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.