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

Abstract

The sigma 1 receptor (σR) 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 σR are poorly understood, and molecular interactions of selective ligands with σR have not been elucidated. The recent crystallographic determination of σR 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 σR and its interaction with BiP. Haloperidol, PD144418, and 4-PPBP enhanced σR homomer BRET signals in a dose dependent manner, suggesting their significant effects in stabilizing σR multimerization, whereas (+)-pentazocine and several other ligands do not. In non-denaturing gels, (+)-pentazocine significantly decreased whereas haloperidol increased the fraction of σR multimers, consistent with the results from the homomer BRET assay. Further, BRET assays examining heteromeric σR-BiP interaction revealed that (+)-pentazocine and haloperidol induced opposite trends of signals. From molecular modeling and simulations of σR in complex with the tested ligands, we identified initial clues that may lead to the differed responses of σR 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 σR-ligand binding and its impact on interaction of σR with client proteins.

High-throughput screening discovers antifibrotic properties of haloperidol by hindering myofibroblast activation

“…In each binding site the highest-and the lowest-ranked haloperidol conformations, according to both Glide Score and Emodel score (38,39), are A and C, respectively (Supplemental Table 3). In its highest-ranked binding pose, haloperidol forms interactions with almost all the residues of the binding cavity (Supplemental Figure 6E and Supplemental Table 4), in agreement with those reported by Yano et al (40).…”

Structural basis for σ1 receptor ligand recognition

“…This is consistent with prior data, which suggest that σ1 receptor agonists bias the receptor towards lower molecular weight states, while antagonists bias it towards higher molecular weight states 4,[22][23][24] . Additionally, molecular modeling by Yano et al predicted that (+)-pentazocine and other multimer-impeding ligands would occupy this space differentially from haloperidol and other multimer-promoting ligands 24 , which is consistent with our structural results. Importantly, crystallographic studies by necessity favor conformationally stable, low-energy states, and so the structures shown here may not represent a fully activated state of the receptor.…”

“…Currently, the biochemical basis for agonism or antagonism at the σ 1 receptor is largely unknown, which complicates the unambiguous assignment of efficacy class for σ 1 ligands. The most well-documented biochemical difference between the two ligand classes is that antagonists increase the receptor's oligomeric state, while agonists decrease the oligomeric state 4,[22][23][24] . The structural data we present here show that these ligands occupy a different region of the binding pocket ( Fig.…”

“…Specifically, knockdown or antagonism of σ 1 receptor can potentiate G-protein coupled receptor (GPCR) signaling 2,3 , while agonists of the σ 1 receptor result in an IP3 receptor-dependent intracellular calcium flux 19 and inhibition of sodium 18 and potassium 20,21 channel currents. The σ 1 receptor exists in multiple oligomeric states, and reports suggest agonists causes a shift to monomeric or low molecular weight species, while antagonists bias the receptor towards high molecular weight species 4,[22][23][24] . However, the dominant physiologically relevant oligomeric forms and the precise way in which oligomerization is tied to agonist binding are unknown.…”

Virtual screening identifies novel high-affinity σ1 receptor ligands

“…Despite decades of study, structural data for the σ 1 receptor have only recently become available 11 . The receptor crystallized as a homotrimer, though it is known to exist in multiple oligomeric states 12-14 . Each monomer has an occluded ligand binding site containing Glu172 and Asp126 11 , which are essential for ligand binding 15 .…”