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The receptor binding of a library of 187 steroids to five steroid hormone receptors (estrogen, progestin, androgen, mineralocorticoid, and glucocorticoid) has been analyzed by correspondence factor analysis (CFA) in order to illustrate how the method could be used to derive structure-activity-relationships from much larger libraries. CFA is a cartographic multivariate technique that provides objective distribution maps of the data after reduction and filtering of redundant information and noise. The key to the analysis of very complex data tables is the formation of barycenters (steroids with one or more common structural fragments) that can be introduced into CFA analyses used as mathematical models. This is possible in CFA because the method uses X2-metrics and is based on the distributional equivalence of the rows and columns of the transformed data matrix. We have thus demonstrated, in purely objective statistical terms, the general conclusions on the specificity of various functional and other groups derived from prior analyses by expert intuition and reasoning. A finer analysis was made of a series of A-ring phenols showing the high degree of glucocorticoid receptor and progesterone receptor binding that can be generated by certain C-11-substitutions despite the presence of the phenolic A-ring characteristic of estrogen receptor-specific binding.

In view of the multifactorial nature of the endocrine dysfunctions that may develop during prostate cancer and the unsuitability of the most widely used statistical methods to study such dysfunction, we have in the present study examined the relationships among 17 biological variables in 26 patients with advanced prostate cancer by two complementary multivariate methods, correspondence factorial analysis (CFA) and a hierarchical automatic classification procedure. The 17 variables included 14 hormones, their precursors or metabolites [LH, FSH, estradiol (E2), testosterone, dihydrotestosterone (DHT), androstenedione (A), androstenediol (Ediol), dehydroepiandrosterone (DHA), DHA-sulphate (DS), cortisol (CORT), 17 alpha-hydroxyprogesterone (17-OH-PROG), pregnenolone (PREG), 17 alpha-hydroxypregnenolone (17-OH-PREG), and androstanediol glucuronide (ADG)], one plasma binding protein, namely, sex-hormone-binding protein (SHBG) and two tumour markers, prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA). The originality of these multivariate methods is that they do not preselect a dependent variable nor perform two-by-two correlations as in stepwise multiple regression analysis but describe the patient population by extracting layers of correlations (from strong to weak) from amid confounding variables. Compared to principal component analysis which is based on covariance, CFA, based on the chi 2-metric, enables the licit representation of both tests and patients on the same factorial maps. From an examination of proximity among variables, it is possible to deduce which tests are related, which groups of patients have similar hormone profiles, and which tests vary most in which patients. The most discriminant factors in this particular population of patients were PSA and PAP levels, which were, however, not strongly correlated and were apparently selectively associated with certain hormones. PAP seemed the more pathological marker; PSA was somewhat anticorrelated to the adrenal androgen (DHA and DS) and PREG levels. The hormones with the lowest variance were A, Ediol and CORT reflecting their key roles in metabolism. A number of patients were hypogonadic. SHBG levels were not closely related to total T levels but anticorrelated with ADG suggesting that, in the patients concerned, SHBG decreases the bioavailable T fraction. There was no correlation between ADG and precursor hormones (PREG, DHA, DS) but a slight anticorrelation between these precursors and DHT. Therefore the source of ADG in these patients does not seem to be increased levels of precursor hormones nor of DHT but increased peripheral tissue metabolism of androgens. In future, descriptive multivariate analyses of large patient cohorts should help to define subpopulations with distinctive hormone profiles for prospective clinical studies.

The following hormones, the plasma protein SHBG, and the tumor markers prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) were assayed at 18 time-points over 1 month during a double-blind randomized study in 36 stage D2 cancer patients receiving either "buserelin+placebo" or "buserelin+nilutamide (Anandron)": LH, FSH, estradiol, testosterone, dihydrotestosterone, androstenedione, 5-androstene-3,17 beta-diol, dehydroepiandrosterone and its sulfate, cortisol, 17 alpha-hydroxyprogesterone, pregnenolone, 17 alpha-hydroxypregnenolone, and 3 alpha-androstanediol glucuronide. Multivariate analysis of the treatment values (over 10,000 assays) by two complementary methods, correspondence factorial analysis (CFA) and the minimum spanning tree (MST) method, identified those variables within the pathways of androgen metabolism that were correlated over time and, in a comparison of the two treatment groups, identified the enzyme targets of nilutamide action in humans. Whereas nilutamide tended to decrease androstenedione slightly, it affected no other variables including cortisol, except for pregnenolone, 17 alpha-OH-pregnenolone, and 17 alpha-OH-progesterone, which were increased. These increases are indicative of weak inhibition of C17,20 lyase by nilutamide, but, according to the multivariate analysis, are insufficient to account for the more marked and rapid fall in PAP and PSA noted on addition of nilutamide to buserelin that must therefore be explained by another mechanism such as androgen receptor blockade by nilutamide.

