Strategy in drug reseabch. Synthesis and study of the psogestational and ovulation inhibitory activity of a series of 11β-substituted-17α-ethynyl-4-estren-17β-ols

Using the strategy based on the Hansch method which analyses effects of substituents on biological activity in terms of their hydrophobic, electronic and steric effects we selectively synthesised a series of 11β-substituted-17α-ethynyl-4-estren-17β-ols that combine ease of synthesis with good discrimination between these factors aiming at finding the compounds with optimum biological activity in that series. The compounds were tested quantitatively in the Clauberg test (rabbit) and the ovulation inhibition test (rat). The differences in biological activity could reasonably be correlated with two steric effects introduced by the 11β-substituent. These were a change in the overall shape of the 11β-substituent and the angular methyl group, and direct steric hindrance of the steroid-receptor protein binding. Some exceptions were found possibly due to metabolic conversion of these compounds to the corresponding 11β-substituted-17α-ethynyl-l,5,5(10)-estratriene-5, 17β-diols.     

Glucocorticoid agonists as well as antagonists are effective inducers of mouse mammary tumor virus RNA in mouse mammary tumor cells treated with inhibitors of ADP-ribosylation

Glucocorticoids increase expression of specific genes by a mechanism involving binding to and "activation" of a specific receptor protein. Other steroids, such as RU 486, bind to the glucocorticoid receptor but the resultant steroid-receptor complex is unable to activate glucocorticoid sensitive genes. In the present study we have observed that steroid regulation of the glucocorticoid-regulated mouse mammary tumor virus (MMTV) genome in cultured mouse mammary tumor cells is altered by treatment of the cells with inhibitors of (ADP-ribose)n synthetase. The ability of glucocorticoid agonists to increase MMTV is about 2-fold increased by the inhibitor treatment. Interestingly, RU 486 and other steroids that are normally inactive in control cells are very good inducers of MMTV in the treated cells. This alteration in MMTV expression is associated with a 37% increase in nuclear binding of the glucocorticoid, triamcinolone acetonide, and also RU 486 in the inhibitor-treated cells. Steroids that do not bind to the glucocorticoid receptor are not inducers in control or in treated cells. The results point to a role for ADP-ribosylation of proteins as a negative regulator of MMTV expression and suggest a mechanism for activation of steroid-sensitive genomes.     

Isolation and characterization of a subchloroplast particle enriched in iron-sulfur protein and P700.

Transcortin-bound gluco- and mineralocorticoids were fractionated on a number of chromatographic systems. Contrary to earlier suggestions of a homogenous unit by competition binding and Scatchard analysis, a polymorphic nature of the globulin was evident with corticosterone on Sephadex A-50 columns (components in 0.4 and 0.6 m KCl) and with synthetic steroids (triamcinolone acetonide, dexamethasone) or natural mineralocorticoids (aldosterone, 18-hydroxy-deoxy-corticosterone) on diethylaminoethyl cellulose-52 gels (species in 0.001 and 0.06 m phosphate). Besides the major component of molecular weight 55,000, a heavier shoulder in the 67,000 molecular weight region was obtained with cortisol, dexamethasone, triamcinolone, and aldosterone from Sephadex G-200 columns, on which binding was reduced in the presence of high salt (0.4 m KCl). Triamcinolone- and dexamethasone-bound components eluted at lower salt concentrations from the DE-52 column than natural steroid-corticosteroid-binding globulin complexes. The various features of serum carrier binding are discussed in terms of steroid-receptor association in hormone-specific target tissues.     

Testicular cytosol factors inhibit the binding of steroid-receptor complexes to chromatin

