AU2004208990B2

AU2004208990B2 - Restoring function to gonadotropin releasing hormone receptor mutants

Info

Publication number: AU2004208990B2
Application number: AU2004208990A
Authority: AU
Inventors: P. Michael Conn
Original assignee: Oregon Health Science University
Current assignee: Oregon Health Science University
Priority date: 2003-01-29
Filing date: 2004-01-27
Publication date: 2010-11-11
Anticipated expiration: 2024-01-27
Also published as: EP1599494A4; WO2004069859A3; AU2004208990A1; EP1599494A2; CA2514449A1; WO2004069859A2

Description

WO 2004/069859 PCT/US2004/002290 RESTORING FUNCTION TO GONADOTROPIN RELEASING HORMONE RECEPTOR MUTANTS CROSS REFERENCE TO RELATED APPLICATION 5 This application claims the benefit of U.S. Provisional Application No. 60/443,691 filed January 29, 2003, herein incorporated by reference in its entirety. ACKNOWLEDGMENT OF GOVERNMENT SUPPORT Some of the work described in this patent application was funded by NIH grants 10 HD-19899, RR-00163, HD-18185 and TW/HD-00668. The government of the United States may have certain rights in this invention. FIELD This disclosure relates to methods of screening agents for their ability to restore 15 functionality to a mutated gonadotropin-releasing hormone receptor, increase the amount of wild-type GnRHR on the cell surface, or both, and methods of using identified agents to treat a subject having hypogonadism. BACKGROUND 20 Disease-causing receptor mutations were widely believed to lose function as a result of inability to engage in receptor-ligand or receptor-effector binding interactions. However, recent observations indicate that receptor misfolding and subsequent misrouting is a mechanism that results in loss of receptor function (Janovick et al., J. Clin. Endocrinol. Metab., 87:325 5-62, 2002; Leafios-Miranda et al., J. Clin. Endo. 25 Metab., 87:4825-8, 2002; Conn et al., Molecular Interventions, 2:308-16, 2002). Gonadotropin-releasing hormone (GnRH) plays a central role in neural regulation of reproductive function. This decapeptide is produced by specialized neurons found in the mediobasal hypothalamus, the axons of which project to the median eminence. From there, GnRH enters the portal circulation and binds to the WO 2004/069859 PCT/US2004/002290 -2 gonadotropin-releasing hormone receptor (GnRHR) on pituitary gonadotropes, stimulating the synthesis and release of the gonadotropins LH and FSH. Sequence analysis of the GnRHR is consistent with the seven transmembrane domain motif, characteristic of the G protein-coupled receptors superfamily (Ulloa-Aguirre and Conn. 5 G protein-coupled receptors and the G protein family. In: Handbook of Physiology Endocrinology. Section 7 Cellular Endocrinology Conn PM, ed. New York: Oxford University Press, 87-124, 1998). Human GnRHR (hGnRHR) is coupled to the Gqi I system; after GnRH binding, the activated GnRHR-Gqni1 protein complex activates the membrane-associated enzyme phospholipase Cp, leading to inositol 1,4,5-trisphosphate 10 (IP) production and the release of intracellular calcium (Kaiser et al., Endocr. Rev. 18:46-70, 1997). Some forms of congenital hypogonadotropic hypogonadism (HH) result from mutational defects in GnRHR. At least 14 mutations of GnRHR are associated with HH. One is a truncation mutant; eight are compound heterozygotes (de Roux et al., N. 15 Engl. J. Med. 337:1597 - 1602, 1997; Layman et a., Nature Genetics 18:14-15, 1998; Caron et al., J. Clin. Endocrinol. Metab. 84:990-6, 1999; de Roux et al., J. Clin. Endocrinol. Metab. 84:567-72, 1999; Kottler et al., J. Clin. Endocrinol. Metab. 85:3002-8, 2000; Beranova et al., J. Clin. Endocrinol. Metab. 86:1580-8, 2001; Pralong et a/., J. Clin. Endocrinol. Metab. 84: 3811-6, 1999; Costa et al., J. Clin. 20 Endocrinol. Metab. 86:2680-6, 2001); and five are compound homozygotes (Pralong et a/., J. Clin. Endocrinol. Metab. 84: 3811-6, 1999; Soderlund et al., Clin. Endocrinol. (Oxf) 54:493-8, 2001; Pitteloud. et a/., J. Clin. Endocrinol. Metab. 86:2470-5, 2001). These mutations are widely distributed across the entire sequence of the GnRHR. Expression in heterologous cell systems that express each naturally-occurring GnRHR 25 mutant separately show that some mutants are totally non-functional (E 9 "K, A 129 1, R1 39 H, S 168 R, C 20 0 y, S 21 7 R, L 266 R, C 2 79 Y, and L 31 4 X) while others retain a modest degree of function (N' 0 K, TI 32 , Q 106 R, R 262 Q, and Y 2 84 C). It was previously believed that these mutations interfere with ligand binding or preclude interaction with effector proteins.

WO 2004/069859 PCT/US2004/002290 -3 Mutant E 9 0 K has been 'rescued' by deleting K 19 1 (which, when present, decreases expression of hGnRHR at the plasma membrane) or by adding a C-terminal sequence (Maya-Nunez et al., J. Clinical Endocrinol. Metab. 87:2144-9, 2002). In addition, previous approaches to correct defective receptors include genetic approaches, such as 5 increased receptor expression to produce larger numbers of receptors (Cheng et al., Am. J. Physiol. 268:L615-24, 1995). However, rescue by these genetic approaches is not presently practical for in vivo use. Other methods of correcting defective receptors include the use of non-specific protein stabilizing agents to stabilize extant molecules rendered incompetent by genetic defects, such as polyols and sugars (Back et al. 10 Biochemistry 18:5291-6, 1979; Brown et al., J. Clin. Invest. 99:1432-44, 1997; Brown et al., Cell Stress Chaperones 1:117-24, 1996). However, such methods are non specific. Therefore, there is a need for a method to restore function to mutant GnRHR molecules that can be used therapeutically in vivo. In addition, there is a need for 15 methods that allow one to screen for therapeutically effective agents that can be used to treat subjects having diseases associated with mutated GnRHR. SUMMARY The inventor has demonstrated that compounds from several different chemical 20 families (herein referred to as "pharmacoperones") can restore function to many gonadotropin-releasing hormone receptor (GnRHR) mutants. Therefore, agents from at least three different chemical classes (indoles, quinolones, and macrolides) can restore function to mutant GnRHR by allowing defective receptors to route to the plasma membrane, bind ligand and couple to effectors. A model is proposed whereby 25 chemically distinct agents serve as molecular scaffolding, cause mutant GnRH receptors to fold correctly and thereby avoid detection by the cellular quality control apparatuses. The correctly folded receptor can then traffic to the cell membrane, and be available for ligand binding.

WO 2004/069859 PCT/US2004/002290 -4 The agents that were successful in restoring function to one GnRHR mutant usually rescued all mutants (that could be rescued by any of the peptidomimetics). However, function was not restored to particular GnRHR mutants (human: S 168 R and

S

21 7 R; rat des260-265 -GnRHR and C229A-GnRHR) by any agent, indicating that receptors 5 these were either grossly deformed or that loss-of-function was due to inability to bind ligand. In addition, the efficacy of each agent (measured by the ability of a fixed dose to restore function to the mutant receptor) was proportional to the binding affinity of the molecules for the WT receptor. In one example, agents that restore function to a mutant GnRHR are GnRHR 10 antagonists, and therefore may compete with the natural ligand. Such agents are believed to stabilize the ligand binding site of the mutant receptors. In another example, agents that restore function to a mutant GnRHR do not compete for the natural ligand binding site. Therapeutic agents that bind outside the natural ligand binding site of GnRHR do not interfere with subsequence activation of the receptor with an agonist. 15 In particular examples, the disclosed pharmacoperones have one or more of the following characteristics: specificity for the receptor being rescued, ability to arrive at the correct cellular locus (enter the cell, get to the endoplasmic reticulum, and remain stable long enough to bind the nascent molecule), and ability to dissociate from the molecule (or, in the alternative, not compete with the physiological ligand) after it 20 arrives at the appropriate target locus (such as the cell surface). Based on these observations, disclosed herein are methods for screening for agents that can restore function to a mutant GnRHR. In particular examples, the methods include contacting the agent with a mutant GnRI-IR under conditions that allow interaction between the agent and the receptor, and then determining whether 25 functionality was restored to the receptor. Several methods are disclosed for determining whether functionality was restored to the mutant GnRHR by the agent, for example determining an amount of inositol phosphate (IP) production, determining an amount GnRHR agonist binding, determining an amount of surface-bound mutant GnRHR-ligand complex internalized, or combinations thereof.

WO 2004/069859 PCT/US2004/002290 -5 In other examples, the screening methods include contacting a GnRHR antagonist with GnRHR under conditions that allow interaction between the agent and the receptor, and screening the antagonist for ability to restore GnRHR function. In certain examples of the screening method, the inhibitory concentration (IC 5 o) of the 5 agent is determined. Agents having a particular IC 50 , such as indole or quinolone derivatives having an IC 50 of less than 5 nM (for example less than 3 nM, less than 2.5 nM, or less than 2 nM), or macrolide derivatives having an IC 50 of less than 700 nM (for example less than 100 nM, less than 20 nM, or less than 2 nM), are selected for further study. 10 In addition, methods'are disclosed for restoring function to a mutant GnRHR by contacting the identified agents, such as an indole-, quinilone-, or macrolide-derivative, with the mutant receptor. In particular examples, a therapeutically effective amount of such an agent is used to treat a subject having a mutant GnRHR, for example a subject having HH. 15 The inventor has also determined that normal expression of human GnRHR, but not rat GnRHR, was increased by the agents that were successful in rescuing mutant GnRHR. The presence of Lys 1 9 1 and other features (Janovick et al., Endocrine 22:317 28, 2003) in the primate sequence limits the percentage of the synthesized receptor that reaches the plasma membrane to about 50%. However, the pharmacoperones of the 20 present disclosure function to override this inhibition, possibly due to structural stabilization, but are unable to do so in the rodent sequence. Therefore, agents that can restore function to a mutant GnRHR can also be used to increase expression of wild type human GnRHR at the plasma membrane. Methods are disclosed for administering a therapeutically effective amount of 25 the disclosed pharmacoperones that can restore function to a mutant GnRHR, to increase expression of a wild-type hGnRHR on the cell surface. In particular examples, such agents are administered to a subject having hypogonadism, such as men and women suffering from erectile dysfunction or menopause, respectively.

