FRET Studies of Quinolone-Based Bitopic Ligands and Their Structural Analogues at the Muscarinic M₁ Receptor

Abstract

Aiming to design partial agonists as well as allosteric modulators for the M muscarinic acetylcholine (MAChR) receptor, two different series of bipharmacophoric ligands and their structural analogues were designed and synthesized. The hybrids were composed of the benzyl quinolone carboxylic acid (BQCA)-derived subtype selective allosteric modulator 3 and the orthosteric building block 4-((4,5-dihydroisoxazol-3-yl)oxy)-N,N-dimethylbut-2-yn-1-amine (base of iperoxo) 1 or the endogenous ligand 2-(dimethylamino)ethyl acetate (base of acetylcholine) 2, respectively. The two pharmacophores were linked via alkylene chains of different lengths (C4, C6, C8, and C10). Furthermore, the corresponding structural analogues of 1 and 2 and of modified BQCA 3 with varying alkyl chain length between C2 and C10 were investigated. Fluorescence resonance energy transfer (FRET) measurements in a living single cell system were investigated in order to understand how these compounds interact with a G protein-coupled receptor (GPCR) on a molecular level and how the single moieties contribute to ligand receptor interaction. The characterization of the modified orthosteric ligands indicated that a linker attached to an orthoster rapidly attenuates the receptor response. Linker length elongation increases the receptor response of bitopic ligands, until reaching a maximum, followed by a gradual decrease. The optimal linker length was found to be six methylene groups at the MAChR. A new conformational change is described that is not of inverse agonistic origin for long linker bitopic ligands and was further investigated by exceptional fragment-based screening approaches.

Ein photoschaltbarer Ligand zur Regulierung der Rezeptoraktivierung

“…Daraus (zu Details siehe Lit. ) kann geschlossen werden, das Photoiperoxo 12 eine spezifische Affinität zum Rezeptor besitzt und antagonistisch wirkt. Dies wurde in Studien zur Calcium‐Freisetzung nach Ligandbindung (Abbildung S2 c in den SI) mithilfe eines Calciumionen‐ und DAG‐sensitiven Fluoreszenztests bestätigt.…”

“…Bemerkenswerterweise zeigt sich eine verlangsamte Aktivierungskinetik im Vergleich zu Iperoxo. Vergleichbare Resultate hatten sich bereits für das Derivat 18 mit dem linearen Alkin‐Linker gezeigt (Abbildung f), dessen Kinetik deutlich langsamer ist als für vergleichbare dualstere Liganden mit Polymethylen‐Linker. Dies legt nahe, dass die Struktur der Linker‐Einheit und die Rezeptoraktivierungskinetik eng zusammenhängen.…”

“…Durch die Reduktion der Lichtexposition wurde die durch das trans ‐Isomer verursachte Gq‐Aktivierung nicht beeinflusst, während das Signal des cis ‐Isomers signifikant um mehr als 30 % reduziert wurde. Anschließend nutzten wir eine duale Ca 2+ /DAG‐Fluoreszenzsonde, mit 488 nm und 562 nm Anregungswellenlängen, welche simultan oder separat genutzt werden kann, mit dem Vorteil, dass eine Anregungswellenlänge im Absorptionsmaximum von BQCAAI liegt, während die andere dies nicht tut (Abbildung a). Die Messung des Calciumsignals (ohne DAG) zeigt das Auftreten eines schnellen Signals für das trans ‐Isomer (Abbildung d, rot), während das cis ‐Isomer selbst nach 200 s kein Signal induziert (Abbildung e, rot).…”

Novel BQCA‐ and TBPB‐Derived M1 Receptor Hybrid Ligands: Orthosteric Carbachol Differentially Regulates Partial Agonism

“…[19] This approachh as the advantage that it is not reliant on the endogenous neurotransmitter and that biased signaling by activation of as pecific downstream signali sp ossible via as ingle molecule. [21][22][23][24] Furthermore,t he design of the connecting linker represents ac hallenge not only for dualsteric GPCR ligands, but for any kind of bivalent ligand.F or this reason,r andomw alk as well as computational approaches have been used to guide linker design. For this purpose, the connection points have to be chosen carefully in order to avoid alteration of essential functional groups responsible for receptor binding and function.…”

“…[20] In earlier studies, av ariety of dualsteric M 1 ligands have been developed, includingap hotoswitchable BQCA-iperoxoh ybrid. [21][22][23][24] Furthermore,t he design of the connecting linker represents ac hallenge not only for dualsteric GPCR ligands, but for any kind of bivalent ligand.F or this reason,r andomw alk as well as computational approaches have been used to guide linker design. [25] Because the orthosteric and allosteric binding pockets of the M 1 receptor are separated by the so-called "tyrosine lid" formed by three tyrosine residues (Figure1), dualsteric ligands have to bridge these two sites by al inker.W eh ave chosen three different linker lengths for this study.F irst, aC 3 -alkyl chain linker represents the shortestp ossible linker length to bypasst he tyrosine lid.…”