AU5426596A

AU5426596A - Modulators for new members of the steroid/thyroid superfamily of receptors

Info

Publication number: AU5426596A
Application number: AU54265/96A
Authority: AU
Inventors: Ronald M Evans; Barry M Forman
Original assignee: Salk Institute for Biological Studies
Current assignee: Salk Institute for Biological Studies
Priority date: 1995-05-16
Filing date: 1996-04-17
Publication date: 1996-11-29
Also published as: WO1996036230A1

Description

Modulators for New Members of the Steroid/Thyroid Superfamily of Receptors

FIELD OF THE INVENTION

The present invention relates to intracellular receptors, and modulators therefor. In a particular aspect, the present invention relates to methods for the identification of compounds which function as modulators (or precursors thereof) for specific members of the intracellular receptor family. In other aspects, the present invention relates to various uses for the compounds so identified.

BACKGROUND OF THE INVENTION

A central problem in eukaryotic molecular biology continues to be the elucidation of molecules and mechanisms that mediate specific gene regulation. As part of the scientific attack on this problem, a great deal of work has been done in efforts to identify modulators (i.e., endogenous or exogenous inducers and/or repressors) which are capable of mediating specific gene regulation.

Although much remains to be learned about the specifics of gene regulation, it is known that ligands modulate gene transcription by acting in concert with intracellular components, including intracellular receptors and discrete DNA sequences known as hormone response elements (HREs) .

As additional members of the steroid/thyroid superfamily of receptors are identified, the search for endogenous or exogenous inducers and/or repressors for such newly discovered receptors has become an important part of the effort to learn about the specifics of gene regulation. The identification of compounds which directly or indirectly interact with intracellular receptors, and thereby affect transcription of hormone-responsive genes, would be of significant value, e.g., for therapeutic applications.

Additional novel intracellular receptors (i.e., members of the steroid/thyroid superfamily of receptors) continue to be identified. Frequently, however, the primary ligand(s) for these novel receptors can not readily be identified. Accordingly, the identification of ligands and/or modulators for such receptors is of great value.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, we have identified modulators for orphan member(s) of the steroid/thyroid superfamily of receptors which is related to the previously described constitutively active receptor- alpha (CAR-α; also known as "MB-67," see Baes et al., in Mol. and Cell. Biology 11:1544-1552 (1994)). Thus, compounds of the general class of androstans have been identified as modulators for a newly discovered isoform of CAR. Compounds discovered in accordance with the present invention can be employed in a variety of applications, e.g., for the modulation of processes mediated by an isoform of CAR or CAR-like species, to increase the libido of a subject (especially a subject undergoing therapy using a 5α-reductase inhibitor) , in a screening assay for the presence of receptors involved in the modulation of libido, and the like.

Also provided in accordance with the present invention are methods for the identification of compounds which modulate processes mediated by an isoform of CAR or

CAR-like species, as well as novel compositions derived therefrom. BRIEF DESCRIPTION OF THE FIGURE

Figure 1 illustrates the suppression of an isoform of CAR by 5 -androstane derivatives.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided methods for modulating the activity of a CAR or CAR-like isoform, said method comprising administering an effective amount of a steroid-like compound having the structure I, as set forth below:

wherein:

R = R = O; or R = hydrogen and

R 2 is α-OR, wherein R is selected from hydrogen, lower alkyl, acyl or trimethylsilyl;

R 3 and R4 are each independently hydrogen or lower alkyl;

R = R = O; or R and R are both hydrogen; or R is absent when there is a double bond between C and C ; X, Y, Z and A are each independently selected from hydroxy, alkoxy (of a lower alkyl group) , mercapto (of a lower alkyl group) , halogen, trifluoromethyl, cyano, nitro, amino, carboxyl, carbamate, sulfonyl, or sulfonamide; a falls in the range of 0 up to 4; b falls in the range of 0 up to 4; c falls in the range of 0 up to 4; and d falls in the range of 0 up to 3.

As employed herein, the phrase "CAR or CAR-like isoform" refers to a member of the steroid/thyroid superfamily of receptors which is optionally constitutively active, and has at least 75 % overall amino acid identity (up to 86 % sequence similarity) with the receptor set forth in SEQ ID N0:1 (CAR-α) , at least 88 % amino acid identity (up to 91 % sequence similarity) in the DNA binding domain thereof, with respect to the DNA binding domain of the receptor set forth in SEQ ID N0:1, and at least 74 % amino acid identity (up to 87 % sequence similarity) in the ligand binding domain thereof, with respect to the ligand binding domain of the receptor set forth in SEQ ID N0:1.

As employed herein, the phrase "modulating the activity of a CAR or CAR-like isoform" refers to the ability of a modulator (e.g., a ligand or precursor thereof) for an isoform of CAR or a CAR-like species to induce expression of gene(s) maintained under hormone expression control, or to repress expression of gene(s) maintained under such control.

As employed herein, the phrase "processes mediated by an isoform of CAR or a CAR-like species" refers to biological, physiological, endocrinological, and other bodily processes which are mediated by receptor or receptor combinations which are responsive to natural or synthetic androstans. Modulation of such processes can be accomplished in vitro or in vivo . In vivo modulation can be carried out in a wide range of subjects, such as, for example, humans, rodents, sheep, pigs, cows, and the like.