WO 2004/069859 PCT/US2004/002290 -6 BRIEF DESCRIPTION OF THE FIGURES FIGS. 1-3 are graphs showing the efficacy (assessed by IP production) of each of (1) indoles, (2) quinolones and (3) erythromycin macrolides on restoration of 5 function to the GnRHR mutant E90L FIGS. 4-6 are bar graphs showing the effect of 1 pg/ml of each (4) indole, (5) quinolone and (6) erythromycin macrolide in restoring function to each GnRHR mutant. In each figure shows IP production in the presence of 10-7 M buserelin. For clarity in FIGS. 4-6, the SEM bars were omitted. The standard deviation was typically less than 10 10% of the corresponding mean. FIG. 7 is a bar graph showing the unrescued (no drug) coupling of receptor in the absence (upper graph) or presence (lower graph) of 10~7 M buserelin. 15 DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations and Terms The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein and in the appended claims, the singular forms "a" 20 or "an" or "the" include plural references unless the context clearly dictates otherwise. For example, reference to "an indole" includes a plurality of such indoles and reference to "the gonadotropin-releasing hormone receptor" includes reference to one or more gonadotropin-releasing hormone receptors and equivalents thereof known to those skilled in the art, and so forth. Similarly, the word "or" is intended to include "and" 25 unless the context clearly indicates otherwise. Hence "comprising A or B" means including A, or B, or A and B. Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

WO 2004/069859 PCT/US2004/002290 -7 Agent: Any polypeptide, compound, small molecule, organic compound, salt, polynucleotide, pharmacologic of biologic chaperone, or other molecule of interest. Analog: An agent (such as an organic chemical compound) that is structurally 5 similar to another, but differs slightly in composition, for example the replacement of one atom by an atom of a different element or functional group. For example, an analog of In3, ((2S)-2-[5-[2-(2-azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2-(3,5 dimethylphenyl)-lH-indol-3-yl]-N-(2-pyridin-4-ylethyl)propan-1-amine, is structurally similar to In3, and has a similar effect on restoring function to a GnRHR mutant such as 10 E 90 K. Binding: A specific interaction between two molecules, such that the two molecules interact. Binding can be specific and selective, so that one molecule is bound preferentially when compared to another molecule. In one example, specific binding is identified by a disassociation constant (Kd). In another example, specific binding of an 15 antagonist for a receptor is identified by an inhibitory concentration (IC 5 o). G protein-coupled receptor (GPCR): A superfamily of proteins, characterized by seven transmembrane alpha-helices, that signal through interaction with a family of heterotrimeric GTP-binding proteins, referred to as G proteins. Examples include, but are not limited to, beta-adrenergic receptor (betaAR), cystic fibrosis transmembrane 20 conductance regulator (CFTR), gonadotropin-releasing hormone receptor (GnRHR), rhodopsin, and vasopressin receptor (V2R). Gonadotropin releasing hormone receptor (GnRHR): GnRH receptors belong to the family of G protein-coupled receptor proteins and have been localized to the anterior pituitary, brain and reproductive organs as well as many steroid-dependent 25 tumor tissues. In humans, after GnRH ligand binding, the activated GnRHR-G 1 1 protein complex activates the membrane-associated enzyme phospholipase Cp, leading to inositol 1,4,5-trisphosphate (IP) production and the release of intracellular calcium. The term GnRHR includes any GnRHR gene, cDNA, RNA, or protein from any organism and includes a GnRHR that can normally traffic to the cell surface and bind WO 2004/069859 PCT/US2004/002290 GnRH. Examples of wild-type GnRHR nucleic acid sequences include, but are not limited to, GenBank Accession No. AFOO 1950 (human cDNA) and GenBank Accession No. S59525 (rat mRNA). Examples of GnRHR amino acid sequences include, but are not limited to: Genbank Accession Nos: AAB71348 (human) and AAB2642 (rat). In 5 one example, a GnRHR sequence includes a full-length wild-type (or native) sequence, as well as GnRHR allelic variants, variants, fragments, homologs or fusion sequences that retain wild-type function. GnRHR agonist: Agents that can bind to GnRHR and initiate the physiological and pharmacological responses characteristic of GnRHR. Examples include native 10 GnRHR ligands, such as GnRH, as well as other agents that can mimic the action of GnRH. Particular examples include buserelin (D-tert-butyl-Ser?, des-Gly'", Pro 9 , ethylamide-GnRH) and leuprolide (D-Leu 6 , Pro 9 , des-Glyl"-ethylamide-GnRH). GnRHR antagonist: Agents that bind to the ligand-binding site of GnRHR and interfere with binding of a ligand or agonist to the GnRHR binding site, thereby 15 resulting in decreased GnRHR-associated responses normally induced by the ligand or agonist. The inhibitory activity of an antagonist for a receptor is represented as an inhibitory concentration (IC 5 o), wherein better inhibitors have lower IC 5 0 values. Antagonism can be competitive and reversible (it binds reversibly to a region of the receptor in common with the agonist.) or competitive and irreversible (antagonist binds 20 covalently to the agonist binding site, and no amount of agonist can overcome the inhibition). GnRR ligand: Agents that can bind to the ligand-binding site of GnRHR. In some examples, such agents bind reversibly. The native GnRHR ligand is GnRH. Includes GnRHR agonists that bind at the ligand-binding site. 25 Hypogonadism: An underactivity of the sex glands. In men, hypogonadism is a condition that occurs when the testicles do not produce enough testosterone. In women, hypogonadism is a condition that occurs when the ovaries do not produce enough estrogen. Primary hypogonadism occurs when there is a problem with the testicles/ovaries themselves. Secondary hypogonadism occurs when there is a problem WO 2004/069859 PCT/US2004/002290 -9 with the pituitary gland. The hypothalamus secretes GnRH to stimulate the pituitary gland. In response, the pituitary gland secretes other hormones (follicle stimulating hormone (FSH) and luteinizing hormone (LH)). In turn, the ovaries (female) and testes (male) to secrete hormones (estrogen and testosterone, respectively) responsible for 5 normal sexual development in puberty. Any disruption in this cascade causes a deficiency of the sex hormones and halts normal pubertal sexual maturation. One example of secondary hypogonadism is hypogonadotropic hypogonadism. Hypogonadism can occur during fetal development, at puberty, or in adults. Symptoms associated with hypogonadism include: erectile dysfunction in men (the 10 inability to achieve or maintain an erection), infertility, decreased sex drive, decrease in beard and growth of body hair, decrease in size or firmness of the testicles, decrease in muscle mass and increase in body fat, enlarged male breast tissue, hot flashes, mood swings, irritability, depression, fatigue, osteoporosis (decreased bone density), delayed puberty, and combinations thereof. 15 There are several causes of hypogonadism, including: Klinefelter's syndrome (males with an extra X chromosome, which causes abnormal development of the testicles), undescended testicles, hemochromatosis (too much iron in the blood), testicular trauma, cancer treatment such as chemotherapy or radiation therapy, normal aging (older men and women generally have lower levels of testosterone and estrogen, 20 respectively, although the decline of the hormone varies greatly among people), pituitary disorders, medications including psychiatric drugs and agents used to treat heartburn and gastroesophageal reflux disease, and genetic mutations. Currently available treatments include male or female hormone replacement (testosterone replacement therapy (TRT) and hormone replacement therapy (HRT), 25 respectively), which can include administration of androgens such as testosterone and estrogen. Pituitary hormones, such as GnRH, can also help increase testosterone and estrogen levels. The present disclosure provides additional methods that can be used to treat hypogonadism, such as administration of agents that restore function to mutant WO 2004/069859 PCT/US2004/002290 -10 GnRHR, and administration of agents that increase the presence of wild-type GnRHR on the cell surface. Particular examples of hypogonadism include hypotestosteronism (a disorder characterized by lower than normal plasma levels of testosterone) and 5 hypoestrogeneism (a disorder characterized by lower than normal plasma levels of estrogen). Hypogonadotropic hypogonadism (HH): A congenital disorder characterized by failure of gonadal function secondary to deficient gonadotropin secretion, resulting from either a pituitary or hypothalamic defect, and is commonly seen in association 10 with structural lesions or functional defects affecting this region. HH presents as a wide clinical spectrum, characterized by delayed sexual development and by inappropriately low or apulsatile gonadotropin and sex steroid levels, in the absence of anatomical or functional abnormalities of the hypothalamic-pituitary axis. This disorder is genetically heterogeneous and can be sporadic or familial (X-linked or autosomal). At least 14 15 mutations of the GnRHR are associated with HH. Other genes currently recognized to be involved include KAL-1 (associated with X-linked Kallmann Syndrome), pituitary transcription factors (HESX1, LHX3, and PROP-1), orphan nuclear receptors (DAX-1, associated with X-linked adrenal hypoplasia congenital, and SF- 1), and three genes also associated with obesity (leptin, leptin receptor, and prohormone convertase 1). 20 Indole (2,3-benzopyrrole): A heterocyclic compound having the chemical formula CsH 7 N, and derivatives thereof, such as In30, ((2S)-2-[5-[2-(2 azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2-(3,5-dimethylphenyl)-lH-indol 3-yl]-N- {2-[4-(methylsulfinyl)phenyl]ethyl}propan- 1-amine); In31b, ((2S)-N-[2-(4 carboxyphenyl)ethyl]-2-[5-[1,1-dimethyl-2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2 25 (3,5-dimethylphenyl)-lH-indol-3-yl]propan-1-aminium trifluoroacetate); and In3, ((2S) 2-[5-[2-(2-azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2-(3,5-dimethylphenyl) 1H-indol-3-yl]-N-(2-pyridin-4-ylethyl)propan-1-amine. Exemplary indole antibiotics are disclosed in U.S. Patent Nos. 6,486,192; 5,380,723; and 4,076,831, all herein WO 2004/069859 PCT/US2004/002290 - 11 incorporated by reference. In a particular example, an indole includes indole derivatives that have an IC 50 of about less than 3 nM for hGnRHR. Inhibitory concentration (IC 50 ): An inhibitory concentration is a concentration that inhibits an effect (such as a receptor-mediated effect). The amount of inhibitory 5 agent needed to reduce an effect by 50% is an IC 5 o. In one example, the IC 5 0 of a GnRHR antagonist is the concentration of antagonist needed to reduce an amount of ligand binding (such as GnRH) to the receptor by 50%, compared to an amount of ligand binding in the absence of the antagonist. Lower IC 50 values suggest better inhibition. 10 Inositol phosphate (IP): Includes all phosphorylated states of inositol, such as inositol- 1-phosphate (IP 1 ), inositol-4,5-diphosphate (IP 2 ), inositol-1,4,5-triphosphate

(IP

3 ), and inositol-1,3,4,5-triphosphate (IP 4 ). In one example, IPs are produced following activation of a GnRHR. Activation of GnRHR, for example by binding of a GnRHR ligand or agonist to cell-surface GnRHR, leads to activation of phospholipase 15 C, which catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) into

IP

3 and 1,2-diacylglycerol (DG). IP 3 then acts as a second messenger stimulating the release of calcium. Label: An agent capable of detection, for example by ELISA, spectrophotometry, flow cytometry, or microscopy. For example, a label can be attached to a protein, thereby 20 permitting detection of the protein. Examples of labels include, but are not limited to, radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent agents, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring 25 Harbor, New York, 1989) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998). Macrolide: Any of a large group of antibiotics containing a macrolide ring linked glycosidically to one or more sugars. Members of the macrolide antibiotic group include erythromycin, carbomycin, azithromycin, and clarithromycin, and derivates WO 2004/069859 PCT/US2004/002290 - 12 thereof. Examples of particular erythromycin-derived macrolides include A-7662.0, (Erythromycin A); A-64755.0 (11-deoxy-11-[carboxy-phenylethylamino]-6-0-methyl erythromycin A 11,12-(cyclic carbamate)); A-177775.0, (3'-N-desmethyl-3'-N cyclopentyl- 11 -deoxy- 11 -[carboxy-(3,4-dichlorophenylethylamino)]-6-0-methyl 5 erythromycin A 11,12-(cyclic carbamate)); and A-222509.0, 3',3'-N-desmethyl-3',3'-N cyclopropylmethyl-11-deoxy-11-[carboxy-(3-chloro,4-fluoro-phenylethylamino)]-6-0 methyl-erythromycin A 11,12-(cyclic carbamate)). Additional macrolide antibiotics are disclosed in U.S. Patent Nos. 5,786,338; 5,169,956; and 4,374,764, all herein incorporated by reference. In a particular example, a macrolide includes macrolide 10 derivatives that have an IC 50 of about less than 700 nM for hGnRHR. Mammal: This term includes both human and non-human mammals. Similarly, the terms "patient," "subject," and "individual" includes living multicellular vertebrate organisms, such as human and veterinary subjects. Mimetic: A molecule (such as an organic chemical compound) that mimics the 15 activity of an agent, such as the activity of a GnRHR antagonist on trafficking of mutant GnRHR to the cell surface. Peptidomimetic and organomimetic embodiments are within the scope of this term, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three dimensional arrangement of the peptide backbone and component amino acid 20 sidechains in the peptide, resulting in such peptido- and organomimetics of the peptides having substantial specific activity. For computer modeling applications, a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with computer modeling software (using computer assisted 25 drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165-174 and Principles of Pharmacology (ed. Munson, 1995), chapter 102 for a description of techniques used in computer assisted drug design.

