Allosteric Activation of the Follicle-stimulating Hormone (FSH) Receptor by Selective, Nonpeptide Agonists

Abstract

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 (EC50's = 20 μm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 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 (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 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. 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 (EC50's = 20 μm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 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 (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 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) 2The abbreviations used are: FSH, follicle-stimulating hormone; TSH, thyroid-stimulating hormone; -R, receptor; GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; TM, transmembrane; DMEM, Dulbecco's modified Eagle's medium; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography; CRE, cyclic-AMP response element. 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 ∼30,000 Da that consists of a common α-subunit joined noncovalently to a hormone-specific β-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 (1Braun T. Schofield P.R. Sprengel R. EMBO J. 1991; 10: 1885-1890Crossref PubMed Scopus (314) Google Scholar, 2Christophe S. Robert P. Maugain S. Bellet D. Koman A. Bidart J.-M. Biochem. Biophys. Res. Commun. 1993; 196: 402-408Crossref PubMed Scopus (22) Google Scholar) 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 Gs initiates the cAMP signaling cascade (3Johnson G.L. Dhanasekaran N. Endocr. Rev. 1989; 10: 317-331Crossref PubMed Scopus (121) Google Scholar). The ability of the FSH-R to activate adenylyl cyclase was exploited to generate a Chinese hamster ovary (CHO) FSH-R reporter cell line (4Albanese C. Cristin-Maitre S. Sluss P.M. Crowley W.F. Jameson J.L. Mol. Cell. Endocrinol. 1994; 101: 211-219Crossref PubMed Scopus (54) Google Scholar, 5George S.E. Bungay P.J. Naylor L.H. Biochem. Pharmacol. 1998; 56: 25-30Crossref PubMed Scopus (19) Google Scholar, 6George S.E. Bungay P.J. Naylor L.H. J. Neurochem. 1997; 69: 1278-1285Crossref PubMed Scopus (59) Google Scholar). Screening of a collection of diverse chemotypes led to the identification of compounds that activated this reporter cell line but were inactive on CHO cells lacking the human FSH-R. The activity of these compounds on receptor chimeras, which contained only a small portion (TM1 through TM3) of the FSH-R, suggests action through a site distinct from the large N-terminal extracellular domain, the region reported to be the predominant site of hormone binding (1Braun T. Schofield P.R. Sprengel R. EMBO J. 1991; 10: 1885-1890Crossref PubMed Scopus (314) Google Scholar, 2Christophe S. Robert P. Maugain S. Bellet D. Koman A. Bidart J.-M. Biochem. Biophys. Res. Commun. 1993; 196: 402-408Crossref PubMed Scopus (22) Google Scholar). Reported effects of FSH and FSH-R defects provide evidence for the importance of this glycoprotein hormone and its receptor on female fertility. Mutations in FSH-R have been linked to clinical cases of infertility. Nonfunctional FSH-Rs have been found to cause resistant ovary syndrome, which results in infertility in women (7Aittomaki K. Lucena J.L.D. Pakarinen P. Sistonen P. Tapanainen J. Gromoll J. Kaskikari R. Sankila E.M. Lehvaslaiho H. Engel A.R. Nieschlag E. Huhtaniemi L. de la Chapelle A. Cell. 1995; 82: 959-968Abstract Full Text PDF PubMed Scopus (839) Google Scholar). The FSH-β gene knock-out female mouse is infertile because of inhibition of folliculogenesis prior to antral follicle formation (8Kumar T.R. Wang Y. Lu N. Matzuk M.M. Nat. Genet. 1997; 15: 201-204Crossref PubMed Scopus (1105) Google Scholar). In addition, FSH-R gene knock-out female mice are also infertile because of arrested folliculogenesis (9Dierich A. Sairam M.R. Monaco L. Fimia G.M. Gansmuller A. LeMeur M. Sassone-Corsi P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13612-13617Crossref PubMed Scopus (723) Google Scholar). In males, FSH regulates the function of testicular Sertoli cells. However, the importance of FSH in male fecundity is less clearcut, as clinical reports of mutations to either FSH or its receptor describe reduced fertility but not complete sterilization. Human menopausal gonadotropin, a 1:1 mixture of FSH and luteinizing hormone, has been used successfully clinically to induce ovarian stimulation in both in vivo and in vitro fertilization therapy. Human menopausal gonadotropin is purified from the urine of postmenopausal women and must be administered by injection. Recombinant human FSH has recently become available for parenteral human therapy. Treatment typically involves multiple injections, which can have a negative impact on patient compliance and initiation of this course of treatment. Consequently, patients being treated for infertility may prefer an orally administered small molecule that mimics the activity of FSH. Therefore, we have set out to identify nonpeptide synthetic agonists to the hFSH-R for potential use in novel infertility treatments. A class of thiazolidinone agonists was identified through unbiased combinatorial chemistry library screening. Additional optimization through analog library screening and parallel synthesis led to the development of a potent FSH-R-selective and -dependent agonist with full efficacy to FSH. The thiazolidinone agonists appear to activate the FSH-R through an allosteric mechanism, which was determined by the use of human FSH-R/TSH-R chimeras. Chinese hamster ovary (CHO) K-1 cells were used to engineer a clonal cell line that stably expresses the human FSH-R. cDNA for the human FSH-R was cloned into the α+12CA5-KH expression vector as described (10Koller K.J. Whitehorn E.A. Tate E. Ries T. Aguilar B. Chernov-Rogan T. Davis A.M. Dobbs A. Yen M. Barrett R.W. Anal. Biochem. 