Testicular and prostatic androgen-receptor complexes as well as uterine estradiol-receptor complexes, partially purified by ammonium sulfate precipitation (15-37%), were bound to germ cell chromatin. At equivalent concentrations, less testicular androgen-receptor complexes bound to chromatin than did the other two steroid-receptor complexes. Addition of a partially purified testicular androgen-receptor preparation with prostatic androgen-receptor or uterine estradiol-receptor preparation to the binding interaction mixture reduced the binding of either of the latter two steroid-receptor complexes to chromatin. These data suggest the presence of inhibitory factor(s) in the testicular receptor preparations. Testicular cytosols were fractionated by ammonium sulfate precipitation into fractions A (15-37% saturation), B (37-50%) and C (50-75%). All fractions inhibit binding of these steroid-receptor complexes to chromatin. Fractions A and B appear to be heat labile, while fraction C was more stable. Further fractionation of A and C fractions on DEAE cellulose yielded A1 and C1 (filtrates) as well as A2 and C2 (0.3 M NaCl eluents), respectively. Subfractions A1, A2, and C2 contained inhibitory factors for the binding of steroid-receptor complexes to chromatin while C1 showed no effect. These data demonstrated that testicular cytosol contains a variety of inhibitory factors which affect the binding of both androgen-receptor and estradiol-receptor complexes to chromatin.     

Characterization and quantification of the androgen and glucocorticoid receptors in cytosol from rat skeletal muscle

The binding of the radioactive synthetic hormonal steroids [3H]dexamethasone (9 alpha-fluoro-11 beta, 17 alpha, 21-trihydroxy-16 alpha-methyl-1,4-pregnadiene-3,20-dione) and [3H]methyltrienolone (17 beta-hydroxy-17 alpha-methyl-4,9,11-estratien-3-one) to cytosol from rat skeletal muscle was studied using dextran-coated charcoal to separate unbound and receptor-bound steroid. The rates of association, dissociation, and degradation of the complexes of dexamethasone and methyltrienolone with receptor were highly dependent on temperature. The temperature dependence of association was greater for dexamethasone, and that of degradation was greater for methyltrienolone. Dissociation rates were insignificant for both steroid-receptor complexes compared to association and degradation rates. The apparent equilibrium dissociation constants for the binding of dexamethasone and methyltrienolone to their receptor binding sites were about 7 and 0.3 nM, respectively, regardless of temperature (0. 15 or 23 degrees C). The lack of influence of temperature on the equilibrium constants indicate that the binding was of hydrophobic character, and the corresponding free energy changes upon binding of dexamethasone and methyltrienolone to their respective binding sites were -41 and -49 kJ mol-1 under equilibrium conditions at 0 degrees C. The apparent maximum number of binding sites determined from Scatchard plots under these conditions was about 1900 fmol/g of tissue, 3500 fmol/mg of DNA or 30 fmol/mg of protein in the case of the dexamethasone receptor, and the corresponding figures for the methyltrienolone were about 100 fmol/g of tissue, 200 fmol/mg of DNA or 2 fmol/mg of protein. The ligand specificities of the binding sites for dexamethasone and methyltrienolone were typical of a glucocorticoid and an androgen receptor, respectively. Both steroid-receptor complexes were retained on DNA-cellulose columns, and were eluted by NaCl at an ionic strength of 0.1. The DNA-cellulose step purified about 20 times, and was used to allow gel exclusion chromatography and electrofocusing. Both steroid-receptor complexes were excluded from a column of Sephadex G-150. Electrofocusing in preparative columns gave reproducible patterns consisting of three peaks for each receptor. The apparent isoelectric points were 5.4, 5.6 and 6.2 for the glucocorticoid receptor, and 5.9, 6.2 and 8.5 for the androgen receptor.     

Nuclear acceptor sites for androgen-receptor complexes in seminal-vesicle epithelium.

An assay method in vitro was developed and applied to quantify acceptor binding of steroid-receptor complexes in nuclei from isolated epithelium of guinea-pig seminal vesicle. Steroid-receptor complex prepared from 1-day-castrated animals was incubated with purified nuclei from 1-28 day-castrated animals in a medium containing 0.15 M-KCl. Free and bound steroid-receptor complexes were measured and the data were submitted to Scatchard analysis. With nuclei from 1-day-castrated animals the Kd for binding of cytosolic [3H]dihydrotestosterone-receptor complexes was found to be 0.83 X 10(-10) M and the capacity for binding was 0.35 pmol/mg of nuclear DNA. Scatchard analysis consistently disclosed only a single line of constant slope and gave the same kinetic constants for nuclei obtained from animals castrated up to 28 days before assay. Administration of 2 mg of dihydrotestosterone, 3 alpha-androstanediol or androsterone or 100 microgram of oestradiol-17 beta 1 h before killing of the 1-day-castrated animals that provided the nuclei resulted in a significant decrease in nuclear acceptor binding of the steroid-receptor complex compared with untreated animals. Thus our assay method disclosed nuclear acceptor sites that may be involved in responses to androgens (and oestrogens) in vivo. We conclude that there is a class of nuclear accept or sites of high affinity and limited capacity that may be occupied by steroid-receptor complexes in vivo.     

