Asoprisnil (J867): a selective progesterone receptor modulator for gynecological therapy

Asoprisnil is a novel selective steroid receptor modulator that shows unique pharmacodynamic effects in animal models and humans. Asoprisnil, its major metabolite J912, and structurally related compounds represent a new class of progesterone receptor (PR) ligands that exhibit partial agonist and antagonist activities in vivo. Asoprisnil demonstrates a high degree of receptor and tissue selectivity, with high-binding affinity for PR, moderate affinity for glucocorticoid receptor (GR), low affinity for androgen receptor (AR), and no binding affinity for estrogen or mineralocorticoid receptors. In the rabbit endometrium, both asoprisnil and J912 induce partial agonist and antagonist effects. Asoprisnil induces mucification of the guinea pig vagina and has pronounced anti-uterotrophic effects in normal and ovariectomized guinea pigs. Unlike antiprogestins, asoprisnil shows only marginal labor-inducing activity during mid-pregnancy and is completely ineffective in inducing preterm parturition in the guinea pig. Asoprisnil exhibits only marginal antiglucocorticoid activity in transactivation in vitro assays and animal models. In male rats, asoprisnil showed weak androgenic and anti-androgenic properties. In toxicological studies in female cynomolgus monkeys, asoprisnil treatment abolished menstrual cyclicity and endometrial atrophy. Early clinical studies of asoprisnil in normal volunteers demonstrated a dose-dependent suppression of menstruation irrespective of the effects on ovulation, with no change in basal estrogen concentrations and no antiglucocorticoid effects. Unlike progestins, asoprisnil does not induce breakthrough bleeding. With favorable safety and tolerability profiles thus far, asoprisnil appears promising as a novel treatment of gynecological disorders, such as uterine fibroids and endometriosis.     

A structural and in vitro characterization of asoprisnil: a selective progesterone receptor modulator

Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P4). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P4-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P4-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P4-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P4, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.     

11-(pyridinylphenyl)steroids--a new class of mixed-profile progesterone agonists/antagonists

Recently, a new class (often referred to as SPRMs: selective progesterone receptor modulators) of progesterone receptor ligands with mixed agonist/antagonist properties has been described. Such compounds are envisaged, for example, as treatment for endometriosis, uterine fibroids, and leiomyomas. Existing SPRMs include Asoprisnil 1 and Uliprisnil acetate 2. In our hands, however, these compounds proved to have a predominantly or exclusively antagonistic in vitro profile, which may make this type of compound less attractive, for example, as contraceptives. We therefore aimed at a class of mixed-profile compounds that would show a more evenly balanced agonist/antagonist profile. A novel class of 11beta-[4-(heteroaryl)phenyl]-substituted pregnanes was identified that displayed the desired balance. Contrary to known SPRMs, this novel class of MPP (mixed-profile progestagen) was found to have a truly mixed activity, including a sizeable agonist component.     

Molecular properties and preclinical pharmacology of JNJ-1250132, a steroidal progesterone receptor modulator that inhibits binding of the receptor to DNA in vitro

Progesterone receptor modulators have diverse potential therapeutic uses, including the treatment of endometriosis, uterine fibroids and breast cancer. Here we describe the molecular properties and preclinical pharmacology of a new steroidal progestin antagonist, JNJ-1250132. The compound is a high affinity ligand for the progesterone receptor, possessing cross-reactivity with other steroid receptors comparable to that of steroidal antagonists such as mifepristone. It inhibits progestin-inducible alkaline phosphatase gene expression in T47D human breast cancer cells, and also inhibits their in vitro proliferation. It inhibits gestation in rats and progesterone-dependent endometrial transformation in rabbits with efficacies comparable to mifepristone. Like mifepristone, it is a glucocorticoid antagonist in vivo. In cell-free DNA binding assays, the compound inhibits binding of the human progesterone receptor to a progesterone response element, and thus is similar to onapristone in this regard. In contrast, as judged by proteolytic analysis, JNJ-1250132 induces a receptor conformation more similar to that induced by mifepristone, which promotes receptor binding to DNA. Therefore, JNJ-1250132 has unique effects on the progesterone receptor that may translate into a novel clinical profile.     

Molecular Changes Underlying Genistein Treatment of Wound Healing: A Review

Estrogen deprivation is one of the major factors responsible for many age-related processes including poor wound healing in postmenopausal women. However, the reported side-effects of estrogen replacement therapy (ERT) have precluded broad clinical administration. Therefore, selective estrogen receptor modulators (SERMs) have been developed to overcome the detrimental side effects of ERT on breast and/or uterine tissues. The use of natural products isolated from plants (e.g., soy) may represent a promising source of biologically active compounds (e.g., genistein) as efficient alternatives to conventional treatment. Genistein as natural SERM has the unique ability to selectively act as agonist or antagonist in a tissue-specific manner, i.e., it improves skin repair and simultaneously exerts anti-cancer and chemopreventive properties. Hence, we present here a wound healing phases-based review of the most studied naturally occurring SERM.     