WO 2004/069859 PCT/US2004/002290 - 13 Mutant GnRHR: A GnRHR sequence that includes at least one amino acid substitution or deletion compared to a wild-type sequence, which results in the development of HH in a subject. Examples of mutant GnRHR include, but are not limited to, human N OK, T 32I, E K, Q10 R, A 129D, R 139H, C200Y, R262 Q,L 66R, C 279Y, 5 and Y 284 C mutations, as well as rat des325-327, desL237-L241 and C278A mutations. In a particular example, mutant GnRHR does not include the human S 68R or S217R mutations, or the rat des260-65-GnRHR or C 229 A mutation. Peripheral Administration: Administration outside of the central nervous system. Peripheral administration does not include direct administration to the brain. 10 Peripheral administration includes, but is not limited to intravascular, intramuscular, subcutaneous, inhalation, oral, rectal, transdernal or intra-nasal administration. Quinolone: A heterocyclic compound having the chemical formula C 9

H

7 N, and derivatives thereof, such as Q89, (7-chloro-2-oxo-4-{2-[(2S)-piperidin-2-yl]ethoxy} -N pyrimidin-4-yl-3-(3,4,5-trimethylphenyl)-1,2-dihydroquinoline-6-carboxamide); Q76, (N 15 (7-chloro-3-(3,5-dimethylphenyl)-2-oxo-4-{2-[(2S)-piperidin-2-yl]ethoxy} -1,2 dihydroquinolin-6-yl)-N'-cyclopropylurea); and Q08, ((2S)-2-(2-{[7-chloro-6-[(6,7 dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)carbonyl]-3-(3,5-dimethylphenyl)-2-oxo-1,2 dihydroquinolin-4-yl]oxy} ethyl)piperidinium trifluoroacetate). Includes synthetic antibacterial agents (quinolone antibiotics) such as nalidixic acid, cinoxacin, rosoxacin, 20 and the fluorinated 4-q's. Additional quinolone antibiotics are disclosed in U.S. Patent Nos. 5,990,122; 5,646,163; and 5,385,906, all herein incorporated by reference. In a particular example, a quinolone includes quinolone-derivatives that have an IC 50 of about less than 3 nM for hGnRHR. Restore function: An agent, such as a GnRHR pharmacoperone, is said to restore 25 function to a mutant GnRHR when contact of the agent with a mutant GnRHR results in increased GnRHR-type activity of the mutant receptor, as compared to binding in the absence of the agent. Restoring function to a mutant GnRHR does not require restoration of 100% of wild-type activity.

WO 2004/069859 PCT/US2004/002290 - 14 In one example, increasing GnRHR-type activity of the mutant receptor increases binding of a mutant GnRHR to a GnRHR agonist (such as buserelin), as compared to binding in the absence of the agent. For example, binding of a GnRHR agonist to a mutant GnRHR having restored function increases at least 25% when compared to 5 binding of a mutant GnRHR to GnRHR agonist in the absence of the agent. In other non limiting examples, binding of a GnRHR agonist to a mutant GnRHR having restored function increases at least 50%, such as at least 60%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 100%, or even such as at least 200%, when compared to binding of a mutant GnRHR to GnRHR agonist in the absence of an agent. 10 Such binding can be performed using the methods disclosed herein (see Example 1). In another or in an additional example, an agent (such as a GnRHR antagonist), is said to restore function to a mutant GnRHR when contact of the agent with a mutant GnRHR results in an increase in GnRH agonist-stimulated inositol phosphate (IP) production, as compared to IP production in the absence of the agent. For example, IP 15 production by a mutant GnRHR having restored function increases at least 25% when compared to IP production by mutant GnRHR in the absence of the agent. In other non limiting examples, IP production by a mutant GnRHR having restored function increases at least 50%, such as at least 60%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 100%, such as at least 200%, or even such as at least 500% 20 when compared to IP production by mutant GnRHR in the absence of the agent. Determination of IP production can be performed using the methods disclosed herein (see Example 1). In another or in an additional example, an agent is said to restore function to a mutant GnRHR when contact of the agent with a mutant GnRHR results in increased 25 surface-bound receptor-ligand complex internalized, as compared to an amount of surface-bound receptor-ligand complex internalized in the absence of the agent. For example, surface-bound receptor-ligand complex internalized by a mutant GnRHR having restored function increases at least 25% when compared to IP production by a mutant GnRHR in the absence of the agent. In other non-limiting examples, surface-bound WO 2004/069859 PCT/US2004/002290 - 15 receptor-ligand complex internalized by a mutant GnRHR having restored function increases at least 50%, such as at least 60%, such as at least 75%, such as at least 80%, such as at least 90%, such as at least 100%, or even such as at least 200% when compared to an amount of surface-bound receptor-ligand complex internalized by a mutant GnRHR 5 in the absence of the agent. Determination of surface-bound receptor-ligand complex internalized can be performed using the methods disclosed herein (see Example 2). Subject: Living multicellular vertebrate organisms, a category that includes, both human and veterinary subjects for example, mammals, rodents, and birds. Therapeutically effective amount: An amount of an agent (alone or in 10 combination with other therapeutically effective agents) sufficient to achieve a desired biological effect, for example an amount that is effective to increase binding of a GnRHR agonist to a mutant GnRHR by at least a desired amount, increase GnRH agonist-stimulated inositol phosphate (IP) production by a mutant GnRHR by at least a desired amount, increase surface-bound mutant GnRHR-ligand complex internalized by 15 at least a desired amount, or combinations thereof, such as in the cell of a subject to whom it is administered. In a particular example, it is an amount of an agent effective to increase binding of a GnRHR agonist to a mutant GnRHR by at least a desired amount, such as an increase by at least 25%, at least 50%, at least 75%, at least 20%, or even at least 200% 20 as compared to an amount of binding prior to treatment. In other or additional examples, it is an amount effective to increase GnRH agonist-stimulated IP production by a mutant GnRHR by at least a desired amount, such as increase by at least 25%, at least 50%, at least 75%, at least 20%, or even at least 200% as compared to an amount of IP production prior to treatment. In other or additional examples, it is an amount 25 effective to increase surface-bound mutant GnRHR-ligand complex internalized by at least a desired amount, such as increase by at least 25%, at least 50%, at least 75%, at least 20%, or even at least 200% as compared to an amount of complex internalized prior to treatment.

WO 2004/069859 PCT/US2004/002290 -16 In some examples, it is an amount of an agent that can restore function to a mutant GnRHR (alone or in combination with other therapeutically effective agents) that can improve signs or symptoms of HH due to a GnRHR mutation. In other examples, it is an amount of an agent (alone or in combination with other 5 therapeutically effective agents) that can increase an amount of wild-type GnRHR at the cell surface, for example by at least 10%, at least 20%, or even at least 50% as compared to the absence of the agent. In particular examples, a therapeutically effective amount improves signs or symptoms of hypogonadism, for example such a condition associated with decreased functional GnRHR at the cell surface. 10 An effective amount of an agent that restores function to a mutant GnRHR, such as a GnRHR antagonist, can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount of agent may be dependent on the source of agent administered, the subject being treated, the severity and type of HH being treated, and the manner of administration. For example, 15 a therapeutically effective amount of an agent that restores function to a mutant GnRHR can vary from about 1 pg/kg body weight to about 20 jg/kg body weight per day, about 1 pg/kg body weight to about 10 pg/kg body weight per day, about 10 pg/kg body weight to about 20 jg/kg body weight per day, or about 1-2 jg agent/kg body weight/day. In particular examples, a therapeutically effective amount of an agent, such 20 as an antagonist, peaks in the serum, then returns to negligible levels indicating that the agent has been removed from the rescued receptor. I have the To assess restoration of mutant GnRHR function, or increased expression of wild-type human GnRHR at the cell surface, the methods disclosed herein can be used to compare a subject before and after treatment. For example, binding of a GnRHR 25 agonist to a mutant GnRHR, GnRH agonist-stimulated IP production, and the surface bound mutant GnRHR-ligand complex internalized can be determined using the methods described in Examples 1 and 2.

WO 2004/069859 PCT/US2004/002290 - 17 Translocation: The transport of an agent, such as a wild-type or mutant GnRHR, from one part of a cell to another part of the cell. In one example, it includes the transport of a wild-type or mutant GnRHR across a cell membrane. All of the examples provided herein are non-limiting examples provided merely 5 for illustration and not limitation. Screening for Agents that Restore Functionality to Mutant Receptors Provided herein are methods that can be used to screen agents for their ability to restore function to a mutant GnRHR. In particular examples, the mutant GnRHR is a 10 human GnRHR including a N' K, T I, E90K Q1 R, A129D, R 139H,C "Y, R2Q, L66 R, C29Y or a Y28C amino acid substitution. In other examples, the mutant GnRHR is a rat GnRHR including a Des325-327, DesL237-L241 or a C278A mutation. In one example, the agents to be screened are GnRHR antagonists. Exemplary antagonists include, but are not limited to derivatives, analogs, and mimetics of indole, 15 quinolone, and macrolide antibiotics, such as Q89, (7-chloro-2-oxo-4-{2-[(2S) piperidin-2-yl]ethoxy}-N-pyrimidin-4-yl-3-(3,4,5-trimethylphenyl)-1,2 dihydroquinoline-6-carboxamide); Q76, (N-(7-chloro-3-(3,5-dimethylphenyl)-2-oxo-4 {2-[(2S)-piperidin-2-yl]ethoxy}-1,2-dihydroquinolin-6-yl)-N-cyclopropylurea); Q08, ((2S)-2-(2-{[7-chloro-6-[(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)carbonyl]-3 20 (3,5-dimethylphenyl)-2-oxo- 1,2-dihydroquinolin-4-yl]oxy} ethyl)piperidinium trifluoroacetate); In30, ((2S)-2-[5-[2-(2-azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2 oxoethyl]-2-(3,5-dimethylphenyl)-lH-indol-3-yl]-N-{2-[4 (methylsulfinyl)phenyl] ethyl}propan- I-amine); In3, ((2S)-2-[5-[2-(2 azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2-(3,5-dimethylphenyl)-1H-indol 25 3-yl]-N-(2-pyridin-4-ylethyl)propan-1-amine; A-64755.0 (11-deoxy- 11-[carboxy phenylethylamino]-6-0-methyl-erythromycin A 11,12-(cyclic carbamate)); A-177775.0, (3'-N-desmethyl-3'-N-cyclopentyl- 11 -deoxy- 11 -[carboxy-(3,4 dichlorophenylethylamino)]-6-0-methyl-erythromycin A 11,12-(cyclic carbamate)); and A-222509.0, 3',3'-N-desmethyl-3',3'-N-cyclopropylmethyl-11-deoxy-11-[carboxy- WO 2004/069859 PCT/US2004/002290 - 18 (3-chloro,4-fluoro-phenylethylamino)]-6-0-methyl-erythromycin A 11,12-(cyclic carbamate)). In some examples, indoles and quinolones to be screened have an IC 50 of less than 5 nM for wild-type human GnRHR, for example less than 4 nM, less than 3 nM, 5 less than 2.5 nM, less than 2.3 nM, less than 2 nM, or less than 1 nM. In other examples, the macrolide antibiotic to be screened, such as an erythromycin-derived macrolide, has an IC 50 of less than 700 nM for wild-type human GnRHR, for example less than 100 nM, less than 25 nM, less than 20 nM, or less than 5 nM. In one example, the method includes screening an indole, quinolone, or 10 macrolide (including derivatives, analogs, or mimetics thereof) for an ability to restore functionality to a mutant GnRHR. The method includes include contacting the indole, quinolone, or macrolide with the mutant GnRHR under conditions that allow interaction between the indole, quinolone, or macrolide and the mutant GnRHR and under conditions that restore function to the mutant GnRHR, then incubating the indole, 15 quinolone, or macrolide with the mutant GnRHR for a time sufficient to allow interaction between the indole, quinolone, or macrolide and the mutant GnRHR. In some examples, the mutant GnRHR is expressed recombinantly in a cell, and the cell is contacted with the agent. Subsequently, a determination is made as to whether functionality was restored to the mutant GnRHR. Agents that restore functionality to 20 mutant GnRHR can be selected for further study. Several types of assays can be used (alone or in combination) to determine whether functionality was restored to the mutant GnRHR. In one example, the method includes determining an amount of inositol phosphate (IP) production by the cell, wherein an increase in IP production as compared to an amount of IP production in the 25 absence of the indole, quinolone, or macrolide indicates that the agent restored functionality to the mutant GnRHR. In particular examples, IP production increases by at least 25%, such as at least 50%, at least 75%, at least 100%, at least 200%, or even at least 1000% in the presence of the agent, as compared to an amount of IP production in the absence of the agent. Any method used by those skilled in the art can be used to WO 2004/069859 PCT/US2004/002290 - 19 measure IP production. In one example, IP production is measured as follows. Following incubating the agent (such as an indole, quinolone, or macrolide) with the cell, the cell is washed to remove the agent, then incubated with [ 3 H]inositol (a precursor of IPs) for a time sufficient to allow uptake of the inositol by the cell. The 5 cell is then contacted the cell with a GnRHR agonist for a time sufficient to allow stimulation of IP production, and the amount of IP produced determined, for example by disrupting the cells and the amount of radioactivity incorporated into total IPs is determined by liquid scintillation spectroscopy (for example, see the method described in Huckle and Conn, Methods Enzymol. 141:149-55, 1987). Exemplary GnRHR 10 agonists include buserelin (D-tert-butyl-Ser 6 , des-Glyl", Pro 9 , ethylamide-GnRH), leuprolide (D-Leu 6 , Pro 9 , des-Glyl-ethylamide-GnRH), and GnRH. Another assay that can be used to determine whether functionality was restored to the mutant GnRHR includes determining an amount of GnRHR agonist binding to a mutant GnRHR on a surface of the cell, wherein an increase in binding as compared to 15 an amount of binding in the absence of the indole, quinolone, or macrolide indicates that the agent restored functionality to the mutant GnRHR. In particular examples,, GnRHR agonist binding increases by at least 25%, such as at least 50%, at least 75%, at least 100%, at least 200%, or even at least 1000% in the presence of the agent, as compared to an amount of binding in the absence of the agent. Any method used by 20 those skilled in the art can be used to measure GnRHR agonist binding. In one example, GnRHR agonist binding is measured as follows. Following incubating the agent (such as an indole, quinolone, or macrolide) with the cell, the cell is washed to remove the agent, then incubated with a GnRHR agonist (such as a GnRHR ligand) of the for a time sufficient to allow binding of the agonist to GnRHR present on the cell 25 surface. Subsequently, the amount of GnRHR agonist binding is determined, for example by detecting a label present on the agonist. In one example, the GnRHR agonist includes a label, such as a radiolabel or fluorophore. In a particular example, the GnRHR agonist includes 1 25 I-buserelin.

