Abstract

Background and Purpose
The development of biased agonism provides a promising avenue to improve the pharmacological properties of fentanyl derivatives, but the molecular mechanism underlying ligand bias still remains ambiguous. Therefore we sought to find out the critical sites of μ-receptor governing ligand bias and clarify corresponding molecular mechanism for designing and synthesizing effective analgesics with reduced adverse effects.
Experimental Approach
Critical sites governing ligand bias were identified both by computational prediction and cell assay-based bias analysis on wild-type and site-directed mutant μ-opioid receptor. Then molecular dynamics simulations of wild-type and mutant μ-opioid receptor were conducted to investigate the mechanism of bias activation.
Key Results
D3.32A and H6.52L mutation disrupted the binding of fentanyl derivatives with μ-opioid receptor. W6.48L mutation drove most fentanyl derivatives to β-arrestin-bias but promote sufentanil to cAMP signaling-bias. The result of molecular dynamics simulation showed that W6.48 and Y7.43 were paired activation switches of ligand bias at μ-opioid receptor.
Conclusion and Implications
D3.32 and H6.52 were critical residues in driving morphine and fentanyl derivatives to bind with μ-opioid receptor. W6.48 was a pivotal residue in governing the bias signaling and the interactions of ligands with W6.48 and Y7.43 were the structural determinants for the signaling bias of μ-opioid receptor, which will be conducive for better design and synthesis of effective opioid analgesics with the reduced adverse effects.
Fentanyl derivatives | ligand bias | μ-opioid receptor | molecular dynamics simulations | biased mechanism
Morphine and fentanyl derivatives have been the world’s most widely and frequently used opioid analgesics for decades(1 ). However, various adverse effects threaten the medication safety of morphine and fentanyl derivatives, such as gastrointestinal disorder, tolerance, dependence and respiratory depression etc(2 ). Notably, morphine and fentanyl derivatives have been evolved in the global public health threat recent years for an unprecedented rise of death due to respiratory depression caused by overdose(3 ,4 ). Consequently, unremitting efforts have been directed towards seeking novel opioid analgesics with improved therapeutic profiles.
A recent development in theory of biased agonism at μ-opioid receptor (MOR) provides a promising avenue for therapeutic improvement. It is reported that both desirable and adverse effects of morphine and fentanyl derivatives are attributed to the activation of MOR(5 ). As other G-protein-coupled receptors (GPCRs), two parallel signaling pathways mediated the function of MOR through its activation, one is G protein-dependent signaling and the other is β-arrestin-dependent signaling. A series of β-arrestin-knockout mice experiments displayed enhanced and prolonged morphine-induced antinociception with attenuated respiratory suppression(6-10 ). Recent research indicated that β-arrestin-biased compounds of morphine and fentanyl derivatives are devoted to the respiratory depression while G protein-bias pathway is responsible for the antinociception(11 ). These findings showed that MOR agonists preferentially biased towards G protein signaling over β-arrestin signaling exert analgesia with reduced unwanted fatal side effects. The discovery of several G-protein biased compounds proves the rationality and practicality of biased agonism theory, such as PZM21 and TRV130(12 ) (13 ), which were promising potential novel analgesics.
The molecular mechanism underlying biased agonism still remains ambiguous. Recent crystal structure analyses of MOR displayed the binding modes of MOR with agonists(14 ,15 ), which suggested that different ligands may induce diverse multiple stable receptor conformations that leads to variety degrees of activation of downstream signaling pathways. Jeffrey S. Smith advanced a ternary complex model to explain the factors resulted in the development of biased response, allosterically interpret the interactions of receptors with ligand and transducer(16 ). The intermediate conformational state of receptor induced by ligand decides either transducer-G protein or β-arrestin is needed for stabilization of the ternary complex. Researches of other GPCR, such as neuropeptide Y1 and Y4 receptor, CXC-chemokine receptor 4 and 7, suggested that distinct amino acid within the receptor can influence the downstream signaling bias(17 ,18 ). Hence, there may be some crucial residues in MOR within core binding region accounting for biased agonism, and these residues are the breakthrough to explore the mechanism underlying the development of signaling bias, which will direct the design and syntheses of desirable biased ligands. In this study, the mutation experiments and cell-based G-protein and β-arrestin assays are in combination with computational modeling studies were used to identify the key residues governing ligand bias and to clarify the molecular mechanism underlying biased agonism.