Influence of domain interactions on conformational mobility of the progesterone receptor detected by hydrogen/deuterium exchange mass spectrometry.

Structural and functional details of the N-terminal activation function 1 (AF1) of most nuclear receptors are poorly understood due to the highly dynamic intrinsically disordered nature of this domain. A hydrogen/deuterium exchange (HDX) mass-spectrometry-based investigation of TATA box-binding protein (TBP) interaction with various domains of progesterone receptor (PR) demonstrate that agonist-bound PR interaction with TBP via AF1 impacts the mobility of the C-terminal AF2. Results from HDX and other biophysical studies involving agonist- and antagonist-bound full-length PR and isolated PR domains reveal the molecular mechanism underlying synergistic transcriptional activation mediated by AF1 and AF2, dominance of PR-B isoform over PR-A, and the necessity of AF2 for full AF1-mediated transcriptional activity. These results provide a comprehensive picture elaborating the underlying mechanism of PR-TBP interactions as a model for studying nuclear receptor (NR)-transcription factor functional interactions.

Progesterone decreases levels of the adhesion protein E-cadherin and promotes invasiveness of steroid receptor positive breast cancers.

Progestins are reported to increase the risk of invasive breast cancers in postmenopausal women receiving hormone therapy with estrogen plus progestin. We report here that estrogen and progesterone receptor positive (ER+PR+) rat mammary tumors arising in the presence of estrogen and progesterone exhibit increased invasiveness and decreased expression of E-cadherin protein compared with tumors growing in the presence of estrogen alone. A similar decrease of E-cadherin expression was observed in human ER+PR+ invasive ductal carcinoma compared with ductal carcinoma in situ. In agreement with findings in the rat, estrogen plus progestin R5020 treatment decreased E-cadherin expression in vitro in T47D human breast cancer cells. Decrease of E-cadherin protein was mediated by progesterone receptor B (PRB) and dependent on the activation of the Wnt pathway. These results suggest that progesterone signaling via PRB contributes to tumor invasiveness and can provide an important therapeutic target for treatment of invasive ER+PR+ breast cancers.

GATA2 is expressed at critical times in the mouse uterus during pregnancy.

In mammals, such as mouse and human, timely production of the progesterone receptor (PR) in the proper uterine compartments is critical for preparing the uterus for the initiation and maintenance of pregnancy. Developmentally, the expression of GATA2, a member of the six member zinc-finger family of transcription factors, has been shown to be necessary for multiple non-related tissues, such as the hematopoietic system, adipose maturation and the urogential system. We recently identified Gata2 as a potential progesterone target gene in the mouse uterus; however, the expression of the GATA genes in the mouse uterus during pregnancy has not been demonstrated. In the present study, we examined the expression of GATA2 protein during the phases of pregnancy, including early pregnancy where progesterone (P4) signaling is critical in order to facilitate the window of receptivity for embryo implantation and during the decidualization of the uterine stroma, a process of cellular proliferation and differentiation which is necessary for maintenance of the invading embryo until placentation occurs. Here, we report that GATA2 protein is expressed in the uterine luminal and glandular epithelium pre-implantation, spatio-temporally co-localizing with that of the PR. Additionally, GATA2 continues to be expressed in the decidualized stroma throughout early pregnancy indicating a role in the maintenance of decidual cells. Based on these findings, we conclude that GATA2 is expressed during critical phases of early pregnancy, similar to that of the PR, and that it may play a major role in mediating P4 signaling in the mouse uterus.

Imaging progesterone receptor in breast tumors: synthesis and receptor binding affinity of fluoroalkyl-substituted analogues of tanaproget.