WO 2004/069859 PCT/US2004/002290 -20 Another assay that can be used to determine whether functionality was restored to the mutant GnRHR includes determining an amount of internalized surface-bound mutant GnRHR-ligand complex, wherein an increase in internalization as compared to an amount of internalization in the absence of the indole, quinolone, or macrolide 5 indicates that the agent restored functionality to the mutant GnRHR. In particular examples, internalization of surface-bound mutant GnRHR-ligand complex increases by at least 25%, such as at least 50%, at least 75%, at least 100%, at least 200%, or even at least 1000% in the presence of the agent, as compared to an amount of internalization in the absence of the agent. Any method used by those skilled in the art can be used to 10 measure internalization of surface-bound mutant GnRHR-ligand complex. In one example, internalization of surface-bound mutant GnRHR-ligand complex is measured as follows. Following incubating the agent (such as an indole, quinolone, or macrolide) with the cell, the cell is washed to remove the agent, then incubated with a GnRHR agonist (such as buserelin) for a time sufficient to allow binding of the agonist to 15 GnRHR present on the cell surface, thereby forming a GnRHR-agonist complex, and allowing the complex to internalize into the cell. At the desired time, the agonist is removed from the cell surface, and the amount of GnRHR-agonist complex present in the cell determined, for example by detecting a label present on the agonist. In a particular example, the GnRHR agonist includes 12 5 I-buserelin, and internalization is 20 determined by quantitating the amount of radioactivity present. Methods are provided for screening indoles, quinolones, and macrolides (including derivatives, analogs, and mimetics thereof) for an ability to restore function to a mutant GnRHR. In one example, the method includes contacting the indole, quinolone, or macrolide with a wild-type GnRHR under conditions that allow 25 interaction between the indole, quinolone, or macrolide and the wild-type GnRHR, in the presence or absence of a GnRHR agonist, and then determining an inhibitory concentration (IC 5 0 ) of the indole, quinolone, or macrolide for the wild-type GnRHR. Methods for determining the IC 50 of an antagonist are known, and include determining the specificity of the agent for GnRHR, for example by measuring IP production (as WO 2004/069859 PCT/US2004/002290 -21 described above) in the presence of a range of GnRHR agonist concentrations (such as about 10 1 M - 10 7 M) and in the presence of a single concentration of agent (such as about 1 jg/ml). In particular examples, indoles and quinolones having an IC 50 of less than 3 nM for wild-type GnRHR, and macrolides with an IC 5 0 of less than 700 nM for 5 wild-type GnRHR, are selected as candidate agents which can restore function to a mutant GnRHR. As described in the examples below, agents identified that were able to restore function to mutant GnRHR, were also able to increase the presence of wild-type human GnRHR on the cell surface. Therefore, the methods described above can also be used 10 to screen for agents that increase trafficking of wild-type GnRHR to the cell surface. In particular examples, wild-type hGnRHR is incubated with the agent, and the amount of IP production, GnRHR agonist binding, internalization of wild-type GnRHR-ligand complex, or combinations thereof, determined, and compared to an amount in the absence of the agent. Such agents can be used to treat subjects having hypogonadism, 15 for example to alleviate symptoms associated with erectile dysfunction, infertility, decreased libido, decrease in beard and growth of body hair, decrease in size or firmness of the testicles, decrease in muscle mass and increase in body fat, enlarged male breast tissue, hot flashes, mood swings, irritability, depression, fatigue, osteoporosis, delayed puberty, or combinations thereof. 20 One skilled in the art will appreciate that similar methods can be used to identify agents that restore function to other mutated receptors, such as other G-coupled protein receptors. Restoring Function to Misrouted Receptors and 25 Increasing Wild-type Receptors on the Cell Surface Methods are disclosed for restoring function to receptors that are misrouted, for example due to misfolding. Examples of disorders (and the corresponding protein) caused by misrouted proteins, includes, but is not limited to: cystic fibrosis (CFTR chloride channel), systemic amyloidosis (amyloid fibrils, light chain variable domains), WO 2004/069859 PCT/US2004/002290 -22 I-cell disease, nephrogenic diabetes insipidus (aquaporin-2, V-2 receptor), cancer (p53 protein), retinitis pigmentosa (rhodopsin, carotenoid receptors), emphysema and alpha 1 antitrypsin deficiency liver disease (alpha 1 antitrypsin), Alzheimer's Disease (amyloid, tau protein), Creutzfeldt-Jakob (amyloid), spongiform encephalopathies (prion 5 glycoprotein (PrP)), sickle cell anemia (hemoglobin), Parkinson's Disease (alpha synclein, Parkin, ubiquitin C) and cataracts (lens crystallins). In one example, the mutant receptor is a mutant gonadotropin releasing hormone receptors (GnRHRs). In particular examples, the mutant human GnRHR includes a N10K, T32, E90K, Q106R, A129D, R139H, c200Y, R262% 266R, C279Y or a y284C amino 10 acid substitution. In other examples, the mutant GnRHR is a rat GnRHR including a Des325-327, DesL237-L241 or a C278A mutation. In some examples, the mutant GnRHR is expressed in a cell but is not transported to the cell surface. In order to restore function to such receptors, the method includes contacting the cell with a therapeutically effective amount of an agent that 15 increases transport of the mutant GnRHR to the cell surface. By allowing the mutant GnRHR to correctly route to the plasma membrane, an increased number of receptors are available at the cell surface for ligand binding. In one example, the agent binds to the GnRHR at a non-ligand binding site, with high affinity. Exemplary agents include GnRHR antagonists, such as indoles, quinolones, macrolides, or combinations thereof. 20 Examples of GnRHR antagonists that at least partially restore function to mutant GnRHR include several members of the indole, quinolone, and macrolide chemical groups, such as Q89, Q76, Q08, In30, In3, A-64755.0, A-177775.0, and A-222509. In one example, the agent includes an indole antibiotic, quinolone antibiotic, macrolide antibiotic, or combinations thereof. 25 In some examples where the GnRHR antagonist binds to the receptor at its ligand binding site, thereby interfering with binding of the ligand (gonadotropin releasing hormone, GnRH), the pharmacological chaperone or "pharmacoperone" is removed from the receptor so that the rescued GnRHR can more effectively bind ligand and couple to its effector protein. In some examples, such antagonists bind to the WO 2004/069859 PCT/US2004/002290 - 23 GnRHR, but do so reversibly, thereby providing a mechanism for removing the therapeutic antagonist. In examples where a GnRHR antagonist does not bind to the receptor at its ligand binding site, and therefore does not interfere with binding of the ligand once the 5 receptor reaches the cell surface, removal of the antagonist may be less desirable before activating the receptor with an agonist. In a particular example, the disclosed methods of restoring function to a mutant GnRHR include contacting a therapeutically effective amount of an indole, quinolone, macrolide, or combinations thereof (including derivatives, analogs, or mimetics thereof) 10 identified using the screening methods described above with the mutant GnRHR, wherein contacting restores function to the mutant GnRHR. Restoring function to the mutant GnRHR can include increasing binding of a GnRHR agonist to a mutant GnRHR by at least 25%, increasing GnRH agonist-stimulated IP production by a mutant GnRHR by at least 25%, increasing surface-bound mutant GnRHR-ligand 15 complex internalized by at least 25%, or combinations thereof. In particular examples, the mutant GnRHR is present in a subject, and the method includes administering a therapeutically effective amount of the identified indole, quinolone, or macrolide (or combinations thereof) to the subject. The indole, quinolone, or macrolide can be administered in a pharmaceutically acceptable carrier, alone or in the presence of 20 additional therapeutic agents. In one example, the subject has hypogonadotropic hypogonadism (HH). Methods are also provided for increasing the amount of wild-type human GnRHR on the cell surface. In some examples, the method includes contacting a therapeutically effective amount of an indole, quinolone, macrolide or combinations 25 thereof (including derivatives, analogs, or mimetics thereof) identified using the screening methods described above with the wild-type human GnRHR (hGnRHR), wherein contacting increases wild-type hGnRHR expression at a cell surface. In one example, the amount of wild-type hGnRHR on the cell surface increases by at least 10%, for example at least 25%, at least 50%, at least 100%, or even at least 150%, in WO 2004/069859 PCT/US2004/002290 -24 the presence of the agent, as compared to an amount present in the absence of the agent. In some examples, the wild-type hGnRHR is present in a subject, and the indole, quinolone, macrolide or combinations thereof is administered to the subject in a therapeutically effective amount. In particular examples, the subject has 5 hypogondadism, and may have one or more of the following symptoms: erectile dysfunction, infertility, decreased libido, decrease in beard and growth of body hair, decrease in size or firmness of the testicles, decrease in muscle mass and increase in body fat, enlarged male breast tissue, hot flashes, mood swings, irritability, depression, fatigue, osteoporosis, delayed puberty, or combinations thereof. 10 One skilled in the art will appreciate that similar methods can be used to restore function to other mutated receptors, thereby correcting diseases for which the etiology is misrouted or misfolded proteins. Examples of such diseases include, but are not limited to, cystic fibrosis, nephrogenic diabetes insipidus, hypercholesterolemia, 15 cataracts, Alzheimer's, and retinitis pigmentosa. EXAMPLE 1 Pharmacological Rescue of GnRNR Mutants 20 This example describes methods used to demonstrate that agents from several different chemical classes can restore functionality to several rat and human GnRH mutant receptors. One skilled in the art will appreciate that similar methods can be used to screen other agents of interest, such as other indoles, quinolones, and erythromycin derived macrolides. Furthermore, similar methods can be used to screen and restore 25 function to other mutant G-protein-coupled receptors. The following chemical structures (collectively referenced as "the agents") were utilized; those of the quinolone class are prefaced by the letter "Q" and those of the indole class by the letters "In" and were produced by Merck and Company (Ashton et al., Bioorg. Med. Chem. Lett. 11:1727-3 1, 2001; Ashton et al. Bioorg. Med. Chen. Lett.

