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 hGn-RHR 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.

Challenges and Opportunities of Trapping Ligands

“…Many more studies are required to better understand the mechanisms regulating various analog-specific phenomena such as orthosteric or allosteric receptor binding, effects of various antagonists in constitutively active systems, analog-induced receptor signaling, trafficking, and degradation. Nonetheless, the data of Kohout et al (2007) strongly suggest that using inherent receptor trapping properties may lead to design of novel insurmountable antagonists within GPCRs and other receptor superfamilies. Perhaps an era of highly specific and efficacious insurmountable receptor antagonists is just beginning.…”

“…It is noteworthy that the evolutionary rate of gonadotropin receptors is considerably slower than that of their natural ligands. In contrast to gonadotropin receptors, significant functional differences between human and Old World monkey GnRH receptors, as described by Kohout et al (2007), may suggest adaptation of GnRH receptor to simultaneous modifications in other components of reproductive system during primate evolution. Consistent with more rigorous control of reproduction in apes and humans, significantly lower expression of human than rodent GnRH receptors was recently correlated with the presence of primate-specific lysine 191 residue, increased rate of receptor misfolding, and decreased receptor signaling .…”

“…It determines the longer drug effect in vitro or in vivo and is commonly considered a key parameter predicting duration of drug efficacy. Accordingly, Kohout et al (2007) showed a direct correlation between the degree of insurmountability and the dissociation rate of TAK-013 from the receptor. It is possible that the trapping mechanism may function not only for nonpeptide ligands but also may be responsible for delayed dissociation of many peptide and protein ligands.…”

“…Such a trapping mechanism could be both ligand-and receptor species-specific. Although analogous models were previously proposed for other G protein-coupled receptors, the study by Kohout et al (2007) provides an important advance in the GnRH antagonists field and an illustration of the fact that preclinical studies using animal models with nonhuman receptors may have very limited value in predicting drug efficacy in human disease. There are many examples showing that highaffinity protein, peptide, or nonpeptide agonists or antagonists have also enhanced clinical efficacy.…”

“…Examples of the first two instances are presented in the article by Kohout et al (2007). The authors determined amino acid residues involved in TAK-013 insurmountability at the human GnRH receptor that are absent in the Rhesus macaque GnRH receptor molecule.…”