As employed herein, "lower alkyl" refers to straight or branched chain alkyl groups having in the range of about 1 up to 4 carbon atoms; "alkyl" refers to straight or branched chain alkyl groups having in the range of about 1 up to 12 carbon atoms; "substituted alkyl" refers to alkyl groups further bearing one or more substituents such as hydroxy, alkoxy (of a lower alkyl group) , mercapto (of a lower alkyl group) , aryl, carboxyl, heterocyclic, halogen, trifluoro ethyl, cyano, nitro, amino, carboxyl, carbamate, sulfonyl, sulfonamide, and the like.

As employed herein, "acyl" refers to alkyl- carbonyl groups.

Presently preferred compounds employed in the practice of the present invention include those wherein R 1 of structure I is hydrogen and R 2 i.s α-OR (wherei.n R i.s as defined above, with R = hydrogen or acyl being especially preferred) ; compounds wherein R of structure I is methyl; compounds wherein R 4 of structure I is methyl; compounds according to structure I wherein R = R = 0; compounds wherein R and R of structure I are both hydrogen; compounds wherein R of structure I is absent, and there is a double bond between C 16 and C17, and the like.

In accordance with another embodiment of the present invention, there are provided methods for the identification of compounds which modulate the activity of a CAR or CAR-like isoform (as defined herein) , said method comprising: contacting host cell(s) containing receptor- encoded DNA and a suitable hormone response element linked to reporter-encoded DNA with test compound, and determining the effect of test compound on the level of expression of said reporter.

Optionally, the receptor-encoded DNA employed in the practice of the present invention will also encode one or more exogenous transactivation domains, such as, for example, the T. or τ₂ transactivation domains described in United States Patent No. 5,217,867, which is incorporated by reference herein in its entirety.

Those of skill in the art can readily determine suitable response elements for use in the practice of the present invention, such as, for example, the response elements described in United States Patent No. 5,091,518 and PCT published application no. WO 92/16546, both of which are hereby incorporated by reference herein.

Identification methods according to the present invention involve the use of a functional bioassay system, wherein the CAR or CAR-like isoform (as defined herein) and a reporter plasmid are cultured in suitable host cells in the presence of test compound. Evidence of transcription (e.g., expression) of reporter gene is then monitored to determine the presence of an activated receptor-ligand complex. Accordingly, the functional bioassay system utilizes two plasmids: an "expression" plasmid and a "reporter" plasmid. The expression plasmid can be any plasmid which contains and is capable of expressing DNA encoding the CAR or CAR-like isoform receptor protein, in a suitable host cell. The reporter plasmid can be any plasmid which contains an operative hormone response element functionally linked to an operative reporter gene.

Exemplary reporter genes include chloramphenicol transferase (CAT) , luciferase (LUC) , beta-galactosidase (?-gal) , and the like. Exemplary promoters include the simian virus (SV) promoter or modified form thereof (e.g., ΔSV) , the thymidine kinase (TK) promoter, the mammary tumor virus (MTV) promoter or modified form thereof (e.g., ΔMTV) , and the like [see, for example, Mangelsdorf et al., in Nature _34_5:224-229 (1990), Mangelsdorf et al., in Cell 6.6:555-561 (1991), and Berger et al., in J. Steroid Biochem. Molec. Biol. £1:733-738 (1992)]. The plasmids pGMCAT, pGHCAT, and the like, are examples of reporter plasmids which contain an operative hormone responsive promoter/enhancer element functionally linked to an operative reporter gene, and can therefore be used in the above-described functional bioassay (see Example 1 for details on the preparation of these plasmids) . In pGMCAT, the operative hormone responsive promoter/enhancer element is the MTV LTR; in pGHCAT it is the functional portion of the growth hormone promoter. In both pGMCAT and GHCAT the operative reporter gene is the bacterial gene for chloramphenicol acetyltransferase (CAT) .

As used herein in the phrase "operative response element functionally linked to an operative reporter gene", the word "operative" means that the respective DNA sequences (represented by the terms "hormone response element" and "reporter gene") are operational, i.e., work for their intended purposes; the word "functionally" means that after the two segments are linked, upon appropriate activation by a ligand-receptor complex, the reporter gene will be expressed as the result of the fact that the "hormone response element" was "turned on" or otherwise activated.

In practicing the above-described functional bioassay, the expression plasmid and the reporter plasmid are co-transfected into suitable host cells. The transfected host cells are then cultured in the presence and absence of a test compound to determine if the test compound is able to produce activation of the promoter operatively linked to the hormone response element of the reporter plasmid. Thereafter, the transfected and cultured host cells are monitored for induction (i.e., the presence) of the product of the reporter gene sequence.

Cells contemplated for use in the practice of the present invention include transformed cells, non- transformed cells, neoplastic cells, primary cultures of different cell types, and the like. Exemplary cells which can be employed in the practice of the present invention include liver cell lines (e.g., Hep-G2) , primary hepatocytes, adipocyte or pre-adipocyte cell lines (e.g., 3T3-L1 cells, 3T3-442-A cells, OB17 cells, and the like) , as well as CV-1 cells, HuTuδO cells, F9 cells, NTERA2 cells, NB4 cells, HL-60 cells, 293 cells, Hela cells, NIH-3T3 cells, and the like. Preferred host cells for use in the functional bioassay system are COS cells and CV-1 cells. COS-1 (referred to as COS) cells are monkey kidney cells that express SV40 T antigen (Tag) ; while CV-1 cells do not express SV40 Tag. The presence of Tag in the COS-1 derivative lines allows the introduced expression plasmid to replicate and provides a relative increase in the amount of receptor produced during the assay period. CV-1 cells are presently preferred because they are particularly convenient for gene transfer studies and provide a sensitive and well-described host cell system.

