Ayako Mutsumi

Studies on aromatase inhibition with 4-androstene-3,6,17-trione: Its 3β-reduction and time-dependent irreversible binding to aromatase with human placental microsomes

The metabolism of 4-androstene-3,6,17-trione (AT), previously described as a suicide substrate for aromatase, and its irreversible binding to aromatase were studied by using human placental microsomes. AT was rapidly converted into 3 beta-reduced metabolite (3-OHAT) with an enzyme other than aromatase in the microsomes in the presence of NADPH under either aerobic or anaerobic conditions. The conversion was efficiently prevented by a steroid 5 alpha-reductase inhibitor. 3-OHAT was characterized as a competitive (Ki = 6.5 microM) and irreversible inhibitor of aromatase. Both 14C-labeled AT and 3-OHAT were demonstrated to be irreversibly bound to aromatase probably through a sulfur atom of the enzyme in time-dependent manners in the presence of NADPH, being accompanied with time-dependent losses of the enzyme activity. It was shown that the process of an apparent time-dependent loss of aromatase activity caused by AT even under conditions allowing its 3 beta-reduction should principally depend on the action of the parent inhibitor AT itself and not on that of the metabolite 3-OHAT.

19-Hydroxy-4-androsten-17-one: potential competitive inhibitor of estrogen biosynthesis.

3-Deoxy steroids having a 4-ene system were found to be competitive inhibitors of human placental aromatase. 19-Hydroxy-4-androsten-17-one (2) potently inhibits the enzyme with an apparent Ki of 12.5 nM, but does not produce a time-dependent inactivation of the enzyme.

Mechanism for aromatase inactivation by a suicide substrate, androst-4-ene-3,6,17-trione: The 4β,5β-epoxy-19-oxo derivative as a reactive electrophile irreversibly binding to the active site☆

Aromatase is a cytochrome P450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione to estrone through three sequential oxygenations of the 19-methyl group. Androst-4-ene-3,6,17-trione (1) is a suicide substrate of aromatase. The inactivation mechanism for steroid 1 has been studied to show that the inactivation reaction proceeds through the 19-oxo intermediate 3. To further clarify the mechanism, 4 beta, 5 beta-epoxyandrosta-3,6,17,19-tetraone (6) was synthesized as a candidate for a reactive electrophile involved in irreversible binding to the active site of aromatase, upon treatment of compound 3 with hydrogen peroxide in the presence of NaHCO3. The epoxide 6 inhibited human placental aromatase in a competitive manner (Ki = 30 microM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH (K1 = 88 microM, kinact = 0.071 min-1). NADPH and BSA both stimulated the inactivation rate without a significant change of the K1 in either case (kinact: 0.133 or 0.091 min-1, in the presence of NADPH or BSA, respectively). The substrate androst-4-ene-3,17-dione protected the inactivation, but a nucleophile, L-cysteine, did not. When both the epoxide 6 and its 19-methyl analog 4 were subjected separately to reaction with N-acetyl-L-cysteine in the presence of NaHCO3, the 19-oxo steroid 6 disappeared from the reaction mixture more rapidly (T1/2 = 40 sec) than the 19-methyl analog 4 (T1/2 = 3.0 min). The results clearly indicate that the 4 beta, 5 beta-epoxy-19-oxo compound 6, which is possibly produced from 19-oxo-4-ene steroid 3 through the 19-hydroxy-19-hydroperoxide intermediate, is a reactive electrophile that irreversibly binds to the active site of aromatase.

A time-dependent inactivation of aromatase by 19-oxygenated Androst-4-ene-3,6,17-triones

19-Hydroxyandrost-4-ene-3,6,17-trione (19-OHAT), its 19-oxo derivative (19-oxo AT) and 4 beta, 5 beta-epoxyandrostane-3,6,17-trione (5) were synthesized as possible intermediates involved in a mechanism-based inactivation of aromatase caused by androst-4-ene-3,6,17-trione (AT). These compounds, inhibited the enzyme in a competitive manner with Kis of 0.61, 7.5 and 5.1 microM for 19-OHAT, 19-oxo AT, and compound 5. The two 19-oxygenated steroids showed a time-dependent, pseudo-first order rate of inactivation of aromatase with kinacts of 0.222 and 0.076 min-1 for 19-OHAT and 19-oxo AT, respectively, while compound 5 did not. NADPH and oxygen were required for the inactivation. Androstenedione blocked the inactivation, while L-cysteine partially prevented that of 19-OHAT and almost completely that of 19-oxo AT. When the 19-oxygenated steroids were separately subjected to reaction with N-acetyl-L-cysteine, these rapidly disappeared from the reaction mixture with t1/2 of 25 min (19-OHAT) and 20 s (19-oxo AT). This finding indicates that L-cysteine prevents inactivation by a chemical dependent elimination of the inhibitors from the incubate. These results suggest that the 19-oxygenation rather than the 4,5-epoxidation may be involved in the time-dependent inactivation by AT.

