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S Moslemi

Publications and source records attributed to S Moslemi.

11 recordsLinked to original sources
PubMed2000

New aromatase inhibitors. Synthesis and biological activity of aryl-substituted pyrrolizine and indolizine derivatives.

We report herein the design and the synthesis of some aryl-substituted pyrrolizine and indolizine derivatives, on the basis of a hypothetical pharmacophore structure designed to fit the catalytic site of the human cytochrome P450 aromatase. The in vitro biological evaluation of these compounds allowed us to point out two new potent non-steroidal aromatase inhibitors, MR 20494 and MR 20492, with IC50 values in the range of 0.1 microM.

Aromatase Inhibitors↗
PubMed1998

Design and synthesis of a new type of non steroidal human aromatase inhibitors.

The structure-activity relationship study of one of recently described aromatase inhibitors, compound 1 (MR20814), allowed us to design some related derivatives as potential new inhibitors. Among those we synthesized, chlorophenylpyridylmethylenetetrahydroindolizinone 5 (MR20492) exhibited in vitro a ten-fold higher inhibition of the enzyme (IC50 = 0.2 +/- 0.0 microM and Ki = 10.3 +/- 3.3 nM).

Aromatase Inhibitors↗
PubMed1997

Purification and characterization of equine testicular cytochrome P-450 aromatase: comparison with the human enzyme.

Cytochrome P-450 aromatase was purified by five chromatographic steps from adult stallion testis. It was first separated from NADPH-cytochrome P-450 reductase (reductase) on omega-aminohexyl-Sepharose 4B then purified to homogeneity on concanavalin A-Sepharose 4B, hydroxyapatite-Sepharose 4B, DEAE-Sepharose CL-6B and on a second hydroxyapatite-Sepharose 4B. On the other hand, purifications of the equine testicular and rat liver reductases, which allowed the reconstitution of aromatase activity in vitro, were achieved for each species in one chromatographic step on an adenosine 2',5'-diphosphate-agarose affinity column. Analysis on SDS/PAGE indicated single bands with apparent molecular masses of 53, 82, and 80 kDa for purified equine testicular cytochrome P-450 aromatase (eAROM), equine testicular reductase and rat liver reductase respectively. eAROM shows a time- and concentration-dependent activity that was stable for at least 2 months when stored at -78 degrees C. It is a highly hydrophobic protein composed from 505 residues and direct sequencing of its N-terminal part showed good homology when compared with human aromatase. When deglycosylated by N-glycosidase-F the apparent molecular mass of eAROM was decreased from 53 to 51 kDa as revealed by electrophoresis, its activity, however, was not impaired. eAROM exhibits much higher affinity for androgens than for 19-norandrogens, Km values were approximately 3, 16 and 170 nM for androstenedione (A), testosterone (T) and 19-nortestosterone (19-NT) respectively. However, it aromatizes 19-norandrostenedione (19-NA) slightly more efficiently than A, the estrone (E1) formed was 4.27 vs 3.54 pmol min-1 micrograms-1 respectively (P < 0.01). After incubation of eAROM with radiolabelled A and separation of steroids on HPLC, E1, 19-hydroxyandrostenedione (19-OHA) and 19-oxoandrostenedione (19-oxoA) were accumulated in the incubation medium in a time-dependent manner. The presence of 4-hydroxyandrostenedione (4-OHA), a suicide inhibitor of aromatase, cause a time-dependent inactivation of the enzyme. Whereas the activity of eAROM was unchanged in the presence of K+ (up to 250 mM), it was increased in the presence of EDTA (up to 50 mM) and decreased in the presence of DTT or Mg2+ (from 25 mM). We conclude that: (a) eAROM is a glycoprotein, however, deglycosylation by N-glycosidase-F does not appear to impair its activity, (b) eAROM aromatizes really both androgens and 19-norandrogens having a higher affinity for androgens, (c) the intermediary compounds of aromatization 19-OHA and 19-oxoA appear to be synthesized by the same active site that synthesizes E1 as the final product, (d) the inhibition of eAROM by increasing concentrations of Mg2+ and the stimulation of its activity by EDTA, taken together, indicate the importance of negatively charged residues in the polypeptide chain of equine aromatase, which play a role in enzymatic activity.

