Pharmacological Characterization of Low Molecular Weight Biased Agonists at the Follicle Stimulating Hormone Receptor.

Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, ß-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or ß-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for ß-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or ß-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.

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.

Discovery of substituted benzamides as follicle stimulating hormone receptor allosteric modulators.

Follicle-stimulating hormone (FSH), acting on its receptor (FSHR), plays a pivotal role in the stimulation of follicular development and maturation. Multiple injections of protein formulations are used during clinical protocols for ovulation induction and for in vitro fertilization that are followed by a selection of assisted reproductive technologies. In order to increase patient convenience and compliance several research groups have searched for orally bioavailable FSH mimetics for innovative fertility medicines. We report here the discovery of a series of substituted benzamides as positive allosteric modulators (PAM) targeting FSHR. Optimization of this series has led to enhanced activity in primary rat granulosa cells, as well as remarkable selectivity against the closely related luteinizing hormone receptor (LHR) and thyroid stimulating hormone receptor (TSHR). Two modulators, 9j and 9k, showed promising in vitro and pharmacokinetic profiles.

Follicle-Stimulating Hormone Induces Lipid Droplets via Gαi/o and ß-Arrestin in an Endometrial Cancer Cell Line.

Follicle-stimulating hormone (FSH) and its G protein-coupled receptor, FSHR, represents a paradigm for receptor signaling systems that activate multiple and complex pathways. Classically, FSHR activates Gαs to increase intracellular levels of cAMP, but its ability to activate other G proteins, and ß-arrestin-mediated signaling is well documented in many different cell systems. The pleiotropic signal capacity of FSHR offers a mechanism for how FSH drives multiple and dynamic downstream functions in both gonadal and non-gonadal cell types, including distinct diseases, and how signal bias may be achieved at a pharmacological and cell system-specific manner. In this study, we identify an additional mechanism of FSH-mediated signaling and downstream function in the endometrial adenocarcinoma Ishikawa cell line. While FSH did not induce increases in cAMP levels, this hormone potently activated pertussis toxin sensitive Gαi/o signaling. A selective allosteric FSHR ligand, B3, also activated Gαi/o signaling in these cells, supporting a role for receptor-mediated activation despite the low levels of FSHR mRNA. The low expression levels may attribute to the lack of Gαs/cAMP signaling as increasing FSHR expression resulted in FSH-mediated activation of the Gαs pathway. Unlike prior reports for FSH-mediated Gαs/cAMP signaling, FSH-mediated Gαi/o signaling was not affected by inhibition of dynamin-dependent receptor internalization. While chronic FSH did not alter cell viability, FSH was able to increase lipid droplet size. The ß-arrestins are key adaptor proteins known to regulate FSHR signaling. Indeed, a rapid, FSH-dependent increase in interactions between ß-arrestin1 and Gαi1 was observed via NanoBiT complementation in Ishikawa cells. Furthermore, both inhibition of Gαi/o signaling and siRNA knockdown of ß-arrestin 1/2 significantly reduced FSH-induced lipid droplet accumulation, implying a role for a Gαi/o/ß-arrestin complex in FSH functions in this cell type. As FSH/FSHR has been implicated in distinct hormone-dependent cancers, including endometrial cancer, analysis of the cancer genome database from 575 human endometrial adenocarcinoma tumors revealed that a subpopulation of samples expressed FSHR. Overall, this study highlights a novel mechanism for FSHR signal pleiotropy that may be exploited for future personalized therapeutic approaches.

Rescue of Cell Surface Expression and Signaling of Mutant Follicle-Stimulating Hormone Receptors.

Mutations in G protein-coupled receptors (GPCRs) underlie numerous diseases. Many cause receptor misfolding and failure to reach the cell surface. Pharmacological chaperones are cell-permeant small molecules that engage nascent mutant GPCRs in the endoplasmic reticulum, stabilizing folding and "rescuing" cell surface expression. We previously demonstrated rescue of cell surface expression of luteinizing hormone receptor mutants by an allosteric agonist. Here we demonstrate that a similar approach can be employed to rescue mutant follicle-stimulating hormone receptors (FSHRs) with poor cell surface expression using a small-molecule FSHR agonist, CAN1404. Seventeen FSHR mutations described in patients with reproductive dysfunction were expressed in HEK 293T cells, and cell surface expression was determined by enzyme-linked immunosorbent assay of epitope-tagged FSHRs before/after treatment with CAN1404. Cell surface expression was severely reduced to ≤18% of wild-type (WT) for 11, modestly reduced to 66% to 84% of WT for 4, and not reduced for 2. Of the 11 with severely reduced cell surface expression, restoration to ≥57% of WT levels was achieved for 6 by treatment with 1 µM CAN1404 for 24 h, and a corresponding increase in FSH-induced signaling was observed for 4 of these, indicating restored functionality. Therefore, CAN1404 acts as a pharmacological chaperone and can rescue cell surface expression and function of certain mutant FSHRs with severely reduced cell surface expression. These findings aid in advancing the understanding of the effects of genetic mutations on GPCR function and provide a proof of therapeutic principle for FSHR pharmacological chaperones.