The progesterone receptor (PR) is estrogen regulated, and PR levels in breast tumors can be used to predict the success of endocrine therapies targeting the estrogen receptor (ER). Tanaproget is a nonsteroidal progestin agonist with very high PR binding affinity and excellent in vivo potency. When appropriately radiolabeled, it might be used to image PR-positive breast tumors noninvasively by positron emission tomography (PET). We describe the synthesis and PR binding affinities of a series of fluoroalkyl-substituted 6-aryl-1,4-dihydrobenzo[d][1,3]oxazine-2-thiones, analogues of Tanaproget. Some of these compounds have subnanomolar binding affinities, higher than that of either Tanaproget itself or the high affinity PR ligand R5020. Structure-binding affinity relationships can be rationalized by molecular modeling of ligand complexes with PR, and the enantioselectivity of binding has been predicted. These compounds are being further evaluated as potential diagnostic PET imaging agents for breast cancer, and enantiomerically pure materials of defined stereochemistry are being prepared.

Preclinical and clinical properties of trimegestone: a potent and selective progestin.

Trimegestone (TMG) is a novel, 19-norpregnane progestin with potent and selective properties. In preclinical studies, TMG has been shown to provide high endometrial selectivity. Further, TMG has high affinity and selectivity for the progesterone receptor and lacks the agonist effects of other steroid hormones. In clinical studies, TMG has been shown to have high endometrial safety and an improved bleeding profile along with improved tolerability compared with other progestins. In addition, TMG also does not impede the beneficial effects of estrogen, especially on bone, and does not compromise quality of life. The preclinical findings of lack of mineralocorticoid activity of TMG were supported in clinical findings, with neutral effect on body weight. Similarly, the smaller effect of TMG on the GABA-ergic (gamma-aminobutyric acid) system in preclinical studies is consistent with the improvement of central nervous system-related effects on depressed mood and sleep quality in clinical studies. Low-dose estradiol/TMG regimens provide rapid relief from menopausal symptoms, reducing the number and severity of hot flushes as effectively as 2 mg 17beta-estradiol/1 mg norethisterone acetate. Therefore, it may be concluded that TMG provides a clinically proven option in hormone therapy for both clinicians and patients.

Progesterone receptors: their localization, binding activity and expression in the pig oviduct during follicular and luteal phases.

Estrogens (E) and progesterone (P) are known to require their respective steroid receptors in order to exert structural and functional effects on the oviduct. Cyclic changes in progesterone receptor (PR) localization in the oviductal tissue of female pigs were determined using an immunohistochemical technique with mouse monoclonal antibody mPRI against PR. The variations observed during the estrous cycle in the progesterone receptor (PR) intensity and proportion between ampulla and isthmus probably reflect different response of these regions to progesterone. Immediately before ovulation, during follicular phase, no staining was observed in either the ampulla or the isthmus stroma. However, a low expression of PR in the epithelium of the ampulla was observed. After ovulation, during luteal phase, PR immunostaining was more intense in the whole oviduct. According to immunohistochemical assays, the binding assays for nuclear and cytosolic PR (PRn and PRc, respectively), by using [3H] R5020 at 4 degrees C for 15 h, also showed a higher specific binding during luteal phase. However, the PR mRNA in the oviduct, analyzed by RT-PCR, showed similar levels at both stages of the estrous cycle. Although this methods could not be quantitative, indicate the possibility that a post-transcriptional control could differentially regulate the PR in the pig oviduct.

Expression of the progesterone receptor and progesterone- metabolising enzymes in the female and male human kidney.

Due to high binding affinity of progesterone to the human mineralocorticoid receptor (hMR), progesterone competes with the natural ligand aldosterone. In order to analyse how homeostasis can be maintained by mineralocorticoid function of aldosterone at the MR, especially in the presence of elevated progesterone concentrations during the luteal phase and pregnancy, we investigated protective mechanisms such as the decrease of free progesterone by additional binding sites and progesterone metabolism in renal cells. As a prerequisite for sequestration of progesterone by binding to the human progesterone receptor (hPR) we demonstrated the existence of hPR expression in female and male kidney cortex and medulla at the level of transcription and translation. We identified hPR RNA by sequencing the RT-PCR product and characterised the receptor by ligand binding and scatchard plot analysis. The localisation of renal hPR was shown predominantly in individual epithelial cells of distal tubules by immunohistology, and the isoform hPR-B was detected by Western blot analysis. As a precondition for renal progesterone metabolism, we investigated the expression of steroid-metabolising enzymes for conversion of progesterone to metabolites with lower affinity to the hMR. We identified the enzyme 17alpha-hydroxylase for renal 17alpha-hydroxylation of progesterone. For 20alpha-reduction, different hydroxysteroid dehydrogenases (HSDs) such as 20alpha-HSD, 17beta-HSD type 5 (3alpha-HSD type 2) and 3alpha-HSD type 3 were found. Further, we detected the expression of 3beta-HSD type 2 for 3beta-reduction, 5alpha-reductase (Red) type 1 for 5alpha-reduction, and 5beta-Red for 5beta-reduction of progesterone in the human kidney. Therefore metabolism of progesterone and/or binding to hPR could reduce competition with aldosterone at the MR and enable the mineralocorticoid function.

