Gα<sub>s</sub> signalling of the CB<sub>1</sub> receptor and the influence of receptor number

Finlay, David B.; Cawston, Erin E.; Grimsey, Natasha L.; Hunter, Morag R.; Korde, Anisha; Vemuri, V. Kiran; Makriyannis, Alexandros; Glass, Michelle

doi:10.1111/bph.13866

Cited by 76 publications

(133 citation statements)

References 60 publications

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“…Experiments utilized human CB 1 or CB 2 tagged at the N ‐terminus with three haemagglutinin sequences (HA‐hCB 1 , HA‐hCB 2 ) stably transfected into HEK293. HEK293 were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and zeocin, (250 μg/mL). Cells were maintained in 5% CO 2 at 37°C in a humidified atmosphere.…”

Section: Methodsmentioning

confidence: 99%

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Synthesis and pharmacology of new psychoactive substance 5F‐CUMYL‐P7AICA, a scaffold‐ hopping analog of synthetic cannabinoid receptor agonists 5F‐CUMYL‐PICA and 5F‐CUMYL‐PINACA

Banister

Adams

Kevin

et al. 2018

Drug Testing and Analysis

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Synthetic cannabinoid receptor agonists (SCRAs) are a dynamic class of new psychoactive substances (NPS), with novel chemotypes emerging each year. Following the putative detection of 5F-CUMYL-P7AICA in Australia in 2016, the scaffold-hopping SCRAs 5F-CUMYL-PICA, 5F-CUMYL-PINACA, and 5F-CUMYL-P7AICA were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-quadrupole time-of-flight-MS (LC-QTOF-MS). Since little is known of the pharmacology of 7-azaindole SCRAs like 5F-CUMYL-P7AICA, the binding affinities and functional activities of all compounds at cannabinoid type 1 and type 2 receptors (CB and CB , respectively) were assessed using tritiated radioligand competition experiments and fluorescence-based plate reader membrane potential assays. Despite CB binding affinities differing by over two orders of magnitude (K = 2.95-174 nM), all compounds were potent and efficacious CB agonists (EC = 0.43-4.7 nM), with consistent rank order for binding and functional activity (5F-CUMYL-PINACA >5F-CUMYL-PICA >5F-CUMYL-P7AICA). Additionally, 5F-CUMYL-P7AICA was found to exert potent cannabimimetic effects in mice, inducing hypothermia (6°C, 3 mg/kg) through a CB -dependent mechanism.

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Section: Methodsmentioning

confidence: 99%

“…Experiments utilized human CB 1 or CB 2 tagged at the N-terminus with three haemagglutinin sequences (HA-hCB 1 , HA-hCB 2 ) stably transfected into HEK293. 24…”

Section: In Vitro Cannabinoid Receptor Binding Experimentsmentioning

confidence: 99%

Synthesis and pharmacology of new psychoactive substance 5F‐CUMYL‐P7AICA, a scaffold‐ hopping analog of synthetic cannabinoid receptor agonists 5F‐CUMYL‐PICA and 5F‐CUMYL‐PINACA

Banister

Adams

Kevin

et al. 2018

Drug Testing and Analysis

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104

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“…Experiments utilized human CB 1 or CB 2 tagged at the N ‐terminus with three haemagglutinin sequences (3HA‐hCB 1 , 3HA‐hCB 2 ) stably transfected into HEK293 . HEK293 were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and zeocin (250 μg/mL).…”

Section: Methodsmentioning

confidence: 99%

“…Experiments utilized human CB 1 or CB 2 tagged at the N-terminus with three haemagglutinin sequences (3HA-hCB 1 , 3HA-hCB 2 ) stably transfected into HEK293. 47,48…”

Section: In Vitro Cannabinoid Receptor Binding Experimentsmentioning

confidence: 99%

The chemistry and pharmacology of putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) 5F‐PY‐PICA, 5F‐PY‐PINACA, and their analogs

