Selective and Wash‐Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single‐Molecule Imaging of μ‐Opioid Receptor Dimerization

Abstract

μ‐Opioid receptors (μ‐ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ‐ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ‐OR antagonist E‐p‐nitrocinnamoylamino‐dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single‐molecule imaging of μ‐ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ‐ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ‐ORs interact with each other to form short‐lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ‐OR pharmacology at single‐molecule level.

Investigation of Inactive-State κ Opioid Receptor Homodimerization via Single-Molecule Microscopy Using New Antagonistic Fluorescent Probes

“…The acquired movies were analysed with an automated single particle detection and tracking algorithm in MATLAB environment (Figure 4), and further investigated using custom algorithms, as previously described. 47,48,91,92 and anomalous diffusion exponent (α), derived from one-color SMM measurements using compound 9b…”

“…The recent introduction of innovative microscopy methods to investigate GPCRs in living cells with high spatio-temporal resolution [41][42][43][44] has led to important findings on their organization, signaling and dynamics on the surface of living cells. [45][46][47][48] Within this field, the development of novel fluorescent probes to label wild-type receptors is of high importance. [47][48][49][50][51][52][53][54] However, studies on developing KOR selective fluorescent probes have been particularly scarce.…”

“…[45][46][47][48] Within this field, the development of novel fluorescent probes to label wild-type receptors is of high importance. [47][48][49][50][51][52][53][54] However, studies on developing KOR selective fluorescent probes have been particularly scarce. Most available probes are non-selective derivatives of wide-spectrum OR antagonists like naloxone or βnaltrexamine.…”

“…[77][78][79] Furthermore, Cy5 has been successfully used in recently developed opioid fluorescent probes that have been successfully used both in vitro and in vivo. 47,48,59,[80][81][82] In previous studies [33][34][35][36] it had been shown that the implementation of a linker on the guanidinyl NH leads to molecules which can retain affinity, antagonistic activity and selectivity for the KOR. Inspired by these studies, and also by investigations highlighting the contribution of the linker's length and physicochemical properties in avoiding non-specific binding and preserving the pharmacological profile of the prototype ligand, 60,61,[83][84][85] we designed three sets of fluorescent probes: a) compounds 9a and 9b, which bear a 11-bond aliphatic linker (Scheme 1); b) compound 13, which bears a 17-bond linker with the incorporation of a diglycine moiety (Scheme 2); c) compounds 17a and 17b, which bears a 23-bond linker with the incorporation of a tetraglycine moiety (Scheme 2).…”

Opioid Pharmacology under the Microscope

“…Pharmacology Fast Forward. Published on March 20, 2020as DOI: 10.1124 at ASPET Journals on August 1, 2020 molpharm.aspetjournals.org Downloaded from molecule imaging of receptors bound to a high-affinity fluorescent ligand revealing short-lived MOP-R homodimers in the plasma membrane (Gentzsch et al, 2019).…”