Selective oestrogen receptor modulators

Selective oestrogen receptor modulators (SERMs) are compounds which act like oestrogens in some target tissues but which antagonise their effects in others. The first example of a SERM (referred to as a first-generation compound) was tamoxifen, for which oestrogen-like agonist activity on bone was seen to occur simultaneously with oestrogen antagonist activity on the breast. An unwanted effect of tamoxifen was its oestrogen-like action on the endometrium. Second-generation compounds have since been developed, most notably raloxifene, which has oestrogen-like actions on bone, lipids and the coagulation system, and oestrogen antagonist effects on the breast and uterus. Raloxifene has undergone very extensive, prospective, placebo-controlled, randomised trial evaluation, in which anti-fracture efficacy (to date only for vertebral fracture) has been accompanied by a major reduction in the incidence of new breast cancer. The compound is similar to placebo in its uterine effects, and similar to oestrogen in causing a two- to threefold increase in the risk of venous thromboembolism. Its lipid effects are similar to those of oestrogen, except for a relatively small effect on high-density lipoprotein cholesterol, and no significant effect on triglycerides. Data on cardiovascular event rates are not yet available; data on cognitive function are preliminary and, to date, reassuring. The mechanisms by which the same compound can exert oestrogen agonist effects on one target and antagonist effects on another are still being clarified. Important aspects include the fact that the oestrogen receptor undergoes different conformational changes according to the ligand. Thus the crystal structure of oestradiol bound to the oestrogen receptor differs from that of raloxifene bound to the same receptor. The existence of two oestrogen receptor subtypes may also be relevant. Mechanisms include differing interactions with various domains of the oestrogen receptor, and tissue-specific recruitment of steroid receptor co-activators and co-repressors may underlie some of the tissue-specific effects. The SERMs may be the prototype for other selective steroid receptor modulators, for example the androgen and progesterone receptors. The development of tissue target-specific agents is an exciting advance in endocrine pharmacology and can be extended to agents, such as tibolone, which exert some of their tissue specificity through their metabolites.     

Inhibition of uterine contractility by progesterone and progesterone metabolites: mediation by progesterone and gamma amino butyric acidA receptor systems.

Progesterone and several progesterone metabolites are capable of inhibiting uterine contractility. Some progesterone metabolites have shown little or no affinity for the progesterone receptor but have been found to be potent modulators of the GABAA receptor system. This study examined whether the inhibition of uterine contraction by progesterone and its metabolites was progesterone receptor-mediated or gamma amino butyric acidA (GABAA) receptor-mediated. Uterine contractions were measured in annular rings of uterine tissue, 5 mm in length, from diestrous II rats, under a fixed tension of 1 gram. The steroids tested were 3 beta-hydroxy-5 beta-pregnan-20-one (6 micrograms/ml), 5 beta-pregnane-3,20-dione (10 micrograms/ml), 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha,5 alpha-THP, 27.5 micrograms/ml), and progesterone (40 micrograms/ml). All compounds significantly inhibited spontaneous uterine contractions when compared to controls. No effect was seen by either 16 micrograms/ml of the progesterone antagonist, RU486, or 32 micrograms/ml of the GABAA antagonist, pictrotoxin, when administered alone. However, when uterine tissues were exposed to a combination of the steroid and the antagonist, the effect of 3 beta-hydroxy-5 beta-pregnan-20-one and 3 alpha,5 alpha-THP was blocked by picrotoxin but not by RU486, indicating that the action of these steroids was mediated through the GABAA system. The effect of 5 beta-pregnane-3,20-dione and progesterone was effectively blocked by RU486 but not by picrotoxin, suggesting that their actions were mediated through the progesterone receptor system. These results indicate that multiple mechanisms exist in the uterus for inhibiting uterine contractility by progesterone and its metabolites.     

Endocrine pharmacological characterization of progesterone antagonists and progesterone receptor modulators with respect to PR-agonistic and antagonistic activity