Banister

Kevin

Martin

et al. 2019

Drug Testing and Analysis

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5F‐PY‐PICA and 5F‐PY‐PINACA are pyrrolidinyl 1‐(5‐fluoropentyl)ind (az)ole‐3‐carboxamides identified in 2015 as putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS). 5F‐PY‐PICA, 5F‐PY‐PINACA, and analogs featuring variation of the 1‐alkyl substituent or contraction, expansion, or scission of the pyrrolidine ring were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–quadrupole time‐of‐flight–mass spectrometry (LC–QTOF–MS). In competitive binding experiments against HEK293 cells expressing human cannabinoid receptor type 1 (hCB1) or type 2 (hCB2), all analogs showed minimal affinity for CB1 (pKi < 5), although several demonstrated moderate CB2 binding (pKi 5.45–6.99). In fluorescence‐based membrane potential assays using AtT20‐hCB1 or ‐hCB2 cells, none of the compounds (at 10 μM) produced an effect >50% of the classical cannabinoid agonist CP55,940 (at 1 μM) at hCB1, although several showed slightly higher relative efficacy at hCB2. Expansion of the pyrrolidine ring of 5F‐PY‐PICA to an azepane (8) conferred the greatest hCB2 affinity (pKi 6.99) and activity (pEC50 7.54, Emax 72%) within the series. Unlike other SCRA NPS evaluated in vivo using radio biotelemetry, 5F‐PY‐PICA and 5F‐PY‐PINACA did not produce cannabimimetic effects (hypothermia, bradycardia) in mice at doses up to 10 mg/kg.

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“…Homogenate radioligand competition binding assays were performed as previously described (Finlay et al, 2017), except that radioligand ([ 3 H]-CP) was used at its predefined pK D of 1.6 nM (Finlay et al, 2017). Full concentration-displacement curves were performed for each displacer ligand (WIN, AEA, 2-AG, THC, and BAY).…”

Section: Competition Binding Assaysmentioning

confidence: 99%

Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB₁ receptors

Zhu

Finlay

Glass

et al. 2019

British J Pharmacology

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Background and Purpose Receptor internalisation is by nature kinetic. Application of a standard equilibrium dose response model to describe the properties of a ligand inducing internalisation, while commonly used, are therefore problematic. Here, we propose two quantitative approaches to address this issue—(a) a model‐free method and (b) a kinetic modelling approach—and systematically evaluate the performance of these methods against traditional equilibrium methods to characterise the internalisation profiles of cannabinoid CB1 receptor agonists. Experimental Approach Kinetic internalisation assays were conducted using a concentration series of six CB1 receptor ligands. Internalisation rate analysis and snapshot equilibrium analysis were performed. A model‐free method was developed based on the mean residence time of internalisation. A kinetic internalisation model was developed under the quasi‐steady state assumption. Key Results Rates of receptor internalisation depended on both agonist and concentration. Agonist potencies from snapshot equilibrium analysis increased with stimulation time, and there was no single time point at which internalisation profiles could infer agonist properties in a comparative manner. The model‐free method yielded a time‐invariant measure of potency/efficacy for internalisation. The kinetic model adequately described the internalisation of CB1 receptors over time and provided robust estimates of both potency and efficacy. Conclusion and Implications Applying equilibrium analysis to a non‐equilibrium pathway cannot provide a reliable estimate of agonist potency. Both the model‐free and kinetic modelling approaches characterised the internalisation profiles of CB1 receptor agonists. The kinetic model provides additional advantages as a method to capture changes in receptor number during other functional assays.

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Gα_s signalling of the CB₁ receptor and the influence of receptor number

Cited by 76 publications

References 60 publications

Synthesis and pharmacology of new psychoactive substance 5F‐CUMYL‐P7AICA, a scaffold‐ hopping analog of synthetic cannabinoid receptor agonists 5F‐CUMYL‐PICA and 5F‐CUMYL‐PINACA

Synthesis and pharmacology of new psychoactive substance 5F‐CUMYL‐P7AICA, a scaffold‐ hopping analog of synthetic cannabinoid receptor agonists 5F‐CUMYL‐PICA and 5F‐CUMYL‐PINACA

The chemistry and pharmacology of putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) 5F‐PY‐PICA, 5F‐PY‐PINACA, and their analogs

Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB₁ receptors

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Gαs signalling of the CB1 receptor and the influence of receptor number

Cited by 76 publications

References 60 publications

Synthesis and pharmacology of new psychoactive substance 5F‐CUMYL‐P7AICA, a scaffold‐ hopping analog of synthetic cannabinoid receptor agonists 5F‐CUMYL‐PICA and 5F‐CUMYL‐PINACA

Synthesis and pharmacology of new psychoactive substance 5F‐CUMYL‐P7AICA, a scaffold‐ hopping analog of synthetic cannabinoid receptor agonists 5F‐CUMYL‐PICA and 5F‐CUMYL‐PINACA

The chemistry and pharmacology of putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) 5F‐PY‐PICA, 5F‐PY‐PINACA, and their analogs

Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB1 receptors

Contact Info

Product

Resources

About

Gα_s signalling of the CB₁ receptor and the influence of receptor number

Model‐free and kinetic modelling approaches for characterising non‐equilibrium pharmacological pathway activity: Internalisation of cannabinoid CB₁ receptors