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

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• 作者：Staffan Uhlé ; n^a ; ^b ; ^{staffan.uhlen@neuro.uu.se" class="auth_mail" title="E-mail the corresponding author} ; Helgi B. Schiö ; th^b ; Jan Anker Jahnsen^a
• 关键词：Spiroxatrine ; MK912 ; Off-rate ; Residence time ; [3H]-RX821002 ; Kinetics
• 刊名：European Journal of Pharmacology
• 出版年：2016
• 出版时间：5 October 2016
• 年：2016
• 卷：788
• 期：Complete
• 页码：113-121
• 全文大小：1010 K

文摘

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 α_2A- and α_2C-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 k_off constants for various unlabeled ligands.

The results show that the α_2C-selectivity of MK912 depends mainly on a very slow off-rate at the α_2C-adrenoceptor subtype. Regarding the markedly α_2C- over α_2A-selective compound spiroxatrine, its much faster on-rate at α_2C- than α_2A-adrenoceptors explains much of its exceptional α_2C-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 k_off constants of unlabeled drugs.