Cytoplasmic receptor protein for etiochalanolone in chick embryo liver.

Acute intermittent porphyria is a gentic disease associated with changes in the activity of some of the hepatic enzymes in the heme biosynthetic pathway and in the activity of delta4-5alpha-steroid reductase, an enzyme involved in steroid metabolism. Embryonic chick liver has been used as a model system to study the effects of several naturally occurring 5beta steroid metabolites on delta-aminolevulinic acid synthetase, the rate-limiting enzyme in the heme pathway (Granick, S., and Kappas, A. (1967) J. Biol. Chem. 242, 4587-4593). In this study we have identified in vitro a hepatic cytoplasmic receptor which binds [3H]-etiocholanolone (a 5beta-H androgen metabolite). The steroid-receptor complex has an apparent Kd value of 3.5 times 10(-6) M at 0-4degrees; the number of binding sites per cell is 23,000. The macromolecular complex sediments at approximately 4 S in a 5 to 20% sucrose gradient and is unaffected by KCl concentrations up to 0.4 M. The steroid-receptor complex can be destroyed by heat (60degrees) or proteolytic digestion and is inhibited by sulfhydryl-blocking agents. Competition studies revealed that among the nonradioactive steroids tested, all of the primary androgens and the progestins as well as their 5alpha and 5beta metabolites block the binding of [3H]etiochalanolone. Only the glucuronide derivative of etiocholanolone, glucocorticoids and their metabolites, 17beta-estradiol, and cyproterone compete poorly for the receptor. The steroid receptor described here appears to be different from the androgen receptor isolated from rat liver and prostate.     

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[³H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [³H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur.     

Quantitative structure-activity relationships of estrogenic steroids substituted at C14, C15

The uterotropic activity of thirty 3-methoxyestradiol derivatives is measured and discussed on the basis of X-ray crystallographic results and quantitative structure-activity relationship analyses involving hydrophobic substituent constants pi and f as well as steric parameters Pr and L. In addition, estrogenicity is compared to data of interceptive activity and receptor binding affinity. All the biological data exhibit a high degree of intercorrelation. 17 beta-Hydroxysteroids having 14 alpha configuration reveal a generally better capability of high-affinity binding than those being 14 beta configurated. Between the uterotropic activity and the hydrophobicity of C14, C15 substituents, statistically significant correlations are found which suggest a close contact between the steroidal D-ring subsite and the receptor protein (e.g. for 14 alpha steroids: log UDD = -0.996 pi -0.392; n = 9, r = -0.943, s = 0.235, t = -7.5, alpha less than 0.001). The hydrophobic nature of both 14 alpha and 14 beta medium-sized substituents employed is shown by QSAR regressions to exert a stronger influence than steric effects. Furthermore, there are indications to additional hydrogen bonding and steric repulsion phenomena. As to the receptor-binding models discussed in the literature, it is concluded that the receptor protein has a high conformational flexibility to accommodate very different drug structures all having the common phenolic ring A. But, if an appropriate spacing of steroidal key atoms is recognized by the receptor and, consequently, the steroid-receptor complex is formed, the binding is complemented by hydrophobic interactions also in the D-ring region.     

Glucocorticoid-receptor interactions. Discrimination between glucocorticoid agonists and antagonists by means of receptor-binding kinetics.

