Receptor-Binding Affinity of Gonadotropin-Releasing Hormone Analogs: Analysis by Radioligand-Receptor Assay

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Abstract
A recently developed radioligand-receptor assay for pituitary gonadotropin-releasing hormone (GnRH) receptors, employing a radioiodinated superagonist analog as the ligand, was used to analyze the receptor-binding properties of several GnRH analogs. This receptor assay is specific for GnRH and related peptides with agonist or antagonist activity. All superagonist analogs had equilibrium association constants (Ka) between 4- and 8-fold greater than that of the natural GnRH decapeptide (Ka = 6.6 × 108 m−1). Conversely, weak agonists and inactive analogs were bound with affinities 0.003–0.05 times that of GnRH. Weak antagonists, such as [D-Phe2]GnRH and [DPhe2, D-Phe6]GnRH, had lower binding constants, while the most potent antagonist, [D-pGlu1, D-Phe2, D-Trp3,6]GnRH, exhibited 8-fold higher affinity than GnRH for pituitary receptors. There was a general positive correlation between receptor-binding affinity and relative biological activity, whether agonist or antagonist, for the several GnRH analogs tested. However, the observed increases in receptor affinity (4- to 8-fold) did not account for the 30- to 100-fold greater LH-releasing activity of the superagonists. The relatively high bioactivity of such analogs in comparison to their binding constants is attributable to their increased resistance to degradation in addition to their increased binding affinity. Comparison of receptor binding data with structural features of the GnRH analogs revealed that the three Nterminal amino acids are critical for receptor recognition, while the C-terminal des-Gly10 ethylamide modification does not enhance binding affinity. The radioligand assay provides a rapid and reliable method for evaluating the contribution of receptorbinding affinity to the agonist or antagonist actions of new GnRH analogs. This procedure also permits systematic determination of the structural requirements for GnRH receptor recognition. (Endocrinology106: 1154, 1980)