A new, simple and robust radioligand binding method used to determine kinetic off-rate constants for unlabeled ligands. Application at α 2A - and α 2C -adrenoceptors

Abstract

Kinetic on and off rate constants for many receptor ligands are difficult to determine with regular radioligand binding technique since only few of the ligands are available in labeled form. Here we developed a new and simple radioligand binding method for determining the kinetic off-rate constant for unlabeled ligands, using whole cells expressing α2A- and α2C-adrenoceptors. The new method involves pre-incubation with unlabeled ligand, centrifugation of microtiter plates in order to adhere the cells to the bottom surface, and then upside-down centrifugation of the plates for few seconds to wash away the non-bound fraction of the pre-incubated ligand. The final on-reaction assay for the radioligand is then started by quick addition of a relatively fast-associating radioligand to the cells. The curve obtained is defined by a fairly simple mathematical formula that reflects the simultaneous dissociation of pre-incubated ligand and association of the radioligand. The method proved to produce highly reproducible results in determining the koff constants for various unlabeled ligands. The results show that the α2C-selectivity of MK912 depends mainly on a very slow off-rate at the α2C-adrenoceptor subtype. Regarding the markedly α2C- over α2A-selective compound spiroxatrine, its much faster on-rate at α2C- than α2A-adrenoceptors explains much of its exceptional α2C-selectivity. Several new techniques for determining the kinetic component of ligand-receptor interactions at molecular level are currently developing. As a reference, based on standard radioligand binding techniques, the present study describes a simple and robust experimental and mathematical procedure for determining koff constants of unlabeled drugs.

Steviol Rebaudiosides bind to four different sites of the human sweet taste receptor (T1R2/T1R3) complex explaining confusing experiments

“…Nuclear Magnetic Resonance (NMR) is utilized to identify and map sweet ligand and T1R2/T1R3 heterodimer receptor binding sites, employing Saturation Transfer Difference (STD) spectra 12 . The radioligand binding assay determines equilibrium dissociation constants for both radioligands and nonradioactive ligands, providing insights into association and dissociation rates 49,50 , albeit with limitations related to radio ligand depletion 51 .…”