The above-described cells (or fractions thereof) are maintained under physiological conditions when contacted with physiologically active compound. "Physiological conditions" are readily understood by those of skill in the art to comprise an isotonic, aqueous nutrient medium at a temperature of about 37°C.

In accordance with yet another embodiment of the present invention, there is provided a method to increase the libido of a subject, said method comprising inhibiting the activity of CAR or CAR-like isoforms (as defined above) . In a particular aspect the above-described method to increase libido can be carried out by administering to a subject a libido-enhancing amount of a steroid-like compound having the structure I, as described herein.

Since individual subjects may present a wide variation in severity of symptoms and each drug has its unique therapeutic characteristics, the precise mode of administration and dosage employed for each subject is left to the discretion of the practitioner.

In accordance with still another embodiment of the present invention, there are provided physiologically active composition(s) comprising a compound having the structure I, as described herein, in a suitable vehicle rendering said compound amenable to oral delivery, transdermal delivery, intravenous delivery, intramuscular delivery, topical delivery, nasal delivery, and the like.

Pharmaceutical compositions of the present invention can be used in the form of a solid, a solution, an emulsion, a dispersion, a micelle, a liposome, and the like, wherein the resulting composition contains one or more of the compounds of the present invention, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The carriers which can be used include glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. The active compound (i.e., compounds of structure I as described herein) is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of diseases.

Pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of a sweetening agent such as sucrose, lactose, or saccharin, flavoring agents such as peppermint, oil of wintergreen or cherry, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients may also be manufactured by known methods. The excipients used may be, for example, (1) inert diluents such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents such as corn starch, potato starch or alginic acid; (3) binding agents such as gum tragacanth, corn starch, gelatin or acacia, and (4) lubricating agents such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl onostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in the U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874, to form osmotic therapeutic tablets for controlled release.

In some cases, formulations for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.

The pharmaceutical compositions may be in the form of a sterile injectable suspension. This suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides, fatty acids (including oleic acid) , naturally occurring vegetable oils like sesame oil, coconut oil, peanut oil, cottonseed oil, etc. , or synthetic fatty vehicles like ethyl oleate or the like. Buffers, preservatives, antioxidants, and the like can be incorporated as required.

Compounds contemplated for use in the practice of the present invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters of polyethylene glycols, which are solid at ordinary temperatures, but liquify and/or dissolve in the rectal cavity to release the drug. Since individual subjects may present a wide variation in severity of symptoms and each drug has its unique therapeutic characteristics, it is up to the practitioner to determine a subject's response to treatment and vary the dosages accordingly.

Typical daily doses, in general, lie within the range of from about 0.5 μg to about 10 mg per kg body weight, and, preferably within the range of from 50 μg to 1 mg per kg body weight and can be administered up to four times daily. The daily IV dose lies within the range of from about 1 μg to about 10 mg per kg body weight, and, preferably, within the range of from 10 μg to 500 μg per kg body weight.

In an alternate aspect of this embodiment of the present invention, compositions useful for ameliorating the libido-reducing effects of a 5α-reductase inhibitor are provided. Such compositions comprise a libido-enhancing amount of a steroid-like compound having the structure I, as described herein, and a 5α-reductase inhibitor.

Those of skill in the art can readily identify

5α-reductase inhibitors suitable for use in the practice of the present invention. An example of a 5α-reductase inhibitors contemplated for use in the practice of the present invention is finasteride (PROSCAR) .

In accordance with yet another embodiment of the present invention, there is provided a method for ameliorating the libido-reducing effects of a 5α-reductase inhibitor, said method comprising co-administering, to a subject being treated with 5α-reductase inhibitor(s) , a libido-enhancing amount of a steroid-like compound having the structure I, as described herein.

In accordance with a still further embodiment of the present invention, there is provided a method of screening cells or cell extracts to determine the presence of receptors involved in the modulation of libido, said method comprising

contacting cells or cell extracts with a compound having the structure I, as described herein, and thereafter

identifying those cells or cell extracts which bind said compound.

The invention will now be described in greater detail by reference to the following non-limiting examples.

Example 1 Preparation of reporter constructs

Various reporter constructs are used in the examples which follow. They are prepared as follows:

TK-LUC: The MTV-LTR promoter sequence is removed from the MTV-LUC plasmid described by Hollenberg and Evans in Cell 55:899-906 (1988) by tfindlll and Xhol digest, and cloned with the Hindlll-X ol fragment of the Herpes simplex virus thymidine kinase gene promoter (-105 to +51 with respect to the transcription start site, m, isolated from plasmid pBLCAT2, described by Luckow & Schutz in Nucleic Acids Res. 33:5490 (1987)) to generate parental construct TK-LUC. pTK-^RARE._{2 j}-LUC: One, two or three copies of double-stranded beta-retinoic acid response element (βRKRE) oligonucleotides, comprising a direct repeat of two half sites separated by a spacer of five nucleotides, wherein each half site comprises the sequence

N_χ-RGBNNM- , wherein

R is selected from A or G; B is selected from G, C, or T; each N is independently selected from

A, T, C, or G;

M is selected from A or C; and x falls in the range of 0 up to 5; with the proviso that at least 4 nucleotides of said -RGBNNM- sequence are identical with the nucleotides at corresponding positions of the sequence -AGGTCA-, is cloned upstream of the TK promoter of TK-LUC at the Hindlll site.

