Pharmacological profiling of sigma 1 receptor ligands by novel receptor homomer assays

The sigma 1 receptor (σ₁R) is a structurally unique transmembrane protein that functions as a molecular chaperone in the endoplasmic reticulum (ER), and has been implicated in cancer, neuropathic pain, and psychostimulant abuse. Despite physiological and pharmacological significance, mechanistic underpinnings of structure-function relationships of σ₁R are poorly understood, and molecular interactions of selective ligands with σ₁R have not been elucidated. The recent crystallographic determination of σ₁R as a homo-trimer provides the foundation for mechanistic elucidation at the molecular level. Here we report novel bioluminescence resonance energy transfer (BRET) assays that enable analyses of ligand-induced multimerization of σ₁R and its interaction with BiP. Haloperidol, PD144418, and 4-PPBP enhanced σ₁R homomer BRET signals in a dose dependent manner, suggesting their significant effects in stabilizing σ₁R multimerization, whereas (+)-pentazocine and several other ligands do not. In non-denaturing gels, (+)-pentazocine significantly decreased whereas haloperidol increased the fraction of σ₁R multimers, consistent with the results from the homomer BRET assay. Further, BRET assays examining heteromeric σ₁R-BiP interaction revealed that (+)-pentazocine and haloperidol induced opposite trends of signals. From molecular modeling and simulations of σ₁R in complex with the tested ligands, we identified initial clues that may lead to the differed responses of σ₁R upon binding of structurally diverse ligands. By combining multiple in vitro pharmacological and in silico molecular biophysical methods, we propose a novel integrative approach to analyze σ₁R-ligand binding and its impact on interaction of σ₁R with client proteins.