Jacques Gilbert

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.

A chemical classification of nonsteroidal antagonists of sex-steroid hormone action

A highly varied collection of nonsteroids have been reported over the last forty years as being able to exert an antihormonal action versus steroid hormones in vivo. This diversity is partly explained by the manifold molecular targets of these compounds which may be either enzymes or receptors (leading to inhibition of steroid production and action respectively) and by the different possible levels of interference within feedback loops between the central nervous system, pituitary, gonads and other peripheral organs. The present chapter is a chemists classification of some of these structures often in the absence of detailed biochemical data. Nonsteroid antiestrogens (and estrogens) most often share a common feature with diethylstilbestrol and consequently the effects of structural modifications on biological activities can be studied in a rational manner. This is not the case for non-steroidal antiandrogens that we have only been able to classify into conventional chemical groups. Nor have any true lead compounds nor well-defined chemical classes been identified for nonsteroid antiprogestogens. This is however the only hormonal class where natural products play an important role.

Analogies and differences in the modulation of progesterone receptor induction and cell proliferation by estrogens and antiestrogens in MCF-7 human breast cancer cells: Study with 24 triphenylacrylonitrile derivatives

Structure-activity relationships in a homogeneous series of 24 triphenylacrylonitrile derivatives were examined with respect to the stimulation of progesterone receptor induction and cell proliferation in MCF-7 cells. In general, triphenylacrylonitrile derivatives were found to be full or partial agonists for both responses; the partial agonists were also able to antagonize the stimulatory action of estradiol. The agonistic activities of these molecules decreased as the size of the lateral side chain increased, but the side-chains correlated with partial agonism of progesterone receptor induction were bulkier than those correlated with partial agonism of cell proliferation. Agonistic and antagonistic effects on both responses were correlated with affinity for the estrogen receptor. Half maximal effects on the two responses occurred at different concentrations (4-fold) of the compounds. It can be concluded that in MCF-7 cells, triphenylacrylonitrile modulation of progesterone receptor induction and cell proliferation are mediated by the estrogen receptor; the two effects, which occur at different concentrations and with slightly different substituents of the compounds, are differentially modulated.

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.

Dual action of hydroxylated diphenylethylene estrogens on protein kinase C

Protein kinase C (PKC) I (gamma), II (beta) and III (alpha) subspecies are all activated by 1,1-di-(p-hydroxyphenyl)ethylene derivatives (DPE) at micromolar concentrations. This PKC activation depends on the presence of both Ca2+ and phosphatidylserine (PS) but does not require diacylglycerol (DG). DPEs enhance PKC activity at low PS concentrations, but not at saturating PS concentrations. Like DG, DPEs increase the apparent affinity of PKC for PS as well as for Ca2+, but lead to a decrease in the catalytic activity (Vmax). In the presence of saturating DG concentrations, DPEs exhibit an inhibitory action. The derivatives also inhibit the activity of the proteolytic fragment of PKC, protein kinase M. It is concluded that DPEs are mixed-type inhibitors, probably interacting with the catalytic domain of the enzyme.

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.