Evidence for Follicle-stimulating Hormone Receptor as a Functional Trimer.

Follicle-stimulating hormone receptor (FSHR), a G-protein coupled receptor, is an important drug target in the development of novel therapeutics for reproductive indications. The FSHR extracellular domains were observed in the crystal structure as a trimer, which enabled us to propose a novel model for the receptor activation mechanism. The model predicts that FSHR binds Asnα(52)-deglycosylated FSH at a 3-fold higher capacity than fully glycosylated FSH. It also predicts that, upon dissociation of the FSHR trimer into monomers, the binding of glycosylated FSH, but not deglycosylated FSH, would increase 3-fold, and that the dissociated monomers would in turn enhance FSHR binding and signaling activities by 3-fold. This study presents evidence confirming these predictions and provides crystallographic and mutagenesis data supporting the proposed model. The model also provides a mechanistic explanation to the agonist and antagonist activities of thyroid-stimulating hormone receptor autoantibodies. We conclude that FSHR exists as a functional trimer.

Structure of follicle-stimulating hormone in complex with the entire ectodomain of its receptor.

FSH, a glycoprotein hormone, and the FSH receptor (FSHR), a G protein-coupled receptor, play central roles in human reproduction. We report the crystal structure of FSH in complex with the entire extracellular domain of FSHR (FSHR(ED)), including the enigmatic hinge region that is responsible for signal specificity. Surprisingly, the hinge region does not form a separate structural unit as widely anticipated but is part of the integral structure of FSHR(ED). In addition to the known hormone-binding site, FSHR(ED) provides interaction sites with the hormone: a sulfotyrosine (sTyr) site in the hinge region consistent with previous studies and a potential exosite resulting from putative receptor trimerization. Our structure, in comparison to others, suggests FSHR interacts with its ligand in two steps: ligand recruitment followed by sTyr recognition. FSH first binds to the high-affinity hormone-binding subdomain of FSHR and reshapes the ligand conformation to form a sTyr-binding pocket. FSHR then inserts its sTyr (i.e., sulfated Tyr335) into the FSH nascent pocket, eventually leading to receptor activation.

Design and synthesis of the first generation of novel potent, selective, and in vivo active (benzothiazol-2-yl)acetonitrile inhibitors of the c-Jun N-terminal kinase.

Several lines of evidence support the hypothesis that c-Jun N-terminal kinase (JNKs) plays a critical role in a wide range of diseases including cell death (apoptosis)-related disorders (neurodegenerative diseases, brain, heart, and renal ischemia, epilepsy) and inflammatory disorders (multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases). Screening of our internal compound collection for inhibitors of JNK3 led to the identification of (benzothiazol-2-yl)acetonitrile derivatives as potent and selective JNK1, -2, -3 inhibitors. Starting from initial hit 1 (AS007149), the chemistry and initial structure-activity relationship (SAR) of this novel and unique kinase inhibitor template were explored. Investigation of the SAR rapidly revealed that the benzothiazol-2-ylacetonitrile pyrimidine core was crucial to retain a good level of potency on rat JNK3. Therefore, compound 6 was further optimized by exploring a number of distal combinations in place of the chlorine atom. This led to the observation that the presence of an aromatic group, two carbons away from the aminopyrimidine moiety and bearing substituents conferring hydrogen bond acceptor (HBA) properties, could improve the potency. Further improvements to the biological and biopharmaceutical profile of the most promising compounds were performed, resulting in the discovery of compound 59 (AS601245). The in vitro and in vivo anti-inflammatory potential of this new JNK inhibitor was investigated and found to demonstrate efficacy per oral route in an experimental model of rheumatoid arthritis (RA).

Design, synthesis, and biological activity of novel, potent, and selective (benzoylaminomethyl)thiophene sulfonamide inhibitors of c-Jun-N-terminal kinase.