New antiprogestins with partial agonist activity: potential selective progesterone receptor modulators (SPRMs) and probes for receptor- and coregulator-induced changes in progesterone receptor induction properties.

A pharmacologically relevant property of steroid hormone-regulated gene induction is the partial agonist activity of antisteroid complexes. We now report that dexamethasone-mesylate (Dex-Mes) and dexamethasone-oxetanone (Dex-Ox), each a derivative of the glucocorticoid-selective steroid dexamethasone (Dex), are two new antiprogestins with significant amounts of agonist activity with both the A and B isoforms of progesterone receptor (PR), for different progesterone-responsive elements, and in several cell lines. These compounds continue to display activity under conditions where another partial antiprogestin (RTI-020) is inactive. These new antiprogestins were used to determine whether the partial agonist activity of PR complexes can be modified by changing concentrations of receptor or coregulator, as we have recently demonstrated for glucocorticoid receptors (GRs). Because GR and coregulator concentrations simultaneously altered the position of the physiologically relevant dose-response curve, and associated EC(50), of GR-agonist complexes, we also examined this phenomenon with PR. We find that elevated PR or transcriptional intermediary factor 2 (TIF2) concentrations increase the partial agonist activity of Dex-Mes and Dex-Ox, and the EC(50) of agonists, independently of changes in total gene transactivation. Furthermore, the corepressors SMRT (silencing mediator for retinoid and thyroid receptors) and NCoR (nuclear receptor corepressor) each suppresses gene induction but NCoR acts opposite to SMRT and, like the coactivator TIF2, reduces the EC(50) and increases the partial agonist activity of antiprogestins. These comparable responses of GR and PR suggest that variations in receptor and coregulator concentrations may be a general mechanism for altering the induction properties of other steroid receptors. Finally, the magnitude of coregulator effects on PR induction properties are often not identical for agonists and the new antagonists, suggesting subtle mechanistic differences. These properties of Dex-Mes and Dex-Ox, plus the sensitivity of their activity to cellular differences in PR and coregulator concentrations, make these steroids potential new SPRMs (selective progesterone receptor modulators) that should prove useful as probes of PR induction properties.

In vitro characterization of trimegestone: a new potent and selective progestin.

Trimegestone (TMG) is a novel 19-norpregnane progestin under development for hormone replacement therapy and oral contraception. The objective of the current study was to characterize the potency and steroid receptor selectivity of TMG in several in vitro assays and to compare its activity to that of medroxyprogesterone acetate (MPA). TMG and MPA had a similar competitive binding affinity for human and rabbit progesterone receptor (PR). However, TMG had a significantly higher affinity for rat PR (IC(50) = 3.3 nM) than MPA (IC(50) = 53.3 nM). In T47D cells, both compounds increased alkaline phosphatase activity and cell proliferation with comparable potencies (EC(50s) of 0.1 nM and of 0.02 nM, respectively). To further characterize the progestational activity and steroid receptor selectivity, we established an immortalized human endometrial stromal cell line (HESC-T). This cell line lacks endogenous estrogen receptor (ER) and PR but does have functional glucocorticoid receptors (GR). When ER is transiently expressed in the cells, 17beta-estradiol (E(2)) induces PR, allowing the study of PR-regulated genes. In HESC-T cells expressing exogenous ER, and therefore PR, both TMG and MPA increased HRE-tk-luciferase activity tenfold with an EC(50) of 0.2 nM. In HESC-T cells without exogenous ER, and therefore no PR, TMG did not induce HRE-tk-luciferase activity, whereas MPA induced the reporter activity with an EC(50) of about 10 nM. This MPA-induced reporter activity is believed to be mediated through GR. The steroid receptor selectivity of TMG was further evaluated using the HRE-tk-luciferase assay in the human lung carcinoma cell line A549, which contains GR but no PR. In these cells TMG had no effect on luciferase activity, whereas MPA increased the reporter activity in a dose-dependent manner with an EC(50) of approximately 30 nM. Furthermore, HRE-tk-luciferase assay in mouse fibroblast cell line L929, which expresses androgen receptor (AR) but no PR, showed that TMG had weak antiandrogenic activity whereas MPA had androgenic activity. In summary, data from several in vitro assays demonstrate that TMG is a potent progestin with a better receptor selectivity profile than MPA.

