New Technologies for Elucidating Opioid Receptor Function

Abstract

Recent advances in technology, including high resolution crystal structures of opioid receptors, novel chemical tools, and new genetic approaches have provided an unparalleled palette of tools for deconstructing opioid receptor actions in vitro and in vivo. Here we provide a brief description of our understanding of opioid receptor function from both molecular and atomic perspectives, as well as their role in neural circuits in vivo. We then show how insights into the molecular details of opioid actions can facilitate the creation of functionally selective (biased) and photoswitchable opioid ligands. Finally, we describe how newly engineered opioid receptor-based chemogenetic and optogenetic tools, and new mouse lines, are expanding and transforming our understanding of opioid function and, perhaps, paving the way for new therapeutics.

Keywords: DREADDs; biased signaling; chemogenetics; functional selectivity; mouse models; optogenetics.

Figure 1. Identification of G-protein and β-arrestin biased κ-opioid agonists

A. Molecular model of docking pose of U69,593 to KOR and structures of U69593 and ICI 199,441. ICI 199,441 was identified as a β-arrestin biased agonist B. Docking pose of salvinorin A to KOR, structure of salvinorin A and RB-64. RB-64 was identified as a G-protein biased KOR agonist C. Salvinorin A shows all of the prototypical actions of KOR agonists in wild-type (WT) mice although certain side-effects (sedation, impaired coordination and anhedonia) are reduced in β-arrestin2 knock-out (KO mice). RB-64 has analgesic actions and mildly impairs coordination, but is apparently devoid of anhedonia and sedation in WT mice. RB-64 has no effect in KOR KO mice indicating its effects are likely due to KOR agonism.

Figure 2. Molecular insights into opioid receptor actions yield structure based design of new DREADD

A and B show overview and close-up view of the sodium (Na+) site in the 5-opioid receptor (40). In B, residues are enumerated according to the Ballosteros-Weinstein convention (95). C shows the putative location of the Na+ site in the inactive state of the μ-opioid receptor (11), while D shows structural rearrangements leading to the loss of this site in the activated and presumably G-protein coupled state (37). E shows docking results for salvinorin B—an inactive metabolite of salvinorin A to the wild-type κ-opioid receptor (52). F shows how salvinorin B is predicted to interact with the D3.32N mutant opioid receptor the κ-opioid DREADD (KORD)

Figure 3. Summary of modern optogenetic approaches for dissecting opioid peptide and receptor function in vitro and in vivo

A. Cartoon depicting chimeric “Opto-XR” approach in which rhodopsin cDNA is fused with wildtype GPCR cDNA intracellular loops and tail to generate a photo-sensitive receptor system capable of spatiotemporal engagement of canonical GPCR signaling pathways such as Gq, Gs, and Gi or arrestin recruitment in selected cell types when combined with viral and genetic approaches in vivo. Opto-MOR receptors (62) take advantage of similarities between RO4 Gi-coupled opsins and Mu-opioid receptors. B. Adapted from (70)Left, One-letter amino-acid sequences of LE, Dyn-8 and their corresponding photo-switchable CNB-modified analogues CYLE and CYD8. Right, the chemical structure of CYLE. The CNB moiety (photoswitch) is highlighted in red. C. Summary of circuit-based chemogenetic and optogenetic targeting approach and available endogenous opioid cre-driver mice. Stategy is provided using a double-inverted open reading frame (DIO) construct, and fiber optics for optogenetic manipulation. For DREADD-based approaches, injection and then CNO manipulations would occur in vivo or in vitro. Extensions of this approach are also possible, whereby other opsins or DREADDs can be used for multiplexing or inhibition experiments.