Pharmacological selection of cannabinoid receptor effectors: Signalling, allosteric modulation and bias

Manning, Jamie J.; Green, Hayley M.; Glass, Michelle; Finlay, David B.

doi:10.1016/j.neuropharm.2021.108611

Cited by 24 publications

(20 citation statements)

References 159 publications

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“…Allostery is a remarkable phenomenon that is used ubiquitously in nature to achieve homeostasis. The motivation to understand allostery comes from a desire to untangle the native mechanisms of protein function, as well as the ultimate goal of designing allosteric proteins and allosteric drugs. − It is now clear that both structure and dynamics must be accounted for in describing allosteric transitions, and NMR is the single tool that is most ideally suited to provide both types of information . Nature often uses oligomerization and symmetry in allosteric mechanisms, and because homodimers are the most common form of enzyme found in nature, , we have chosen to study homodimeric systems by NMR to gain a deeper understanding of allostery.…”

Section: Introductionmentioning

confidence: 99%

Visualizing an Allosteric Intermediate Using CuAAC Stabilization of an NMR Mixed Labeled Dimer

et al. 2021

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Homodimers are the most abundant type of enzyme in cells, and as such, they represent the most elemental system for studying the phenomenon of allostery. In these systems, in which the allosteric features are manifest by the effect of the first binding event on a similar event at the second site, the most informative state is the asymmetric singly bound (lig 1 ) form, yet it tends to be thermodynamically elusive. Here we obtain milligram quantities of lig 1 of the allosteric homodimer, chorismate mutase, in the form of a mixed isotopically labeled dimer stabilized by Cu (I) -catalyzed azide−alkyne cycloaddition (CuAAC) between the subunits. Below, we outline several critical steps required to generate high yields of both types of unnatural amino acid-containing proteins and overcome multiple pitfalls intrinsic to CuAAC to obtain high yields of a highly purified, fully intact, active mixed labeled dimer, which provides the first glimpse of the lig 1 intermediate. These data not only will make possible NMR-based investigations of allostery envisioned by us but also should facilitate other structural applications in which specific linkage of proteins is helpful.

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Section: Introductionmentioning

confidence: 99%

Visualizing an Allosteric Intermediate Using CuAAC Stabilization of an NMR Mixed Labeled Dimer

et al. 2021

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“…In the midbrain consisting of the PAG, both endocannabinoid concentrations are largest during proestrus relative to most other phases 9 . Given these dynamic patterns, we must also acknowledge that CP55,940 is an orthosteric agonist that competes with the endocannabinoids to non‐selectively bind to CB1R and CB2R 33 . In the current study, the brain regions modulating antinociception were more sensitive to 1 mg/kg of CP55,940 during proestrus compared with metestrus (Figure 3A).…”

Section: Discussionmentioning

confidence: 71%

“… 9 Given these dynamic patterns, we must also acknowledge that CP55,940 is an orthosteric agonist that competes with the endocannabinoids to non‐selectively bind to CB1R and CB2R. 33 In the current study, the brain regions modulating antinociception were more sensitive to 1 mg/kg of CP55,940 during proestrus compared with metestrus (Figure 3A ). Although Bradshaw and colleagues 9 did not measure phase‐mediated endocannabinoid levels in the spine, it may be that during proestrus, CP55,940 out‐competes the endocannabinoids in the spine to activate the cannabinoid receptors.…”

Section: Discussionmentioning

confidence: 72%

“…9 Given these dynamic patterns, we must also acknowledge that CP55,940 is an orthosteric agonist that competes with the endocannabinoids to non-selectively bind to CB1R and CB2R. 33 In the current study, the brain regions modulating antinociception were more sensitive to 1 mg/kg of CP55,940 during proestrus compared Immediately following the tetrad battery of behavioral assays, blood was collected from all mice. Plasma was measured for circulating β-estradiol content.…”

Section: Discussionmentioning

confidence: 83%

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Impact of the mouse estrus cycle on cannabinoid receptor agonist‐induced molecular and behavioral outcomes

