Structure-Activity Relations of Successful Pharmacologic Chaperones for Rescue of Naturally Occurring and Manufactured Mutants of the Gonadotropin-Releasing Hormone Receptor

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Abstract
We expressed a test system of wild-type (WT) rat (r) and human (h) gonadotropin-releasing hormone (GnRH) receptors (GnRHRs), including naturally occurring (13) and manufactured (five) “loss-of-function” mutants of the GnRHR. These were used to assess the ability of different GnRH peptidomimetics to rescue defective GnRHR mutants and determine their effect on the level of membrane expression of the WT receptors. Among the manufactured mutants were the shortest rGnRHR C-terminal truncation mutant that resulted in receptor loss-of-function (des325–327-rGnRHR), two nonfunctional deletion mutants (des237–241-rGnRHR and des260–265-rGnRHR), two nonfunctional Cys mutants (C229A-rGnRHR and C278A-rGnRHR); the naturally occurring mutants included all 13 full-length GnRHR point mutations reported to date that result in full or partial human hypogonadotropic hypogonadism. The 10 peptidomimetics assessed as potential rescue molecules (“pharmacoperones”) are from three differing chemical pedigrees (indoles, quinolones, and erythromycin-derived macrolides) and were originally developed as GnRH peptidomimetic antagonists. These structures were selected for this study because of their predicted ability to permeate the cell membrane and interact with a defined affinity with the GnRH receptor. All peptidomimetics studied with an IC50 value (for hGnRHR) ≤2.3 nM had measurable efficacy in rescuing GnRHR mutants, and within a single chemical class, this ability correlated to these IC50 values. Erythromycin-derived macrolides with IC50 values as high as 669.5 nM showed efficacy as rescue compounds. The ability to rescue a particular receptor was a reasonable predictor of the ability to rescue others, even across species lines, although particular mutants could not be rescued by any of the drugs tested.