PhD Scientific Days 2026

Poster Session 3.W - Pharmaceutical Sciences and Health Technologies

Kinetic Characterization of [³H]ASEM Binding at α7 Nicotinic Acetylcholine Receptor

Name of the presenter

Lóska, Dóra

Institute/workplace of the presenter

Department of Pharmacodynamics

Authors

Dóra Lóska¹, Ferenc Horti², András Boros³, Tamás Tábi¹, Balázs Lendvai³
1: Department of Pharmacodynamics
2: Gedeon Richter Plc.
3: Gedeon Richter Plc., Semmelweis University Richter Department

Text of the abstract

Introduction: The α7 nicotinic acetylcholine receptor (nAChR) is a key pharmacological target in neurological processes. Although positive allosteric modulators (PAMs) have gained increasing attention, their binding characteristics remain incompletely understood. Radioligands are available only for the orthosteric binding site with [11C]ASEM most widely used in human PET studies. Kinetic binding studies are particularly valuable, since they provide mechanistic insight into how fast a ligand binds (kon) and how long it stays bound (koff).
Aims: Our aim was to characterize first the in vitro binding kinetics of [³H]ASEM to human α7 nicotinic acetylcholine receptor and to determine the key kinetic parameters (Kd, koff, kon, t1/2).
Method: Membrane homogenates were prepared from Flp-In 293 cells stably expressing recombinant α7 nACh subunits forming functional homopentameric ion channels. Binding assays were performed at 25 °C using the orthosteric antagonist radioligand [³H]ASEM. Non-specific binding was defined in the presence of 10 µM methyllycaconitine (MLA), and PAMs were applied as pretreatment when required. Association kinetics were assessed over varying incubation times, while dissociation was initiated by excess MLA. Samples were filtered over UniFilter® GF/C, and radioactivity was quantified using a microplate counter.
Results: Kinetic binding analysis of [³H]ASEM at α7 nACh receptors revealed a dissociation rate constant (koff) of 0.462 min⁻¹ and an association rate constant (kon) of 1.0785 nM⁻¹·min⁻¹, corresponding to a dissociation half-life (t1/2) of 1.57 min. These parameters indicate relatively rapid binding kinetics. The equilibrium dissociation constant (Kd) derived from kinetic parameters (koff/kon) was 0.428 nM, while the experimentally determined Kd from equilibrium binding was 0.5654 nM. We began to investigate the effect of a PAM compound (BNC-375) which did not affect the dissociation kinetics of [³H]ASEM.
Conclusion: We determined the kinetic parameters of [³H]ASEM binding at α7 nACh receptor. BNC-375 did not affect [³H]ASEM dissociation kinetics. In addition to the investigation of α7 nACh receptor allosteric modulators, particularly PAMs, which are of significant interest for therapeutic development, we plan to extend these kinetic investigations to agonist radioligand for a more comprehensive assessment of PAMs receptor binding.