WO 2004/069859 PCT/US2004/002290 -25 11:1723-6, 2001; and Ashton et al., Bioorg. Med. Chem. Lett. 11:2597-602, 2001): Q89, (7-chloro-2-oxo-4-{2-[(2S)-piperidin-2-yl]ethoxy} -N-pyrimidin-4-yl-3-(3,4,5 trimethylphenyl)-1,2-dihydroquinoline-6-carboxamide); Q76, (N-(7-chloro-3-(3,5 dimethylphenyl)-2-oxo-4-{2-[(2S)-piperidin-2-yl]ethoxy}-1,2-dihydroquinolin-6-yl)-NV 5 cyclopropylurea); Q08, ((2S)-2-(2- { [7-chloro-6-[(6,7-dimethoxy-3,4 dihydroisoquinolin-2(1H)-yl)carbonyl]-3-(3,5-dimethylphenyl)-2-oxo-1,2 dihydroquinolin-4-yl]oxy} ethyl)piperidinium trifluoroacetate); In30, ((2S)-2-[5-[2-(2 azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2-(3,5-dimethylphenyl)-1H-indol 3-yl]-N- {2-[4-(methylsulfinyl)phenyl]ethyl}propan- 1-amine); In3lb, ((2S)-N-[2-(4 10 carboxyphenyl)ethyl]-2-[5-[1,1-dimethyl-2-(4-methylpiperazin-1-yl)-2-oxoethyl]-2 (3,5-dimethylphenyl)-1H-indol-3-yl]propan-1-aminium trifluoroacetate); In3, ((2S)-2 [5-[2-(2-azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2-(3,5-dimethylphenyl) 1H-indol-3-yl]-N-(2-pyridin-4-ylethyl)propan-1-amine. Erythromycin-derived macrolides were prepared by Abbott Laboratories (Bush 15 et al. (1999) Program and Abstracts of the 81st Annual Meeting of the Endocrine Society, San Diego, CA, Abstract P3-225; Diaz et al. (1999) Program and Abstracts of the 81st Annual Meeting of the Endocrine Society, San Diego, CA, Abstract P3-226) and are prefaced by the letter "A." A-7662.0 , (Erythromycin A); A-64755.0 (11 deoxy-11l-[carboxy-phenylethylamino]-6-O-methyl-erythromycin A 11,12-(cyclic 20 carbamate)); A-1 77775.0, (3'-N-desmethyl-3'-N-cyclopentyl- 11-deoxy- 11-[carboxy (3,4-dichlorophenylethylamino)]-6-0-methyl-erythromycin A 11,12-(cyclic carbamate)); A-222509.0, 3',3'-N-desmethyl-3',3'-N-cyclopropylmethyl-11-deoxy-11 [carboxy-(3 -chloro,4-fluoro-phenylethylamino)] -6-0-methyl-erythromycin A 11,12 (cyclic carbamate)). 25 Wild-type (WT) hGnRHR cDNA in pcDNA3 was subcloned into pcDNA3.1 at KpnI and XbaI restriction enzyme sites. All GnRHR mutants were constructed, sequenced and prepared using standard molecular biology techniques (Janovick et al., J. Clin. Endocrinol. Metab., 87:3255-62, 2002). Naturally occurring mutants of hGnRHR associated with human hypogonadotropic hypogonadism, N 10 K, T 32 1, E 90 K, Q1 06

R,

WO 2004/069859 PCT/US2004/002290 -26

A

12 'D, R 139 H, S1 6 8 R, C 200 Y, S 21 7 R, R 26 2 Q, L 266 R, C 279 Y and Y 2 84 C, were generated. Manufactured mutants of the rGnRHR included the shortest rat GnRHR c-terminal truncation mutant that results in receptor loss-of-function, des 325

-

327 -rGnRHR; two deletion mutants (des 2 37

-

241 -rGnRHR, and des 260-265-rGnRHR) and two Cys mutants 5 (C 22 9 A-GnRHR and C 278 A-GnRHR). Large-scale plasmid DNAs were prepared using a Qiagen Endotree Maxi-prep kit (Qiagen, Valencia, CA). The purity and identity of the amplified plasmid DNAs were further verified by restriction enzyme analysis. WT and mutant hGnRHR were separately transiently expressed in COS-7 cells using standard molecular biology 10 methods (Leafios-Miranda et al., J. Clin. Endo. Metab., 87:4825-8, 2002). Briefly, cells were maintained in growth medium (DMEM) containing 10% fetal calf serum (FCS; Life Technologies, Grand Island, NY) and 20 [ig/ml gentamicin (Gemini Bioproducts, Calabasas, CA) in a 5% CO 2 humidified atmosphere at 37'C. One hundred thousand cells/well were seeded in 24-well plates (Costar, Cambridge, MA). 15 Twenty-four hours after plating, the cells were transfected with 0.05 ptg DNA per well (for IP production assay) or 0.1 pig DNA per well (for saturation binding studies) using 2 pl lipofectamine in 0.25 ml OPTI-MEM containing 1% DMSO (vehicle) or 1 jig/ml of each indole, quinoline or erythromycin macrolide prepared in vehicle. After five hours, 0.25 ml of DMEM containing 20% FCS with or without indole, 20 quinoline or erythromycin macrolide (as indicated) was added to each well. The cells were incubated for an additional 18 hours at 37'C, then washed and fresh growth medium with (1 ptg/ml) or without the therapeutic agent was added to the cells for another 28 hours at 37*C. The cells were then washed twice with DMEM/0.1% BSA/Gentamicin and were preloaded with 3 H-inositol (for IP assays) or DMEM (for 25 internalization studies) for 18 hours prior to stimulation with agonist. During this latter 18 hour period, as well as the period of GnRH stimulation, indole, quinoline and erythromycin macrolide were not present. The quantification of IP production and saturation binding were determined as described previously (Huckle and Conn, Methods Enzymol. 141:149-55, 1987).

WO 2004/069859 PCT/US2004/002290 - 27 To determine the saturation binding, the cells were washed twice with warm DMEM/BSA prior to incubating with the GnRH agonist [ 1 25 1]-buserelin [D-tert-butyl Ser , des-Glym, Pro', ethylamide-GnRH; specific activity, 700 pCi/gg; 230,000 cpm/0.5 ml, pH 7.4; Hoeschst-Roussel Pharmaceuticals (Somerville, NJ); also see Marian et al., 5 Mol. Pharmacol. 19:399-405, 1981] and nonspecific binding was measured in the presence of 1 gM GnRH (NIDDK National Hormone and Peptide Program, Bethesda, MD). Cells were incubated at room temperature for 90 minutes. The medium was removed, plates containing the cells were placed on ice and washed twice with ice-cold PBS. Then, 0.2 M NaOH/0.1% SDS (Costa et al., J Clin. Endocrinol. Metab. 86:2680 10 6, 2001) was added to the wells to solubilize the cells. The sample was transferred to a glass tube and counted in a gamma counter (Packard Instruments; Downers Grove, IL). Specific binding was calculated by subtracting non-specific binding (binding measured in the presence of 1 pM GnRH) from total binding (no GnRH). To measure inositol phosphates (IP) production, anywhere from 4 - 51 hours 15 after the start of transfection with WT or mutant GnHRH, transiently transfected COS-7 cells were washed twice with DMEM (no indole, quinoline or erythromycin macrolide) containing 0.1% bovine serum albumin (BSA), and intracellular inositol lipids were incubated in inositol-free DMEM supplemented with 4 R Ci/ml [ 3 H]myo-inositol for 18 hours at 37 0 C. After the preloading period, cells were washed twice with DMEM 20 (inositol free) containing 5 mM LiCl (no indole, quinoline or erythromycin macrolide) and incubated for 2 hours at 37 0 C in the absence or presence of the indicated doses of GnRH agonist buserelin dissolved in 0.5 ml DMEM (inositol free)-LiCl. At the end of the incubation period, medium was removed, and 1 ml 0.1 M formic acid was added to each well. Cells were then frozen and thawed to disrupt the cell membranes. 25 IP accumulation was measured by Dowex anion exchange chromatography and liquid scintillation spectroscopy, as previously described (Huckle and Conn, Methods Enzymol. 141:149-55, 1987). Briefly, 1 ml aliquots of cell lysates were applied to 0.4 ml columns of Dowex 1-X8 (200-400 mesh, formate form). Free [ 3 H]inositol was eluted with 10 bed volumes of water; labeled IPI, IP 2 , and IP 3 with 8 bed volumes each WO 2004/069859 PCT/US2004/002290 -28 of 0.2, 0.5, and 1.0 M ammonium formate in 0.1 M formic acid, respectively. Radioactivity was determined in the various Dowex fractions by liquid scintillation spectroscopy. Under these chromatographic conditions, [ 3 H]PIs are not eluted from Dowex columns, and can be identified by thin layer chromatography. 5 The IC 50 for each indole, quinoline and erythromycin macrolide was calculated as follows. Cells expressing wild-type GnRHR are incubated in the presence of 1 jig/mi of the indole, quinoline or erythromycin macrolide and in the presence of various concentrations of 125 1-buserelin (10-13 M - 10 7 M), and the amount of IP production measured as described above. The IC 50 concentration for the unlabeled indole, 10 quinoline or erythromycin macrolide in a competition experiment is the concentration required to inhibit the radiolabeled ligand ( 1 25 1-buserelin) to the specific ligand binding site of GnRHR by 50%. Although the IC 50 value can be determined many times by eye, the most accurate method for determining IC 50 values is to use non-linear regression analysis using the equation for a sigmoid plot. 15 Y = Bottom * (Top-Bottom) (1-1 0 x log 1C50) where Top is the top of the curve, Bottom is the bottom of the curve; Y is the amount bound (either as cpm or as % of control); and X is the concentration of unlabeled agent. The IC 5 o determined for each agent is shown in the FIGS. 1-3. 20 FIGS. 1 (indoles), 2 (quinolones) and 3 (erythromycin macrolides) show the efficacy (assessed by IP production) of each agent assayed for restoring function to the

E

90 I mutant. The data shown in FIGS. 1-3 are the means ± SEM from triplicate determinations. For each chemical class, the data are presented with the lowest IC 5 0 value (for the hGnRHR, shown in figures) first. The data indicate that a concentration 25 of 1 ptg/ml is, in most cases, the dose of agent that elicits an optimum response. FIGS. 4 (indoles), 5 (quinolones) and 6 (erythromycin macrolides) show the effect of the 1 pg/ml concentration of each agent in restoring function to each mutant.

