A negative allosteric modulator demonstrates biased antagonism of the follicle stimulating hormone receptor

Abstract

High quality gamete production in males and females requires the pituitary gonadotropin follicle stimulating hormone (FSH). In this report a novel chemical class of small molecule inhibitors of FSH receptor (FSHR) is described. ADX61623, a negative allosteric modulator (NAM), increased the affinity of interaction between 125I-hFSH and human FSHR (hFSHR) five fold. This form of FSHR occupied simultaneously by FSH and ADX61623 was inactive for cAMP and progesterone production in primary cultures of rat granulosa cells. In contrast, ADX61623 did not block estrogen production. This demonstrates for the first time, biased antagonism at the FSHR. To determine if ADX61623 blocked FSH induction of follicle development in vivo, a bioassay to measure follicular development and oocyte production in immature female rats was validated. ADX61623 was not completely effective in blocking FSH induced follicular development in vivo at doses up to 100 mg/kg as oocyte production and ovarian weight gain were only moderately reduced. These data illustrate that FSHR couples to multiple signaling pathways in vivo. Suppression of one pool of FSHR uncouples Gαs and cAMP production, and decreases progesterone production. Occupancy of another pool of FSHR sensitizes granulosa cells to FSH induced estradiol production. Therefore, ADX61623 is a useful tool to investigate further the mechanism of the FSHR signaling dichotomy. This may lead to a greater understanding of the signaling infrastructure which enables estrogen biosynthesis and may prove useful in treating estrogen dependent disease.

Novel pathways in gonadotropin receptor signaling and biased agonism

“…These authors have identified a biased NAM that has the property to partially block cAMP response to FSH stimulation, to completely abrogate FSH-induced progesterone production in primary granulosa cells and yet has no effect on estrogen production in these cells. Interestingly, other compounds that behave as full NAM have been identified within the same chemical series [86]. These data are in line with the existence of multiple active conformations of the receptors, each potentially activating distinct intracellular signaling mechanisms.…”

“…Additionally, some analogs of this class could activate both pathways. Another very interesting example of biased signaling at the FSH-R by small molecules has been recently reported by Dias and collaborators [86]. These authors have identified a biased NAM that has the property to partially block cAMP response to FSH stimulation, to completely abrogate FSH-induced progesterone production in primary granulosa cells and yet has no effect on estrogen production in these cells.…”

“…Despite these challenges, advances in combinatorial chemistry have recently led to the discovery of several families of small molecules acting as agonists or antagonists at the FSH-R (Fig. 3) [79][80][81][82][83][84][85][86][87]. Two families of small molecules have been reported to exert selective agonist effects at the LH-R as well [88,89].…”

“…All the other available families of molecules failed to compete with the native hormone in binding assays. Noteworthy, a recently reported modulator at the FSH-R has been shown to enhance FSH affinity for the receptor, suggesting that this compound induces the stabilization of a conformer of the FSH-R such that the ECD binding site gains affinity towards its cognate hormone [86]. Further, an allosteric modulator, originally designed for the LH-R, has shown that it also binds the FSH-R without competing for the natural ligand binding site, and promotes cell-surface plasma membrane expression of the receptor, acting as a pharmacological chaperone [91].…”

“…Strikingly, recent data demonstrate that a range of pharmacological profiles can be achieved by discrete modifications of a single small molecule core structure [85,86,90]. Indeed, small molecule allosteric agonists of the FSH-R could be converted to antagonists at the FSH-R [83,85].…”