Discovery of a novel class of aldol-derived 1,2,3-triazoles: potent and selective inhibitors of human cytochrome P450 19A1 (aromatase)

The discovery of a novel five-component 1,2,3-triazole-containing pharmacophore that exhibits potent and selective inhibition of aromatase (CYP 450 19A1) is described. All compounds are derived from an initial aldol reaction of a phenylacetate derivative with an aromatic aldehyde. Structure-activity data generated from both syn- and anti-aldol adducts provides initial insights into the requirements for both potency and selectivity.     

Biochemical functions of RIB5 and RIB6 gene products participating in riboflavin biosynthesis in the yeast Pichia guilliermondii

The biosynthesis of riboflavin precursor 6,7-dimethyl-8-ribityllumazine was studied in extracts of Pichia guilliermondii yeast mutants of rib5 and rib6 genotypes with impaired synthesis of proteins P1 and P2, respectively. It was shown that synthesis of 6,7-dimethyl-8-ribityllumazine took place in extracts of rib5 mutant (active P1 protein) in the presence of 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and the compound formed from ribose-5-phosphate by extracts of rib6 mutant (active P2 protein). No lumazine was formed in extracts of rib6 mutant from pyrimidine substrate and ribose-5-phosphate preincubated with extracts of rib5 mutant. Hence, P1 protein (the product of RIB5 gene) participates in the biosynthesis of 6,7-dimethyl-8-ribityllumazine from 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and aliphatic intermediate which is formed from ribose-5-phosphate, under the action of P2 protein (the product of RIB6 gene).     

On the inhibitory action of 29 drugs having side effect gynecomastia on estrogen production

To examine the influence on aromatase and sulfatase pathways in estrogen pool by drugs reported to cause gynecomastia as the side effect, 29 ethical drugs were incubated with human placental microsomes as an enzyme source. The percent inhibition of drugs on aromatase pathway was obtained by sum of the velocity constants of two products, estrone (E1) and estradiol (E2) from testosterone (T) as the substrate, and that on sulfatase pathway was obtained as the velocity constant of production of E1 from estrone sulfate (E1S). Although several drugs including ketoconazole showed a significant inhibition effect on aromatase pathway at their non-clinical over-dose concentration (100 microM), no influence on the inhibition was observed in any drugs at their approximately therapeutic concentration (1 microM). However, several drugs including spironolactone gave the product ratio (E2/E1) having higher value than that of the control, the result means spironolactone inhibits the conversion of E2 to E1. No inhibitory effect of the drugs tested on estrogen production from E1S (sulfatase pathway) was confirmed. The results suggest the possibility that the tested drugs known to cause gynecomastia have no inhibitory effect essentially on aromatase and sulfatase pathways.     

Diverse function of aromatase and the N-terminal sequence deleted form

The diverse function of human placental aromatase including estradiol 6-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6,7-³H₂,4-¹⁴C]estradiol, 7-ethoxycoumarin, and [N-methyl-³H₃]cocaine. 6-Hydroxy[7-³H,4-¹⁴C]estradiol was isolated as the metabolite of estradiol and the ³H-water release method based on the 6-³H label was established. The initial rate kinetics of the 6-hydroxylation gave K_m of 4.3 μM, V_max of 4.02 nmol min⁻¹mg⁻¹, and turnover rate of 0.27 min⁻¹. Testosterone competed dose-dependently with the 6-hydroxylation and showed the K_i of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed K_m of 200 μM, V_max of 12.5 nmol min⁻¹mg⁻¹ and turnover rate of 1.06 min⁻¹. The N-demethylation of cocaine was analysed by the ³H-release method, giving K_m of 670 μM, V_max of 4.76 nmol min⁻¹mg⁻¹, and turnover rate of 0.49 min⁻¹. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min⁻¹mg⁻¹ (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min⁻¹.     