Alternatively, response elements having a similar structure to that set forth above, except having a spacer of only four nucleotides, can be used. Thus, response elements comprising a direct repeat of two half sites separated by a spacer of four nucleotides, wherein each half site comprises the sequence N_χ-RGBNNM-, as described above, can be used in place of the βRARE described above.

CMX-?GAL: The coding sequence for the E. coli ?-galactosidase gene is isolated from plasmid pCHllO [see Hall et al., J. Mol. Appl. Genet. 2:101-109 (1983)] by iϊindlll and BamEI digest, and cloned into pCMX eucaryotic expression vector [see Umesono et al., supra] . 15

Example 2 Screening for CAR or CAR-like isoforms

A. With PCR-qenerated probe

A probe spanning the DNA-binding domain of the CAR-encoding DNA described by Baes et al. (Mol. and Cell.

Biol. 14.:1544-1552 (1994); i.e., nucleic acid residues 303 to 545 of SEQ ID N0:1) is prepared by PCR. The probe is labeled by the random-primer labeling method or by PCR using 32P nucleotides. The labeled probe is then used to probe a lambda-gtll mammalian liver cDNA library (e.g., mouse liver cDNA library or other readily available library, such as are commercially available from Clontech or Stratagene) to identify related receptors. The hybridization mixture contains 35% formamide, IX Denhart's, 5X SSPE (IX SSPE = 0.15 M NaCl, lOmM Na₂HP0₄ ImM EDTA) , 0.1% SDS, 10% dextran sulfate, 100 μg/ml denatured salmon sperm DNA and 10⁶ cpm of [³²P]-labelled probe. Duplicate nitrocellulose filters are hybridized for 16h at 42°C, washed once at 25°C for 15 min with 2X SSC (IX SSC=0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS and then washed twice at 55°C for 30 min. in 2X SSC, 0.1% SDS. The filters are autoradiographed for 3 days at -70°C using an intensifying screen.

After several rounds of screening, several positive clones are obtained. Sequence analysis of at least one of the positive clones indicates that this clone encodes a novel member of the steroid/thyroid superfamily of receptors, having approximately 75 % overall amino acid identity with the receptor set forth in SEQ ID N0:1, approximately 88 % amino acid identity in the DNA binding domain thereof, with respect to the DNA binding domain of the receptor set forth in SEQ ID N0:1, and approximately 74 % amino acid identity in the ligand binding domain thereof, with respect to the ligand binding domain of the receptor set forth in SEQ ID N0:1.

If the initial clone isolated is a partial clone, then an insert of the above-identified positive clone (labeled with ³²P) is also used as a probe to rescreen the same library or additional library(ies) . Hybridization conditions for such rescreening comprise a hybridization mixture containing 50% formamide, IX Denhart's, 5X SSPE, 0.1% SDS, 100 μg/ml denatured salmon sperm DNA and 10 cpm of [³²P]-labelled probe. Duplicate nitrocellulose filters are hybridized for 16h at 42°C, washed once at 60°C for 15 min with 0.1X SSC (IX SSC = 0.15 M NaCl, 0.015 M sodium citrate), 0.1% SDS and then washed twice at 60°C for 30 min. in 0.1X SSC, 0.1% SDS. The filters are autoradiographed for 3 days at -70°C using an intensifying screen.

After several rounds of screening, several positive clones are obtained.

B. With synthetic oli onucleotides

A lambda-gtll mammalian liver cDNA library is screened in duplicate with a 32P-labeled synthetic oligonucleotide:

TGYGARGGNT GYAARGGNTC TTT (SEQ ID NO:3), under low-stringency conditions (i.e., 1M NaCl/0.05mM Tris-HCl, pH 8.0/5mM EDTA/150 units of heparin per ml/0.05%, sodium pyrophosphate/100 μg of yeast RNA per ml/0.1% (wt/vol) NaDodS0₄ at 46°C) and washed at high stringency, as described by Burglin et al., in Nature 241:239-243 (1989). In the above oligonucleotide, Y is selected from C or T, R is selected from A or G, and N is any one of A, G, C or T. Thus, the oligonucleotide employed is a mixture of all possible DNA sequences encoding the amino acid sequence: CEGCKGFF (SEQ ID NO:4), wherein each letter above is the conventional single letter abbreviation for amino acid residues, i.e., C is cysteine,

E is glutamic acid, G is glycine, K is lysine and F is phenylalanine.

Example 3 Screening assay for modulators of CAR or CAR-like isoforms

CV-1 cells are co-transfected with a vector encoding the CAR isoform isolated as described in Example 2 (incorporated into a CMV-driven expression vector) , and pTK-?RARE-LUC at a ratio of about 100 ng of receptor- encoding DNA per 10 cells. The usual amounts of DNA per 10⁵ cells are 100 ng of CDM8-CAR, 300 ng of pTK-ffRARE-LUC, and 500 ng of CMX-?GAL. Typically, transfections are performed in triplicate. The plates are then incubated for 2-3 hours at 37°C.

The cells are washed with fresh medium. Fresh medium containing one concentration of a serial dilution of agonist is added to each well. A typical agonist dilution series extends from 10 -5M through 10-11M. A solvent control is performed for each agonist. The cells are incubated at 37°C for 1-2 days.

The cells are rinsed twice with buffered saline solution. Subsequently, cells are lysed, in situ , by adding 200 μl of lysis buffer. After 30 minutes incubation at room temperature, 40 μl aliquots of cell lysate are transferred to 96-well plates for luciferase reporter gene assays and -galactosidase transfection controls [see Heyman et al.. Cell 8:397-406 (1992)].