The response profiles of 36 para-substituted diphenylethylenes (DPEs) and triphenylacrylonitriles (TPEs) have been compared by multivariate analysis. The responses measured were (a) relative binding affinity (RBA) for the cytosol estrogen receptor (ER), (b) ability to promote the growth of the human MCF7 breast cancer cell-line, (c) cytotoxicity in MCF7 cells, and (d) ability to stimulate or inhibit protein kinase C (PKC) III activity under three different conditions of enzyme activation. The prime object of the analysis was to observe the simultaneous influence of diverse combinations of substituents on all these in vitro responses. To do this, the minimum spanning tree (MST) method was used to organize the molecules into a network in which proximate molecules are closely related with regard to their responses whereas remote molecules are distinct. The MST of this population of molecules had four main branches. E2 and its TPE mime were located in a central position within the trunk whereas the tips of the branches tended toward molecules of different specificity, i.e., cytotoxic molecules that bind to ER and interfere with PKC, noncytotoxic molecules that also bind to ER and interfere with PKC but promote cell growth, molecules only active on PKC, and molecules active on all parameters except PKC stimulation. A parallel MST analysis of the relationships among the response parameters themselves confirmed previous conclusions: For this population of molecules, RBAs for ER are fairly closely related to ability to promote MCF7 cell growth and only little to cytotoxicity (Bignon et al. J. Med. Chem. 1989, 32, 2092). Cytotoxicity is much more clearly correlated with inhibition of diacylglycerol-stimulated PKC activity than with RBAs for ER. PKC inhibition differs substantially depending upon whether the substrate is H1 histone or protamine sulfate.

We have performed a systematic study of the interaction of 36 di- and tri-phenylethylene derivatives (DPEs and TPEs) with protein kinase C (PKC). The results were submitted to a multivariate analysis in order to identify the structural features that might be implicated in interference with the activity of three PKC subspecies under three enzyme activation conditions. Four groups of test-compounds, each with common chemical features, could be distinguished clearly. The first group comprised all TPEs substituted with at least one basic dialkylaminoethoxy side-chain. These inhibited type alpha, beta and gamma PKC subspecies activated by Ca2+ and phosphatidylserine (PS) with or without diolein (DO) at micromolar concentrations but did not inhibit protamine sulfate phosphorylation. The other effectors, which all possessed a 1,1-bis-(p-hydroxyphenyl) ethylene moiety, influenced PKC activity at high concentrations (30-200 microM) and could be divided into two groups. One group constituted PKC inhibitors in the TPE series and inhibited PKC activated by Ca2+, PS and DO, as well as protamine sulfate phosphorylation. The other group constituted dual-type inhibitors/activators in the DPE series and stimulated PKC in the presence of Ca2+ and low PS concentrations but inhibited the enzyme in the simultaneous presence of DO. The fourth group of compounds was inactive and had, for the most part, one or two substituents with weak steric hindrance. In agreement with previous data for six lead compounds, this study suggests that, in these chemical series, a basic amino side-chain leads to interaction with phospholipid and the regulatory domain of PKC, whereas a 1,1-bis-(p-hydroxyphenyl) ethylene moiety leads to interaction with the catalytic domain of the enzyme.