Studies were conducted to assess progesterone antagonists (PAs) and progesterone receptor modulators (PRMs) with respect to PR agonistic and antagonistic activities in vivo. These properties are not always adequately reflected in transactivation in vitro models. Studies were performed in pregnant rats, estrogen-primed rabbits (McPhail -Test), and cycling and pregnant guinea pigs. Tested compounds included mifepristone (RU486), onapristone, J867, J956, J1042, and ZK137316. J-compounds induced sub-maximum endometrial transformation and, paradoxically, inhibited effects of progesterone in rabbits. Mifepristone, onapristone, and ZK137316 behaved as 'pure' antagonists in this species. Inhibition of uterine PGF(2alpha) secretion and inhibition of luteolysis in cycling guinea pigs were more sensitive parameters of PR-agonistic and antagonistic properties. 'Pure' PAs inhibited uterine PGF(2alpha) secretion and luteal regression completely. The PR agonist R5020 reversed both effects which demonstrates a PR mediation. Agonistic PRMs (J-substances and mifepristone) showed no or blunted antiluteolytic effects compared to the 'pure' PR antagonist onapristone. When tested in pregnant guinea pigs for their labor-inducing potential, PR agonistic PRMs had much reduced or abolished abortifacient activity compared to mifepristone (mifepristone > J956 > J867/J912 > J1042). However, in cycling animals, superior antiovulatory and antiproliferative properties of the J-substances were seen. Antiovulatory effects of 'pure' and agonistic PRMs are probably due to different mechanisms. The relevance of rodent studies for antiovulatory and uterine antiproliferative effects for the human is still uncertain. The non-abortifacient PRM J1042 induced stromal compaction and inhibition of endometrial proliferation in monkeys, but this effect was not stronger than that of the 'purer' PAs. 'Pure' PAs are important pharmacological tools analogous to PRKO models to study the role of PR in the menstrual cycle and in pregnancy.     

Priming effects of novel nonsteroidal progesterone receptor modulators CP8816 and CP8863 on the development of adenomyosis in the mouse uterus

The possibility of therapeutic application of novel nonsteroidal progesterone receptor modulators CP8816 and CP8863 for preventing the development of uterine adenomyosis was investigated in mice. First priming effects of CP8816 on 17beta-estradiol (E2)-induced cell division in uterine tissues were examined. As a result, pretreatment with CP8816 or progesterone significantly suppressed the elevation of the mitotic activity in the luminal epithelial cells of mice treated with E2 later. Priming with CP8816 had little effect on the stromal cells, but progesterone priming caused an increase of stromal mitotic activity in mice treated with E2 later. To evaluate the inhibitory effect of these compounds on the development of adenomyosis induced experimentally by pituitary grafting, 7-week-old female mice were isografted with a single anterior pituitary in the uterus and divided into four groups. Two groups of mice were given daily subcutaneous injections of 1 mg of CP8816 or the vehicle alone for 6 weeks from the day after the grafting. Remaining two groups of mice were given oral administration of 1 mg of CP8863 or the vehicle only for 5 weeks starting one week after the grafting. The incidence of adenomyosis was significantly lower in the groups of mice treated with CP8816 and CP8863 than in the respective control groups. The mechanism by which CP compounds inhibited the development of adenomyosis might be related to their priming effects, i.e., their inhibitory effect on epithelial cell division and lack of effect on stromal cell division after subsequent exposure to E2.     

Development of p-carborane-based nonsteroidal progesterone receptor antagonists

Progesterone receptor (PR) regulates various physiological processes, including the female reproductive system, and development of nonsteroidal PR antagonists is considered desirable for clinical application, as they are expected to have reduced side effects. We have synthesized a series of nonsteroidal PR antagonists using a 4-cyanophenyl-p-carborane core structure. Among them, compound 14d exhibited potent PR-antagonistic activity (IC₅₀: 27 nM). It showed high binding affinity for PR, but did not bind to androgen receptor or estrogen receptor. This PR-selective antagonist may be a promising lead compound for clinically applicable progesterone receptor modulators.     

X-ray structures of progesterone receptor ligand binding domain in its agonist state reveal differing mechanisms for mixed profiles of 11β-substituted steroids

We present here the x-ray structures of the progesterone receptor (PR) in complex with two mixed profile PR modulators whose functional activity results from two differing molecular mechanisms. The structure of Asoprisnil bound to the agonist state of PR demonstrates the contribution of the ligand to increasing stability of the agonist conformation of helix-12 via a specific hydrogen-bond network including Glu(723). This interaction is absent when the full antagonist, RU486, binds to PR. Combined with a previously reported structure of Asoprisnil bound to the antagonist state of the receptor, this structure extends our understanding of the complex molecular interactions underlying the mixed agonist/antagonist profile of the compound. In addition, we present the structure of PR in its agonist conformation bound to the mixed profile compound Org3H whose reduced antagonistic activity and increased agonistic activity compared with reference antagonists is due to an induced fit around Trp(755), resulting in a decreased steric clash with Met(909) but inducing a new internal clash with Val(912) in helix-12. This structure also explains the previously published observation that 16α attachments to RU486 analogs induce mixed profiles by altering the binding of 11β substituents. Together these structures further our understanding of the steric and electrostatic factors that contribute to the function of steroid receptor modulators, providing valuable insight for future compound design.     

