES, pH 7.0, 2 mM TCEP, 0.02% [wt/vol] NaN₃, 0.01% [wt/vol] BSA, and 0.1 mM orthovanadate) for 1 h at 30°C. Reactions were performed in 384-well plates for fluorescence (Greiner) with 10 ng RSK CTKD per well. The reactions were started by the addition of 100 μM ATP and 1 μM STK1 substrate, giving a 10 μl reaction solution. After 20 min at room temperature, the reaction was stopped with 5 μl anti-phospho STK antibody labeled with Eu³⁺-cryptate (FRET donor) and 5 μl streptavidin-XL665 (FRET acceptor), both prepared in EDTA. In inhibition studies, 10 ng constitutively active RSK CTKDs were mixed with DMSO or a concentration series of ruxolitinib, SB-203580, TG-100-115, or fmk in kinase buffer for a 30-min incubation prior kinase reaction (as described above). Evaluation of the time-dependent effect of fmk on RSK2-T577E-D694* was performed by incubation with a concentration series of fmk in kinase buffer (50 mM succinic acid, pH 6.0, 2.5 mM TCEP, 0.02% [wt/vol] NaN₃, 0.01% [wt/vol] BSA and 0.1 mM orthovanadate) for 1, 2, 4, 6 min or 2, 5, 15, 30 min (depending on concentration series) followed by kinase reaction. IC₅₀ values were calculated by non-linear regression using sigmoid concentration response. The observed rate constant of fmk inhibition, k_obs, at each concentration was determined from the slope of a semi-logarithmic plot of inhibition versus time. k_obs was re-plotted against inhibitor concentration and fitted to a hyperbolic equation, k_obs = k_inact[inhibitor]/(K_i + [inhibitor]) (22 (link)). Data analysis was performed using GraphPad Prism 9. All experiments were repeated at least three times (n = 3) and presented as means ± SD. The P-values were determined by t test.

Quantification of intracellular IP1 was performed using the HTRF (Homogeneous Time Resolved Fluorescence) IP1 competitive immunoassay (IP-One Tb kit. Cisbio. France) according to the manufacturer’s instructions. Briefly, 20,000 HEK293T/17 cells or 3.10⁶ platelets were distributed in a 384-well white microplate (Greiner) and incubated with the indicated molecules for 30 min or 2 h at 37 °C in the presence of 50 mM of LiCl to prevent IP1 degradation. After addition of d2-labeled IP1 (acceptor) and anti-IP1-Cryptate (donor) for 1 h, the specific FRET signals were calculated by the fluorescence ratio of the acceptor and donor emission signal (665/620 nm) collected using a modified Infinite F500 (Tecan Group Ltd). Conversion of the HTRF ratio of each sample into IP1 concentrations was performed on the basis of a standard curve to determine the linear dynamic range of the assay.