Pharmacological Programming of Endosomal Signaling Activated by Small Molecule Ligands of the Follicle Stimulating Hormone Receptor.

Follicle-stimulating hormone receptor (FSHR) is a G protein-coupled receptor (GPCR) with pivotal roles in reproduction. One key mechanism dictating the signal activity of GPCRs is membrane trafficking. After binding its hormone FSH, FSHR undergoes internalization to very early endosomes (VEEs) for its acute signaling and sorting to a rapid recycling pathway. The VEE is a heterogeneous compartment containing the Adaptor Protein Phosphotyrosine Interacting with Pleckstrin homology Domain and Leucine Zipper 1 (APPL1) with distinct functions in regulating endosomal Gαs/cAMP signaling and rapid recycling. Low molecular weight (LMW) allosteric FSHR ligands were developed for use in assisted reproductive technology yet could also provide novel pharmacological tools to study FSHR. Given the critical nature of receptor internalization and endosomal signaling for FSHR activity, we assessed whether these compounds exhibit differential abilities to alter receptor endosomal trafficking and signaling within the VEE. Two chemically distinct LMW agonists (benzamide, termed B3 and thiazolidinone, termed T1) were employed. T1 was able to induce a greater level of cAMP than FSH and B3. As cAMP signaling drives gonadotrophin hormone receptor recycling, rapid exocytic events were evaluated at single event resolution. Strikingly, T1 was able to induce a 3-fold increase in recycling events compared to FSH and two-fold more compared to B3. As T1-induced internalization was only marginally greater, the dramatic increase in recycling and cAMP signaling may be due to additional mechanisms. All compounds exhibited a similar requirement for receptor internalization to increase cAMP and proportion of FSHR endosomes with active Gαs, suggesting regulation of cAMP signaling induced by T1 may be altered. APPL1 plays a central role for GPCRs targeted to the VEE, and indeed, loss of APPL1 inhibited FSH-induced recycling and increased endosomal cAMP signaling. While T1-induced FSHR recycling was APPL1-dependent, its elevated cAMP signaling was only partially increased following APPL1 knockdown. Unexpectedly, B3 altered the dependence of FSHR to APPL1 in an opposing manner, whereby its endosomal signaling was negatively regulated by APPL1, while B3-induced FSHR recycling was APPL1-independent. Overall, FSHR allosteric compounds have the potential to re-program FSHR activity via altering engagement with VEE machinery and also suggests that these two distinct functions of APPL1 can potentially be selected pharmacologically.

Discovery and Development of Small Molecule Allosteric Modulators of Glycoprotein Hormone Receptors.

Glycoprotein hormones, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH) are heterodimeric proteins with a common α-subunit and hormone-specific ß-subunit. These hormones are dominant regulators of reproduction and metabolic processes. Receptors for the glycoprotein hormones belong to the family of G protein-coupled receptors. FSH receptor (FSHR) and LH receptor are primarily expressed in somatic cells in ovary and testis to promote egg and sperm production in women and men, respectively. TSH receptor is expressed in thyroid cells and regulates the secretion of T3 and T4. Glycoprotein hormones bind to the large extracellular domain of the receptor and cause a conformational change in the receptor that leads to activation of more than one intracellular signaling pathway. Several small molecules have been described to activate/inhibit glycoprotein hormone receptors through allosteric sites of the receptor. Small molecule allosteric modulators have the potential to be administered orally to patients, thus improving the convenience of treatment. It has been a challenge to develop a small molecule allosteric agonist for glycoprotein hormones that can mimic the agonistic effects of the large natural ligand to activate similar signaling pathways. However, in the past few years, there have been several promising reports describing distinct chemical series with improved potency in preclinical models. In parallel, proposal of new structural model for FSHR and in silico docking studies of small molecule ligands to glycoprotein hormone receptors provide a giant leap on the understanding of the mechanism of action of the natural ligands and new chemical entities on the receptors. This review will focus on the current status of small molecule allosteric modulators of glycoprotein hormone receptors, their effects on common signaling pathways in cells, their utility for clinical application as demonstrated in preclinical models, and use of these molecules as novel tools to dissect the molecular signaling pathways of these receptors.

Novel efficient routes to heparin monosaccharides and disaccharides achieved via regio- and stereoselective glycosidation.

A new methodology for the synthesis of heparin building blocks has been developed. We describe novel efficient routes to both L-iduronic acid and D-glucuronic acid acceptors. Glycosylation with thioglycosides donors gave corresponding disaccharides in a regio- and stereoselective fashion. An improved approach to synthesizing azido-glucose thioglycoside donor to render azido-sugar from mannose via nucleophilic substitution is described. [reaction: see text]