Allosteric and Orthosteric Binding Modes of Two Nonpeptide Human Gonadotropin-Releasing Hormone Receptor Antagonists

Abstract

Nonpeptide antagonists of the human gonadotropin-releasing hormone receptor (GnRH-R) have been the subject of considerable interest because of their potential as a new class of oral therapeutics for the treatment of sex hormone-dependent diseases and infertility. While many classes of competitive GnRH-R antagonists have been described, we present here the first characterization of an allosteric nonpeptide GnRH-R antagonist. Previously, 5-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-ylmethyl)furan-2-carboxylic acid (2,4,6-trimethoxyphenyl)amide (here called Furan-1) had been demonstrated to be a potent GnRH-R antagonist both in vitro and in vivo. Using mutagenesis, the binding sites for Furan-1 and another potent nonpeptide antagonist (NBI-42902) have been mapped and are shown to be adjacent but nonoverlapping. Furan-1 is shown to affect the binding kinetics of radiolabeled peptide agonists as well as radiolabeled NBI-42902, and the kinetic data fit the allosteric ternary complex model. Furan-1 is considerably negatively cooperative with the nonpeptide antagonist and extremely negatively cooperative with the peptide agonist [125I-His5,d-Tyr6]GnRH so that it is nearly indistinguishable from an orthosteric competitive compound. Taken together, these data were used to develop a model of the nonpeptides bound to the GnRH-R binding site consistent with the current data.

Amiloride Derivatives and a Nonpeptidic Antagonist Bind at Two Distinct Allosteric Sites in the Human Gonadotropin-Releasing Hormone Receptor

“…In this assay, HMA and FD-1 showed the same effects when the endogenous ligand GnRH was used (data not shown), even though the binding sites of triptorelin and GnRH are not identical (Fromme et al, 2001). Furthermore, FD-1 seemed to be a more potent allosteric inhibitor than Furan-1 (Sullivan et al, 2006). To prove that the allosteric characteristics of FD-1 were specific for this nonpeptidic antagonist only, TAK-013 was also examined.…”

“…6). CMPD-1 has recently been shown to be an allosteric inhibitor for the GnRH receptor too (Sullivan et al, 2006). Previously, that same compound (named Furan-1 or CMPD-1) had been demonstrated to be a potent nonpeptidic antagonist (Anderes et al, 2003), whereas its allosteric effects occur at higher concentrations.…”

“…1). Amiloride derivatives have been well described as allosteric inhibitors for different GPCRs at concentrations in the high micromolar range (Gao and IJzerman, 2000), whereas FD-1 is a derivative of a recently described allosteric inhibitor for the GnRH receptor (Sullivan et al, 2006). The ability of a compound to modulate the dissociation rate of 125 I-triptorelin was used as a measure for allosteric modulation.…”

Silicon Analogues of the Nonpeptidic GnRH Antagonist AG‐045572: Syntheses, Crystal Structure Analyses, and Pharmacological Characterization

“…The first aqueous wash solution (A) was extracted with CH 2 Cl 2 (20 mL), and the resulting organic extract was used to extract the second aqueous wash solution (B), and the organic extract was separated, followed by a second extraction of the aqueous was solutions A and B with a fresh portion of CH 2 Cl 2 (20 mL), using the same protocol as described for the first extraction sequence. All organic extracts were combined and dried over anhydrous Na 2 SO 4 (the remaining Na 2 SO 4 and solids that did not dissolve in either phase were removed by filtration). The solvent was removed under reduced pressure, and the residue was dried in vacuo (10 mbar, 20 8C, 30 min) and then purified by column chromatography on silica gel (stationary phase A, 250 g; column dimensions, 70 3.5 cm; eluent, EtOAc/ n-hexane 55:45 (v/v)).…”

“…The resulting two-phase mixture was stirred at 0 8C for 5 min and then at 20 8C for a further 15 min. The organic phase was separated, the aqueous layer was extracted with CH 2 Cl 2 (3 50 mL), and the organic extracts were combined and dried over anhydrous Na 2 SO 4 . The solvent was removed under reduced pressure, and the solid residue was dried in vacuo (0.01 mbar, 20 8C, 1 h) and dissolved in Et 2 O (140 mL).…”