In vitro pharmacological characterization of positive allosteric modulators of the delta opioid receptor

The aim of this thesis is the pharmacological characterization of a series of positive allosteric modulators (PAMs) targeting the δ-opioid peptide (DOP), receptor, a G protein- coupled receptor (GPCR) endogenously activated by the peptide Leu-Enkephalin (Leu-ENK) and implicated in the regulation of pain, mood, and emotional behavior. Upon activation, GPCRs couple to heterotrimeric G proteins and β-arrestins. It is possible that G proteins and β-arrestins can trigger different downstream signaling waves – with linked varied biological output. The DOP receptor belongs to the opioid receptor family, which also includes the μ- and κ-opioid receptors, and is predominantly expressed in the brain regions involved in pain modulation and affective control. Activation of the DOP receptor has been also associated with potential antidepressant and anxiolytic effects, with fewer adverse outcomes compared to μ- opioid receptor stimulation, making it an attractive therapeutic target for chronic pain and mood disorders. Unfortunately, orthosteric activation of the DOP receptor has been linked to pro-convulsant effects. In this study, the reference agonist Leu-Enkephalin and five putative PAMs—MIPS-3614, MIPS-3515, MIPS-3423, MIPS-3430, and MIPS-3793—were evaluated using the Bioluminescence Resonance Energy Transfer (BRET) assay. In this system, the donor (luciferase) and the acceptor (GFP) were fused respectively to the DOP receptor and β-arrestin2, enabling the quantification of receptor–β-arrestin interactions in living SH-SY5Y cells expressing DOP-β-arrestin2. Our analyses showed that proposed DOP PAMs mediate a robust leftward shift of the concentration-response curve to Leu-ENK. Future studies are necessary to understand whether this type of modulation is capable of evoking an altered signaling fingerprint downstream of the receptor. Thanks to allosteric modulation, it may be possible to increase DOP receptor activity – ameliorating pain - without incurring the side effects typical of the DOP and other classical opioid receptors.