Uterine epithelial morphology and progesterone receptors in a mifepristone-treated viviparous lizard Pseudemoia entrecasteauxii (Squamata: Scincidae) during gestation

Structural and functional changes to the uterus associated with maintenance of pregnancy are controlled primarily by steroid hormones such as progesterone. We tested the hypothesis that progesterone regulates uterine structural changes during pregnancy in the viviparous skink, Pseudemoia entrecasteauxii, by treating pregnant females with the progesterone receptor antagonist mifepristone at different stages of pregnancy. Expression and distribution of progesterone receptor was determined using Western blot and immunohistochemistry. During early pregnancy, mifepristone treatment resulted in altered uterine epithelial cell surface morphology and high embryo mortality, but did not affect females at mid and late stages of pregnancy. Females treated with mifepristone in early pregnancy exhibited abnormal uterine epithelial cell morphology such as lateral blebbing and presence of wide gaps between cells indicating loss of intercellular attachment. Chorioallantoic membranes of the embryo were not affected by mifepristone treatment. Two isoforms (55 kDa and 100 kDa) of progesterone receptor were identified using immunoblots and both isoforms were localized to the nucleus of uterine epithelial cells. The 55 kDa isoform was expressed throughout pregnancy, whereas the 100 kDa isoform was expressed during mid and especially late pregnancy. In P. entrecasteauxii, mifepristone may prevent successful embryo attachment in early pregnancy through its effects on uterine epithelial cells but may have little effect on pregnancy once the maternal-embryo structural relationship is established.     

Compound profiling using a panel of steroid hormone receptor cell-based assays

A major focus in the current discovery of drugs targeting nuclear receptors (NRs) is identifying drugs with reduced side effects by improving selectivity, not only from other receptors but also by selective modulation of the NR of interest. Cellular assays not only provide valuable information on functional activity, potency, and selectivity but also are ideally suited for differentiating partial agonists and antagonists. The ability to partially activate a receptor is believed to be closely tied to the ability to selectively modulate the NR, resulting in expression of a subset of the normally regulated genes. To this end, the authors have built a complete panel of cell-based steroid hormone receptor assays for the androgen receptor, estrogen receptor alpha, estrogen receptor beta, glucocorticoid receptor, mineralocorticoid receptor, and progesterone receptor by stably engineering a Gal4 DNA-binding domain/nuclear receptor ligand-binding domain fusion protein into an upstream activation sequence beta-lactamase reporter cell line. Each assay was validated with known agonists and antagonists for correct pharmacology and high-throughput compatibility. To demonstrate the utility of these assays, the authors profiled 35 pharmacologically relevant compounds in a dose-response format against the panel in both agonist and antagonist modes. The results demonstrated that selective estrogen receptor modulators can be identified and differentiated, as well as mixed and partial agonists and antagonists easily detected in the appropriate assays. Importantly, a comparison of the chimeric assays with full-length reporter gene assay data from the literature shows a good degree of correlation in terms of selectivity and pharmacology of important ligands. Taken together, these steroid hormone receptor assays provide good selectivity, sensitivity, and appropriate pharmacology for high-throughput screening and selectivity profiling of modulators of steroid hormone receptors.     

Bull sperm acrosome reaction induced by gamma-aminobutyric acid (GABA) is mediated by GABAergic receptors type A

The effect of gamma-aminobutyric acid (GABA) on the bull sperm acrosome reaction was evaluated, and the interaction of progesterone, a physiologic inducer of the acrosome reaction, with the GABA receptor was explored. The acrosome reaction was stimulated by GABA in a dose-dependent manner. This effect was inhibited completely by bicuculline, a GABA A receptor antagonist, but GABA B and C receptor antagonists had no effect. Accordingly, muscimol, a GABA A receptor agonist, stimulated the acrosome reaction to the same extent as GABA, whereas baclofen (GABA B receptor agonist) and CACA (GABA C receptor agonist), had no effect. Preincubation with progesterone followed by the addition of GABA resulted in a significant increase in the percentage of acrosome reacted spermatozoa compared with progesterone or GABA alone. Taking into account that this increase was less than a simple addition of effects, it might be suggested that GABA and progesterone act through the same receptor and/or use the same mechanism of action. To test this hypothesis, the abilities of GABA and progesterone to induce acrosome reaction were tested in the presence of bicuculline, which suppressed both stimulatory effects. Given that the GABA A receptor is linked to the Cl(-) channel, we tested whether picrotoxin, a blocker of this channel, could modulate the effects of progesterone or GABA. Cl(-) channel blocker picrotoxin dramatically reduced the GABA and progesterone-initiated AR. In conclusion: GABA and progesterone stimulate the acrosome reaction in bull spermatozoa acting through a classical GABA A receptor. The mechanism of action requires the functional integrity of the Ca(2+) Cl(-) channel.