The kinetics of binding to the molybdate-stabilized glucocorticoid receptor of rat thymus cytosol were determined at 0 degrees C for a number of glucocorticoid agonists and antagonists. Equilibrium constants derived from the rate constants for association and dissociation were in good agreement with those determined directly or by competition under equilibrium conditions. Kinetics parameters for the slowly dissociating form of binding detected by a non-equilibrium dextran/charcoal competitive binding assay reflected the nature and extent of functional-group substitution on the steroid nucleus, but bore no relation to the classification of steroids as glucocorticoid agonists or antagonists. It is concluded that the binding of antagonists that is detected by such methods is agonist-like binding, which is not relevant to their antiglucocorticoid actions. Both agonists and antagonists displayed Michaelis--Menten association kinetics, but this behaviour was much more pronounced for antagonists. This is attributed to the existence of a second form of steroid-receptor complex, which escapes detection by the usual assay methods as a result of a high rate of dissociation and which is quantitatively antagonist-specific under equilibrium conditions. Direct evidence for the existence of two forms of antagonist-receptor complex was provided by results showing that the dissociation of the glucocorticoid antagonist progesterone from the receptor was biphasic.     

Studies on the translocation inhibitor of 3H-dexamethasone-receptor complex.

Recent reports on the binding of glucocorticoid-receptor complexes to rat liver nuclei suggested the presence of components which inhibited the binding. The inhibitory component(s) of the receptor translocation was observed not only in the cytosol of the liver but also in cytosols of the kidney, the spleen and the thymus. The cytoplasmic levels of the inhibitor in these tissues were not modified by the administration of Dexamthasone (DEX). The liver inhibitor was macromolecular and clearly separated from the DEX-receptor complex on DEAE-cellulose chromatography. The mechanism of the inhibition seemed to be an interaction between the inhibitor and the steroid-receptor complex. In addition, the inhibition seemed to be less specific for the bindings of different steroid-receptor complexes to nuclei. The bindings of hepatic 3H-DEX-receptor complex by nuclei derived from livers of adrenalectomized and DEX-treated rats, in the presence or absence of the translocation inhibitor, were similar.     

‘Sephadex’ Column Chromatography as an Adjunct to Competitive Protein Binding Assays of Steroids

THE remarkably specific binding properties of certain proteins make possible competitive protein binding (CPB) assays for many hormones^1,2. Various kinds of binding proteins have been used, including plasma proteins, tissue proteins, antibodies (radio-immunoassay) and enzymes (radio-enzymatic assay), but no protein has been found which binds only a single substance—each can be shown to bind a series of closely related analogues. To measure a single ligand in the complex mixtures present in biological fluids, it is therefore often necessary to separate the competing analogues. This is especially true in the use of the plasma proteins for the assay of steroids, for each binds two distinct groups of steroid—the corticosteroid binding globulin (CBG) binds corticoids and progestins and the sex hormone binding globulin (SHBG) binds androgens and oestradiol. Because all the bound steroids are of biological interest, however, it is often desirable to be able to measure all of them, so that the diversity of binding can be an advantage if the steroids can be adequately separated before assay.     

Nuclear binding of progesterone in hen oviduct. Role of acidic chromatin proteins in high-affinity binding.

The multiple classes of binding sites for the progesterone-receptor complex in hen oviduct muclei were found to be of chromatin origin. The highest-affinity, and presumably most physiologically important class, is localized in oviduct chromatin and contains approx. 6000-10000 sites per nucleus. None of these sites is detected in spleen chromatin. Two new techniques were used for assaying rapidly the binding of steroid-receptor complexes to soluble deoxyribonucleoproteins in vito. The extent of high-affinity binding by the nucleo-acidic protein fraction from spleen chromatin is as great as that by the nucleo-acidic protein from oviduct chromatin. Consequently the tissue-specific nuclear binding of the progesterone receptor is found not to be a consequence of the absence of the nuclear binding sites (acceptors) from chromatin of non-target tissue (spleen), but rather a result of complete masking of these sites. In the target-tissue (oviduct) chromatin, approx. 70% of the high-affinity acceptor sites are also masked. Acidic proteins, and not histones, appear to be responsible for the masking of these acceptor sites. In addition, acidic proteins represent (or at least are an essential part of) these high-affinity sites in the oviduct nucleus. Pure DNA displays a few high-and many low-affinity binding sites. In support of previous work with immature chicks, the acidic protein fraction of the nucleo-acidic results thus support the hypotheis that protein complexed with DNA, and not DNA alone, represent the high-affinity binding sites for the steroid-receptor complexes in nuclear chromatin. The lower-affinity classes of binding sites may represent DNA and/or other nuclear components.     