Residues in the ligand binding domain that confer progestin or glucocorticoid specificity and modulate the receptor transactivation capacity.

To localize regions conferring ligand binding specificity of the human glucocorticoid (hGR) and progesterone (hPR) receptors, we constructed chimeras comprising the DNA-binding domain of the yeast transcription factor GAL4, linked to the ligand binding domain of hGR or hPR. Replacement of a sequence of hGR encompassing helices H6 and H7 with the homologous sequence from hPR creates a chimeric protein GP3, which binds the progestin RU 27987 with high affinity, and results in a concomitant loss of glucocorticoid binding [dexamethasone (DEX), RU 43044]. Moreover, GP3 is not able to mediate RU 27987-induced transactivation. A detailed mutational analysis of this sequence and the study of the recently solved hPR crystal structure revealed five residues that confer progestin responsiveness to GR or modulate ligand binding and transcriptional activation. Notably, the simultaneous presence of residues Ser637 and Phe639 on GP3, lining the ligand binding pocket, is specifically involved in RU 27987 binding. The absence of residues Asp641, Gln642, and Leu647 on GP3 is accountable for the lack of glucocorticoids binding on GP3. Unlike residue 642, residues 641 and 647 are not in direct contact with the ligand and most likely act through steric-mediated interactions. The presence of Gln642 and Leu647 are determinant for transcriptional activation in response to DEX and RU 27987, respectively. DEX-dependent transactivation is further enhanced by the presence of Leu647.

Evidence for progesterone receptors in the human fetoplacental vascular tree.

The presence of progesterone receptors (PR) throughout the human term fetoplacental vascular tree was investigated. By reverse transcription-polymerase chain reaction (RT-PCR), we showed expression of PR mRNAs in stem villi vessels, chorionic arteries and veins, and umbilical arteries and veins. Binding studies and Scatchard analysis revealed a single class of high-affinity binding sites for (3)H-R5020 (promegestone) in cytosolic extracts of all placental vessels, with K(d) values in the range of 2.5-4 nM. High levels of PR were detected in placental vessels compared to other vascular tissues. Thus, maximum binding capacities of stem villi vessels, chorionic arteries and veins, and umbilical arteries and veins were 247 +/- 25, 377 +/- 58, 295 +/- 40, 371 +/- 118, and 672 +/- 144 fmol/mg protein, respectively. Endothelial cell elimination in chorionic arteries did not significantly modify the number of PR. RT-PCR and binding studies also assessed PR expression in cultured placental vascular smooth muscle cells isolated from stem villi vessels. All these data suggested that most of the PR of fetoplacental vessels were from the media. In conclusion, we report here the first evidence of the presence of PR in the muscular layer of human term fetoplacental vessels. This finding, together with the high progesterone concentrations in cord blood, suggests that the interactions between the PR and its ligand may play a role in the physiology and physiopathology of human fetoplacental vascularization.

The pharmacological profile of a novel norpregnance progestin (trimegestone).