WO 2004/069859 PCT/US2004/002290 -29 For reference, FIGS. 7 and 8 show the unrescued coupling of the receptor in the absence (figure 7) or presence (figure 8) of 10- M buserelin, a GnRHR agonist. The member of each drug class with the lowest affinity for the human GnRHR is oriented closest to the viewer. 5 EXAMPLE 2 GnRHR Internalization in the Presence of GnRH peptide Antagonist This example describes a method that can be used to determine an amount of GnRHR internalization in the presence of a potential therapeutic agent, such as a 10 GnRHR antagonist. Such a-method can be used to screen agents for their ability to restore function to mutant GnRHR. Cells are transfected with mutant or wild-type GnRHR as described in Example 1. In some examples, 12 well plates were used and 2 x 105 cells were plated per well, thereby allowing each plate to serve as one time point. The agent, such as a GnRHR 15 antagonist or vehicle alone is incubated as described in Example 1 for the IP assay (Example 2). Following transfection, a radioligand acid wash method (Marian et al., Mol. Pharmacol. 19:399-405, 1981; Heding et al., Endocrinology 141:299-306, 2000) is used to measure internalization of the mutant or WT human GnRHRs. This method distinguishes internalized and non-internalized receptor. Briefly, cells are washed twice 20 with 0.5 ml DMEM containing 0.1% BSA, then incubated with [ 1 25 1]-buserelin (see Example 1). At the desired time, the iodinated ligand is removed and the plate placed on ice. Cells are washed twice with 0.5 ml ice cold PBS, then 0.5 ml acid wash solution (50 mM acetic acid and 150 mM NaCl, pH 2.8) is added to each well and incubated for 12 minutes on ice. 25 To determine the surface-bound iodinated ligand, the acid wash is collected and counted on a Packard gamma counter (Downers Grove, IL). To determine the internalized radioligand-receptor complex, the cells are solubilized in 0.5 ml PBS containing 0.1% Triton-X100, collected and counted. Nonspecific binding for all conditions is determined using the same method but in the presence of 10 ptM unlabeled WO 2004/069859 PCT/US2004/002290 -30 GnRH. Nonspecific binding is subtracted from the surface-bound and internalized radioligand, and the amount of internalized radioligand is expressed as the percent internalized of the total bound at each time point. 5 EXAMPLE 3 Indole, Quinolone, and Macrolide Mimetics Also disclosed are biologically active, non-peptide organic molecules that mimic the action of the indoles, quinolones, and macrolides described in Example 1 that can restore function to a GnRHR mutant, such as N 10K, T321, E90K, Q106R, A129D, 10 R139H, C200Y, R262Q, L266R, C279Y, or Y284C. The ability of a mimetic of the indoles, quinolones, and macrolides described in Example 1 to restore function to a GnRHR mutant can be determined using the methods disclosed herein. A person of ordinary skill in the art will appreciate that certain structural changes can be made to compounds of the present disclosure, as long as such structural 15 changes do not alter the biological activity of the indoles, quinolones, and macrolides disclosed herein, that is, their ability to restore function to GnRHR mutants. For example, the indole derivative IN3 includes a number of alkyl substituents, specifically methyl groups, such as the gem dimethyl groups and the methyl group at the chiral center adjacent to the indole. These alkyl substituents may vary in length and position 20 on the molecule, and typically are selected from the group consisting of lower (such as ten carbon atoms or fewer) aliphatic groups, more particularly lower alkyl groups, including straight and branched chains, as well as all biologically active stereoisomers. Also, the methyl groups of the 3,5-dimethylbenzene ring can be varied to be other lower aliphatic groups, most likely lower alkyl groups, and the relative positioning of such 25 lower aliphatic groups can be other than 3,5. Additionally, the number of lower aliphatic groups can vary from 1-5. Moreover, IN3 includes an amide functionality, and such amide can vary from the [2.2.2] bicycloaminooctane moiety of IN3. For example, other cyclic compounds, as well as acyclic amides, may be included. Also, with reference to such amides, the WO 2004/069859 PCT/US2004/002290 -31 carbonyl oxygen can be replaced with other heteroatoms, most notably sulfur. Also the nitrogen atom of the heterocyclic amines also can vary, and potentially can be replaced with an atom selected from the group consisting of oxygen and sulfur. Furthermore, regioisomers of the oxygen, nitrogen and sulfur heterocycles can replace the 5 heterocyclic amine moieties. Thus, the indole moiety may be replaced by fused bicyclic aromatic moieties, such as benzimidazole, benzofuran, and benzothiophene derivatives. Particular aromatic heterocycles are selected from the group consisting of furan, pyrrole, thiophene, oxazole, imidazole, thiazole, quinoline, isoquinoline, pyrimidine, purine, benzofuran, benzothiophene, and derivatives thereof. The hydrogen atom 10 bonded to the nitrogen atom of the aliphatic amine also can be replaced with lower aliphatic substituents, with such substituents typically being selected from the group consisting of lower alkyl groups, with methyl groups being a likely such substituent. The substitution pattern of the pyridine derivative can be varied, for example a 2 pyridine or a 3-pyridine derivative can be used in place of the 4-pyridine moiety of IN3. 15 Additionally, the pyridine moiety can be replaced with another aromatic group, such as a five membered, six membered, or fused aromatic heterocycle. Finally, the number of methylene units spacing particular functional groups and moieties of 1N3 can be varied. For example, IN3 includes 2 methylene units that space the amine portion of IN3 from the pyridine ring. The number of methylene units may vary from about 1-10, more 20 typically from about 2-5. EXAMPLE 4 Screening Assays This example describes methods that can be used to screen agents for their 25 ability to restore functionality to a mutant receptor, such as GnRHR having one or more amino acid substitutions or deletions that lead to the development of HH in a subject. As disclosed in the Example 1, agents from several different chemical families, including indoles, quinolones, and macrolides, were able to restore function to many GnRHR mutants. Therefore, screening assays can be used to identify and analyze other WO 2004/069859 PCT/US2004/002290 - 32 agents, such as other GnRHR antagonists, for example other derivatives of indoles, quinolones, and macrolides, that can also restore function to mutant GnRHR. However, the present disclosure is not limited to the particular methods disclosed herein. Agents identified via the disclosed assays can be useful, for example, in 5 restoring function to mutant GnRHR molecules, for example in treating a subject, having HH. In addition, agents identified via the disclosed assays can be useful, for example, in increasing cell-surface expression of wild-type GnRHR, for example in treating a subject having hypogonadism. Assays for testing the effectiveness of the identified agents, are discussed below. 10 Exemplary agents that can be screened include, but are not limited to, any peptide or non-peptide composition in a purified or non-purified form, such as peptides made of D-and/or L-configuration amino acids (in, for example, the form of random peptide libraries; see Lam et al., Nature 3 54:82-4, 1991), phosphopeptides (such as in the form of random or partially degenerate, directed phosphopeptide libraries; see, for 15 example, Songyang et al., Cell 72:767-78, 1993), antibodies, and small or large organic or inorganic molecules. A test agent can also include a complex mixture or "cocktail" of molecules. Particular test agents include indole, quinolone, and macrolide derivatives and mimetics. The basic principle of the assay systems used to identify agents that restore 20 function to mutant GnRHR, increase expression of wild-type GnRHR to the cell surface, or both, involves preparing a reaction mixture containing the wild-type or mutant GnRHR protein and the agent under conditions and for a time sufficient to allow the GnRHR and agent to interact and bind and restore function to the mutant GnRHR (or increase expression of the wild-type protein on the cell surface, or both). Controls 25 are incubated without the test agent or with a placebo. Exemplary controls include agents known not to bind to or restore function to GnRHR, such as A-7662.0, Q08, and IN3 lb. The ability of the agent to restore function to the mutant GnRHR (or increase expression of the wild-type protein on the cell surface, or both) is then determined.