The "activated" hepatic glucocorticoid-receptor complex. Its generation and properties.

The glucocorticoid receptor-glucocorticoid complex of the hepatic cytosol need undergo an "activation" to enable its binding to nuclei, chromatin, or stripped DNA. The conditions of this activation have been studied using native calf thymus DNA absorbed to cellulose. At low ionic strength, activation is very slow at 0 degrees, but, takes place rapidly at 25 degrees, reaching completion at 1 hour. Addition of 10 mm CaCl2 or 150 mm NaCl increases the rate of activation of the receptor at 0 degrees. Neither magnesium nor manganese ions can replace calcium with respect to enabling activation of the steroid-receptor complex to occur at low temperatures. Isofocusing studies reveal that the major component of the unactivated steroid-receptor complex has an isoelectric point of 7.1. Incubation of the steroid-receptor complex at 25 degrees for 30 min leads to its conversion to a form with an isoelectric point of 6.1 concurrent with the development of its ability to bind to DNA-cellulose. Sucrose density gradient analysis reveals that no detectable alteration in the sedimentation coefficient of the steroid-receptor complex occurs during its activation. MnCl2 (20mm) effeciently precipitates the unactivated hormone-receptor complex and to a lesser degree, precipitates the activated hormone-receptor complex.     

Glucocorticoid binding and mechanism of resistance in some clones of mouse myeloid leukemic cells resistant to induction of differentiation by dexamethasone.

Several clones of dexamethasone-resistant cells, which could not differentiate even in a high concentration of dexamethasone, were isolated from glucocorticoid-sensitive myeloid leukemic cells. Some of them were shown to be deficient in steroid binding to specific cytoplasmic receptors, while the others contained glucocorticoid-specific cytoplasmic receptors that might be the same as those in sensitive cells. One of the resistant clones was found to be almost completely deficient in nuclear acceptor sites for cytoplasmic steroid-receptor complexes. The remaining clones were also characterized by significantly reduced amounts of nuclear-bound glucocorticoid. These results suggest that resistibility to glucocorticoids in the resistant clones of myeloid leukemic cells is due mainly to a defect in some steps of intracellular transfer of the steroid. Dexamethasone-sensitive cells, which could differentiate in the presence of dexamethasone, could be also induced to differentiate by protein factor(s) in ascitic fluid. Although all the resistant cells showed a low response to ascitic fluid, some of them showed 10-fold enhancement of phagocytic activity which is a typical character of differentiated cells. These results suggest that response to steroids is not directly correlated with that to protein inducer(s).     

Adrenal steroids as modulators of nerve cell function

Adrenal steroids modulate the function of nerve cells. Some, but not all actions of these steroids take place after binding to intracellular receptor systems and translocation of the steroid-receptor complex into the cell nucleus. Studies on the rat brain revealed heterogeneity of receptors. One population of receptor sites is present in abundance in extrahypothalamic limbic brain regions, e.g. neurons of the hippocampus, septum and amygdala. This neuronal receptor system displays a stringent binding specificity towards corticosterone, which is the naturally occurring glucocorticoid of the rat. Focussing the studies on the corticosterone receptor system in hippocampal neurons has provided further insight in the understanding of some of the actions of the steroid. Certain hippocampus-associated behaviors and indices of neurotransmission (serotonin) were disturbed after removal of the adrenals, but selectively restored after replacement with a low dose of corticosterone. The specificity, localization and dose-dependency of the corticosterone action on behavior and neurotransmission corresponds to the properties of its receptor system. The responsiveness to corticosterone is altered after changes in number of receptor sites. Chronic stress or high doses of exogenous corticosterone cause a long-term reduction. Other factors involved in regulation of receptor number are the neurotransmitter serotonin and neuropeptides related to ACTH and vasopressin. These substances restore changes in number of hippocampal corticosterone receptor sites due to aging, endocrine or neural deficiencies. Our results show that the number of corticosterone receptors is a sensitive index for brain functioning. Thus, the receptor system mediates some of the modulatory actions of corticosterone on nerve cell function and it may adjust its capacity under the influence of neural and endocrine factors.