Trimegestone is a novel norpregnane progestin that is being developed, in combination with estradiol, for the treatment of menopausal symptoms. The pharmacological characteristics of trimegestone have been evaluated in both in vitro and in vivo test systems, and compared with reference progestins. Interaction with hormonal steroid receptors from animal tissues and with human recombinant receptors in vitro has demonstrated that trimegestone has a high specificity and potency for the progesterone receptor, no affinity for the estrogen receptor, and weak affinity for androgen, glucocorticoid and mineralocorticoid receptors. With respect to progestomimetic activity in vivo, trimegestone was more potent than reference progestins in the endometrial transformation test in the rabbit, preventing the uterotrophic effect of estradiol in the immature mouse bioassay, and had more effect on traumatic deciduoma formation and greater oral antiovulatory activity in the rat. In vivo, trimegestone effectively maintained pregnancy in the rat, but was devoid of any uterotrophic activity. Trimegestone showed no androgenic, glucocorticoid, antiglucocorticoid or mineralocorticoid activity, but did show some antiandrogenic and antimineralocorticoid activity at higher doses. Administration of trimegestone to ovariectomized rats, in combination with estradiol, inhibited the uterotrophic effects of estradiol. At doses up to 1 mg/kg intravenously and 30 mg/kg orally, trimegestone was not associated with any unwanted pharmacological effects. Overall, the results show trimegestone to have a favorable pharmacological profile with potent progestomimetic activity.

Effect of estrogen agonists and antagonists on induction of progesterone receptor in a rat hypothalamic cell line.

Estrogen is essential in the hypothalamus for the central regulation of reproduction. To understand the molecular mechanism(s) of estrogen action in the hypothalamus, immortalized rat embryonic hypothalamic cell lines were characterized for steroid receptors and subcloned. Scatchard analysis of the D12 subclone demonstrated one high affinity estrogen receptor-binding site (Kd = 31.3+/-1.9 pM) with a Bmax of 30.8+/-0.8 fmol/mg. Estrogen receptor-alpha protein was identified by Western blot and gel shift analyses. Treatment with estradiol (48 h) stimulated progesterone receptor (PR) messenger RNA expression and binding to [3H]R5020, a synthetic progestin. Because the agonist or antagonist activity of estrogen mimetics can be cell type dependent, the activities of various estrogen mimetics were determined in D12 cells. ICI 182,780 (IC50 = 0.63 nM), raloxifene (IC50 = 1 nM), enclomiphene (IC50 = 77 nM), and tamoxifen (IC50 = 174 nM) inhibited the induction of PR by estradiol, and none of these compounds significantly stimulated PR when given alone. In contrast, 17alpha-ethynyl estradiol (EC50 = 0.014 nM), zuclomiphene (EC50 = 100 nM), and genistein (EC50 = 17.5 nM) functioned as estrogen agonists in these cells. In addition, the estrogen-induced progesterone receptor activated a progesterone response element reporter construct in response to progestins. Thus, the D12 rat hypothalamic cell line provides a useful model for characterizing tissue-selective estrogenic compounds, identifying estrogen- and progesterone-regulated hypothalamic genes, and understanding the molecular mechanisms of steroid action in various physiological processes mediated by the hypothalamus.

Hormone-dependent interaction between the amino- and carboxyl-terminal domains of progesterone receptor in vitro and in vivo.

