Discussion

In this study, we intended to find out the critical residues of μ-opioid receptor governing ligand bias by integrating computational simulations and cell assay based bias analysis. We first tried to predict the key residues influencing ligand bias at MOR by computer modeling. Then the site-directed mutation on MOR was conducted and CHO cell lines stably expressing wild-type and mutant MORs were established in order to characterize the binding affinities and functional activities of morphine and fentanyl derivatives with wild-type and mutant MORs. Based on results from functional assays, the bias profiles of morphine and fentanyl derivatives were obtained by operational model analysis. We found that 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 of MOR. The interactions among ligand and W6.48, Y7.43 were responsible for the signaling bias. The stabilization of Y7.43 by ligand and the stable interactions of ligand with W6.48 and Y7.43 were propitious to the β-arrestin signal, while the stabilization of W6.48 is propitious to biased G-protein signal.
Recent years, unremitting efforts continually put into the research about structures of GPCR. People were trying to illustrate the mechanism underlying the activation of receptor through analysis of the structural characteristics of active receptors. It is indicated that the ligand-induced comformation of receptor is conducive to the bias signaling(24 ,25 ). In this work, we found that the residue W6.48 and Y7.43 were paired activation switches of ligand bias at MOR, which is consistent with the computational result of Chen’s work(26 ). And we proposed that the interaction between W6.48 and Y7.43 with fentanyl derivatives lead to two contrary states of transmembrane helix. When H6 moved towards H5, the “close” state impeded the activation of G-protein; When H6 moved far away from H5, the “open” state is in favour of the G-protein-dependent signaling. This is in agreement with the previous structural researches of MOR and other GPCRs. It is reported that, compared with the structure of MOR-Nb39(15 ), the TM6 of MOR-Gi is outward from TM5(14 ). The structures of other active GPCRs, such as β2 adrenergic receptor and glucagon like peptide-1 receptor, revealed that activation always involves the relative outward movements of TM5 and TM6, which allows cavity structure for accommodating the α subunit of G-protein(27-29 ).
Opioids were used to alleviate or cure pain for centuries, but the opioid side-effects still limited their use in clinic. A great number of efforts were put in to solve the fatal hypoventilation and other adverse effects, but no completely ideal ligand was found. People have ever tried to design µ or κ subtype-selective ligands(30 ,31 ), as well as allosteric modulators(32 ), to obtain a pain-killer without those unwanted effects, but the result is not satisfactory. Recently, the bias agonism stands a chance of separating analgesia and adverse effects for opioid ligands, the discovery of potential drugs TRV130 and PZM21 was a good signal. However, there is still a long way to go. On one hand, the mechanism of biased signaling was not clear enough. We are not sure which structural features contribute to the specific conformation of the receptor that facilitate G-protein-biased signaling. On the other hand, there is controversy in the quantification and assessment of ligand bias, the method of bias analysis was not compeletely developed. In addition, TRV130 was not approved to use clinically for its little difference of hypoventilation versus morphine in phase Ⅲ study(33 ). In this study, we found the critical sites affecting ligand bias and tried to explain the mechanim of ligand bias at MOR, which contributed to better design and discovery of opioid ligands with reduced adverse effects.