The biological activities of promegestone (R 5020), used world-wide as a radioligand for the progestin receptor, are described. Promegestone is a potent progestin devoid of androgenic side-effects and has marked anti-estrogenic activity. Its therapeutical effectiveness (0.125 mg or 0.250 mg) in luteal insufficiency has been established in a double-blind clinical trial versus dydrogesterone (10 or 20 mg) which revealed its significantly superior potency (p less than 0.001). On the basis of the pharmacological data described, its use in cancer therapy according to a sequential protocol for the treatment of hormone-dependent breast cancer and in psychiatry to palliate the psychological disorders of the premenstrual syndrome and the perimenopause seems promising.

The relative binding affinities of 48 steroids for four classes of hormone receptor (progestin, PR; androgen, AR; glucocorticoid, GR; mineralocorticoid, MR) have been analyzed by correspondence analysis. The steroids were, for the most part, derivatives of nortestosterone, differing by their degree of unsaturation, by the presence or absence of a 17 alpha-ethynyl group, and by the length of the C-13 alkyl substituent. Derivatives of norprogesterone were included as reference compounds. Distribution maps visualizing the results of the mathematical analysis revealed that the majority of the test steroids were within the zone of influence of AR and PR and had limited affinity for GR and MR. Overall lack of specificity and enhanced affinity for GR and MR were induced by increasing unsaturation and by the presence of a C-13 ethyl group. The general and specific conclusions of the analysis confirm and extend previous intuitive and partial interpretations of the data. Correspondence analysis, however, has the advantage of taking into account the sum total of the available information, without any preconceived notion of the relative importance of a specific structural feature or biological parameter and, furthermore, enables simultaneous representation on a single graph of the receptor and steroid fields. The present example demonstrates the use of this type of methodology in processing routine screening data involving multiple parameters.

The relative binding affinities of over 30 steroids have been measured for the cytosol glucocorticoid receptor (GR) of thymus, liver, and hepatoma tissue culture cells and for progestin, androgen, and mineralocorticoid receptors. The data have been analyzed by correspondence analysis to reveal the singularities among the receptors of different hormonal classes, the similarities in GR of different origins, and the different specificities of the ligands. Additional data on new steroids have been injected into the system as well as results on a further parameter, namely the induction of tyrosine aminotransferase (TAT) activity, to illustrate the power and flexibility of the methodology. The analysis has confirmed previous correlations between GR binding and TAT response but also highlighted the antiglucocorticoid activity of progestins. This method should prove to be a substantial aid to the interpretation of increasingly complex data, in particular with regard to the action of existing and newly synthesized steroids on glucocorticoid systems of differential sensitivity.

The design of a new drug is conditioned by knowledge of the biochemical mechanisms involved in the etiology of the disease to be treated. With regard to endocrine pathologies, such knowledge can be obtained in the clinic from systematic assays of urinary and plasma hormones, enzyme activities and target tissue receptor concentrations. The present paper describes the results of our assays of plasma 3 alpha-androstanediol glucuronide, 5 alpha-reductase and androgen receptor in prostate cancer patients. The activity of the nonsteroid antiandrogen anandron is discussed in relation to these parameters: anandron may inhibit slightly adrenal androgen biosynthesis but, in particular, counters the action of these adrenal androgens on the prostate. It does not inhibit rat prostate 5 alpha-reductase activity but interacts with androgen receptor to exert an antiandrogen action.

Paromomycin binds specifically to a single type of binding site on the 70-S streptomycin-sensitive Escherichia coli ribosome. This site is different from that of dihydrostreptomycin since paromomycin binds to streptomycin-resistant ribosomes and sine dihydrostreptomycin does not compete for paromomycin binding. Paromomycin binding, unlike dihydrostreptomycin binding, is independent of changes in ribosome concentration but influenced by magnesium ion concentration. Moreover, paromomycin does not bind to the 30-S subunit of the streptomycin-sensitive ribosome, except in the presence of dihydrostreptomycin, which probably induces the conformational changes necessary for a paromomycin binding site. This induction does not occur with streptomycin-resistant ribosomes. Neither antibiotic binds to the 50-S subunit. In general, binding of the one antibiotic increases the number of sites available for binding of the other. Both antibiotics exhibit marked non-specific binding at high antibiotic/ribosome ratios. Competition studies have enabled the classification of other aminoglycosides according to their ability to compete for the paromomycin and dihydrostreptomycin binding sites. Derivatives structurally related to paromomycin compete for its binding, the degree of competition being related to antibacterial activity, but do not compete for dihydrostreptomycin binding; they, on the contrary, increase the number of dihydrostreptomycin binding sites. Neither gentamicin nor kanamycin derivatives, which induce a high level of misreading, nor kasugamycin and spectinomycin, which do not induce misreading, compete for paromomycin or dihydrostreptomycin binding sites. Other sites may be involved in the binding of these aminoglycosides and in inducing misreading.

