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

Abstract

Abstract The present work is the last of a three‐part study investigating a panel of 30 systematically designed synthetic cannabinoid receptor agonists (SCRAs) including features such as the 4‐pentenyl tail and varying head groups including amides and esters of l ‐valine (MMB, AB), l ‐ tert ‐leucine (ADB), and l ‐phenylalanine (APP), as well as adamantyl (A) and cumyl moieties (CUMYL). Here, we evaluated these SCRAs for their capacity to activate the human cannabinoid receptor 1 (CB 1 ) via indirect measurement of G protein recruitment. Furthermore, we comparatively evaluated the results obtained from three in vitro assays, based on the recruitment of β‐arrestin 2 (βarr2 assay) or Gα i protein (mini‐Gα i assay), or binding of [ 35 S]‐GTPγS. The observed efficacies ( E max ) varied depending on the conducted assay. Statistical analysis suggests that the population means of the relative intrinsic activity (RA i ) significantly differ for the [ 35 S]‐GTPγS assay and the other two assays, but the population means of the βarr2 and mini‐Gα i assays were not statistically different. Our data suggest that differences observed between the βarr2 and mini‐Gα i assays are the best predictor for ‘biased agonism’ towards βarr or G protein recruitment in our study. SCRAs carrying an ADB or MPP moiety as a head group tended to produce elevated E max values in the βarr2 assay, which might result in a tendency of these compounds to cause pronounced tolerance in users—a hypothesis that should be evaluated further by future studies. In general, a comparison of efficacies derived from different assays is difficult and should only be conducted very cautiously.

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