Kim

Zagzoog

Black

et al. 2022

Pharmacology Res & Perspec

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Sexual dimorphisms are observed in cannabinoid pharmacology. It is widely reported that female animals are more sensitive to the cataleptic, hypothermic, antinociceptive, and anti‐locomotive effects of cannabinoid receptor agonists such as CP55,940. Despite awareness of these sex differences, there is little consideration for the pharmacodynamic differences within females. The mouse estrus cycle spans 4–5 days and consists of four sex hormone‐mediated phases: proestrus, estrus, metestrus, and diestrus. The endocannabinoid system interacts with female sex hormones including β‐estradiol, which may influence receptor expression throughout the estrus cycle. In the current study, sexually mature female C57BL/6 mice in either proestrus or metestrus were administered either 1 mg/kg i.p. of the cannabinoid receptor agonist CP55,940 or vehicle. Mice then underwent the tetrad battery of behavioral assays measuring catalepsy, internal body temperature, thermal nociception, and locomotion. Compared with female mice in metestrus, those in proestrus were more sensitive to the anti‐nociceptive effects of CP55,940. A similar trend was observed in CP55,940‐induced catalepsy; however, this difference was not significant. As for cannabinoid receptor expression in brain regions underlying antinociception, the spine tissue of proestrus mice that received CP55,940 exhibited increased expression of cannabinoid receptor type 1 relative to treatment‐matched mice in metestrus. These results affirm the importance of testing cannabinoid effects in the context of the female estrus cycle.

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“…Current literature surrounding allosteric agonists in combination with orthosteric agonists is prone to misinterpretation due to experimental design and analysis failing to consider the magnitude of allosteric agonism in the absence of an orthosteric ligand. 34 One example of this misinterpretation is when PAM concentration−response curves are performed in the presence of the EC 20 orthosteric agonist, without considering the activity of the allosteric compound alone. This can lead to the inability to distinguish allosteric agonism from allosteric effects that are specific to the presence of an orthosteric agonist, 17,35−37 resulting in the incorrect designation of allosteric agonists as allosteric modulators.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

In Vitro Characterization of 6-Methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011) at the Type 1 Cannabinoid Receptor: Allosteric Agonist or Allosteric Modulator?

Green

Finlay

Ross

et al. 2022

ACS Pharmacol. Transl. Sci.

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Orthosteric activation of CB1 is known to cause a plethora of adverse side effects in vivo. Allosteric modulation is an exciting therapeutic approach and is hoped to offer improved therapeutic potential and a reduced on-target side effect profile compared to orthosteric agonists. This study aimed to systematically characterize the in vitro activity of the positive allosteric modulator ZCZ011, explicitly considering its effects on receptor regulation. HEK293 cells expressing hCB1 receptors were used to characterize ZCZ011 alone and in combination with orthosteric agonists. Real-time BRET approaches were employed for G protein dissociation, cAMP signaling, and β-arrestin translocation. Characterization also included ERK1/2 phosphorylation (PerkinElmer AlphaLISA) and receptor internalization. ZCZ011 is an allosteric agonist of CB1 in all pathways tested, with a similar signaling profile to that of the partial orthosteric agonist Δ9-tetrahydrocannabinol. ZCZ011 also showed limited positive allosteric modulation in increasing the potency and efficacy of THC-induced ERK1/2 phosphorylation, β-arrestin translocation, and receptor internalization. However, no positive allosteric modulation was observed for ZCZ011 in combination with either CP55940 or AMB-FUBINACA, in G protein dissociation, nor cAMP inhibition. Our study suggests that ZCZ011 is an allosteric agonist, with effects that are often difficult to differentiate from those of orthosteric agonists. Together with its pronounced agonist activity, the limited extent of ZCZ011 positive allosteric modulation suggests that further investigation into the differences between allosteric and orthosteric agonism is required, especially in receptor regulation end points.

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Pharmacological selection of cannabinoid receptor effectors: Signalling, allosteric modulation and bias

Cited by 24 publications

References 159 publications

Visualizing an Allosteric Intermediate Using CuAAC Stabilization of an NMR Mixed Labeled Dimer

Visualizing an Allosteric Intermediate Using CuAAC Stabilization of an NMR Mixed Labeled Dimer

Impact of the mouse estrus cycle on cannabinoid receptor agonist‐induced molecular and behavioral outcomes

In Vitro Characterization of 6-Methyl-3-(2-nitro-1-(thiophen-2-yl)ethyl)-2-phenyl-1H-indole (ZCZ011) at the Type 1 Cannabinoid Receptor: Allosteric Agonist or Allosteric Modulator?

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