The glycoprotein hormones (LH, FSH, and TSH) are critical to the maintenance of physiological homeostasis and control of reproduction. However, despite an obvious utility for synthetic pharmacological agents, there are few reports of selective, nonpeptide agonists or antagonists to receptors for these hormones. We have identified and characterized a novel synthetic molecule capable of inhibiting the action of FSH. This compound, 7-[4-[Bis-(2-carbamoyl-ethyl)-amino]-6-chloro-(1,3,5)-triazin-2-ylamino)-4-hydroxy-3-(4-methoxy-phenylazo)-naphthalene]-2-sulfonic acid, sodium salt (compound 1), is a selective, noncompetitive inhibitor of the human (h) and rat (r) FSH receptors (FSHRs). Compound 1 selectively inhibited binding of [(125)I]hFSH with an IC(50) value of 5.4 +/- 2.3 micro M. Radioligand-binding assays were performed using the baculovirus expressed extracellular domain of hFSHR (BV-tFSHR) to demonstrate site-specific interaction. Compound 1 competed for [(125)I]hFSH binding to BV-tFSHR with an IC(50) value of 10 +/- 2.8 micro M. Functionally, compound 1 inhibited hFSH-induced cAMP accumulation and steroidogenesis in vitro with an IC(50) value of 3 +/- 0.6 micro M. Competition of compound 1 for binding to other glycoprotein hormone receptors and other G protein-coupled receptors demonstrated select activity for FHSRs. Compound 1 inhibited ovulation in immature and cycling adult rats. These data provide proof of concept that selective, small molecule antagonists can be designed for glycoprotein hormone receptors.
The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC 50's ؍ 20 M) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC 50 ؍ 2 nM) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC 50 ؍ 980 nM) and estradiol production from primary rat ovarian granulosa cells (EC 50 ؍ 10.5 nM). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy. Follicle-stimulating hormone (FSH)2 is a glycoprotein hormone produced by the anterior pituitary that plays a key role in stimulating ovulation and spermatogenesis. Like other members of the glycoprotein hormone family (luteinizing hormone, chorionic gonadotropin, and thyroid-stimulating hormone (TSH)), FSH is a heterodimeric protein of ϳ30,000 Da that consists of a common ␣-subunit joined noncovalently to a hormone-specific -subunit. FSH activity is mediated through binding to the FSH receptor (FSH-R), which belongs to family I of the large 7-transmembrane-spanning, G protein-coupled receptor (GPCR) superfamily. The glycoprotein hormone receptors are unique among the members of the GPCR family, because they recognize protein hormones and are dominated by a large N-terminal extracellular region (366 amino acids in the case of the FSH-R) that is the predominant site of hormone binding (1, 2) and is required for signal transduction.The binding of FSH to its receptor results in the activation of adenylyl cyclase through heterotrimeric G proteins. Interaction of the activated FSH-R with G s initiates the cAMP signaling cascade (3). The ability of the FSH-R to activate adenylyl cyclase was exploited to generate a Chinese hamster ovary (CHO) FSH-R reporter cell line (4 -6). Screening of a collection...
Under physiological conditions, FSH is secreted into the circulation as a complex mixture of several isoforms that vary in the degree of glycosylation. Although it is well established that the glycosylation of FSH is important for the serum half-life of the hormone and coupling of the receptor to adenylate cyclase, little is known concerning how physiologically occurring glycosylation patterns of this hormone affect receptor signaling. In this study, we have examined the biological activity of deglycosylated human FSH (DeGly-phFSH), recombinant mammalian-expressed hFSH (CHO-hFSH), and insect cell-expressed hFSH (BV-hFSH, alternatively glycosylated) as compared with that of purified human pituitary FSH (phFSH) using a Chinese hamster ovarian cell line stably expressing the hFSH receptor (3D2 cells). Differentially glycosylated forms of FSH did not bind to the FSH receptor in the same manner as phFSH. Although all hormones showed similar potency in competing for [125I]phFSH binding to the hFSH receptor, competition curves for deglycosylated and insect cell-produced FSH were steeper. Similarly, glycosylation of FSH had a profound effect on bioactivity of the hormone. Purified hFSH produced a sigmoidal dose-dependent stimulation in cAMP production, whereas DeGly-phFSH and BV-hFSH induced biphasic (bell-shaped) dose-response curves. BV-hFSH also elicited biphasic effects on steroidogenesis in primary cultures of rat granulosa cells. The cellular response to BV-hFSH was dependent on the degree of receptor-transducer activation. BV-hFSH bioactivity was strictly inhibitory when combined with the ED80 of phFSH. Lower concentrations of phFSH resulted in a gradual shift from inhibition to a biphasic activity in the presence of the ED20 of phFSH. Biphasic responses to BV-hFSH were attributed to activation of different G protein subtypes, since treatment of 3D2 cells with cholera toxin or pertussis toxin differentially blocked the two phases of BV-hFSH bioactivity. These data suggest that alternative glycosylation of FSH leads to a functionally altered form of the hormone. Functionally different hormones appear to convey distinct signals that are transduced by the receptor-transduction system as either stimulatory or inhibitory intracellular events via promiscuous, glycosylation-dependent G protein coupling. Promiscuity in signaling of the FSH receptor, in turn, may represent a potentially novel mechanism for FSH action, whereby the gonad may respond in diverse ways to complex hormonal signals such as those presented by circulating FSH isoforms.
Indole, as a typical N-heteroaromatic compound existed in coking wastewater, can be used for bio-indigo production. The microbial production of indigo from indole has been widely reported during the last decades using culture-dependent methods, but few studies have been carried out by microbial communities. Herein, three activated sludge systems stimulated by different aromatics, i.e. naphthalene plus indole (G1), phenol plus indole (G2) and indole only (G3), were constructed for indigo production from indole. During the operation, G1 produced the highest indigo yield in the early stage, but it switched to G3 in the late stage. Based on LC-MS analysis, indigo was the major product in G1 and G3, while the purple product 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one was dominant in G2. Illumina MiSeq sequencing of 16S rRNA gene amplicons was applied to analyze the microbial community structure and composition. Detrended correspondence analysis (DCA) and dissimilarity tests showed that the overall community structures of three groups changed significantly during the operation (P<0.05). Nevertheless, the bacteria assigned to phylum Proteobacteria, family Comamonadaceae, and genera Diaphorobacter, Comamonas and Aquamicrobium were commonly shared dominant populations. Pearson correlations were calculated to discern the relationship between microbial communities and indigo yields. The typical indigo-producing populations Comamonas and Pseudomonas showed no positive correlations with indigo yields, while there emerged many other genera that exhibited positive relationships, such as Aquamicrobium, Truepera and Pusillimonas, which had not been reported for indigo production previously. The present study should provide new insights into indigo bio-production by microbial communities from indole.
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