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T Ojasoo

Publications and source records attributed to T Ojasoo.

At least 37 records · Page 2Linked to original sources
PubMed1992

A model for the determination of the 3D-spatial distribution of the functions of the hormone-binding domain of receptors that bind 3-keto-4-ene steroids.

A method of comparing the hydrophobic clusters of proteins (hydrophobic cluster analysis, HCA) has revealed that the 3D-folding pattern of the hormone-binding domain (HBD) of steroid hormone receptors (SHRs) may have an unexpectedly high degree of analogy with the known 3D-crystal structures of proteins belonging to the serine proteinase inhibitor (SERPIN) superfamily, e.g. alpha 1-antitrypsin and ovalbumin. The present paper briefly reviews some of the biochemical evidence that supports the structural validity of the SERPIN model and shows how the model can be used to establish hypothetical 3D-locations for functions attributed to different amino-acids or peptide sequences of the HBD: i.e. heat-shock protein binding, transcription activation, phosphorylation, steroid binding, but also ATP-binding. Indeed, the model has enabled the identification of a Rossmann-fold in SHRs that might bind ATP. Visualization of all these functions should help to interpret the chain of concerted events induced by steroid binding.

Amino Acid Sequence↗
PubMed1991

Influence of di- and tri-phenylethylene estrogen/antiestrogen structure on the mechanisms of protein kinase C inhibition and activation as revealed by a multivariate analysis.

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.

Animals↗
PubMed1990

Hydroxylated triphenylacrylonitriles adopt a unique orientation within the binding site of the estrogen receptor.

The relative binding affinities of a series of twelve para-hydroxylated triphenylethylenes (TPEs) for the estradiol receptor (ER) of calf uterus cytosol were measured by a competition method. The results obtained under equilibrium conditions support the hypothesis of the additivity of the energies corresponding to each of the hydrogen-bond type interactions of di- or tri-hydroxylated TPEs with the estradiol binding site of ER and strongly suggest that, whichever ring is hydroxylated, the orientation of the TPE in the steroid binding site is always the same. A hydroxyl group in a given position always interacts with the same location within the site. Mono-hydroxylation of the highly hydrophobic non-substituted TPE skeleton led to a large increase in relative binding affinity for ER which could be explained by a dual mechanism whereby the interaction specific to the hydroxyl is accompanied by a temperature- or time-dependent binding process that is not related to the hydroxylation position.

Animals↗
PubMed1989

Hydrophobic cluster analysis (HCA) of the hormone-binding domain of receptor proteins.

A new technique of protein sequence analysis, namely, Hydrophobic Cluster Analysis (HCA), has been used to align and compare the sequences of proteins belonging to the receptor superfamily (steroid, thyroid hormone and retinoic acid receptors) and serpin superfamily (corticosteroid binding globulin (CBG) and alpha 1-antitrypsin (alpha 1-AT]. By matching up clusters of hydrophobic amino-acids that oftenmost correspond to identifiable secondary structures (alpha-helices, beta-strands etc.), it has been possible to deduce the following information on the secondary structures of these proteins: CBG is structurally related to alpha 1-AT (HCA score greater than 80%), the structures of the hormone-binding domains of the steroid receptors that bind 3-keto-delta 4-steroids are closely interrelated (greater than 80%) but less closely related to that of the estrogen receptor (ER) (approximately 75%), vitamin D, retinoic acid and thyroid hormone receptors are structurally closely related (greater than or equal to 80%). Their secondary structures are, however, also related to that of the steroid receptors (approximately 70%), and a high degree of analogy exists between the structures of serpins and of the hormone-binding domains of members of the steroid superfamily (60-70%). HCA has clearly shown that a previous local sequence alignment of the estrogen receptor with other steroid receptors and cytochromes P450 has to be reconsidered. The published consensus steroid binding sequence previously identified in cytochromes is in fact 80 amino-acids upstream from its previously defined position. Other regions of contiguous sequence identity have also been identified which may be involved in the hydrophobic core of the protein or in steroid binding. Their positions have been indicated using the crystal structure of alpha 1-AT as a model.

Amino Acid Sequence↗
PubMed1989

Non-michaelian behavior of 5 alpha-reductase in human prostate.