WO 2004/069859 PCT/US2004/002290 - 33 The ability of the agent to increase binding of a GnRHR agonist to a mutant GnRHR, increase GnRH agonist-stimulated IP production by a mutant GnRHR, increase surface-bound mutant GnRHR-ligand complex internalized, or combinations thereof, by at least a desired amount, such as an increase of at least 10%, at least 20%, 5 at least 50%, at least 100%, or even at least 200% in the presence of the agent as compared to an amount of activity in the absence of the agent, indicates that the agent can be used to restore function to a mutant GnRHR, and is therefore possibly an agent that can be used to treat subjects having HH. In addition, the ability of the agent to increase expression of the wild-type 10 protein on the cell surface by at least a desired amount, such as an increase of at least 10%, at least 20%, or even at least 50% in the presence of the agent as compared to an amount of cell-surface expression in the absence of the agent, indicates that the agent can be used to treat a subject having hypogonadism, such as secondary hypogonadism. Methods that can be used to assess these GnRHR activities are described in 15 Examples 1 and 2. Briefly, cells expressing a GnRHR protein (wild-type or mutant) are contacted with the agent. The amount of agent administered can be determined by skilled practitioners. In some examples, several different doses of the potential therapeutic agent can be administered, to identify optimal dose ranges. Following incubation with the agent, assays are conducted to determine the amount of binding of a 20 GnRHR agonist to a mutant GnRHR, the amount of GnRH agonist-stimulated IP production by a mutant GnRHR, the amount of surface-bound mutant GnRHR-ligand complex internalized, the amount of wild-type GnRHR on the cell surface, or combinations thereof, using the methods described in Examples 1 and 2. The ability of an agent, such as those identified using the methods provided 25 above, to restore function to a mutant GnRHR, to increase cell-surface expression of wild-type GnRHR, or both, can be assessed in animal models. An animal model of hypogonadism, such as HH, can be made using standard methods known in the art (for example, see U.S. Patent Nos. 6,037,521; 6,323,390; and 6,576,811, all herein incorporated by reference). Briefly, a gene-targeting vector is generated using standard WO 2004/069859 PCT/US2004/002290 - 34 molecular biology methods. The targeting vector is transfected into ES cells, for example by electroporation. Surviving clones are selected, and analyzed by Southern blot to identify homologous recombinants. Homologous recombinants are expanded, re-verified by Southern blot, and used to generate mouse chimeras by blastocyst 5 injection. The resulting chimeras are crossed, and the resulting progeny tested to identify those that are heterozygous for the mutation. Heterozygotes are crossed to generate homozygous mutants. The resulting homozygous mutant animal models can also be used to screen agents for an ability to ameliorate symptoms associated with hypogonadism. In 10 addition, such animal models can be used to determine the LD 5 o and the ED 50 in animal subjects, and such data can be used to determine the in vivo efficacy of potential agents. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, such as baboons, monkeys, and chimpanzees, can be used to generate an animal model of hypogonadism if needed. 15 The appropriate animal is inoculated with the agent identified in the examples above, alone or in combination with other therapeutic agents. The amount of agent administered can be determined by skilled practitioners. In some examples, several different doses of the potential therapeutic agent can be administered to different test subjects, to identify optimal dose ranges. Subsequent to the treatment, animals are 20 observed for the symptoms associated with hypogonadism. A decrease in the symptoms associated with hypogonadism in the presence of the agent provides evidence that the agent is a therapeutic agent that can be used to restore function to mutant GnRHR or increase expression of wild-type GnRHR, or both. 25 EXAMPLE 5 Treatment of Hypogonadism This example describes methods that can be used to increase expression of a wild-type hGnRHR on the cell surface, for example to treat, or reduce the symptoms of hypogonadism, such as erectile dysfunction, infertility, decreased sex drive, decrease in WO 2004/069859 PCT/US2004/002290 - 35 beard and growth of body hair, decrease in size or firmness of the testicles, decrease in muscle mass and increase in body fat, enlarged male breast tissue, hot flashes, mood swings, irritability, depression, fatigue, osteoporosis, delayed puberty, or combinations thereof. 5 As described in Example 1, several indoles, quinolones and macrolides increased expression of wild-type GnRHR on the surface of the cell. Therefore, use of those agents, as well as other indoles, quinolones or macrolides, such as agents identified using the methods described in Example 4, can be administered to a subject at a therapeutically effective dose, thereby relieving the symptoms associated with 10 hypogonadism (for example as sometimes found in older men and women). Such agents can also be administered with other therapeutic agents, such as testosterone and estrogen. By increasing trafficking of wild-type GnRHR to the surface, more receptors are available for ligand binding. If the agent administered is a GnRHR antagonist, in some examples the 15 antagonist is removed from the GnRHR at the cell surface, to facilitate binding of the receptor to its ligand, GnRH. Methods for removing GnRHR at the cell surface include using an antagonist that reversibly binds to the receptor, and allowing the antagonist to fall off of the receptor over time. 20 EXAMPLE 6 Treatment of Hypogonadotropic Hypogonadism (HI) This example describes methods than can be used to treat or reduce the symptoms of a disorder associated with expression of mutant GnRHR, such as hypogonadotropic hypogonadism. 25 As described in Example 1, several indoles, quinolones and macrolides increased IP production and agonist binding at the cell surface. Therefore, use of those agents, as well as agents identified using the methods described in Example 4, can be administered to a subject at a therapeutically effective dose, thereby reliving the symptoms associated with HH due to a mutant GnRHR. Such agents can also be WO 2004/069859 PCT/US2004/002290 -36 administered with other therapeutic agents, such as testosterone and hCG. By restoring at least partial function to mutant GnRHR, GnRHR is are available at the cell surface for ligand binding. If the agent administered is a GnRHR antagonist, in some examples the 5 antagonist is removed from the GnRHR at the cell surface as described in Example 5. EXAMPLE 7 Calculation of IC 50 This example describes a method that can be used to determine the inhibitory 10 concentration (IC 5 o) of an agent, such as a GnRHR antagonist. However, one skilled in the art will appreciate that other methods can be used. Agents can be tested over a range of concentrations (for example, 10~1 to 108 pLM). For example, serial 1 0-fold dilutions of each agent are prepared in a vehicle (such as DMSO) and stored on ice. Cells, such as COS-7 cells, are transfected with wild-type 15 or mutant hGnRHR as described in Example 1. Cells are transfected with about 0.01 pg - 0.05 mg DNA for inositol phosphate production, or 0.1 mg DNA for saturation binding studies. Cells expressing wild-type GnRHR are incubated in the presence of the agent, such as 1 [tg/ml, and in the presence of various concentrations of 125,I buserelin (10- M - 107M), and IP production or saturation binding measured as 20 described in Example 1. The most accurate method for determining IC 50 values is to use non-linear regression analysis, as described in Example 1. EXAMPLE 8 Pharmaceutical Compositions and Modes of Administration 25 This example provides methods and pharmaceutical compositions that can be used to administer a pharmacological chaperone (pharmacoperone) (alone or in combination with other therapeutic agents) that can restore function to mutant GnRHR, to increase expression of wild-type GnRHR at the cell surface, or both. In particular examples, the pharmacoperone is a GnRHR antagonist. Administration of such WO 2004/069859 PCT/US2004/002290 - 37 compositions to a subject can begin whenever treatment of symptoms associated with decreased binding of GnRH to its receptor, for example due to expression of a mutant GnRHR, is desired. While compositions that include a pharmacoperone may typically be used to treat human subjects, they can also be used to treat similar or identical 5 diseases in other vertebrates such as other primates, farm animals such as swine, cattle and poultry, and sport animals and pets such as horses, dogs and cats. The pharmaceutical compositions that include a pharmacoperone can be formulated in unit dosage form, suitable for individual administration of precise dosages. A therapeutically effective amount of a pharmacoperone can be administered 10 in a single dose, or in multiple doses, for example daily, during a course of treatment. Compositions that include a pharmacoperone can be administered whenever the effect (such as decreased symptoms of HH) is desired. A time-release formulation can also be utilized. A therapeutically effective amount of a composition that includes a 15 pharmacoperone can be administered as a single pulse dose, as a bolus dose, or as pulse doses administered over time. In pulse doses, a bolus administration of a composition that includes a pharmacoperone is provided, followed by a time-period wherein no pharmacoperone is administered to the subject, followed by a second bolus administration. In specific, non-limiting examples, pulse doses of compositions that 20 include a pharmacoperone are administered during the course of a day, during the course of a week, or during the course of a month. The therapeutically effective amount of a composition including a pharmacoperone can depend on the molecule utilized, the subject being treated, the severity and type of the affliction, and the manner of administration, and should be 25 decided according to the judgment of the practitioner and each subject's circumstances. Therapeutically effective amounts of compositions that include a pharmacoperone are those that restore function to a mutant GnRHR by a desired level, or that increase expression of wild-type GnRHR on the cell surface, or both. In vitro assays can be employed to identify optimal dosage ranges. Effective doses can be extrapolated from WO 2004/069859 PCT/US2004/002290 - 38 dose-response curves derived from in vitro or animal model test systems. For example, a therapeutically effective amount of a pharmacoperone can vary from about 0.001 pg per kilogram (kg) body weight to about 20 mg per kg body weight, such as about 1 pg to about 5 mg per kg body weight, such as about 2 pg to about 0.5 mg per kg body 5 weight, or about 5 pg to about 1 mg per kg body weight. The exact dose is readily determined by one of skill in the art based on the potency of the specific compound (such as fludrocortisone or a mimetic thereof) utilized, the age, weight, sex and physiological condition of the subject. The compositions or pharmaceutical compositions can be administered by any 10 route, including intravenous, intraperitoneal, subcutaneous, sublingual, transdermal, intramuscular, oral, topical, transmucosal, vaginal, nasal, rectal, by pulmonary inhalation, or combinations thereof. Compositions useful in the disclosure may conveniently be provided in the form of formulations suitable for parenteral (including intravenous, intramuscular and subcutaneous), nasal, topical, or oral administration. 15 The term "parenteral" refers to non-oral modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. In some examples, compositions that include a pharmacoperone are administered in combination with (such as before, during, or following) a 20 therapeutically effective amount of one or more other therapeutic agents, such as a steroid (for example estrogens and androgens) or other agents that alleviate symptoms associated with hypogonadism, in a single composition or solution for administration together. In other cases, it may be more advantageous to administer the additional agent separately from the pharmacoperone. Compositions that include a pharmacoperone can 25 be administered simultaneously with the additional agent(s), or administered sequentially. In one example, a composition that includes a pharmacoperone is formulated and administered with estrogen or androgen as a single dose. Therapeutic compositions can be provided as parenteral compositions, such as for injection or infusion. Such compositions are formulated generally by mixing a WO 2004/069859 PCT/US2004/002290 -39 pharmacoperone at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, for example one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. In addition, a 5 pharmacoperone can be suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate/acetic acid buffers. The active ingredient, optionally together with excipients, can also be in the form of a 10 lyophilisate and can be made into a solution prior to parenteral administration by the addition of suitable solvents. Solutions such as those that are used, for example, for parenteral administration can also be used as infusion solutions. A form of repository or "depot" slow release preparation can be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream 15 over many hours or days following transdermal injection or delivery. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. The compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, 20 as a sparingly soluble salt. Pharmacoperones can be utilized as free bases, as acid addition salts or as metal salts. The salts ideally are pharmaceutically acceptable, and include metal salts, for example alkali and alkaline earth metal salts, such as potassium or sodium salts. Numerous pharmaceutically acceptable acid addition salts are available. Such products 25 are readily prepared by procedures well known to those skilled in the art. Pharmaceutical compositions that include a pharmacoperone as an active ingredient can be formulated with an appropriate solid or liquid carrier, depending upon the particular mode of administration chosen. The product can be shaped into the desired formulation. In one example, the carrier is a parenteral carrier, such as a WO 2004/069859 PCT/US2004/002290 -40 solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, glycerol and dextrose solution. Non aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. Other carriers include, but are not limited to: fillers, such as sugars, for 5 example lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, also binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyffolidone, and/or, if desired, disintegrators, such as the above-mentioned 10 starches, also carboxymethyl starch, cross-linked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, such as Remington's Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal ofParenteral Science and Technology, Technical Report No. 15 10, Supp. 42:2S, 1988. If desired, the disclosed pharmaceutical compositions can also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Excipients that can be included in the disclosed compositions 20 include flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof. Compositions including a pharmacoperone can be administered by sustained release systems. Suitable examples of sustained-release systems include suitable 25 polymeric materials (such as, semi-permeable polymer matrices in the form of shaped articles, for example films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt). Sustained-release compositions can be administered orally, parenterally, intracistemally, intraperitoneally, WO 2004/069859 PCT/US2004/002290 -41 topically (as by powders, ointments, gels, drops or transdermal patch), or as an oral, otic, or nasal spray. Sustained-release matrices include polylactides (U.S. Patent No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolyiners 22:547-556, 1983, poly(2-hydroxyethyl methacrylate)); 5 (Langer et al., J. Biomed. Mater. Res.15:167-277, 1981; Langer, Chem. Tech. 12:98 105, 1982, ethylene vinyl acetate (Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions include liposomes containing a pharmacoperone (see generally, Langer, Science 249:1527-1533, 1990; Treat et al., in Liposomes in the 10 Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-65, 1989). Liposomes containing a pharmacoperone thereof can be prepared by known methods: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A. 82:3688-92, 1985; Hwang et al., Proc. Nati. Acad. Sci. U.S.A. 77:4030-4034, 1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; 15 Japanese Patent Application No. 83-118008; U.S. Patent No. 4,485,045, U.S. Patent No.. 4,544,545; and EP 102,324. Preparations for administration can be suitably formulated to give controlled release of a pharmacoperone. For example, the pharmaceutical compositions can be in the form of particles comprising a biodegradable polymer and/or a polysaccharide 20 jellifying and/or bioadhesive polymer, an amphiphilic polymer, an agent modifying the interface properties of the particles and a pharmacologically active substance. These compositions exhibit certain biocompatibility features that allow a controlled release of the active substance. See U.S. Patent No. 5,700,486. Compositions that include a pharmacoperone can be delivered by way of a pump 25 (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:201, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574, 1989) or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution can also be employed. One factor in selecting an appropriate dose is the result obtained, as measured by the methods disclosed here, as are deemed appropriate by the WO 2004/069859 PCT/US2004/002290 - 42 practitioner. Other controlled release systems are discussed in Langer (Science 249:1527-33, 1990). In one example, the pump is implanted (for example see U.S. Patent Nos. 6,436,091; 5,939,380; and 5,993,414). Implantable drug infusion devices are used to 5 provide patients with a constant and long-term dosage or infusion of a drug or any other therapeutic agent. Such device can be categorized as either active or passive. Active drug or programmable infusion devices feature a pump or a metering system to deliver the agent into the patient's system. An example of such an active infusion device currently available is the Medtronic SynchroMedTM programmable 10 pump. Passive infusion devices, in contrast, do not feature a pump, but rather rely upon a pressurized drug reservoir to deliver the agent of interest. An example of such a device includes the Medtronic IsoMedTM. For oral administration, the pharmaceutical compositions can take the form of, for example, powders, pills, tablets, or capsules, prepared by conventional means with 15 pharmaceutically acceptable excipients such as binding agents (such as pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (such as lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (such as magnesium stearate, talc or silica); disintegrants (such as potato starch or sodium starch glycolate); or wetting agents (such as sodium lauryl sulphate). The tablets can be 20 coated by methods well known in the art. For administration by inhalation, the compounds for use according to the present disclosure can be conveniently delivered in the fonn of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon 25 dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of for example gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

WO 2004/069859 PCT/US2004/002290 -43 For inhalation, the composition of the present disclosure can also be administered as an aerosol or a dispersion in a carrier. In one specific, non-limiting example, a pharmacoperone (alone or in combination with other therapeutic agents or pharmaceutically acceptable carriers), is administered as an aerosol from a conventional 5 valve, such as, but not limited to, a metered dose valve, through an aerosol adapter also known as an actuator. A suitable fluid carrier can be also included in the formulation, such as, but not limited to, air, a hydrocarbon, such as n-butane, propane, isopentane, amongst others, or a propellant, such as, but not limited to a fluorocarbon. Optionally, a stabilizer is also included, and/or porous particles for deep lung delivery are included 10 (for example, see U.S. Patent No. 6,447,743). In the disclosed methods of treating disorders that result from expression of mutant GnRHR or from decreased expression of wild-type GnRNR at the cell surface, the method includes administering to a subject having hypogonadism a therapeutically effective amount of a pharmacoperone identified using the methods disclosed herein, 15 such as an indole, quinolone, or macrolide. Pharmacoperones can be administered in a single or divided dose. Suitable single or divided doses include, but are not limited to about 0.01, 0.1, 0.5, 1, 3, 5, 10, 15, 30, or 50 pg/kg/day. The disclosure also provides a pharmaceutical pack or kit including one or more containers filled with one or more of the ingredients of the pharmaceutical 20 compositions. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. Instructions for use of the composition can also be included. 25 The disclosure provides compositions that include pharmacoperones, for example a composition that includes at least 50%, for example at least 90%, of a pharmacoperone in the composition. Such compositions are useful as therapeutic agents when constituted as pharmaceutical compositions with the appropriate carriers or diluents.