Amino Acid Sequence↗
PubMed1997

Inhibition and inactivation of equine aromatase by steroidal and non-steroidal compounds. A comparison with human aromatase inhibition.

In order to approach the detailed structure-function relationships of aromatase, we studied the inhibitory and inactivatory potencies of several steroidal androstenedione analogues (1: 4-hydroxyandrostenedione, 2: 4-acetoxyandrostenedione and 3: 7 alpha-(4'-amino)phenylthio-4-androstene-3, 17-dione) and non-steroidal imidazole derivatives (4: ketoconazole, 5: miconazole and 6: fadrozole) on equine aromatase in placental microsomes, a well established mammalian model. Human placental microsomes and the purified enzyme from equine testis were also used to compare inhibition by 1 and 2. In equine microsomes, all compounds tested exhibited a competitive inhibition, with Ki values of 4.1, 26 and 1.8 nM for 1, 2 and 3, and of 2400, 1.4 and 4 nM for 4, 5, and 6, respectively. The Km for androstenedione, the substrate mainly used in these studies, was 1.8 +/- 0.13 nM. The three non-steroidal derivatives did not inactivate equine aromatase, but 1 and 2 acted as comparable inactivators to a much higher degree than 3. Compound 1 inhibited in a similar manner (89-94%) purified or equine and human microsomal aromatases, whereas 2 inhibited microsomal aromatase more efficiently in the horse than in man (92% and 33% inhibition, respectively). There was only a 40% inhibition with 2 on the purified equine enzyme, which is no more in the natural membrane environment. The comparisons between equine and human microsomal aromatases allow precise functional and structural differences to be observed with these enzymes.

Androstenedione↗
PubMed1996

Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro.

Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water release from labelled estradiol and testosterone. Kinetic analysis of estradiol 2-hydroxylation showed an apparent K(m) of 23 microM and a V(max) of 18 nmol/min/mg, whereas the values for testosterone aromatization were a K(m) of 15.7 nM and a V(max) of 34.6 pmol/min/mg. A specific antiserum raised against purified testicular equine P450arom and known to inhibit aromatase activity [1] was also found to inhibit the estrogen hydroxylase activity of equine placental microsomes in a dose-dependent manner with an IC50 value of 15 microl serum: 0.5 ml incubate. The estrogen hydroxylase activity was inhibited in a dose-dependent manner by two classes of aromatase inhibitors, i.e. steroidal-- (4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione)--and non-steroidal--(fadrozole and miconazole). The IC50 values were approximately 300 and 890 nM for 4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione, and 92 and 285 nM, for fadrozole and miconazole, respectively. Furthermore, 4-hydroxyandrostenedione caused a time-dependent inactivation of estrogen hydroxylase activity. We conclude that equine aromatase is able to use estradiol as a substrate, and converts it to catechol estradiol in vitro, possibly using the active site of aromatization. This is the first demonstration that equine aromatase functions as an estrogen 2-hydroxylase, in addition to transforming androgens into estrogen.

Androstenedione↗
PubMed1995

In vitro 19-norandrogen synthesis by equine placenta requires the participation of aromatase.

Explants of equine full-term placenta have been shown to synthesize 19-norandrogens from labelled androgens. Steroid metabolites were purified by silica-gel column chromatography then analysed and quantified by c18-reverse-phase HPLC coupled to a radioactive flow detector. 19-Norandrostenedione was subsequently recrystallized to constant specific activity, providing unequivocal evidence of its synthesis by the equine placenta. 19-Norandrostenedione synthesis appeared to be localized in the microsomal fraction. Regardless of the substrate used, formation of 19-norandrogens was far weaker than that of oestrogens; moreover, the yield of 17-oxosteroids produced was much greater than that of 17 beta-hydroxysteroids, suggesting the presence of a dehydrogenase with predominant oxidative activity. Sulphoconjugated steroids formed were less than 0.5% of total steroids. Although 19-nortestosterone could not be generated by equine purified aromatase incubated with labelled testosterone, the synthesis of 19-norandrogens and oestrogens by equine placental explants was blocked by two specific aromatase inhibitors, 4-hydroxyandrostenedione and fadrozole. Our results provide evidence for a placental origin of at least a part of the 19-norandrogens previously identified in the blood of the pregnant mare. Furthermore, it is suggested that 19-norandrogen biosynthesis would involve the enzymatic metabolism of 19-oxygenated androgens formed by equine aromatase.

