Discovery of BAY-298 and BAY-899: Tetrahydro-1,6-naphthyridine-Based, Potent, and Selective Antagonists of the Luteinizing Hormone Receptor Which Reduce Sex Hormone Levels in Vivo.

The human luteinizing hormone receptor (hLH-R) is a member of the glycoprotein hormone family of G-protein-coupled receptors (GPCRs), activated by luteinizing hormone (hLH) and essentially involved in the regulation of sex hormone production. Thus, hLH-R represents a valid target for the treatment of sex hormone-dependent cancers and diseases (polycystic ovary syndrome, uterine fibroids, endometriosis) as well as contraception. Screening of the Bayer compound library led to the discovery of tetrahydrothienopyridine derivatives as novel, small-molecule (SMOL) hLH-R inhibitors and to the development of BAY-298, the first nanomolar hLH-R antagonist reducing sex hormone levels in vivo. Further optimization of physicochemical, pharmacokinetic, and safety parameters led to the identification of BAY-899 with an improved in vitro profile and proven efficacy in vivo. BAY-298 and BAY-899 serve as valuable tool compounds to study hLH-R signaling in vitro and to interfere with the production of sex hormones in vivo.

Estrogen receptor beta is involved in skeletal muscle hypertrophy induced by the phytoecdysteroid ecdysterone.

SCOPE: The phytoectysteroid ecdysterone (Ecdy) was reported to stimulate protein synthesis and enhance physical performance. The aim of this study was to investigate underlying molecular mechanisms particularly the role of ER beta (ERß). RESULTS: In male rats, Ecdy treatment increased muscle fiber size, serum IGF-1 increased, and corticosteron and 17ß-estradiol (E2) decreased. In differentiated C2C12 myoblastoma cells, treatment with Ecdy, dihydrotestosterone, IGF-1 but also E2 results in hypertrophy. Hypertrophy induced by E2 and Ecdy could be antagonized with an antiestrogen but not by an antiandrogen. In HEK293 cells transfected with ER alpha (ERα) or ERß, Ecdy treatment transactivated a reporter gene. To elucidate the role of ERß in Ecdy-mediated muscle hypertrophy, C2C12 myotubes were treated with ERα (ALPHA) and ERß (BETA) selective ligands. Ecdy and BETA treatment but not ALPHA induced hypertrophy. The effect of Ecdy, E2, and BETA could be antagonized by an ERß-selective antagonist (ANTIBETA). In summary, our results indicate that ERß is involved in the mediation of the anabolic activity of the Ecdy. CONCLUSION: These findings provide new therapeutic perspectives for the treatment of muscle injuries, sarcopenia, and cachectic disease, but also imply that such a substance could be abused for doping purposes.

Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta.

Estrogens control transcriptional responses through binding to two different nuclear receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta). Since these two ER subtypes are thought to mediate different biological effects, there is intense interest in designing subtype-selective ER ligands. In this study, we evaluated the ERalpha and ERbeta selectivity of 19 known estrogens and antiestrogens using reporter cell lines previously developed in our laboratory. The HELN-ERalpha and HELN-ERbeta cells stably express full-length ERalpha and ERbeta, respectively, and are derived from HELN cells (HeLa cells stably transfected with an ERE-driven luciferase plasmid). We report that 16alpha-LE2, PPT and 3beta,5alpha-GSD have a high ERalpha-selective agonist potency while 8beta-VE2, DPN, genistein and biochanin A show ERbeta selectivity with 8beta-VE2 being the most potent and selective ERbeta agonist. We also tested ER antagonists and we showed that raloxifene and RU486 are ERalpha and ERbeta-selective antiestrogens, respectively. In all cases, selectivity is due to differences in binding affinities as indicated by whole-cell ligand-binding assays. Very interestingly, we demonstrate that a combination of genistein and raloxifene produces a full-ERbeta specific response. Together these results demonstrate the usefulness of our stably transfected cell lines to characterize ER ligands and indicate that treatments combining agonist/antagonist ligands produce full-ERbeta selectivity.

In vivo properties of an anti-GnRH Spiegelmer: an example of an oligonucleotide-based therapeutic substance class.

Spiegelmers are high-affinity l-enantiomeric oligonucleotide ligands that display high resistance to enzymatic degradation compared with d-oligonucleotides. The target binding properties of Spiegelmers can be designed by an in vitro-selection process starting from a random pool of oligonucleotides. Applying this method, a Spiegelmer with high affinity (K(D) = 20 nM) for the peptide hormone, gonadotropin-releasing hormone (GnRH) was isolated. The Spiegelmer acts as an antagonist to GnRH in Chinese hamster ovary cells stably expressing the human GnRH receptor, and its activity is unchanged by linking to 40-kDa polyethylene glycol. In a castrated rat model the Spiegelmer further demonstrated strong GnRH antagonist activity, which is more pronounced and persists longer with the polyethylene glycol-linked derivative. Furthermore, in rabbits the anti-GnRH Spiegelmer was shown to have a very low, possibly negligible immunogenic potential. These studies suggest that Spiegelmers could be of substantial interest in the development of new pharmaceutical approaches against GnRH and other targets.

GnRH binding RNA and DNA Spiegelmers: a novel approach toward GnRH antagonism.

Mirror-image oligonucleotide ligands (Spiegelmers) that bind to the pharmacologically relevant target gonadotropin-releasing hormone I (GnRH) with high affinity and high specificity have been identified using the Spiegelmer technology. GnRH is a decapeptide that plays an important role in mammalian reproduction and sexual maturation and is associated with several benign and malignant diseases. First, aptamers that bind to D-GnRH with dissociation constants of 50-100 nM were isolated out of RNA and DNA libraries. The respective enantiomers of the DNA and RNA aptamers were synthesized, and their binding to L-GnRH was shown. These Spiegelmers bind to L-GnRH with similar affinity to that of the corresponding aptamers that bind to D-GnRH. We further demonstrated dose-dependent inhibition of GnRH-induced Ca(2+) release in Chinese hamster ovary cells that were stably transfected with the human GnRH receptor.