1997; 250: 51-60Crossref PubMed Scopus (25) Google Scholar) and transfected into CHO-K1 cells by electroporation. After G418 selection, cells were stained with fluorescein isothiocyanate-labeled 12CA5 antibody, and those expressing the FSH-R were collected by fluorescence-activated cell sorting. Individual clones were examined for the ability to produce cAMP in response to purified human FSH (Cortex Biochem). Clone 3D2 was identified as a stable clone that responded to purified human FSH with an increase in cAMP production. In addition, the cloned human FSH-R gene was transfected along with a 6XCRE-luciferase plasmid (11McDonnell J. Latif M.L. Rees E.S. Bevan N.J. Hill S.J. Br. J. Pharmacol. 1998; 125: 717-726Crossref PubMed Scopus (37) Google Scholar) into CHO K-1 cells by electroporation. Following G418 selection, cells were stained with fluorescein isothiocyanate-labeled 12CA5 antibody, and those expressing the FSH-R were collected by fluorescence-activated cell sorting. Clone 1D7 was identified as a stable clone giving a robust luciferase reporter gene response to human FSH. Reporter cell lines expressing each of the chimeric receptors described below were generated in the same manner as for the CHO FSH-R 6XCRE-luciferase cell line. A mouse adrenocortical tumor cell line (Y1 cells) purchased from American Type Culture Collection (Manassas, VA) was engineered to stably express the full-length human FSHR cDNA (provided by Dr. Kerry Koller) as described previously (12Arey B.J. Deecher D.C. Shen E.S. Stevis P.E. Meade E.H. Jr-Wrobel J. Frail D.E. Lopez F.J. Endocrinology. 2002; 143: 3822-3829Crossref PubMed Scopus (54) Google Scholar). The FSH-R α+T8-12CA5-KH plasmid construct was available from previous work (10Koller K.J. Whitehorn E.A. Tate E. Ries T. Aguilar B. Chernov-Rogan T. Davis A.M. Dobbs A. Yen M. Barrett R.W. Anal. Biochem. 1997; 250: 51-60Crossref PubMed Scopus (25) Google Scholar). TSH-R was amplified by PCR from cDNA made from a human thyroid gland poly(A)+ RNA library (Clontech) using oligonucleotide primers generated from the GenBank™ sequence M32215, and cloned into the α+T8-12CA5-KH plasmid at the XhoI and NotI restriction sites. The correct sequence was verified by automated DNA sequencing. Chimeric constructs were made combining the FSH-R and TSH-R. Oligomers were designed to engineer restriction sites into the of the first and of each amino were the restriction sites were construct contained the TSH-R from amino acids and the FSH-R from amino acids of the proteins. sites were at amino acids for the amino acids FSH-R/TSH-R construct contained the FSH-R from amino acids and the TSH-R from amino acids of the proteins. sites were at amino acids for the amino acids construct contained the FSH-R from amino acids and the TSH-R from amino acids of the proteins. sites were at amino acids for the amino acids construct contained the TSH-R from amino acids the FSH-R from amino acids and the TSH-R from amino acids The same site as in FSH-R/TSH-R was and sites were at amino acids of the amino acids were at into a in Human FSH, human (Cortex or thiazolidinone compounds were in and the was at in the of After an of was and the was in a CHO cells transfected with the human FSH receptor were at into a and at in the of After with cells were with compounds for at in the of the the was by the of of The were and for cAMP by were using either or E.M. J. 1995; Scopus Google Scholar). were from by = nm) or by with as described previously and in synthetic and purified by The production of estradiol by primary rat granulosa cells and progesterone by cells with purified human FSH or compounds was determined as described previously (12Arey B.J. Deecher D.C. Shen E.S. Stevis P.E. Meade E.H. Jr-Wrobel J. Frail D.E. Lopez F.J. Endocrinology. 2002; 143: 3822-3829Crossref PubMed Scopus (54) Google Scholar). Screening for FSH-R a Reporter screening of combinatorial small molecule for compounds that the binding of to the human FSH-R was for small molecule of the FSH we a reporter gene (4Albanese C. Cristin-Maitre S. Sluss P.M. Crowley W.F. Jameson J.L. Mol. Cell. Endocrinol. 1994; 101: 211-219Crossref PubMed Scopus (54) Google Scholar, 5George S.E. Bungay P.J. Naylor L.H. Biochem. Pharmacol. 1998; 56: 25-30Crossref PubMed Scopus (19) Google Scholar, 6George S.E. Bungay P.J. Naylor L.H. J. Neurochem. 