Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M₂ Receptor

Abstract

Fluorescently labeled dibenzodiazepinone-type muscarinic acetylcholine receptor (MR) antagonists, including dimeric ligands, were prepared using red-emitting cyanine dyes. Probes containing a fluorophore with negative charge showed high M R affinities (pK i (radioligand competition binding): 9.10-9.59). Binding studies at M 1 and M 3 -M 5 receptors indicated a M 2 R preference. Flow cytometric and high-content imaging saturation and competition binding (M 1 R, M 2 R and M 4 R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM 2 R cells). Results from dissociation and saturation binding experiments (M 2 R) in the presence of allosteric M 2 R modulators (dissociation: W84, LY2119620 and alcuronium; saturation binding: W84) were consistent with a competitive mode of action between the fluorescent probes and the allosteric ligands. Taken together, these lines of evidence indicate that these ligands are useful fluorescent molecular tools to label the M 2 R in imaging and binding studies, and suggest that they have a dualsteric mode of action.

UR-DEBa242: A Py-5-Labeled Fluorescent Multipurpose Probe for Investigations on the Histamine H3 and H4 Receptors

“…Over the last decades, fluorescent ligands have increasingly become valuable complementary tools to radioligands for investigations on ligand-receptor-interactions at GPCRs. 115,[135][136][137][138][139][140] In general, fluorescent probes are not affected by the above-mentioned disadvantages of radioligands (see section 1.2.6.1). 13 A fluorescent ligand basically consists of a pharmacophore, a linker and a fluorophore, whereas the precursors of the fluorophore (e.g.…”

“…13 Compared to radioligands, fluorescent ligands are superior molecular tools in imaging studies on receptor localization and trafficking in tissue or distinct cells by applying e.g. confocal General introduction -11-microscopy 136 or high content imaging 139 . Moreover, fluorescent ligands can be applied in receptor binding experiments, such as saturation-, kinetic-and competition binding assays.…”

“…Various techniques were employed, for instance flow cytometry 115,136,139 or fluorescence polarization 141 , but also Förster/bioluminescence resonance energy transfer (FRET/BRET) 142,143 .…”

Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins

“…Subsequently, flow cytometry experiments on M 2 R‐CHO cells allowed assessing the nonspecific binding lower than 10 % at K D concentrations. Finally, competition experiments via confocal microscopy allowed to identify the three fluorescent ligands ( 38‐40 ) exhibiting a dualsteric binding mode [107] …”

Data_Sheet_1.DOCX

“…The fluorophore is usually separated from the targeting functionality via a spacer arm; this is often required to maintain biological activity of the targeting portion of the probe and may also aid in water solubility. Several dye-conjugated antagonists of neuronal receptors and transporters have been reported in the literature (Madras et al, 1990;Bakthavachalam et al, 1991;Jacobson et al, 1995;Rayport and Sulzer, 1995;Cha et al, 2005;Eriksen et al, 2009;She et al, 2020). However, many commonly used fluorescent dyes have low quantum yields and suboptimal photostability, thereby limiting their utility in experiments that require prolonged monitoring of protein membrane dynamics at video rates (Resch-Genger et al, 2008).…”