Stereoselective Syntheses of (-)-Podorhizol Lignan and its Derivatives: erythro and threo Preferential Aldol Condensation of Potassium Enolate from γ-Butyrolactone with Alkoxybenzaldehyde

(-)-Podorhizol (1) was stereoselectively synthesized by erythro preferential aldol condensation of 3,4,5-trimethoxy- benzaldehyde with potassium enolate from (+)-(R)-3- (3,4-methylenedioxybenzyl)-4-butanolide (2) (erythro:threo=85:15). Erythro selectivity was observed in the aldol condensation of many alkoxybenzaldehydes with potassium enolate from (+)-γ-butyrolactone 2. However, benzaldehydes having methoxy groups at both the 2 and 6 positions gave threo selectivity in the aldol condensation with potassium enolate from (+)-γ-butyrolactone 2.     

Immunoaffinity purification of aromatase cytochrome P-450 from human placental microsomes, metabolic switching from aromatization to 1 beta and 2 beta-monohydroxylation, and recognition of aromatase isozymes

Microsomal estrogen synthetase (aromatase) cytochrome P-450 was purified from fresh human placental microsomes by monoclonal anti-aromatase P-450 antibody-Sepharose 4B chromatography. The purified P-450 showed a single band of 55 kDa on SDS-polyacrylamide gel electrophoresis and the aromatase specific activity on reconstitution was 70 nmol/min/mg protein. The purified P-450 was stable with a t 1/2 of approximately 2 years on storage at -90 degrees C and showed Km = 43 nM for androstenedione aromatization. However, it was unstable under spectral measurement conditions in the presence of sodium dithionite and carbon monoxide and the carbon monoxide difference spectra showed a maximum at 450 nm and a specific content of 9.1 nmol of P-450/mg protein, giving a turnover number of approximately 7.7 per min for the purified aromatase. The one-step immunochemical purification method gave a 490-fold increase of specific activity with 55% yield of aromatase activity of the original microsomes. Analysis of androgen metabolism by the purified aromatase and an apparent large kinetic isotope effect found at the secondary positions when using [19(-3)H3, 4(-14)C] androgens revealed metabolic switching from the first 19-hydroxylation to 1 beta- and 2 beta- monohydroxylation by aromatase. Substrate specificity for [19(-3)H3]androstenedione and testosterone was indicated by differences in the extent of metabolic switching (18% and 30%) and in the 2 beta/1 beta ratio (60/40 and 10/90, respectively). The mouse monoclonal antibody used for immunoaffinity purification suppresses aromatase activity of human placenta, but was totally ineffective for aromatase in goldfish brain and rat ovary. Rabbit polyclonal antibodies to human placental aromatase P-450 suppressed both human placental and rat ovarian aromatase but were ineffective for goldfish brain aromatase. The study indicates that they are isozymes of aromatase based on different structures of P-450.     

Regulation of steroid and steroid sulfate production and aromatase activity in cultured human granulosa-luteal cells

The regulation of the production of steroids and steroid sulfates and the activity of aromatase in human luteinized granulosa cells were investigated. The cells were cultured for 48 h in the presence or absence of hCG and FSH. Basal production of pregnenolone (Pre, 0.3 +/- 0.03 ng/micrograms protein) and progesterone (P, 19.3 +/- 1.7 ng/micrograms protein) were high compared with that of other steroids beyond P in the steroidogenic pathway. The concentration of 17 alpha-hydroxyprogesterone (17-OHP) was lower 0.17 +/- 0.06 ng/micrograms and that of other steroids in the 4-ene and 5-ene pathways and steroid sulfates less than 0.05 ng/micrograms. Both hCG and FSH (100 ng/ml) stimulated the production of Pre and P 3- to 5-fold, but only minimal stimulation of other steroids and steroid sulfates was observed. Aromatase activity of granulosa-luteal cells was measured from the rate of formation of 3H2O from 1 beta-[3H]androstenedione (1 beta[3H]A) after exposing the cells to hCG, FSH or estradiol (E2) for 48 h. Basal aromatase activity was relatively low, but hCG and FSH stimulated aromatase 8- and 4-fold, respectively. The incubation of granulosa-luteal cells with E2 did not affect basal aromatase activity, but E2 augmented FSH-stimulated aromatase 1.4-fold (P less than 0.025). The results suggest that there is low 17 alpha-hydroxylase and steroid sulfokinase activity in human granulosa-luteal cells. Aromatase activity in these cells is regulated by both hCG and FSH, and intra-ovarian estrogens may regulate granulosa cell aromatase activity.     

