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.
Subject(s)
Phenylurea Compounds/metabolism , Pyrimidinones/metabolism , Receptors, LHRH/antagonists & inhibitors , Receptors, LHRH/chemistry , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Humans , Ligands , Macaca , Models, Molecular , Molecular Sequence Data , Phenylurea Compounds/pharmacology , Protein Structure, Tertiary , Pyrimidinones/pharmacology , Structure-Activity RelationshipABSTRACT
SAR studies of 1,3,5-triazine-2,4,6-triones as human gonadotropin-releasing hormone receptor antagonists resulted in potent compounds. The best compound from the series had a binding affinity of 2 nM.
