|Title:||μ-Opioid receptor signalling mechanisms: quantifying bias and kinetics|
|Publisher:||University of Sydney.|
Faculty of Medicine.
|Abstract:||Opioids such as morphine remain among the mainstay treatments for management of moderate to severe pain. However, the long-term use of opioids is limited by the development of severe adverse effects such as tolerance and addiction. Numerous studies have been devoted to understand the molecular and cellular mechanisms that are responsible for MOPr signalling, short-term regulatory events and cellular adaptations that lead to opioid tolerance in order to develop analgesics with safer therapeutic profiles. A large body of evidence indicates that activity of different opioids at the same receptor does not always stimulate a similar set of signalling pathways. Indeed agonists have various efficacies that can be different when the receptor interacts with distinct signalling effectors, a phenomenon termed agonist bias or functional selectivity. This discovery offers new approaches for development of novel pathway-selective drugs that stabilize particular conformations of the receptor while preferably activating signalling pathways associated with therapeutic outcomes but not those responsible for adverse effects. Although this concept is very interesting, the identification and quantification of biased agonism still remains a challenge. Several analytical methods have been developed to address this issue, but these methods are not applicable for all the agonists and systems. In this study, different analytical approaches for bias quantification were compared for a range of MOPr agonists. MOPr agonists showed distinct bias for different pathways. For example, Bilaid-C2, a novel MOPr agonist, had similar effectiveness to DAMGO for G-protein activation but showed significantly lower effectiveness for Ser375 phosphorylation and βarrestin-2 recruitment and more importantly was not able to promote receptor internalization. In contrast, morphine did not display bias towards any pathways and had significantly lower effectiveness for all pathways compared to DAMGO. The bias values determined in this study can be used for future studies to translate the bias into physiological responses. Furthermore, this study showed that operational model is the optimal approach to determine bias when the agonist affinity value was fixed to the functional dissociation constant. The next part of the study investigated the off-rate kinetics of MOPr agonists for potassium current, Ser375 phosphorylation and βarrestin-2 recruitment. Present study illustrated that the kinetics of MOPr for these signalling pathways were ligand-dependent. Furthermore, there was a robust positive correlation between off-rate kinetics; agonists with greater time constant for G-protein deactivation exhibited relatively slower Ser375 dephosphorylation and βarrestin-2 unbinding, suggesting that higher agonist affinity for GIRK activation reflects sustained occupancy of MOPr in the phosphorylated state with the greater affinity to interact with βarrestin-2. In addition, the results provide strong evidence that duration of receptor occupancy contributes to development of bias. Slowly dissociating agonists, e.g. endomorphins, display greater bias towards endocytosis versus G-protein activation, whereas rapidly dissociating agonists are more biased towards G-protein activation and away from βarrestin-2 recruitment and internalization. In summary, these findings underscore the prominent role of binding kinetics in characterizing of bias profile. The present study demonstrated that off-rate kinetics of all signalling readouts are agonist dependent, suggesting that phosphorylation and arrestin binding require agonist-bound receptors. Moreover, duration of receptor occupancy is highly associated with agonist ability to stimulate arrestin-linked pathways and could be used as a major determinant of biased agonism.|
|Type of Work:||PhD Doctorate|
|Type of Publication:||Doctor of Philosophy Ph.D.|
|Appears in Collections:||Sydney Digital Theses (Open Access)|
|SIANATI Setarah - Final thesis.pdf||Final Thesis||6.58 MB||Adobe PDF|
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