Androstenedione↗
PubMed1993

Different in vitro metabolism of 7 alpha-methyl-19-nortestosterone by human and equine aromatases.

The ability of human and equine placental microsomes to aromatize 7 alpha-methyl-19-nortestosterone (MNT) was studied. Kinetic analysis indicates that MNT shares the androgen-binding site of human and equine placental microsomal aromatases. Human placental microsomal estrogen synthetase had about a 2.5-fold higher relative affinity for MNT than the equine placental enzyme (KiMNT/Km androstenedione of 32 versus 87). However, MNT was not metabolized by human placental microsomes, whereas it was very actively metabolized by equine placental microsomes. Further studies using purified equine cytochrome P-450arom indicated that the presence of a 7 alpha-methyl group and the absence of a C19 methyl group did not impair its conversion by the purified enzyme. The product of this reaction was separated and identified as 7 alpha-methylestradiol by gas chromatography coupled to mass spectrometry.

Animals↗
PubMed1989

Androgen and 19-norandrogen aromatization by equine and human placental microsomes.

The ability of equine and human placental microsomes to aromatize testosterone and 19-nortestosterone was studied. When 3 microM [1 beta,2 beta-3H]testosterone was used as substrate, the specific activity of equine placental microsomal aromatase was 2.5 times higher than that of the human microsomal enzyme. Although 19-nortestosterone was aromatized 67 times more rapidly by equine than by human aromatase, we found that equine aromatase exhibited a markedly weaker affinity for this substrate than did the human enzyme. Competitive inhibition of testosterone aromatization by 19-nortestosterone occurred with both equine and human aromatases. While having no effect on mare placental microsomes, Na+ and K+ (500 mM) stimulated testosterone aromatization by human placental microsomes by 73 and 52% respectively. If indeed a single enzyme is responsible for the aromatization of testosterone and 19-nortestosterone, which seems to be the case in both equine and human placental aromatase, our results show that differences in the structure of the active sites exist between equine and human aromatases.

Animals↗
PubMedDate not supplied

MR 20492 and MR 20494: two indolizinone derivatives that strongly inhibit human aromatase.

In this study, we describe the synthesis of a new family of indolizinone derivatives designed to fit an extrahydrophobic pocket within the active site of aromatase and to strongly inhibit human aromatase. This could help improve the specificity of the inhibitors. Equine aromatase, very well characterized biochemically, is used as a comparative model. Indeed, in a previous comparison between both human and equine aromatases, we described the importance of the interaction between the inhibitor and this pocket for the indane derivative MR 20814. MR 20492 and MR 20494 are more potent inhibitors of human aromatase (Ki/Km: 1.0+/-0.3 and 0.5+/-0.3, respectively). The Ki/Km for MR 20494 is slightly higher than that obtained for fadrozole (0.1+/-0.0) and Ki/Km for both indolizinone derivatives are lower than those obtained for 4-hydroxyandrostenedione (1.9+/-0.8) and MR 20814 (8.1+/-.7). These new compounds are not enzyme inactivators. Moreover, as indicated by the higher Ki/Km values obtained with equine enzyme (9.0+/-0.6 and 6.1+/-1.6 for MR 20492 and MR 20494, respectively), both human and equine aromatase active sites appear to be structurally different. Difference absorption spectra study (350-500 nm) revealed that MR20492 and MR20494 were characterized by a combination of type-I and -II spectra with both enzymes. This result could be due to the isomerization of the molecule in polar solvent (Z and E forms). The evaluation of these new molecules, as well as 4-hydroxyandrostenedione and fadrozole, on aromatase activity in transfected 293 cell cultures evidenced a strong inhibition (IC50: 0.20+/-0.03 microM, 0.20+/-0.02 microM and 0.50+/-0.40 microM for MR 20494, fadrozole and 4-OHA, respectively) except for MR 20492 (3.9+/-0.9 microM) and MR 20814 (10.5+/-0.6 microM). These results proved that these molecules formed part of a promising family of potent inhibitors and that they penetrate 293 cells, without evidencing any cytotoxicity in Hela cells with MTT assay. This is thus encouraging for the development of new drugs for the treatment of estrogen-dependent cancers, these molecules also constitute new tools for understanding the aromatase active site.

Animals↗