The data are expressed as relative light units

(RLUs) per O.D. unit of ?-galactosidase per minute. The triplicates are averaged for each concentration and plotted as normalized RLUs against the dose of agonist or as fold induction vs the dose of agonist. The results of testing with a variety of different compounds are presented in the following table:

Compound Quantity Relative light units

Vitamin K1/K2 @ 2.5 μM 7.6 reverse triiodothyronine 5 μM 8.8

Anhydro-retinol 50 μM 6.8

14-OH-retroretinol 1.4 μM 8.0

Taurocholic acid + @ 200 μM 5.8 Taurodeoxycholic acid

Dehydroepiandrostenone 50 μM 5.9

Vitamin E 50 μM 6.8

This example demonstrates the androstans (such as androstenol) are effective at reducing the constitutive activity of the CAR isoform employed herein.

The selectivity of a modulator for a particular receptor can be measured by comparing the activation/repression of that receptor with the activation/repression of some other related receptor with the same modulator.

Example 4

Dose response of CAR or CAR-like isoforms to modulators therefor

Effector plasmid, reporter plasmid, and yff-galactosidase control plasmid are co-transfected into CV-1 cells at a ratio of about 1:3:5, using a liposome- mediated method, employing N-{l-(2,3-dioleoyloxy)propyl- N,N,N-trimethy1 ammonium methyl sulfate} (i.e., D0TAP (Boehringer Manheim) according to manufacturer's instructions in Dulbecco's modified Eagle's medium (DMEM) with 10% delipidated hormone-depleted fetal calf serum. After about 2-3 hours, the cells are washed twice with fresh DMEM and test compound is added to the media to the final molar concentration indicated in Figure 1. After 24-48 hours of incubation, the media is removed and the cells are lysed. Aliquots are assayed for luciferase and yff-galactosidase activity. Luciferase activity is normalized to optical density units of yβ-galactosidase per minute of incubation.

The data are expressed in Figure 1 as the normalized response to solvent or test compound, relative to induction of the same construct incubated in solvent alone.

Review of Figure 1 reveals that the androstans

(such as androstenol, androstenol-3-acetate, 5α-androstan-3α-ol, and the like) are effective at suppressing the constitutive activity of CAR or CAR-like isoforms, with androstenol and 5α-androstan-3α-ol being the presently preferred androstans for use in the practice of the present invention.

While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Evans, Ronald M. Forman, Barry M.

(ii) TITLE OF INVENTION: MODULATORS FOR NEW MEMBERS OF THE STEROID/THYROID SUPERFAMILY OF RECEPTORS

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Pretty, Schroeder, Brueggemann & Clark

(B) STREET: 444 South Flower Street, Suite 2000

(C) CITY: Los Angeles

(D) STATE: CA

(E) COUNTRY: USA

(F) ZIP: 90071

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/442,464

(B) FILING DATE: 16-MAY-1995

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Reiter, Stephen E.

(B) REGISTRATION NUMBER: 31,192

(C) REFERENCE/DOCKET NUMBER: P41 9881

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 619-546-4737

(B) TELEFAX: 619-546-9392

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1450 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: both

(D) TOPOLOGY: both

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 273..1319

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

GTGAGCTTGC TCCTTAAGTT ACAGGAACTC TCCTTATAAT AGACACTTCA TTTTCCTAGT 60

CCATCCCTCA TGAAAAATGA CTGACCACTG CTGGGCAGCA GGAGGGATGA TAATCCTAAC 120

TCCAATCACT GGCAACTCCT GAGATCAGAG GAAAACCAGC AACAGCGTGG GAGTTTGGGG 180

AGAGGCATTC CATACCAGAT TCTGTGGCCT GCAGGTGACA TGCTGCCTAA GAGAAGCAGG 240 AGTCTGTGAC AGCCACCCCA ACACGTGACG TC ATG GCC AGT AGG GAA GAT GAG 293

Met Ala Ser Arg Glu Asp Glu 1 5

CTG AGG AAC TGT GTG GTA TGT GGG GAC CAA GCC ACA GGC TAC CAC TTT 341 Leu Arg Asn Cys Val Val Cys Gly Asp Gin Ala Thr Gly Tyr His Phe 10 15 20

AAT GCG CTG ACT TGT GAG GGC TGC AAG GGT TTC TTC AGG AGA ACA GTC 389 Asn Ala Leu Thr Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Thr Val 25 30 35

AGC AAA AGC ATT GGT CCC ACC TGC CCC TTT GCT GGA AGC TGT GAA GTC 437 Ser Lys Ser lie Gly Pro Thr Cys Pro Phe Ala Gly Ser Cys Glu Val 40 45 50 55

AGC AAG ACT CAG AGG CGC CAC TGC CCA GCC TGC AGG TTG CAG AAG TGC 485 Ser Lys Thr Gin Arg Arg His Cys Pro Ala Cys Arg Leu Gin Lys Cys 60 65 70

TTA GAT GCT GGC ATG AGG AAA GAC ATG ATA CTG TCG GCA GAA GCC CTG 533 Leu Asp Ala Gly Met Arg Lys Asp Met lie Leu Ser Ala Glu Ala Leu 75 80 85

GCA TTG CGG CGA GCA AAG CAG GCC CAG CGG CGG GCA CAG CAA ACA CCT 581 Ala Leu Arg Arg Ala Lys Gin Ala Gin Arg Arg Ala Gin Gin Thr Pro 90 95 100