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.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1989

Effect of triphenylacrylonitrile derivatives on estradiol-receptor binding and on human breast cancer cell growth.

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.

Animals↗
PubMed1988

11 beta-substituted steroids, an original pathway to antihormones.

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.

Animals↗
PubMed1988

Binding of steroids to the progestin and glucocorticoid receptors analyzed by correspondence analysis.

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.

Animals↗
PubMed1987

Towards the mapping of the progesterone and androgen receptors.

At a time when the secondary structures of receptor proteins are being predicted from sequence data by modeling techniques, knowledge of the ligand characteristics compatible with high-affinity binding to the receptor and with efficient receptor function is indispensable. We have already compared progesterone receptor (PR) ligands in attempts to map the PR hormone-binding site. In the present study, the relative binding affinities (RBAs) of 33 steroid ligands for the cytosol androgen receptor (AR) of rat prostate, measured in a routine screening system, have been compared. Special emphasis has been given to the effects of modifications (unsaturation, methylation, substitution by halogens) that might influence AR recognition by the ring A carbonyl and also to the consequences of these changes on binding specificity. Nonsteroid antiandrogens are reputed to compete with labelled testosterone (or methyltrienolone) binding to AR. Their RBAs, however, are very low compared to those of steroid antiandrogens. It is feasible that such molecules might occupy and interact with the AR site that binds hormone. The solvent accessible surface of one Anandron conformer is highly similar to that of testosterone and this conformer can be adequately superimposed upon the structure of testosterone and of antiandrogenic Des-A steroid derivatives. The nitro group might assume the role of the ring A carbonyl of steroids; reduction of this group to an amine or a hydroxylamine completely suppresses binding. These observations, however, do not eliminate the hypothesis of interference with AR function, and consequent antiandrogenic activity, by interaction with other (adjacent) sites on AR.

Androgen Antagonists↗
PubMed1987

Design of antiandrogens and their mechanisms of action: a case study (anandron).

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.

Androgen Antagonists↗
PubMed1986

Correspondence analysis applied to steroid receptor binding.

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.

Chemical Phenomena↗
PubMed1984

Influence of new hydroxylated triphenylethylene (TPE) derivatives on estradiol binding to uterine cytosol.

Twelve homologous triphenyl acrylonitrile derivatives with a p-OH or p-CH3 group on one or more of the phenyl rings were synthesized in order to assess the relative influence of each position on binding to the estrogen receptor (ER) and on inhibition of prostaglandin synthetase (PGS). Their relative binding affinities (RBAs) for [3H]estradiol (E2)-labeled ER were compared at 0 and 25 degrees C in mouse and rat uterus cytosol with those of tamoxifen derivatives, cyclofenil and diethylstilbestrol. RBAs in both species were closely correlated (r = 0.92) although the RBAs were about twice as high in the mouse as in the rat. The unsubstituted skeleton had an RBA of much less than 0.1 (estradiol = 100). An OH-group in R1 or R2 (Fig. 1) engendered very low affinity whereas an OH-group in R gave rise to a compound with an RBA equivalent to that of E2, emphasizing the importance of this position in the interaction with ER. Compounds with an additional OH-group in R1 or R2 were significantly better competitors than E2. No further increase in RBA was noted with the trihydroxy derivative. The effect of the introduction of a hydrophobic CH3-group decreased affinity as expected in R, but also in position R1 unless a second OH-group was present in R2. None of the 12 test-compounds competed significantly for binding to the "anti-estrogen binding site" in rat kidney supernatant. Although polar groups were not necessary for inhibition of PGS, inhibition was enhanced by the presence of a hydroxy group in R or R1 (but not R2). Even greater inhibition was obtained by the further introduction of a CH3-group in R1 or R respectively. The conformations of these derivatives are compared to those of known estrogen ligands and anti-inflammatory agents in order to obtain further information on these protein recognition sites.

Acrylonitrile↗
PubMed1983

Additional conformational data for the mapping of the progestin binding site: crystal structures of 21-(phenylseleno)progesterone and 17 alpha-(phenylseleno)progesterone.

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.

Crystallization↗
PubMed1983

[Promegestone, a new progestin].

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.

Animals↗