Several lines of evidence support the hypothesis that c-Jun N-terminal kinases (JNKs) play a critical role in a wide range of disease states including cell death (apoptosis)-related and inflammatory disorders (epilepsy, brain, heart and renal ischemia, neurodegenerative diseases, multiple sclerosis, rheumatoid arthritis, and inflammatory bowel syndrome). The screening of a compound collection led to the identification of a 2-(benzoylaminomethyl)thiophene sulfonamide (AS004509, compound I) as a potent and selective JNK inhibitor. Chemistry and structure--activity relationship (SAR) studies performed around this novel kinase-inhibiting motif indicated that the left and central parts of the molecule were instrumental to maintaining potency at the enzyme. Accordingly, we investigated the JNK-inhibiting properties of a number of variants of the right-hand moiety of the molecule, which led to the identification of 2-(benzoylaminomethyl)thiophene sulfonamide benzotriazole (AS600292, compound 50a), the first potent and selective JNK inhibitor of this class which demonstrates a protective action against neuronal cell death induced by growth factor and serum deprivation.

Discovery of new molecules for future treatment of infertility.

The introduction of recombinant gonadotrophins for the treatment of infertility has been an important advance in improving the quality and consistency of therapeutics offered to patients seeking care from fertility specialists. Over the past decade, a number of investigators have discovered small molecules that mimic the effects of FSH and LH. Despite extensive medicinal chemistry efforts from many institutes, including Serono Research Institute, and reasonable in-vitro activity, receptor-targeted agonists have not yet been successfully developed for clinical use, based upon results generated in animal models of follicular stimulation (FSH-like) or ovulation induction [human chorionic gonadotrophin (HCG)-like]. A different approach to gonadotrophin mimicry was identified that modifies intracellular signalling pathways common to gonadotrophins. Phosphodiesterase type 4 enzyme inhibitors and selective prostaglandin E receptor (EP2/EP4) agonists have been demonstrated to mimic the effects of HCG to induce ovulation following oral administration. Multiple approaches with small molecules have been attempted to activate the FSH receptor and initiate cAMP-dependent mechanisms. Pharmacodynamic effects of FSH on follicular growth were demonstrated by inhibiting a different enzyme pathway with a small molecule, albeit in the presence of very low concentrations of circulating FSH. These results raise the possibility that in the future orally active agents can be used in combination with injectable gonadotrophins or perhaps independent of gonadotrophins for first-line interventions for infertility.

MAP kinase phosphatase 3 (MKP3) interacts with and is phosphorylated by protein kinase CK2alpha.

Mitogen-activated protein (MAP) kinases play a central role in controlling a wide range of cellular functions following their activation by a variety of extracellular stimuli. MAP kinase phosphatases (MKPs) represent a subfamily of dual specificity phosphatases, which negatively regulate MAP kinases. Although ERK2 activity is regulated by its phosphorylation state, MKP3 is regulated by physical interaction with ERK2, independent of its enzymatic activity (Camps, M., Nichols, A., Gillieron, C., Antonsson, B., Muda, M., Chabert, C., Boschert, U., and Arkinstall, S., (1998) Science 280, 1262-1265; Farooq, A., Chaturvedi, G., Mujtaba, S., Plotnikova, O., Zeng, L., Dhalluin, C., Ashton, R., and Zhou, M. M. (2001), Mol. Cell 7, 387-399; Zhou, B., and Zhang, Z. Y. (1999) J. Biol. Chem. 274, 35526-35534). The interaction of ERK2 and MKP3 allows the reciprocal cross-regulation of their catalytic activity. Indeed, MKP3 acts as a negative regulator on ERK2-MAP kinase signal transduction activity, representing thus a negative feedback for this MAPK pathway. To identify novel proteins able to complex MKP3, we used the yeast two-hybrid system. Here we report that MKP3 and protein kinase CK2 form a protein complex, which can include ERK2. The phosphatase activity of MKP3 is then slightly increased in vitro, whereas in transfected cells, ERK2 dephosphorylation is reduced. In addition, we demonstrated that CK2 selectively phosphorylates MKP3, suggesting cross-regulation between CK2alpha and MKP3, as well as a modulation of ERK2-MAPK signaling by CK2alpha via MKP3.