GTG CAA CTG AGT AAG GAG CAA GAA GAG CTG ATC CGG ACA CTC CTG GGG 629 Val Gin Leu Ser Lys Glu Gin Glu Glu Leu lie Arg Thr Leu Leu Gly 105 110 115

GCC CAC ACC CGC CAC ATG GGC ACC ATG TTT GAA CAG TTT GTG CAG TTT 677 Ala His Thr Arg His Met Gly Thr Met Phe Glu Gin Phe Val Gin Phe 120 125 130 135

AGG CCT CCA GCT CAT CTG TTC ATC CAT CAC CAG CCC TTG CCC ACC CTG 725 Arg Pro Pro Ala His Leu Phe lie His His Gin Pro Leu Pro Thr Leu 140 145 150

GCC CCT GTG CTG CCT CTG GTC ACA CAC TTC GCA GAC ATC AAC ACT TTC 773 Ala Pro Val Leu Pro Leu Val Thr His Phe Ala Asp lie Asn Thr Phe 155 160 165

ATG GTA CTG CAA GTC ATC AAG TTT ACT AAG GAC CTG CCC GTC TTC CGT 821 Met Val Leu Gin Val lie Lys Phe Thr Lys Asp Leu Pro Val Phe Arg 170 175 180

TCC CTG CCC ATT GAA GAC CAG ATC TCC CTT CTC AAG GGA GCA GCT GTG 869 Ser Leu Pro lie Glu Asp Gin lie Ser Leu Leu Lys Gly Ala Ala Val 185 190 195

GAA ATC TGT CAC ATC GTA CTC AAT ACC ACT TTC TGT CTC CAA ACA CAA 917 Glu lie Cys His lie Val Leu Asn Thr Thr Phe Cys Leu Gin Thr Gin 200 205 210 215

AAC TTC CTC TGC GGG CCT CTT CGC TAC ACA ATT GAA GAT GGA GCC CGT 965 Asn Phe Leu Cys Gly Pro Leu Arg Tyr Thr lie Glu Asp Gly Ala Arg 220 225 230

GTG GGG TTC CAG GTA GAG TTT TTG GAG TTG CTC TTT CAC TTC CAT GGA 1013 Val Gly Phe Gin Val Glu Phe Leu Glu Leu Leu Phe His Phe His Gly 235 240 245

ACA CTA CGA AAA CTG CAG CTC CAA GAG CCT GAG TAT GTG CTC TTG GCT 1061 Thr Leu Arg Lys Leu Gin Leu Gin Glu Pro Glu Tyr Val Leu Leu Ala 250 255 260 GCC ATG GCC CTC TTC TCT CCT GAC CGA CCT GGA GTT ACC CAG AGA GAT 1109 Ala Met Ala Leu Phe Ser Pro Asp Arg Pro Gly Val Thr Gin Arg Asp 265 270 275

GAG ATT GAT CAG CTG CAA GAG GAG ATG GCA CTG ACT CTG CAA AGC TAC 1157 Glu lie Asp Gin Leu Gin Glu Glu Met Ala Leu Thr Leu Gin Ser Tyr 280 285 290 295

ATC AAG GGC CAG CAG CGA AGG CCC CGG GAT CGG TTT CTG TAT GCG AAG 1205 lie Lys Gly Gin Gin Arg Arg Pro Arg Asp Arg Phe Leu Tyr Ala Lys 300 305 310

TTG CTA GGC CTG CTG GCT GAG CTC CGG AGC ATT AAT GAG GCC TAC GGG 1253 Leu Leu Gly Leu Leu Ala Glu Leu Arg Ser lie Asn Glu Ala Tyr Gly 315 320 325

TAC CAA ATC CAG CAC ATC CAG GGC CTG TCT GCC ATG ATG CCG CTG CTC 1301 Tyr Gin lie Gin His He Gin Gly Leu Ser Ala Met Met Pro Leu Leu 330 335 340

CAG GAG ATC TGC AGC TGAGGCCATG CTCACTTCCT TCCCCAGCTC ACCTGGAACA 1356 Gin Glu He Cys Ser 345

CCCTGGATAC ACTGGAGTGG GAAAATGCTG GGACCAAAGA TTGGGCCGGG TTCAAAGGGA 1416

GCCCAGTGGT TGCAATGAAA GACTAAAGCA AAAC 1450

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 348 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ala Ser Arg Glu Asp Glu Leu Arg Asn Cys Val Val Cys Gly Asp 1 5 10 15

Gin Ala Thr Gly Tyr His Phe Asn Ala Leu Thr Cys Glu Gly Cys Lys 20 25 30

Gly Phe Phe Arg Arg Thr Val Ser Lys Ser He Gly Pro Thr Cys Pro 35 40 45

Phe Ala Gly Ser Cys Glu Val Ser Lys Thr Gin Arg Arg His Cys Pro 50 55 60

Ala Cys Arg Leu Gin Lys Cys Leu Asp Ala Gly Met Arg Lys Asp Met 65 70 75 80

He Leu Ser Ala Glu Ala Leu Ala Leu Arg Arg Ala Lys Gin Ala Gin 85 90 95

Arg Arg Ala Gin Gin Thr Pro Val Gin Leu Ser Lys Glu Gin Glu Glu 100 105 110

Leu He Arg Thr Leu Leu Gly Ala His Thr Arg His Met Gly Thr Met 115 120 125

Phe Glu Gin Phe Val Gin Phe Arg Pro Pro Ala His Leu Phe He His 130 135 140 His Gin Pro Leu Pro Thr Leu Ala Pro Val Leu Pro Leu Val Thr His 145 150 155 160

