Innenrücktitelbild: Selective and Wash‐Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single‐Molecule Imaging of μ‐Opioid Receptor Dimerization (Angew. Chem. 15/2020)

Abstract

Hochselektive fluoreszierende Liganden abgeleitet aus dem Schlafmohn, wurden von D. Calebiro, M. Decker et al. in ihrem Forschungsartikel auf S. 6014 entwickelt, um zu untersuchen, ob μ‐Opioidrezeptoren (μ‐ORs) Dimere auf der Plasmamembran bilden können. Die Verbindungen weisen eine lange Verweilzeit und hohe Affinität auf, und eine kleine Fraktion kurzlebiger Homodimere konnte mithilfe dieser chemischen Werkzeuge durch Einzelmolekülmikroskopie nachgewiesen werden.

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

“…CD86 is an unrelated transmembrane protein, which is not expected to interact with KOR. CD86 was labeled with silicon rhodamine (SiR) via a SNAP tag fused at its N terminus. − …”

“…Then, by measuring the colocalization times between KOR- 9b and CD86-SNAP molecules, we were able to determine the expected distribution of random colocalizations. The frequency and duration of receptor–receptor interactions were then estimated by deconvolution of the KOR- 9a and KOR- 9b colocalization times with those obtained with KOR- 9b and CD86-SNAP as previously described. ,, The deconvolved colocalization times of KOR- 9a with KOR- 9b were virtually superimposable to those obtained with the control CD86-SNAP (Figure ). These data suggest that KORs bound to 9a and 9b ligands either do not undergo significant dimerization under our experimental conditions or the interactions are so short-lived (<100 ms) that they cannot be detected by our method.…”

“…The cells were incubated with saturating concentrations of the fluorescent probes ( 9a: 10 nM; 9b: 100 nM), rapidly washed, and immediately imaged by TIRF microscopy. The acquired movies were analyzed with an automated single-particle detection and tracking algorithm in the MATLAB environment (Figure ) and further investigated using custom algorithms, as previously described. ,,, …”

“…The recent introduction of innovative microscopy methods to investigate GPCRs in living cells with high spatio-temporal resolution − has led to important findings on their organization, signaling, and dynamics on the surface of living cells. − Within this field, the development of novel fluorescent probes to label wild-type receptors is of high importance. − However, studies on developing KOR-selective fluorescent probes have been particularly scarce. Most available probes are nonselective derivatives of wide-spectrum OR antagonists like naloxone or β-naltrexamine. − The very few high-affinity KOR fluorescent ligands available have been identified serendipitously by screening of compounds originally designed to target other OR subtypes .…”

“…The selection of a suitable fluorescent dye is crucial for fluorescent ligand design. The cyanine dyes Cy5 (excitation wavelength: 647 nm) and Cy3 (excitation wavelength: 555 nm) are excellent candidates for probe design, exhibiting a nontoxic profile, pH insensitivity, and photostability. − Furthermore, Cy5 has been successfully used in recently developed opioid fluorescent probes that have been successfully used both in vitro and in vivo. ,,,− …”