Full transcriptional activation by steroid hormone receptors requires functional synergy between two transcriptional activation domains (AF) located in the amino (AF-1) and carboxyl (AF-2) terminal regions. One possible mechanism for achieving this functional synergy is a physical intramolecular association between amino (N-) and carboxyl (C-) domains of the receptor. Human progesterone receptor (PR) is expressed in two forms that have distinct functional activities: full-length PR-B and the amino-terminally truncated PR-A. PR-B is generally a stronger activator than PR-A, whereas under certain conditions PR-A can act as a repressor in trans of other steroid receptors. We have analyzed whether separately expressed N- (PR-A and PR-B) and C-domains [hinge plus ligand-binding domain (hLBD)] of PR can functionally interact within cells by mammalian two-hybrid assay and whether this involves direct protein contact as determined in vitro with purified expressed domains of PR. A hormone agonist-dependent interaction between N-domains and the hLBD was observed functionally by mammalian two-hybrid assay and by direct protein-protein interaction assay in vitro. With both experimental approaches, N-C domain interactions were not induced by the progestin antagonist RU486. However, in the presence of the progestin agonist R5020, the N-domain of PR-B interacted more efficiently with the hLBD than the N-domain of PR-A. Coexpression of steroid receptor coactivator-1 (SRC-1) and the CREB binding protein (CBP), enhanced functional interaction between N- and C-domains by mammalian two-hybrid assay. However, addition of SRC-1 and CBP in vitro had no influence on direct interaction between purified N- and C-domains. These results suggest that the interaction between N- and C-domains of PR is direct and requires a hormone agonist-induced conformational change in the LBD that is not allowed by antagonists. Additionally, coactivators are not required for physical association between the N- and C-domains but are capable of enhancing a functionally productive interaction. In addition, the more efficient interaction of the hLBD with the N-domain of PR-B, compared with that of PR-A, suggests that distinct interactions between N- and C-terminal regions contribute to functional differences between PR-A and PR-B.

3,3'4,4'-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells.

3,3',4,4'-Tetrachlorobiphenyl (tetraCB) binds to the aryl hydrocarbon receptor (AhR), and several reports have demonstrated that AhR agonists exhibit antiestrogenic and antitumorigenic activities in human breast cancer cells, the rodent uterus and breast. In contrast, a recent study showed that 3,3',4,4'-tetraCB bound the estrogen receptor (ER) and exhibited ER agonist activities, and we therefore have reinvestigated the estrogenic and antiestrogenic activities of 3,3',4,4'-tetraCB. Our results showed that 3,3',4,4'tetraCB and a structurally related analog, 3,3',4,4',5-pentaCB, did not bind the mouse uterine or human ER, did not induce proliferation of MCF-7 or T47D human breast cancer cells or induce reporter gene activity in cells transfected with E2-responsive constructs derived from the creatine kinase B (pCKB) or cathepsin D (pCD) gene promoters. Moreover, 3,3',4,4'-tetraCB and 3,3',4,4',5-pentaCB did not induce an increase in uterine wet weight, peroxidase activity or progesterone receptor binding in the 21-25-day-old female B6C3F1 mouse uterus. In contrast, both compounds inhibited 17beta-estradiol (E2)-induced cell proliferation and transactivation in MCF-7/T47D cells and uterine responses in B6C3F1 mice; surprisingly inhibition of E2-induced reporter gene activity was not observed in T47D cells transfected with pCKB, and this was observed as a cell-specific response with other AhR agonists. Additionally, 3,3',4,4'-tetraCB significantly inhibited mammary tumor growth in female Sprague-Dawley rats initiated with 7,12-dimethylbenzanthracene. Our results indicate that 3,3',4,4'-tetraCB does not exhibit ER agonist activity but exhibits a broad spectrum of antiestrogenic responses consistent with ligand-mediated AhR-ER crosstalk.

The steroid promegestone is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor that interacts with the lipid-protein interface.

17,21-Dimethyl-19-nor-pregn-4,9-diene-3,20-dione (promegestone) was used to characterize the mechanism of inhibition of nicotinic acetylcholine (ACh) receptors (AChR) by progestin steroids. Promegestone reversibly inhibited ACh-induced currents of Torpedo AChRs expressed in Xenopus oocytes. Between 1-30 microM promegestone produced a concentration-dependent enhancement of the equilibrium binding affinity of [3H]ACh to Torpedo AChR-rich membranes. For AChRs in the presence of agonist (desensitized state) promegestone was a more potent inhibitor of the binding of the noncompetitive antagonist [3H]phencyclidine (IC50 = 9 microM) than of [3H]histrionicotoxin (IC50 approximately 100 microM). To identify AChR domains in contact with the steroid, AChR-rich membranes equilibrated with [3H]promegestone were irradiated at 312 nm, and 3H-labeled amino acids were identified by amino-terminal sequencing of fragments isolated from subunit proteolytic digests. Within AChR alpha-subunit, 70% of 3H was covalently incorporated in a 10-kDa fragment beginning at Asn-339 and containing the M4 membrane spanning segment, and 30% was in a 20-kDa fragment beginning at Ser-173 and containing the M1-M3 segments. Fragments containing the M2 channel domains as well as the M4 segments were isolated from proteolytic digests of AChR subunits and subjected to amino-terminal sequence analysis. No evidence of [3H]promegestone incorporation was detected in any of the M2 segments. The amino acids in the M4 segments labeled by [3H]promegestone were among those previously shown to be in contact with the lipid bilayer (). These results indicate that the steroid promegestone is an AChR noncompetitive antagonist that may alter AChR function by interactions at the lipid-protein interface.