Stereoselective inhibition of human placental aromatase

We have synthesized the (19R)- and (19S)-isomers (2 and 3 respectively) of 10 beta-oxiranylestr-4-ene-3,17-dione. The configurations and conformations of these compounds were established by X-ray crystallographic analysis. Each of these compounds is a powerful competitive inhibitor of human placental microsomal aromatase, and stereoselectivity of inhibition was observed (Ki values for 2 and 3 were 7 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of oxirane oxygen to the heme iron of the enzyme. The (19R)- and (19S)-10 beta-thiiranes (6 and 7) corresponding to 2 and 3 have been synthesized from the oxiranes by a stereospecific process. The thiiranes are very effective competitive inhibitors of placental aromatase, and show even greater stereoselectivity in binding than the oxiranes (Ki values for 6 and 7 were 1 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of thiirane sulfur to heme iron.     

Functional characterization of 102-amino acid-deleted form of human aromatase (delta102-aromatase).

A truncate form of human aromatase cDNA that corresponds to the recently identified rat cortical type aromatase mRNA variant (Yamada-Mouri et al., J. Steroid Biochem. Molec. Biol., 60: 325-329, 1997) has been generated, and the amino-terminus deleted form of the enzyme has been expressed in CHO cells. The resulting product lacking 102 residues from the N-terminus of aromatase (i.e. 102-aromatase) showed an extremely low enzyme activity using an 'In-cell' assay. A strong aromatase activity, however, was observed for the delta102-aromatase using an in vitro method on the solublized preparations. The in vitro activity was dependent on both incubation time and NADPH concentration as well as inclusion of NADPH-cytochrome P450 reductase in the assay mixture. The average turnover rate of aromatization of the reconstituted delta102-aromatase was 6.8 min(-1). The results of the immunosuppression assay suggested that delta102-aromatase still holds the epitope interactive to MAb3-2C2, a monoclonal antibody raised agaist human placental aromatase P450. Furthermore, the IC50 values of MAb3-2C2 were determined to be 24 and 23 microg/ml for the whole homogenate and the 105,000 x g precipitate fractions prepared from the truncated aromatase expressing cells, respectively, whereas an IC50 of 1.3 microg/ml was shown for the full-length human aromatase. These results indicate that the delta102-aromatase P450 can be expressed and is catalytically competent as the full-length enzyme, but the epitope structure for the monoclonal antibody MAb3-2C2 is altered from that of the native enzyme. In addition, the intracellular distribution of delta102-aromatase may be different from that of the wild-type enzyme, explaining why very low activity was measured using an 'In-cell' assay.     

Expression of human placental aromatase in Saccharomyces cerevisiae

A full-length human placental aromatase cDNA clone, Aro 2, was isolated upon screening a human placental cDNA library with an aromatase cDNA probe and an oligonucleotide probe whose sequence was derived from a human aromatase genomic clone. Nucleotide sequence microheterogeneity was found in the 3'-untranslated region among Aro 2 and in two previously described human aromatase cDNA clones. Both the minor sequence differences and the expression of a single protein species in placental tissue suggest the presence of different alleles for aromatase. Northern blot analyses using one cDNA and two oligonucleotide probes are consistent with the two mRNA messages of 2.9 and 2.5 kilobases arising in human placenta as a consequence of differential processing. Several yeast expression plasmids containing the aromatase cDNA we cloned were constructed. The enzyme was expressed in Saccharomyces cerevisiae. The expressed activity was inhibited by the known aromatase inhibitor, 4-hydroxyandrostenedione. A level of 2 micrograms aromatase/mg partially purified yeast microsomes was estimated by analyses of carbon monoxide difference spectra on microsomal fractions from yeast carrying plasmid pHARK/VGAL. Using [1 beta, 2 beta-3H]androst-4-ene-3,17-dione as the substrate, an apparent Michaels-Menken constant (Km) of 34 nM and a maximum velocity (Vmax) of 23 pmol [3H]water formed per min/mg protein were obtained for the yeast synthesized aromatase by transformation with plasmid pHARK/VGAL. The kinetic results are similar to those determined for human placental aromatase, and suggest that the yeast synthesized aromatase will be useful for further structure-function studies.     