1997; 69: 1278-1285Crossref PubMed Scopus (59) Google Scholar) in which the luciferase gene was of a series of cyclic-AMP response to for compounds that activate adenylyl cyclase (3Johnson G.L. Dhanasekaran N. Endocr. Rev. 1989; 10: 317-331Crossref PubMed Scopus (121) Google Scholar). in the CHO FSH-R 6XCRE-luciferase cell responded in a manner to with human FSH. efficacy for human FSH in this was cells. The for human FSH in this was this FSH-R luciferase reporter to a collection of small molecule combinatorial 20 using synthesis E.M. 1995; PubMed Scopus Google Scholar, E.M. Barrett R.W. J. 1994; PubMed Scopus Google Scholar). were typically by synthesis on with a J. 1995; Scopus Google and compounds were in compounds at an of compounds were for agonist activity using the CHO FSH-R 6XCRE-luciferase reporter cell line. was screening of a library Individual compounds from were and in the CHO FSH-R reporter and 2 were found to activate the luciferase reporter gene but (EC50 20 μm) through of the of through a combinatorial library is that analog can be generated in a for compounds with identify potent an analog library was generated using the same thiazolidinone in with amino acids at the at the and at the D. Davis A.M. S. K. Barrett R.W. J. PubMed Scopus (54) Google Scholar). The and were using D. M.M. B. E.M. J. PubMed Scopus Google and the were for screening The compounds were on a This library of compounds was as compounds at of of the at the of from this were into and the compounds were by and for activity using the CHO FSH-R reporter from were and their were to the of the of these and were as compounds for and were found to act as full agonists in the reporter with the lead compounds (EC50 of and but an efficacy to that of parallel synthesis was used to generate in identification of compounds (EC50 = 2 (EC50 = and (EC50 = nm) as potent agonists in the reporter FSH agonists identified from combinatorial analog on compounds and 2 in a of CHO FSH-R 6XCRE-luciferase clone 1D7 to human FSH and small molecule reporter were as described human compound compound compound compound of Recombinant and compounds and were also examined for their ability to cAMP production in a CHO cell line transfected with the human FSH-R cAMP with an 2 not compounds and to to cAMP or luciferase production in a of cell CHO cells and reporter CHO cells expressing the or the human receptor not thiazolidinone was an agonist of the rat FSH-R, compound was for its ability to activate the FSH-R found in primary of rat ovarian granulosa cells N. 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This cell line produced a increase in progesterone stimulation with FSH in an of was less potent (EC50 = nm) but an efficacy to purified human FSH in stimulating progesterone production the recombinant human FSH-R. purified human FSH compound progesterone production on Y1 cells not of FSH was used in the synthesis of in the library and in chemistry that resulted in formation of of each The and could be by and are each a of The of these compounds were in the reporter and found to be at less from in which the of was by or the were using that the the was for the of this compound class with the FSH-R to of FSH-R/TSH-R and of chimeric receptors is a to identify the activity of hormone receptor chimeras have been used to the for binding and signaling M. R. J. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. D. P. B. Proc. Natl. Acad. Sci. 1991; PubMed Scopus Google Scholar, Y. D. B. Biochem. Biophys. Res. Commun. 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The of this cell line to human was In these CHO FSH-R/TSH-R 6XCRE-luciferase clone did to with an of this cell line did not at to compound at to of FSH, TSH, and thiazolidinone compounds on CHO cell lines expressing chimeric receptors Chimeric receptors were from cDNA for the human FSH-R and human and CHO 6XCRE-luciferase reporter cell lines were generated expressing each of these chimeric receptors as described were in with purified human FSH, purified human TSH, or thiazolidinone compounds for at and luciferase activity was using as described not in a CHO 6XCRE-luciferase clone responded to with an of cells responded to thiazolidinone with an of and to thiazolidinone with an of this cell which the N-terminal domain, is to to and the site of action of compound the chimeric receptor was This construct FSH-R through the first extracellular domain, first first and first extracellular with the of the receptor being TSH-R CHO clone responded to FSH with an of and responded to only at to compound with an of that the FSH contained in the are to high affinity binding of FSH and the thiazolidinone compound the receptor was in which the N-terminal FSH-R from was with the N-terminal of the human TSH-R only FSH-R to the first and the first extracellular CHO 6XCRE-luciferase clone cells did not to FSH at to However, these cells responded to with an of and they responded to compound with an of and to compound with an of thiazolidinone agonists to activate FSH-R by in an allosteric region (TM1 through the first extracellular from the N-terminal The GPCR family of cell receptors is to to A large of the current is of compounds that with is that and discovered members of this family for binding have been for high screening for and agonists of for a orally available of FSH action by screening for compounds that the binding of FSH to CHO cells expressing the FSH from CHO FSH-R cells and a we combinatorial small molecule a of compounds at an of were to compounds that inhibition of FSH the identification of a small molecule that can the a large protein and its cell receptor a has been the in the for small molecule of receptors with a small molecule FSH-R was discovered using a binding (12Arey B.J. Deecher D.C. Shen E.S. Stevis P.E. Meade E.H. Jr-Wrobel J. Frail D.E. Lopez F.J. Endocrinology. 