Microbial models of drug metabolism: microbial transformations of Trimegestone (RU27987), a 3-keto-delta(4,9(10))-19-norsteroid drug.

Screening microorganisms for the biotransformation of the 3-keto-delta(4,9(10))-19-norsteroid RU27987 (Trimegestone) resulted in the isolation of nine identified metabolites, some of them being selectively produced by different strains. Eight metabolites were found to be hydroxylated on various positions of the rings, and one was additionally epoxidized. These microbial metabolites could be used as chromatographic standards and two of them were found identical to the unknown major human metabolites. Moreover, most microbial metabolites were produced in sufficient amounts to be tested for their biological activities. All these features demonstrate the usefulness and versatility of microbial biotransformation systems as a tool for early identification and convenient production of potentially active mammalian and non-mammalian metabolites.

Agonist and antagonists induce homodimerization and mixed ligand heterodimerization of human progesterone receptors in vivo by a mammalian two-hybrid assay.

This study utilizes the mammalian two-hybrid system to examine the role of ligand in the dimerization of human progesterone receptor (hPR). The GAL4 DNA-binding domain and the herpes simplex virus VP16 transactivation domain were fused to the amino terminus of full-length hPR (both the A and B isoforms) to produce chimeric proteins. PR dimerization was detected by the ability of cotransfected GAL4/PR and VP16/PR chimeras in COS cells to induce expression of a reporter gene under the control of GAL4-binding sites (pG5CAT). Hormone agonist-dependent interactions were observed between the two like isoforms of PR (A-A and B-B) and between PR-A and PR-B (A-B), indicating that hormone can stimulate the formation of the three possible dimeric forms of PR within cells. In contrast, neither type I (ZK98299) nor type II (RU486, ZK112993) progestin antagonists stimulated interaction between these same hybrid PR proteins. However, activation of the VP16/PR chimera by antagonists on a progesterone response element-controlled reporter gene (DHRE-E1b-CAT) was only a fraction (4-13%) of that stimulated by agonist R5020. One possibility for the failure to detect an induction in the two-hybrid assay is antagonist-induced repression of the activity of the VP16/PR fusion protein rather than a failure of antagonists to stimulate interaction between the hybrid proteins. To test this idea, an UP-1 carboxyl-terminal truncation mutant of PR was used to construct the two-hybrid proteins. PR-UP-1 selectively binds antagonists, but not agonists, and is fully activated in response to antagonists. Both types of progestin antagonists stimulated interactions between GAL4/PR(UP-1) and VP16/PR(UP-1) hybrid proteins, indicating that antagonists are capable of stimulating PR dimerization in cells and do not function by disrupting or preventing dimerization. To determine whether PR bound to an antagonist can dimerize in whole cells with PR bound to agonist, GAL4/PR(UP-1) was paired in the two-hybrid assay with a VP16/PR fusion protein harboring a point mutation in PR at amino acid 722 (Gly-Cys) that specifically binds progestin agonist but not antagonist. Neither R5020 nor RU486 alone stimulated interaction between these ligand-specific PR hybrid proteins. However, strong interaction was detected by addition of both agonist and antagonists, indicating the formation of mixed ligand heterodimers and that both PR partners require ligand for dimerization to occur. Based on electrophoretic gel mobility shift assays (EMSAs), these heterodimers appear to have substantially reduced DNA binding activity. Progestin antagonists inhibit agonist activation of PR at concentrations that are too low to be accounted for by a simple competition mechanism for binding to PR. We propose that antiprogestin inactivation of PR in trans by heterodimerization contributes to the biological potency of these compounds.

