Identification of psychedelic new psychoactive substances (NPS) showing biased agonism at the 5-HT2AR through simultaneous use of β-arrestin 2 and miniGαq bioassays

Abstract

Identification of psychedelic new psychoactive substances (NPS) showing biased agonism at the 5-HT2AR through simultaneous use of betaarrestin 2 and miniG alpha(q) bioassays.

Systematic evaluation of a panel of 30 synthetic cannabinoid receptor agonists structurally related to MMB‐4en‐PICA, MDMB‐4en‐PINACA, ADB‐4en‐PINACA, and MMB‐4CN‐BUTINACA using a combination of binding and different CB1 receptor activation assays. Part III: The G protein pathway and critical comparison of different assays

“…It can be hypothesized that the similarity of the results from the βarr2 and mini‐Gα i assay is due to the highly similar assay principle (both live cell‐based, assessing recruitment to the same CB 1 construct, with luminescence detection), counterbalancing potential effects of the type of recruitment. It must be noted, though, that using a similar assay set‐up to assess the recruitment of psychedelic compounds to the 5‐HT 2A R (albeit using mini‐Gα q rather than mini‐Gα i in conjunction with βarr2) did lead to compounds showing widely different responses (and, hence, being categorized as being ‘biased’) 35 . Furthermore, it can be assumed that because the βarr2 and the [ 35 S]‐GTPγS assay rely on substantially different assay principles, differences observed for a substance between these assays should not necessarily be interpreted as ‘biased agonism’.…”

“…When the AUC values of the highest concentration of a compound were more than 20% lower than the values of the next dilution, these data points were excluded from the set to avoid skewing the concentration–response graph 34 . The drop in signal of these high concentrations may be due to cell toxicity or solubility issues, as previously hypothesized for different receptor systems 35,36 …”

“…34 The drop in signal of these high concentrations may be due to cell toxicity or solubility issues, as previously hypothesized for different receptor systems. 35,36 The data from both experiments were normalized to the maximum signal of CP-55,940 (arbitrarily set at 100%), which was used as reference compound. Curve-fitting of concentration-response curves was performed using GraphPad Prism 8 (version 8.0.2; GraphPad Software Inc., La Jolla, CA, USA), via nonlinear regression (three parameters, Hill slope = 1; required for the implementation of the intrinsic relative activity model as described below), to determine EC 50 (a measure of potency) and E max (a measure of efficacy) values.…”

A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes

“…We observed excellent signal amplitudes at all four receptor subtypes, which was of particular importance for the weakly expressed recombinant H 4 R. Moreover, we are the first to provide time-resolved courses of agonist-mediated functional responses using mini-G sensors with split-NanoLuc complementation for an entire receptor family, the subtypes of which couple to three different types of mini-G proteins (mGs, mGsi and mGsq). As the presented biosensor is becoming available for an increasing number of GPCRs [23][24][25][26][27], it will be appealing to extend the application in prospective studies to analyse time-resolved differences of distinct GPCRs coupling to the same minimal G protein. The methodology could also be used to investigate one GPCR coupling to different minimal G proteins (coupling specificity), and to supplement ligand binding studies with kinetic input, for example association and dissociation rate constants (k on/off ) and residence time [32,33].…”

“…By replacing the α5 helix of the minimal Gα s protein (mGs) with the respective sequence of other Gα subunits, mini-G proteins covering all major Gα families were derived and appropriate coupling specificities were demonstrated [22]. The application of BRET and split-luciferase complementation (SLC) techniques to GPCRs and mini-G proteins has created new G protein sensors that monitor functional responses in real-time [23][24][25][26][27]. Of particular note, the dynamic assay ranges benefited from the cytosolic nature of the mini-G proteins, as native, membrane-anchored G proteins produce high baseline values due to their closer proximity to membrane-bound GPCRs [23][24][25].…”