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 II: Structure activity relationship assessment via a β‐arrestin recruitment assay

Abstract

Synthetic cannabinoid receptor agonists (SCRAs) are the second largest class of new psychoactive substances (NPS) and are associated with serious adverse effects and even death. Despite this, little pharmacological data are available for many of the most recent SCRAs. This study consists of three different parts, aiming to systematically evaluate a panel of 30 SCRAs using binding and different in vitro human cannabinoid 1 receptor (CB1) activation assays. The present Part II investigated the SCRA analogs for their CB1 activation via a β‐arrestin recruitment assay. The panel was systematically designed to include key structural sub‐features of recent SCRAs. Thus, the 4‐pentenyl tail of MMB‐4en‐PICA and MDMB‐4en‐PINACA was retained while incorporating varying head groups from other prevalent SCRAs, including amides and esters of L‐valine, L‐tert‐leucine, and L‐phenylalanine, and adamantyl and cumyl moieties. All 30 SCRAs activated CB1, with indazoles generally showing the greatest potency (EC50 = 1.88–281 nM), followed by indoles (EC50 = 11.5–2293 nM), and the corresponding 7‐azaindoles (EC50 = 62.4–9251 nM). Several subunit‐linked structure–activity relationships were identified: (i) tert‐leucine‐functionalized SCRAs were more potent than the corresponding valine derivatives; (ii) no major difference in potency or efficacy was observed between tert‐leucine/valine‐derived amides and the corresponding methyl esters; however, phenylalanine analogs were affected by this change; and (iii) minor structural changes to the 4‐pentenyl substituent had little influence on activity. These findings elucidate structural features that modulate the CB1 activation potential of currently prevalent SCRAs and a systematic panel of analogs, some of which may appear in NPS markets in future.

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

“…Part I 24 depicted the synthesis of 30 SCRAs structurally related to MMB‐4en‐PICA and MDMB‐4en‐PINACA, their chemical characterization, and receptor binding affinity for CB 1 . Part II 26 described their CB 1 agonist activity using a live cell‐based reporter assay monitoring in vitro CB 1 activation via recruitment of βarr2. In the present Part III, CB 1 receptor activation was monitored via investigation of the G protein pathway, using the [ 35 S]‐GTPγS binding assay, as well as a live cell‐based receptor assay based on the recruitment of mini‐Gα i .…”

“…The previously described live cell-based receptor mini-Gα i assay, 27 which was developed similarly to the previously established βarr2 bioassay for CB 1 activation, 26,[28][29][30][31] was used to determine CB 1 activation based on the recruitment of mini-Gα i using the NanoLuc Binary Technology (NanoBiT ® ). 32 The modified human embryonic kidney (HEK) 293T cells (stably expressing CB 1 -LgBiT and SmBiT-mini-Gα i ) were routinely maintained at 37 C, 5% CO 2 , under humidified atmosphere in DMEM GlutaMAX™ supplemented with 10% heat-inactivated FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B.…”

“…Part I describes the synthesis and the affinity for the CB 1 receptor for the 30 test compounds 25 . Part II investigates SCRA‐induced activation of the CB 1 receptor via an in vitro βarr2 recruitment assay, including a comparison of the structure activity relationships of the SCRAs of this panel with reports in literature 26 . The here presented part (Part III) explores CB 1 receptor activation in terms of the G protein pathway and discusses the differences observed between different in vitro assays monitoring CB 1 receptor activation.…”

“…25 For APP-4en-P7AICA (26), maximal receptor activation could not be reached in the [ 35 S]-GTPγS assay due to the relatively weak potency displayed by this compound, which is also evident from the data derived from the βarr 2 (9251 nM) and mini-Gα i assays (9721 nM). Overall, the order of the seven most potent compounds within the whole set differed only marginally between the assay types (Table2).Statistical analysis using Welch's t test of the potencies (EC 50 ) derived from the three different assays showed no statistically significant difference between the EC 50 values for six of the test compounds (MDMB-4en-PINACA[12], AB-4en-PINACA[14], ADB-4en-PINACA[15], Cumyl-4en-PINACA[18], MPP-4en-P7AICA[23], and APP-4en-P7AICA[26]). Interestingly, four of these are amongst the seven most potent compounds in this series of SCRAs.…”

“…25 Part II investigates SCRA-induced activation of the CB 1 receptor via an in vitro βarr2 recruitment assay, including a comparison of the structure activity relationships of the SCRAs of this panel with reports in literature. 26 The here presented part (Part III) explores CB 1 receptor activation in terms of the G protein pathway and discusses the differences observed between different in vitro assays monitoring CB 1 receptor activation. Deionized water was prepared in-house using a Medica ® Pro deionizer from ELGA (Celle, Germany) in Freiburg (Germany) and a Direct-Q water purification system from Millipore (Overijse, Belgium) in Ghent (Belgium), respectively.…”