Phe Ala Asp He Asn Thr Phe Met Val Leu Gin Val He Lys Phe Thr 165 170 175

Lys Asp Leu Pro Val Phe Arg Ser Leu Pro He Glu Asp Gin He Ser 180 185 190

Leu Leu Lys Gly Ala Ala Val Glu He Cys His He Val Leu Asn Thr 195 200 205

Thr Phe Cys Leu Gin Thr Gin Asn Phe Leu Cys Gly Pro Leu Arg Tyr 210 215 220

Thr He Glu Asp Gly Ala Arg Val Gly Phe Gin Val Glu Phe Leu Glu 225 230 235 240

Leu Leu Phe His Phe His Gly Thr Leu Arg Lys Leu Gin Leu Gin Glu 245 250 255

Pro Glu Tyr Val Leu Leu Ala Ala Met Ala Leu Phe Ser Pro Asp Arg 260 265 270

Pro Gly Val Thr Gin Arg Asp Glu He Asp Gin Leu Gin Glu Glu Met 275 280 285

Ala Leu Thr Leu Gin Ser Tyr He Lys Gly Gin Gin Arg Arg Pro Arg 290 295 300

Asp Arg Phe Leu Tyr Ala Lys Leu Leu Gly Leu Leu Ala Glu Leu Arg 305 310 315 320

Ser He Asn Glu Ala Tyr Gly Tyr Gin He Gin His He Gin Gly Leu 325 330 335

Ser Ala Met Met Pro Leu Leu Gin Glu He Cys Ser 340 345

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGYGARGGNT GYAARGGNTC TTT 23 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:4:

Cys Glu Gly Cys Lys Gly Phe Phe 1 5

Claims

That which is claimed is:

1. A method for modulating the activity of an isoform of CAR or a CAR-like species, said method comprising administering an effective amount of a steroid¬ like compound having the structure:

wherein:

R = R = 0; or R = hydrogen and R is α-0R, wherein R is selected from hydrogen, lower alkyl, acyl or tri ethylsilyl;

R 3 and R4 are each independently hydrogen or lower alkyl;

R = R = 0; or R and R are both hydrogen; or R is absent when there is a double bond between C and C ;

X, Y, Z and A are each independently selected from hydroxy, alkoxy (of a lower alkyl group) , mercapto (of a lower alkyl group) , halogen, trifluoromethyl, cyano, nitro, amino, carboxyl, carbamate, sulfonyl, sulfonamide; a falls in the range of 0 up to 4; b falls in the range of 0 up to 4; c falls in the range of 0 up to 4; and d falls in the range of 0 up to 3.

2. A method according to claim 1 wherein said isoform of CAR or CAR-like species is a member of the steroid/thyroid superfamily of receptors having at least 75 % overall amino acid homology with the receptor set forth in SEQ ID N0:1 (CAR-α), at least 88 % amino acid homology in the DNA binding domain thereof, with respect to the DNA binding domain of the receptor set forth in SEQ ID N0:1, and at least 74 % amino acid homology in the ligand binding domain thereof, with respect to the ligand binding domain of the receptor set forth in SEQ ID NO:l.

3. A method according to claim 1 wherein said isoform of CAR or a CAR-like species has at least 75 % overall amino acid identity with the receptor set forth in SEQ ID N0:1 (CAR-α) , at least 88 % amino acid identity in the DNA binding domain thereof, with respect to the DNA binding domain of the receptor set forth in SEQ ID NO:l, and at least 74 % amino acid identity in the ligand binding domain thereof, with respect to the ligand binding domain of the receptor set forth in SEQ ID N0:1.

4. A method according to claim 1 wherein said member has at least 86 % overall amino acid similarity with the receptor set forth in SEQ ID N0:1 (CAR-α) , at least 91 % amino acid similarity in the DNA binding domain thereof, with respect to the DNA binding domain of the receptor set forth in SEQ ID N0:1, and at least 87 % amino acid similarity in the ligand binding domain thereof, with respect to the ligand binding domain of the receptor set forth in SEQ ID N0:1.

5. A method according to claim 1 wherein R is hydrogen and R is α-OR, wherein R is as defined above.

6. A method according to claim 5 wherein R is hydrogen or acyl.

7. A method according to claim 1 wherein R is methyl.

8. A method according to claim 1 wherein R 4 is methyl.

method according to claim 1 wherein

R⁵ = R⁶ = 0.

10. A method according to claim 1 wherein R and R are both hydrogen.

11. A method according to claim 1 wherein R is absent, and there is a double bond between C and C .

12. A method for the identification of compounds which modulate the activity of an isoform of CAR or a CAR-like species, said method comprising: contacting host cell(s) containing receptor- encoded DNA and a suitable hormone response element linked to reporter-encoded DNA with test compound, and determining the effect of test compound on the level of expression of said reporter.

13. A method according to claim 12 wherein said isoform of CAR or a CAR-like species is a member of the steroid/thyroid superfamily of receptors having at least 75 % overall amino acid homology with the receptor set forth in SEQ ID NO:l (CAR-α), at least 88 % amino acid homology in the DNA binding domain thereof, with respect to the DNA binding domain of the receptor set forth in SEQ ID NO:l, and at least 74 % amino acid homology in the ligand binding domain thereof, with respect to the ligand binding domain of the receptor set forth in SEQ ID NO:l.

