title: Trapping of a nonpeptide ligand by the extracellular domains of the gonadotropin-releasing hormone receptor results in insurmountable antagonism.
abstract: Drugs that exhibit insurmountable antagonism are proposed to provide improved clinical efficacy through extended receptor blockade. Long-term suppression of the gonadotropin-releasing hormone receptor (GnRHR) is an important therapeutic approach for a number of sex hormone-dependent diseases. In this study, we describe the mechanism and structural components required for insurmountable activity of a GnRHR antagonist. TAK-013 behaves as an insurmountable antagonist at the human receptor (hGnRHR) but as a surmountable antagonist at the macaque receptor (mGnRHR). Mutation of the eight residues that differ between hGnRHR and mGnRHR identified Ser-203 and Leu-300 in extracellular loops (ECL) 2 and 3 of hGnRHR as essential for the insurmountability of TAK-013. Substitution of the corresponding residues in mGnRHR with Ser and Leu (mGnRHR-P203S/V300L) converts TAK-013 to an insurmountable antagonist. In addition, mutation of Met-24 to Leu in the amino terminus of hGnRHR also ablates the insurmountable antagonism of TAK-013. The mechanism of insurmountability of TAK-013 was determined to be governed by its rate of dissociation from the receptor. Although the association rates of TAK-013 to hGnRHR, mGnRHR, and mGnRHR-P203S/V300L do not differ, the dissociation rate half-life correlates closely with the degree of insurmountability observed (169, 9, and 55 min, respectively). Taken together, these data suggest a model of the GnRHR in which ECL2, ECL3, and the amino terminus engage with TAK-013 upon its binding to the transmembrane region of the receptor. These additional interactions form a "trap door" above TAK-013, restricting its dissociation and thus resulting in its insurmountability.
authors.affiliation: Department of Endocrinology, Neurocrine Biosciences Inc., 12790 El Camino Real, San Diego, CA 92130, USA. tkohout@neurocrine.com
authors.forename: Trudy A
authors.initials: TA
authors.lastname: Kohout
authors.forename: Qiu
authors.initials: Q
authors.lastname: Xie
authors.forename: Shelby
authors.initials: S
authors.lastname: Reijmers
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Finn
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Guo
authors.forename: Yun-Fei
authors.initials: YF
authors.lastname: Zhu
authors.forename: R Scott
authors.initials: RS
authors.lastname: Struthers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D008024:Ligands; D008251:Macaca; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010671:Phenylurea Compounds / Q000378:metabolism* / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011744:Pyrimidinones / Q000378:metabolism* / Q000494:pharmacology; D011966:Receptors, LHRH / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: C470760:5-(N-benzyl-N-methylaminomethyl)-1--(2,6-difluorobenzyl)-6-(4-(3-methoxyureido)phenyl)-3-phenylthieno(2,3-d)pyrimidine-2,4(1H,3H)-dione; D008024:Ligands; D010671:Phenylurea Compounds; D011744:Pyrimidinones; D011966:Receptors, LHRH