Discovery, Optimization, and Characterization of ML417: A Novel and Highly Selective D3 Dopamine Receptor Agonist.

To identify novel D3 dopamine receptor (D3R) agonists, we conducted a high-throughput screen using a ß-arrestin recruitment assay. Counterscreening of the hit compounds provided an assessment of their selectivity, efficacy, and potency. The most promising scaffold was optimized through medicinal chemistry resulting in enhanced potency and selectivity. The optimized compound, ML417 (20), potently promotes D3R-mediated ß-arrestin translocation, G protein activation, and ERK1/2 phosphorylation (pERK) while lacking activity at other dopamine receptors. Screening of ML417 against multiple G protein-coupled receptors revealed exceptional global selectivity. Molecular modeling suggests that ML417 interacts with the D3R in a unique manner, possibly explaining its remarkable selectivity. ML417 was also found to protect against neurodegeneration of dopaminergic neurons derived from iPSCs. Together with promising pharmacokinetics and toxicology profiles, these results suggest that ML417 is a novel and uniquely selective D3R agonist that may serve as both a research tool and a therapeutic lead for the treatment of neuropsychiatric disorders.

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.