Identification of a novel tricyclic antidepressant binding site within opioid receptor using molecular dynamics and functional assays.

Abstract

Tricyclic antidepressants (TCAs) had been used as a first-line treatment for mood disorder, but rapidly vanished due to moderate-to-severe adverse effects. Instead, the TCAs showed some promising analgesic effects towards neuropathic diseases, and an outstanding therapeutic effect to patients who suffer from irritable bowel syndrome. However, the mechanism by which those atypical effect are manifested has been unclear. Among the proposed mechanisms were the well-known, pain-related inhibitory G-protein coupled receptor (GiPCR), namely, the opioid receptor (OR). Here, we confirmed that TCA indeed stimulates OR, and commences the Gi signaling. Based on minimal-energy docking, we have identified a putative TCA binding site within OR. So, we predicted the binding site of TCA based on the previously revealed Cryo-EM structure of the activated OR. Within the binding site, an aspartate residue showed the most significant contribution towards the drug binding, and aspartate-to-arginine mutation of the residue severely decreased the OR-Gαi FRET efficiency once robustly induced by AMI.As an alternative way to monitor the Gi-signaling efficacy, we have utilized a well-known functional relationship between Gαi protein and TRPC4. Namely, we measured TRPC4 current in HEK293 cells in which either WT or mutant ORs are co-expressed. All types of TCA elicited μOR-induced TRPC4 current, as did the δ- and κ-ORs. And as expected, TCA-induced current of TRPC4 was not observed in the Asp-Arg mutant of OR. Our findings demonstrate that OR could be proclaimed as a promising target among numerous binding partners of the TCA, and that TRPC4 activation could be expected as a significant downstream pathway.