14. A method according to claim 12 wherein said response element is a direct repeat of two or more half sites separated by a spacer of four or five nucleotides, wherein each half site comprises the sequence N_χ-RGBNNM-, wherein

R is selected from A or G; B is selected from G, C, or T; each N is independently selected from A, T, C, or G;

M is selected from A or C; and x falls in the range of 0 up to 5; with the proviso that at least 4 nucleotides of said -RGBNNM- sequence are identical with the nucleotides at corresponding positions of the sequence -AGGTCA-.

15. A method to increase the libido of a subject, said method comprising modulating the activity of an isoform of CAR or a CAR-like species.

16. A method according to claim 15 wherein said isoform of CAR or a CAR-like species is a member of the steroid/thyroid superfamily of receptors having at least 75 % overall amino acid homology with the receptor set forth in SEQ ID NO:l (CAR-α), at least 88 % amino acid homology in the DNA binding domain thereof, with respect to the DNA binding domain of the receptor set forth in SEQ ID NO:l, and at least 74 % amino acid homology in the ligand binding domain thereof, with respect to the ligand binding domain of the receptor set forth in SEQ ID NO:l.

17. A method to increase the libido of a subject, said method comprising administering to said subject a libido-enhancing amount of a steroid-like compound having the structure I as follows:

wherein:

R ,1 = R = 0; or R ,1 = hydrogen and

R 2 is α-OR, wherei.n R is selected from hydrogen, lower alkyl, acyl or trimethylsilyl;

R 3 and R4 are each independently hydrogen or lower alkyl;

R = R = O; or R and R are both hydrogen; or R is absent when there is a double bond between C and C ;

X, Y, Z and A are each independently selected from hydroxy, alkoxy (of a lower alkyl group) , mercapto (of a lower alkyl group) , halogen, trifluoro ethyl, cyano, nitro, amino, carboxyl, carbamate, sulfonyl, sulfonamide; a falls in the range of 0 up to 4; b falls in the range of 0 up to 4; c falls in the range of 0 up to 4; and d falls in the range of 0 up to 3.

18. A physiologically active composition comprising a compound having the structure I as follows:

wherein:

R = R = O; or R = hydrogen and

R is αα--OORR,, wwhheerreeiinn RR iiss sseelleecctteeed from hydrogen, lower alkyl, acyl or trimethylsilyl;

R 3 andI RR⁴4 aarree each independently hydrogen or lower alkyl;

R = R t == oO;; or R and R are both hydrogen; or R is absent when there is a double bond

_Λb_et.ween C,-,16 and, C„17;

X, Y, Z and A are each independently selected from hydroxy, alkoxy (of a lower alkyl group) , mercapto (of a lower alkyl group) , halogen, trifluoromethyl, cyano, nitro, amino, carboxyl, carbamate, sulfonyl, sulfonamide; a falls in the range of 0 up to 4; b falls in the range of 0 up to 4; c falls in the range of 0 up to 4; and d falls in the range of 0 up to 3

in a suitable vehicle rendering said compound amenable to oral, transdermal or nasal delivery.

19. A method for ameliorating the libido- reducing effects of a 5α-reductase inhibitor, said method comprising co-administering, to a subject being treated with 5α-reductase inhibitor(s) , a libido-enhancing amount of a steroid-like compound having the structure I as follows:

wherein:

R = R = O; or R = hydrogen and

R 2 is α-OR, wherei.n R i.s selected from hydrogen, lower alkyl, acyl or trimethylsilyl;

R 3 and R4 are each independently hydrogen or lower alkyl;

R = R = O; or R and R are both hydrogen; or R is absent when there is a double bond between C¹⁶ and C¹⁷;

X, Y, Z and A are each independently selected from hydroxy, alkoxy (of a lower alkyl group) , ercapto (of a lower alkyl group) , halogen, trifluoro ethyl, cyano, nitro, amino, carboxyl, carbamate, sulfonyl, sulfonamide; a falls in the range of 0 up to 4; b falls in the range of 0 up to 4; c falls in the range of 0 up to 4; and d falls in the range of 0 up to 3.

20. A method according to claim 19 wherein said 5α-reductase inhibitor is finasteride (PROSCAR) .

21. A composition comprising a 5α-reductase inhibitor and a libido-enhancing amount of a steroid-like compound having the structure I as follows:

I

wherein: R¹ = R² = 0; or R¹ = hydrogen and

R is α-OR, wherein R is selected from hydrogen, lower alkyl, acyl or trimethylsilyl;

R 3 and R4 are each independently hydrogen or lower alkyl; R = R = 0; or R and R are both hydrogen; or R is absent when there is a double bond between C and C ;

22. A composition according to claim 21 wherein said 5α-reductase inhibitor is finasteride (PROSCAR) .

23. Method of screening cells or cell extracts to determine the presence of receptors involved in the modulation of libido, said method comprising

contacting cells or cell extracts with a compound having the structure I as follows:

wherein:

R 1 = R2 = 0; or R1 = hydrogen and R is α-OR, wherein R is selected from hydrogen, lower alkyl, acyl or trimethylsilyl; R 3 and RA are each independently hydrogen or lower alkyl; R = R = O; or R and R⁶ are both hydrogen; or R is absent when there is a double bond between C and C ; X, Y, Z and A are each independently selected from hydroxy, alkoxy (of a lower alkyl group) , mercapto (of a lower alkyl group) , halogen, trifluoromethyl, cyano, nitro, amino, carboxyl, carba ate, sulfonyl, sulfonamide; a falls in the range of 0 up to 4; b falls in the range of 0 up to 4; c falls in the range of 0 up to 4; and d falls in the range of 0 up to 3,

and thereafter

identifying those cells or cell extracts which bind said compound.