44 In view of the many possible embodiments to which the principles of this disclosure may be applied, it should be recognized that the illustrated embodiments are only particular examples of the disclosure and should not be taken as a limitation on the scope of the disclosure. Rather, the scope of the disclosure is in accord with the following claims. I therefore claim all that comes within the scope and spirit of these claims. It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. 2427245_1 (GHMatters)

Claims

1. A method of screening an indole, quinolone, or macrolide for an ability to restore functionality to a mutant gonadotropin-releasing hormone receptor (GnRHR), comprising: contacting the indole, quinolone, or macrolide with the mutant GnRHR under conditions that allow interaction between the indole, quinolone, or macrolide and the mutant GnRHR and restore function to the mutant GnRHR; incubating the indole, quinolone, or macrolide with the mutant GnRHR for a time sufficient to allow interaction between the indole, quinolone, or macrolide and the mutant GnRHR; and determining whether functionality of the mutant GnRHR is improved, wherein indoles, quinolones, or macrolides that improve functionality of mutant GnRHR are selected; and wherein the indole is not (2S)-2-[5-[2-(2-azabicyclo[2.2.2]oct-2-yI)-1,1 dimethyl-2-oxoethyl]-2-(3,5- dimethylphenyl)-IH-indol-3-yl]-N-(2-pyridin-4 ylethyl)propan-1 -amine (1n3).

2. The method of claim 1, wherein the mutant GnRHR is expressed recombinantly in a cell, and contacting the indole, quinolone, or macrolide with the mutant GnRHR comprises contacting the cell with the indole, quinolone, or macrolide.

3. The method of claim 2, wherein determining whether functionality was restored to the mutant GnRHR comprises determining an amount of inositol phosphate (IP) production by the cell, wherein an increase in IP production, as compared to an amount of IP production in the absence of the indole, quinolone, or macrolide indicates that the indole, quinolone, or macrolide restored functionality to the mutant GnRHR.

4. The method of claim 3, wherein following incubating the indole, quinolone, or macrolide with the cell, the method further comprises: washing the cell to remove the indole, quinolone, or macrolide; 24227571 (GHMatters) WO 2004/069859 PCT/US2004/002290 - 46 contacting the cell with [ 3 H]inositol for a time sufficient to allow entry of the [ 3 H]inositol into the cell; and contacting the cell with a GnRHR agonist for a time sufficient to allow stimulation of IP production. 5

5. The method of claim 4, wherein the GnRHR agonist is buserelin (D-tert-butyl-Ser 6 , des-Glyl, Pro 9 , ethylamide-GnRH) or leuprolide (D-Leu

6 , Pro 9 , des-Glyl-ethylamide GnRH). 10 6. The method of claim 2, wherein determining whether functionality was restored to the mutant GnRHR comprises determining an amount of GnRHR ligand binding on a surface of the cell, wherein an increase in GnRHR ligand binding to the cell surface compared to an amount of GnRHR ligand binding in the absence of the agent indicates that the agent restored functionality to the mutant GnRHR. 15

7. The method of claim 6, wherein following incubating the indole, quinolone, or macrolide with the cell, the method further comprises: washing the cell to remove the indole, quinolone, or macrolide; and contacting the cell with GnRHR ligand for a time sufficient to allow binding of 20 the ligand to GnRHR present on the cell surface.

8. The method of claim 6, wherein the GnRHR ligand comprises a label.

9. The method of claim 8, wherein the GnRHR ligand comprises 125 I-buserelin. 25

10. The method of claim 1, wherein the indole and quinolone have an IC 50 of less than 3 nM for wild-type human GnRHR. WO 2004/069859 PCT/US2004/002290 -47

11. The method of claim 1, wherein the macrolide has an IC 50 of less than 700 nM for wild-type human GnRHR.

12. The method of claim 1, wherein the macrolide is a erythromycin-derived macrolide. 5

13. The method of claim 1, wherein the mutant GnRHR is a human GnRHR comprising a N OK, T3I, E90 Q106 R, A D, R 139H, C 200 Y, Q62,L L6R, C279Y or a Y 2 84 C mutation. 10

14. The method of claim 1, wherein the mutant GnRHR is a rat GnRHR comprising a Des325-327, DesL237-L241 or a C 278 A mutation.

15. A method of screening an indole, quinolone, or macrolide for an ability to restore functionality to a mutant gonadotropin-releasing hormone receptor (GnRHR), 15 comprising: contacting the indole, quinolone, or macrolide with a cell expressing the mutant GnRHR under conditions that allow interaction between the indole, quinolone, or macrolide and the mutant GnRHR and under conditions that restore function to the mutant GnRHR; 20 incubating the indole, quinolone, or macrolide with the cell for a time sufficient to allow interaction between the indole, quinolone, or macrolide and for the mutant GnRHR to traffic to a cell surface; washing the cells to remove the indole, quinolone, or macrolide; contacting the cell with a GnRHR ligand for a time sufficient to allow binding of 25 the GnRHR ligand with GnRHR present on a surface of the cell; washing the cell to remove unbound GnRHR ligand; and quantitating GnRHR ligand binding to the cell surface, wherein an increase in labeled GnRHR ligand binding to the cell surface compared to an amount of GnRHR 48 ligand binding in the absence of the indole, quinolone, or macrolide indicates that function has been restored to the mutant GnRHR; wherein the indole is not In3.

16. A method of screening an indole or a quinolone for an ability to restore functionality to a mutant gonadotropin-releasing hormone receptor (GnRHR), comprising: contacting the indole or quinolone with a wild-type GnRHR under conditions that allow interaction between the indole or quinolone and the wild-type GnRHR, in the presence of a GnRHR agonist; and determining an IC 50 of the indole or quinolone for the wild-type GnRHR; and selecting indoles or quinolones with an IC 50 less than 3 nM for wild-type GnRHR, wherein an IC 50 less than 3 nM for wild-type GnRHR indicates that the indole or quinolone can restore functionality to the mutant GnRHR; and wherein the indole is not In3.

17. A method of screening a macrolide for an ability to restore functionality to a mutant gonadotropin-releasing hormone receptor (GnRHR), comprising: contacting the macrolide with a wild-type GnRHR under conditions that allow interaction between the macrolide and the wild-type GnRHR, in the presence of a GnRHR agonist; and determining an IC 50 of the macrolide for the wild-type GnRHR; and selecting macrolides with an IC 50 of less than 700 nM for wild-type GnRHR, wherein an IC 5 0 of less than 700 nM for wild-type GnRHR indicates that the macrolide can restore functionality to the mutant GnRHR.

18. A method of restoring function to a mutant GnRHR that is expressed in a cell but not translocated to the cell surface, comprising contacting the cell with a therapeutically effective amount of an agent that enhances translocation of the GnRHR to the cell surface, wherein the agent is not. In3.

19. Use of an agent that enhances translocation of GnRHR to a cell surface, in the manufacture of a medicament for restoring function to a mutant GnRHR that is expressed in a cell but not translocated to the cell surface, wherein the agent is not In3. 2422757_ 1 (GHMatters) 49

20. A method of increasing wild-type GnRHR expression at a cell surface, comprising contacting the cell with a therapeutically effective amount of an agent that enhances translocation of the wild-type GnRHR to the cell surface, wherein the agent is not In3.

21. Use of an agent that enhances translocation of wild-type GnRHR to a cell surface, in the manufacture of a medicament for increasing wild-type GnRHR expression at the cell surface, wherein the agent is not 1n3.

22. The method of claim 18 or 20, or use of claim 19 or 21, wherein the agent is an indole, quinolone, or macrolide.

23. The method or use of claim 22, wherein the agent is an indole antibiotic, quinolone antibiotic, or macrolide antibiotic.

24. The method of claim 23, wherein the indole, quinolone, or macrolide is Q89, (7-chloro-2-oxo-4- {2-[(2S)-piperidin-2-yl]ethoxy} -N-pyrimidin-4-yl-3-(3, 4,5 trimethylphenyl)-1,2-dihydroquinoline-6-carboxamide); Q76, (N-(7-chloro-3-(3, 5 dimethylphenyl)-2-oxo-4- {2-[(2S)-piperidin-2-yl]ethoxyl- 1,2-dihydroquinolin-6-yl) N'-cyclopropylurea); Q08, ((2S)-2-(2- {[7-chloro-6-[(6,7-dimethoxy-3,4 dihydroisoquinolin-2(I H)-yl)carbonyl]-3-(3,5-dimethylphenyl)-2-oxo- 1,2 dihydroquinolin-4-yl]oxy}ethyl)piperidinium trifluoroacetate); 1n30, ((2S)-2-[5-[2 (2- azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2- (3,5-dimethylphenyl)-lH indol-3-yl]-N- {2-[4-(methylsulfinyl)phenyl]ethyl} propan-l-amine); A-64755.0 (11 deoxy- 1 -[carboxy-phenylethylamino]-6-0-methyl-erythromycin A 11,1 2-(cyclic carbamate)); A-177775.0, (3'-N-desmethyl-3'-N-cyclopentyl- 11 -deoxy- 11 -[carboxy (3,4-dichlorophenylethylamino)]-6-0-methyl-erythromycin A 11,12-(cyclic carbamate)); A-222509.0, 3',3'-N-desmethyl-3',3'-N-cyclopropylmethyl- 11 -deoxy- 11 [carboxy-(3-chloro,4-fluoro-phenylethylamino)]-6-0-methyl-erythromycin A 11,12 (cyclic carbamate)), or a mixture thereof.

25. The method of claim 18 or 20, or use of claim 19 or 21, wherein the agent is identified using the method of claim 1. 2422757_1 (GHMatters) 50

26. The method of claim 18, wherein the mutant GnRHR is present in a subject, and contacting comprises administering the agent to the subject.

27. The method of claim 26, wherein the subject has hypogonadotropic hypogonadism (HH). i

28. The method of claim 18 or 20, wherein the agent is administered in a pharmaceutically acceptable carrier.

29. The method of claim 18 or use of claim 19, wherein restoring function to the mutant GnRHR comprises increasing binding of a GnRiRHR agonist to a mutant GnRHR, increasing GnRH agonist-stimulated IP production by a mutant GnRHR, increasing surface-bound mutant GnRHR-ligand complex internalized, or combinations thereof.

30. The method of claim 20, wherein the wild-type GnRHR is present in a subject, and contacting comprises administering the agent to the subject.

31. The method of claim 30, wherein the subject has hypogonadism.

32. The method of claim 31, wherein the subject has erectile dysfunction, infertility, decreased sex drive, decreased beard and growth of body hair, decreased testicle size or firmness, decreased muscle mass and increased body fat, enlarged male breast tissue, hot flashes, mood swings, irritability, depression, fatigue, osteoporosis, delayed puberty, or combinations thereof.

33. The method of claim 1, 15, 16, 17, 18 or 20, or use of claim 19 or 21, substantially as hereinbefore described with reference to the accompanying examples and/or figures. 2422757_ 1 (GHMatters)