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

The interest in the allosteric modulation of G protein-coupled receptors has grown during 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 gonadotropin-releasing hormone (GnRH) receptor by amiloride analogs [e.g., 5-(N,N-hexamethylene)amiloride (HMA)] and a nonpeptide antagonistic furan derivative (FD-1) was studied. First, the compounds' ability to influence the dissociation of a radiolabeled peptide agonist (I-125-triptorelin) from human GnRH receptors stably expressed in Chinese hamster ovary cell membranes was investigated. HMA and FD-1, but not 5-(N-benzyl-N-methylaminomethyl) 1-(2,6-difluorobenzyl)-6-[4-(3-methoxyureido)phenyl]-3-phenylthieno[2,3-d] pyrimidine-2,4(1H,3H)-dione (TAK-013), another nonpeptide antagonist, were shown to increase the dissociation rate of I-125-triptorelin, revealing their allosteric inhibitory characteristics. The simultaneous addition of HMA and FD-1 resulted in an additive effect on the dissociation rate. Second, in a functional assay, it was shown that HMA was a noncompetitive antagonist and that FD-1 had both competitive and noncompetitive antagonistic properties. Equilibrium displacement studies showed that the inhibition of I-125-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, noncooperative, 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.