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Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications
Published on: January 27, 2014
Comprehensive Signaling Profiles Reveal Unsuspected Functional Selectivity of δ-Opioid Receptor Agonists and Allow
Ahmed Mansour1,2, Karim Nagi3, Paul Dallaire1,2
1Department of Pharmacology and Physiology, Faculty of Medicine, Université de Montréal, Montréal, Quebec H3T 1J4, Canada.
Abstract:
Prolonged exposure to opioid receptor agonists triggers adaptations in the adenylyl cyclase (AC) pathway that lead to enhanced production of cyclic adenosine monophosphate (cAMP) upon withdrawal. This cellular phenomenon contributes to withdrawal symptoms, hyperalgesia and analgesic tolerance that interfere with clinical management of chronic pain syndromes. Since δ-opioid receptors (DOPrs) are a promising target for chronic pain management, we were interested in finding out if cell-based signaling profiles as generated for drug discovery purposes could inform us of the ligand potential to induce sensitization of the cyclase path. For this purpose, signaling of DOPr agonists was monitored at multiple effectors. The resulting signaling profiles revealed marked functional selectivity, particularly for Met-enkephalin (Met-ENK) whose signaling bias profile differed from those of synthetic ligands like SNC-80 and ARM390. Signaling diversity among ligands was systematized by clustering agonists according to similarities in E max and Log(τ) values for the different responses. The classification process revealed that the similarity in Gα/Gβγ, but not in β-arrestin (βarr), responses was correlated with the potential of Met-ENK, deltorphin II, (d-penicillamine2,5)-enkephalin (DPDPE), ARM390, and SNC-80 to enhance cAMP production, all of which required Ca2+ mobilization to produce this response. Moreover, superactivation by Met-ENK, which was the most-effective Ca2+ mobilizing agonist, required Gαi/o activation, availability of Gβγ subunits at the membrane, and activation of Ca2+ effectors such as calmodulin and protein kinase C (PKC). In contrast, superactivation by (N-(l-tyrosyl)-(3S)-1,2,3,4-tetrahydroisoquinoline-3-carbonyl)-l-phenylalanyl-l-phenylalanine (TIPP), which was set in a distinct category through clustering, required activation of Gαi/o subunits but was independent of the Gβγ dimer and Ca2+ mobilization, relying instead on Src and Raf-1 to induce this cellular adaptation.
Insights
Opioid receptor agonists cause cellular changes that worsen pain and tolerance. Understanding delta-opioid receptor signaling diversity is key to developing better pain treatments.
Area of Science:
- Pharmacology
- Cellular Biology
- Neuroscience
Background:
- Prolonged opioid agonist exposure causes adaptive changes in the adenylyl cyclase (AC) pathway, increasing cyclic adenosine monophosphate (cAMP) production upon withdrawal.
- These adaptations contribute to withdrawal symptoms, hyperalgesia, and analgesic tolerance, complicating chronic pain management.
- Delta-opioid receptors (DOPrs) are a promising target for managing chronic pain.
Purpose of the Study:
- To investigate if cell-based signaling profiles can predict the potential of DOPr ligands to induce AC pathway sensitization.
- To explore the functional selectivity of various DOPr agonists and their impact on cellular signaling.
Main Methods:
- Monitoring the signaling of DOPr agonists at multiple cellular effectors.
- Systematizing ligand signaling diversity by clustering agonists based on Emax and Log(τ) values for different responses.
- Analyzing the involvement of G protein subunits (Gα/Gβγ), β-arrestin (βarr), calcium (Ca2+) mobilization, calmodulin, protein kinase C (PKC), Src, and Raf-1 in cellular adaptations.
Main Results:
- DOPr agonists exhibited marked functional selectivity, with Met-enkephalin (Met-ENK) showing a distinct signaling bias compared to synthetic ligands (SNC-80, ARM390).
- Similarity in Gα/Gβγ, but not βarr, responses correlated with the potential of Met-ENK, deltorphin II, DPDPE, ARM390, and SNC-80 to enhance cAMP production, all requiring Ca2+ mobilization.
- Met-ENK-induced superactivation required Gαi/o, Gβγ, Ca2+ effectors (calmodulin, PKC). In contrast, TIPP-induced superactivation required Gαi/o but was independent of Gβγ and Ca2+ mobilization, relying on Src and Raf-1.
Conclusions:
- Cell-based signaling profiles can reveal functional selectivity and predict the potential of DOPr ligands to induce AC pathway sensitization.
- Different DOPr agonists induce distinct signaling pathways leading to cAMP superactivation, highlighting the importance of ligand bias in drug discovery for chronic pain.
- Understanding these diverse signaling mechanisms is crucial for developing effective and safer analgesics targeting DOPrs.
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