To determine the hormone-dependence of a tumor, it is preferable to use highly specific radiolabeled ligands when available, since often more than one class of steroid hormone receptor is present in the tissue specimen, and interference from classes other than the one under study cannot be readily eliminated. In this study, we describe a simple in vitro system used to define the molecular requirements for a highly specific interaction between a steroid and the receptor corresponding to a single class of hormone. It is based on the use of homogenate or crude 105,000 X g supernatant prepared from the target organs considered as end points in routine biological potency tests and on the use of available radioligands not bound by plasma proteins (tags) to single out the receptors. For each receptor singled out in the target organ cytoplasm, the ability of over 700 molecules to decrease bound radioactivity was compared to that of the natural hormone (relative binding affinity) with the use of a dextran-coated charcoal technique to separate bound from unbound steroid. On the basis of the results on 81 molecules, presented in this study, the effect of various substituents on the affinity and specificity of the natural hormones was determined. Molecules interacting markedly with several receptors were submitted to X-ray crystallography in order to establish whether overlap between the various conformations of the natural hormone and of the test molecule might not partly account for lack of specificity.

In a study of a series of 26 triphenylacrylonitrile derivatives (TPEs), we investigated the influence of several possibly interrelated factors on the proliferation of human breast cancer cell lines. (1) Chemical substituents: the test compounds were for the most part para-hydroxylated with increasingly bulky hydrophobic and/or basic side chains [isopropyloxy or (diethylamino)ethoxy] or standard reference compounds. (2) Relative binding affinities (RBAs): they competed diversely for [3H]estradiol (E2) binding to calf uterus cytosol and little, if at all, for binding to the [3H]tamoxifen-labeled antiestrogen binding site (AEBS) in lower speed supernatant. A multiparametric comparison of RBAs recorded for calf, rat, and mouse uterus cytosol estrogen receptor (ER) revealed a possible influence of species-specific receptor conformation and/or environment on binding. (3) Estrogen/antiestrogen potency: their stimulation and inhibition of the proliferation of the ER-positive human breast cancer cell line (MCF7) was measured. Compounds with only hydroxy substituents stimulated proliferation more markedly than methylated derivatives and had a maximum effect at 10(-11)-10(-6) M. Stimulation was related to the RBA for ER. Compounds with isopropyloxy or (diethylamino)ethoxy side chains only weakly stimulated MCF7 cell growth and more powerfully antagonized E2-promoted growth. The extent of inhibition depended upon the bulk of the side chain and could be reversed by 10(-7) M E2. Within the same concentration ranges, the test compounds were without effect on the BT20 ER-negative cell line. (4) Cytostatic and/or cytolytic activity: most compounds could arrest the proliferation of both MCF7 and BT20 cells at concentrations above 3 x 10(-6) M. This activity was thus independent of ER. Nevertheless, those compounds with a charged hydrophobic side chain, which were the most powerful antagonists of E2-promoted cell growth, were also the most cytotoxic. The overall results for all molecules on all parameters were submitted to a multivariate analysis (correspondence analysis) which revealed the progressive influence of increasing substitution by hydroxy and more bulky groups on the generation of antagonist activity and cytotoxicity.