2002; 143: 3822-3829Crossref PubMed Scopus (54) Google Scholar). However, by screening for compounds with agonist small molecule have been identified for the receptor B. D. Y. D. C. P. J. D.E. PubMed Scopus Google Scholar) and the receptor P. L. L. J. 1998; PubMed Scopus Google Scholar). a to small molecule FSH-R agonists have been identified T. D. P. R. E. K.J. D. M.L. PubMed Scopus Google Scholar, T. D. P. R. E. K.J. D. M.L. PubMed Scopus Google Scholar). compounds activate a CHO FSH-R reporter cell line and induce cAMP their site of action a reporter cell line expressing the FSH-R and the luciferase gene a cAMP-responsive and a high reporter for compounds that act as FSH this assay, we in of from collection of combinatorial small molecule The lead thiazolidinone and activity in the using the FSH-R reporter the use of combinatorial we identified potent with activity in the the mechanism of action of these novel FSH we for their ability to compete with the binding of FSH to both cells expressing the FSH-R and to from the compounds were to the binding of FSH. that we is the mechanism of activation of the FSH-R by the thiazolidinone The response is and the of the FSH-R, and in the of FSH. However, a of can be FSH is to with high affinity to the large extracellular on the FSH-R (1Braun T. Schofield P.R. Sprengel R. EMBO J. 1991; 10: 1885-1890Crossref PubMed Scopus (314) Google Scholar, 2Christophe S. Robert P. Maugain S. Bellet D. Koman A. Bidart J.-M. Biochem. Biophys. Res. Commun. 1993; 196: 402-408Crossref PubMed Scopus (22) Google Scholar). However, high affinity binding and signaling appear to on the FSH-R J. K. M. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) and other glycoprotein hormone receptors Endocrinology. 1991; PubMed Scopus Google Scholar). have also been reported for a of A. Pharmacol. PubMed Scopus Google Scholar, A. T. Pharmacol. Rev. 2002; PubMed Scopus Google Scholar, B.J. R. A. J. J. Endocrinology. PubMed Scopus Google Scholar) and have been as for glycoprotein hormone receptors on the of a reported of sites for binding and signal transduction. the of human FSH to the extracellular portion of the FSH-R has been along with that the receptor as a PubMed Scopus Google Scholar). mechanism for FSH stimulation of its receptor high affinity binding to this N-terminal by receptor activation of the FSH with the extracellular of the of the receptor to that for M. T. J. Full Text Full Text PDF PubMed Scopus Google Scholar). A for glycoprotein hormone activation has been in which with high affinity to the N-terminal extracellular and the extracellular from an agonist into a full the portion of the receptor S. D. C. 1998; and PubMed Scopus Google Scholar). The thiazolidinone compounds could with the same site on the of the receptor to a in the this mechanism is suggests the that small molecule could be generated that to the same site but the ability to activate the to small molecule FSH could be oral and for other the thiazolidinone compounds could to an site on the FSH-R to cause an allosteric in receptor or the of G of these of action can be to other members of the GPCR family, suggests that the screening of diverse of small using may be a of FSH-R/TSH-R chimeras were generated to identify the for FSH agonist receptor activation and signaling described The results with the FSH-R/TSH-R and cell lines that response to the hormones with the N-terminal extracellular domain, response to the thiazolidinone compounds with the portion of the on the cell line to FSH the FSH-R N-terminal and response to thiazolidinone By the N-terminal in the of we were to that only a small portion of the FSH-R is required for This through is to a response to the thiazolidinone of the site of action of these compounds be to the compounds are on the first extracellular or with of the This study the first reported small molecule high affinity agonist of the class of glycoprotein hormone receptors that to function through a novel allosteric mechanism. The small molecule FSH receptor agonists described here are a the development of an oral alternative to the current FSH treatments for clinical use to induce ovarian stimulation for both in vivo and in vitro fertilization therapy. In addition, the allosteric mechanism by which these agonists act may the infertility that is to FSH-R mutations in the N-terminal extracellular binding