Received for publication November 16, 2007.
Revised March 13, 2008.
Accepted for publication March 14, 2008.

Amiloride derivatives and a non-peptidic antagonist bind at two distinct allosteric sites in the human gonadotropin-releasing hormone receptor

Abstract

The interest in allosteric modulation of G protein-coupled receptors has grown in the past decade. It has been shown that ligands acting at allosteric sites present in these important drug targets have the ability to modulate receptor conformations and fine-tune pharmacological responses to the orthosteric ligand. In the present study, allosteric modulation of the human GnRH receptor by amiloride analogues (e.g. HMA) and a non-peptide antagonistic furan derivative (FD-1) was studied. Firstly, the compounds' ability to influence the dissociation of a radiolabeled peptide agonist ([¹²⁵I]triptorelin) from human GnRH receptors stably expressed in CHO cell membranes was investigated. HMA and FD-1, but not TAK-013, another non-peptide antagonist, were shown to increase the dissociation rate of [¹²⁵I]triptorelin, revealing their allosteric inhibitory characteristics. The simultaneous addition of HMA and FD-1 resulted in an additive effect on the dissociation rate. Secondly, in a functional assay it was shown that HMA was a non-competitive antagonist and that FD-1 had both competitive and non-competitive antagonistic properties. Equilibrium displacement studies showed that the inhibition of [¹²⁵I]triptorelin binding by FD-1 was not affected by HMA. Furthermore, the potency of HMA to increase radioligand dissociation was not affected by the presence of FD-1. Simulation of the data obtained in the latter experiment also indicated neutral cooperativity between the binding of HMA and FD-1. Taken together, these results demonstrate that HMA and FD-1 are allosteric inhibitors that bind at two distinct, non-cooperative, allosteric sites. This presence of a second allosteric site may provide yet another opportunity for the discovery of new ligands for the human GnRH receptor.

Key words: Neuropeptides, Gonadotropins, Receptor binding studies