Regulation of aromatase induction by nuclear receptor coregulator PELP1

Estradiol (E2), estrogen receptor (ER), ER-coregulators have been implicated in the development and progression of breast cancer. In situ E2 synthesis is implicated in tumor cell proliferation through autocrine or paracrine mechanisms, especially in post-menopausal women. Several recent studies demonstrated activity of aromatase P450 (Cyp19), a key enzyme that plays critical role in E2 synthesis in breast tumors. The mechanism by which tumors enhance aromatase expression is not completely understood. Recent studies from our laboratory suggested that PELP1 (Proline, Glutamic acid, Leucine rich Protein 1), a novel ER-coregulator, functions as a potential proto-oncogene and promotes tumor growth in nude mice models without exogenous E2 supplementation. In this study, we found that PELP1 deregulation contributes to increased expression of aromatase, local E2 synthesis and PELP1 cooperates with growth factor signaling components in the activation of aromatase. PELP1 deregulation uniquely up-regulated aromatase expression via activation of aromatase promoter I.3/II. Analysis of PELP1 driven mammary tumors in xenograft as well as in transgenic mouse models revealed increased aromatase expression. PELP1-mediated induction of aromatase requires functional Src and PI3K pathways. Chromatin immuno precipitation (ChIP) assays revealed that PELP1 is recruited to the Aro 1.3/II aromatase promoter. HER2 signaling enhances PELP1 recruitment to the aromatase promoter and PELP1 plays a critical role in HER2-mediated induction of aromatase expression. Mechanistic studies revealed that PELP1 interactions with orphan receptor ERRα, and histone demethylases play a role in the activation of aromatase promoter. Accordingly, ChIP analysis showed alterations in histone modifications at the aromatase promoter in the model cells that exhibit local E2 synthesis. Immunohistochemical analysis of breast tumor progression tissue arrays suggested that deregulation of aromatase expression occurs in advanced-stage and node-positive tumors, and that cooverexpression of PELP1 and aromatase occur in a sub set of tumors. Collectively, our results suggest that PELP1 regulation of aromatase represent a novel mechanism for in situ estrogen synthesis leading to tumor proliferation by autocrine loop and open a new avenue for ablating local aromatase activity in breast tumors.     

Asymmetric aldol reaction of enol trichloroacetate catalyzed by (S, S)-(EBTHI)TiCl(OMe)

(S,S)-Ethylenebis(tetrahydroindenyl)titanium chloride methoxide, (S, S)-(EBTHI)TiCl(OMe) (3) was synthesized from the corresponding titanium dichloride. The asymmetric aldol reaction of enol trichloroacetate of cyclohexanone 1 with aromatic aldehydes was studied in the presence of a catalytic amount of the chiral titanium complex 3, with the result that the optically active syn aldol adduct 2 was preferentially obtained with up to 91% ee.     

Studies on the role of catecholamines in the regulation of the developmental pattern of hypothalamic aromatase

Experiments were conducted to study the regulation of the developmental pattern of aromatase in the forebrain of the perinatal rat. Two experimental designs were used: aromatase measured in primary cultures of fetal hypothalamic cells and in cell-free preparations of forebrain tissue excised at varying ages. In cultured cells, aromatase decreased logarithmically at a slow rate (t1/2 = 7.8 days). Norepinephrine caused a pronounced dose (4 × 10⁻⁶ M) and time-dependent (2–6 days) drop in aromatase without affecting the levels of 5-reductase or substance P. In isolated tissue, aromatase activity was compared with the concentrations of norepinephrine and dopamine in the forebrain of males vs females at different perinatal ages and in discrete forebrain areas at postnatal day 4. In no case was a sex difference in catecholamines seen. An overall developmental decline in aromatase was associated with developmental increases in catecholamine levels. Acute treatment with the beta-agonist, isoproterenol, had no effect on brain aromatase activity.