The crystal structures of 21-(phenylseleno)progesterone (1), which binds with moderate affinity to the progestin receptor, and 17 alpha-(phenylseleno)progesterone (2), which binds hardly at all, have been studied in an attempt to explain these differences in affinity and to obtain further information on the location of the progestin receptor binding site with respect to the progesterone molecule. The crystal structures were refined by isotropic thermal approximation to R values of 0.082 for 1 and 0.084 for 2. The unusual 17 beta side-chain orientation of 2 with a C16-C17-C20-O20 torsion angle of +13 degrees compared to -7 degrees for progesterone would seem to preclude hydrogen bonding with the progestin receptor binding site and provides strong supporting evidence for the contention that this site is located above the beta face of the molecule. Any rotation of the C21 methyl group into a more appropriate position is furthermore impeded by the presence of the 17 alpha-phenylseleno substituent. On the other hand, some hydrogen bonding can occur in the case of 1 (C16-C17-C20-O20 = 31 degrees) despite the fact that the difference in torsion angle (24 degrees) with respect to progesterone is, in absolute values, greater than that for 2 (20 degrees). This is because the orientation of the 17 beta-acetyl side chain of 1 is directed above the beta face closer to the progestin binding site, as previously defined on the basis of data on a large number of molecules, and because the 21-phenylseleno substituent constitutes only limited steric hindrance to binding. Thus, the difference in affinity of these two compounds is entirely consistent with observations that the H-bond donor is located toward O20 in the beta region of C16.

An in-depth analysis of the kinetics of 5 alpha-reductase in human prostatic tissue gave findings inconsistent with the claim that the enzyme is michaelian. In both hyperplastic and malignant tissue, the time-course of the conversion of testosterone (T) into dihydrotestosterone (DHT) was non-linear under conditions ensuring less than 15% conversion of substrate and cofactor. An initial rapid phase of conversion was followed by a long steady-state phase. This time-dependent change in conversion rate was not due to enzyme denaturation, fast inhibition by substrate or product effects. It resulted from a true slow transient kinetic process induced in the reactive enzyme by the substrates. Under our experimental conditions at pH 5.5, 5 alpha-reductase appeared to undergo a conformational change from an initially highly reactive form to a less reactive form. Since this "hysteretic" behavior was correlated with apparently negative cooperativity in enzyme kinetics, we postulate that, as previously described for other key metabolic enzymes, regulation of 5 alpha-reductase activity in the prostate depends on the molecular flexibility of the enzyme and on changes in the cooperativity of different enzyme forms over time. This original non-michaelian behavior may explain the conflicting kinetics reported so far in the literature for this enzyme. The clinical implications of 5 alpha-reductase hysteresis and its involvement in the damping of DHT production within the prostate are discussed.

An exchange assay for the measurement of total cytoplasmic progestin binding sites has been developed on rabbit uterine cytosol using the highly potent progestin, R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) labelled to a high specific activity. This compound has several advantages over progesterone: it is not bound by plasma corticosteroid binding globulin; it has high affinity for the progestin receptor; it binds virtually as fast as progesterone to the receptor, but the complex formed dissociated 8 times slower; its binding is not displaced by more than 2% by compounds devoid of progestational activity (estrogens, testosterone, dexamethasone, aldosterone). Bound endogenous progesterone was exchanged by tritiated R 5020 in a time compatible with receptor stability. At 0 C, total exchange of filled sites occurred in less than 4 h; at this temperature the R 5020-receptor complex was stable for at least 28 h. The conformation of the R 5020-receptor complex was investigated in sucrose density gradients under various experimental conditions. Unlike progesterone, it was possible to detect a 7S peak in uterine cytosol obtained from rabbits injected with a tracer dose of [3H]R 5020 1 h prior to sacrifice.

The anomeric aminoglycosides, RU 21886 and RU 23468, which both have a 2-deoxystreptamine residue, stabilize 70S ribosomes to similar extents at low magnesium ion concentrations. Only RU 21886, however, has marked antibacterial and bactericidal activity and gives rise to a high level of misreading in cell-free protein synthesizing systems. It would thus appear that the ability to stabilize the association of the two ribosomal subunits does not necessarily lead to errors in translation.

11 beta-substituted steroids form a novel class of derivative for the study of ligand-receptor interactions. The present review describes the synthetic pathways leading to 11 beta-substituted norsteroids and the kinetics and specificity of their interaction with receptors of several hormone classes as determined in a routine screening programme. The biochemical data on the interaction of one of these compounds, RU 38486, a potent antihormone presently in clinical development, with the progestin (PR) and glucocorticoid (GR) receptors are briefly reviewed. The comparison of the 3D-structures of these antagonists with those of potent hormones can help to map the interaction sites with PR and GR and highlights the potential use of these molecules as labelling agents and molecular probes.