Tiiu Ojasoo

Steroid hormone receptors and pharmacology.

Abstract The cumbersome nature of many pharmacological tests has seriously limited the full-scale study of the hormonal activity profile of a large number of molecules. To overcome this handicap, a screening system has been devised based on the fact that (a) interaction with the relevant hormone receptor is the key step in triggering a particular hormone response (e.g. estrogenic, progestomimetic, androgenic, mineralocorticoid or glucocorticoid) and (b) the kinetics of the interaction define the nature of the response, either agonistic or antagonistic. The data we have so far obtained with over 900 compounds have been classified according to binding specificity and kinetics and correlated with observations on the crystalline or calculated conformations of chemically related compounds, in order to pave the way to new structure-activity relationships. However, although tailored compounds can thus be designed, their use as drugs depends, on the one hand, upon their pharmacokinetics and metabolism in man and, on the other, on receptor availability at the time of administration. At the present stage, highly specific ligands derived from this screening system are being used not only as drugs but as tools to study the physiological regulation of hormone receptors in both peripheral and central target tissues.

Steroid flexibility and receptor specificity

Unsaturated Δ4,9 and Δ4,9,11 steroid hormones with a keto group in position 3 and a hydroxy, lactone or oxathiolane function in 17β, with or without methyl substituents in positions 17α, 18 and/or 7α, tend to compete significantly for specific binding to the receptor proteins corresponding to several steroid hormone classes (progestin, androgen, mineralo- and glucocorticoid). If the interaction between ligand and receptor protein is compared to a lock-and-key fit, the presence of several common structural features among ligands binding to the receptor proteins of different hormone classes suggests a close affiliation among these proteins; the ability of any one ligand to bind effectively to more than one receptor implies a degree of conformational adaptability exceeding that of the natural hormones. The molecular flexibility and mobility of several Δ4,9 and Δ4,9,11 unsaturated steroids has been established by X-ray crystallography and by molecular geometry calculations and confronted with the corresponding binding specificity profiles. On the basis of these data, it is suggested that the lock-and-key image of complex formation is too static and that the overall steroid-cum-protein flexibility determines complex kinetics and biological response. Furthermore, in view of the limited amount of energy necessary for between-conformer transition (e.g. < 2 kcal for gestrinone for a flexibility range of 3.3 A) compared to the energy of binding to steroid receptors (KD = 10−9M − −12kcal/mol), the in vivo formation of complexes of varying stability with the different conformers of a steroid is quite feasible.

11β-substituted steroids, an original pathway to antihormones

Abstract 11β-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 β-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.

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.

How to analyze publication time trends by correspondence factor analysis: analysis of publications by 48 countries in 18 disciplines over 12 years

This study is a follow‐up to a published Correspondence Factorial Analysis (CFA) of a dataset of over 6 million bibliometric entries (Dore et al. JASIS, 47(8), 588– 602,1996), which compared the publication output patterns of 48 countries in 18 disciplines over a 12‐year period (1981–1992). It analyzes by methods suitable for investigating short time series how these output patterns evolved over the 12‐year span. Three types of approach are described: (1) the chi2 distances of the publication output patterns from the center of gravity of the multidimensional system—which represents an average world pattern—were calculated for each country and for each year. We noted whether the patterns moved toward or away from the center with time; (2) individual annual output patterns were introduced as supplementary variables into an existing global overview covering the whole time‐span [CFA map of (countries × disciplines)]. We observed how these patterns moved about within the map year by year; (3) the matrix (disciplines × time) was analyzed by CFA to derive time trends for each country. CFA revealed the “inner clocks” governing publication trends. The time scale that best fitted the data was not a linear but an elastic scale. Although different countries laid emphasis on publication in different disciplines, the overall tendency was toward greater uniformity in publication patterns with time.

The use of interaction kinetics to distinguish potential antagonists from agonists

Abstract The kinetics of the interaction between a ligand and the cytosolic steroid hormone receptor is an important factor in determining the nature and amplitude of the induced biochemical and biological responses and can be used as the basis of a screening system to distinguish potential antagonists from potent agonists for every class of steroid hormone.

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.

Non-michaelian behavior of 5α-reductase in human prostate

Abstract An in-depth analysis of the kinetics of 5α-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 pH5.5, 5α-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 5α-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 5α-reductase hysteresis and its involvement in the damping of DHT production within the prostate are discussed.

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.

Relative involvement of protein kinase C and of the estrogen receptor in the cytotoxic action of a population of triphenylethylenes on MCF7 cells as revealed by correspondence factorial (CF) analysis

A multivariate statistical method, correspondence factorial (CF) analysis, was used to examine the correlations among the protein binding and cell proliferation effects of a series of 36 di- and triphenylethylenes (DPEs and TPEs). The analysis was applied to a study which measured their competition for estradiol binding to cytosol estrogen receptor (ER), their influence on protein kinase C (PKC) activity under different conditions of enzyme activation, their ability to promote the growth of a breast cancer cell line and to inhibit growth at high concentrations (cytotoxicity). The CF analysis revealed several levels of correlation. First, it distinguished those molecules within the population that stimulated rather than inhibited PKC activity. Second, it made apparent a strong correlation between cytotoxicity and inhibition of Ca++ and phosphatidylserine-dependent PKC activity, which was most marked when the enzyme had been activated by diacylglycerol indicating that PKC inhibition under physiological conditions might contribute to the overall cytotoxicity of these compounds. Third, a lower level of correlation was established between competition for ER binding and cytotoxicity. Taken together, the results suggest that MCF7 cells might be most sensitive to a cytotoxic effect of TPEs (via PKC and other targets) when they at the same time decrease estrogen-stimulated proliferation via an ER-mediated antiestrogenic effect.