3β-hydroxyandrost-4-en-6-one derivatives as aromatase inhibitors

Abstract The 3-formate (II), 3-acetate (III), 3-bromoacetate (IV), 3-propionate (V), 3-methyl ether (VI), and 3-deoxy-derivative (VII) of 3 β -hydroxyandrost-4-ene-6,17-dione (I) were synthesized and tested in human placental microsomes for their ability to inhibit aromatase. II, III, and VII of this series were potent inhibitors of aromatase with the IC 50 s (1.7 and 3.3 μ M) of the latter two comparable to that (1.2 μ M) of 4-hydroxyandrostenedione. Kinetic studies showed that the three steroids are competitive inhibitors of the enzyme with Kis of 16.0, 5.5, and 0.61 μ M for II, III, and VII. Furthermore, II showed a time-dependent, pseudo-first order rate of inactivation of aromatase with Ki of 20.5 μ M and k inact of 1.54 × 10 −2 min −1 , while III gave a time-dependent, biphasic loss of the enzyme activity. NADPH and oxygen were required for the time-dependent inactivation and the substrate, androstenedione, prevented it.

Androst-5-Ene-7,17-dione: A novel class of suicide substrate of aromatase

5-En-7-one steroid 1 was found to be a potent inhibitor of aromatase. This along with its 19-hydroxy derivative 7 was characterized as suicide substrate of human placental aromatase (k(inact)s of 0.069 and 0.058 min-1 and KIs of 143 nM and 11.1 microM, respectively, for steroids 1 and 7). The results suggest that the 19-oxygenation would be involved in the irreversible inactivation of aromatase by the 5-en-7-one steroids.

Aromatization of 16α-hydroxyandrostenedione by human placental microsomes: effect of preincubation with suicide substrates of androstenedione aromatization

Estrogen synthase (aromatase) catalyzes the aromatization of androstenedione (AD) as well as 16alpha-hydroxyandrostenedione (16alpha-OHAD) leading to estrone and estriol, respectively. We found that several steroid analogs including 4-hydroxyandrostenedione (1), 6-oxoandrostenedione (6-oxoAD, 2) and its 19-hydroxy analog (3), 10beta-acetoxyestr-5-ene-7,17-dione (4), androst-5-ene-4,7,17-trione (5), and 17alpha-ethynyl-19-norteststerone (6), which are known suicide inactivators of AD aromatization, are not effective in inactivating 16alpha-OHAD aromatization in a time-dependent manner. The compounds were tested with the use of human placental microsomes and 1beta-tritiated-16alpha-OHAD as the substrate. The results of the tritium water method of 16alpha-OHAD aromatization was confirmed by the gas chromatography-mass spectrometry (GC-MS) method of estriol formation. The 1beta-tritiated-AD was used to measure AD aromatization as a positive control for these experiments. The compounds were tested at concentrations up to 40-fold higher than the K(i)s determined for inhibition of AD aromatization. These studies suggest that differences exist in the binding site structures responsible for aromatization of 16alpha-OHAD and AD.

Inhibition study of human placental aromatase with 3-deoxy- and 6-oxo-steroids using [1β-3H]16α-hydroxyandrostenedione as a substrate: A comparison to that using [1β-3H]androstenedione

Abstract Inhibition of aromatase activity in human placental microsomes with 3-deoxy-(1, 2, 4, 6 and 8), 6-oxo-(3, 5 and 9), and 16α-bromo- (7) steroids was studied using [1β- 3 H]16α- hydroxyandrostenedione (16α-OHAD) as a substrate and compared to that with the conventional substrate, [1β- 3 H]androstenedione. All the steroids inhibited both 16α-OHAD and [1β- 3 H]androstenedione aromatization in a competitive manner. Based on K i / K m ratios obtained in both series of experiments, their relative inhibitory activities in the series with 16α-OHAD were not necessarily identical with those in the other series. The results would be important to understand the relationship of structure to function of aromatase in human placental microsomes.

Synthesis of 16α,19-dihydroxy-4-androstene-3,17-dione and 3β,16α,19-trihydroxy-5-androsten-17-one and their 17β-hydroxy-16-keto isomers

Abstract 3β, 16α, 19-Trihydroxy-5-androsten-17-one [ 6 ]and 16α, 19-dihydroxy-4-androstene-3, 17-dione [ 14 ]were synthesized from the 5α-bromo-6β, 19-epoxy-17-ketone derivative 1, using the bromination at C-16α of the 17-ketone 1 and the controlled alkaline hydrolysis of the 16α-bromo-17-ketones 2 and 11 as key reactions. Zinc dust reductive cleavage of the 6β, 19-epoxy-16α-hydroxy-17-ketones 4 and 12 , produced by controlled hydrolysis, gave the corresponding 19-alcohol derivatives 6 and 14 , which were rearranged to the 17β-hydroxy-16-ketones 7 and 15 when treated with sodium hydroxide. The 3β, 16α, 17β, 19-tetrol 8 was obtained from the 16α-ketol 6 by reaction with sodium borohydride.