Photoaffinity labelling of the human mineralocorticoid receptor with steroids having a reactive group at position 3, 18 or 21.

The ability of a glucocorticoid (triamcinolone acetonide: TA) and three progesterone derivatives with photoreactive groups at different positions (promegestone: R5020; 18-oxo-18-vinylprogesterone: 18OVP; 21-diazoprogesterone: 21DP) to bind covalently to the human mineralocorticoid receptor (hMR) expressed in Sf9 insect cells was assessed. Sedimentation gradient analysis and exchange assays with aldosterone showed that [3H]TA, a partial mineralocorticoid agonist, and [3H]R5020, a pure antimineralocorticoid, were covalently bound to hMR after UV irradiation, with a labelling efficiency of approx. 3-5%. UV irradiation did not alter the heterooligomeric structure of the hMR, since the irradiated [3H]TA- and [3H]R5020-hMR complexes sedimented at approx. 9-10 S, as did the non-irradiated complexes. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a band labelled by [3H]TA or [3H]R5020, having a molecular mass of 120 kDa. This band was not detected in the presence of an excess of the corresponding unlabelled steroid or when the cytosol was recovered from non-infected Sf9 cells. Electrophoresis of a truncated hMR (hMRDelta(1-351)) photolabelled with [3H]TA revealed a 80 kDa band, compatible with the molecular mass of the truncated hMR. Limited chymotrypsin proteolysis of the [3H]TA photolabelled hMR generated a 30 kDa fragment covalently associated with [3H]TA. As the 30 kDa fragment generated by chymotrypsin has been shown to encompass the entire ligand-binding domain of the hMR (B. Couette, J. Fagart, S. Jalaguier, M. Lombès, A. Souque, M.E. Rafestin-Oblin, Biochem. J. 315 (1996) 421-427), the present experiments provide evidence that [3H]TA is covalently bound to the ligand binding domain of the hMR. Exchange assays with [3H]A also revealed that unlabelled 18OVP and 21DP, two mineralocorticoid agonists bearing photoreactive groups at skeleton positions crucial for the ligand-MR interaction, are covalently bound to hMR with an approx. 30-35% labelling efficiency.

Binding characteristics of novel nonsteroidal antiestrogens to the rat uterine estrogen receptors.

Tamoxifen (TAM), the only antiestrogen currently available for the endocrine therapy of breast cancer behaves as a mixed agonist/antagonist of estrogen action, thus limiting its therapeutic potential. We report the binding characteristics of a novel series of nonsteroidal antiestrogens to the rat uterine estrogen receptor. As measured by competition studies, the affinity of EM-652, the active metabolite of the prodrug EM-800, for the estrogen receptor is 7-11 times higher than that of 17beta-estradiol (E2), ICI 182780, and hydroxy-tamoxifen (OH-TAM), the active metabolite of Tamoxifen. EM-652 is 20x more potent than ICI 164384 and Droloxifene while it is 400 times more potent than Toremifene in displacing [3H]E2 from the rat uterine estrogen receptor. On the other hand, the prodrug EM-800 and Tamoxifen have respectively 150-fold and 410-fold less affinity for the estrogen receptor than the pure antiestrogen EM-652. No significant binding of EM-652, EM-800, TAM or OH-TAM was observed to the rat uterine progesterone receptor at concentrations up to 10,000 nM except for TAM that caused a 50% displacement of labeled R5020 at 4000 nM. No significant binding of EM-652 or EM-800 was observed on the rat ventral prostate androgen receptor or the rat uterine progesterone receptor. The present data demonstrate the high affinity and specificity of the new antiestrogen, EM-652, for the rat uterine estrogen receptor. The antiestrogen EM-652 thus becomes the compound having the highest known affinity for the estrogen receptor. Due to its unique potency and its pure antiestrogenic activity already demonstrated in many systems, this antiestrogen could well offer an important advance for the endocrine therapy of breast cancer, uterine cancer, and other estrogen-sensitive diseases in women.