Affinity labeling of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase: evidence for bifunctional catalysis by a different conformation of the same protein for each enzyme activity.

3 beta-Hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase copurify from human placental microsomes as a single enzyme protein. The affinity-alkylating secosteroid, 5,10-secoestr-4-yne-3,10,17-trione, inactivates the dehydrogenase and isomerase reactions in a time-dependent manner, but which of the two activities is targeted depends on the concentration of secosteroid. At 2-5 microM secosteroid, the dehydrogenase activity is alkylated in a site-specific manner (pregnenolone slows inactivation) that follows first-order inactivation kinetics (KI = 4.2 microM, k3 = 1.31 x 10(-2) min-1). As the secosteroid level increases from 11 to 30 microM, dehydrogenase is paradoxically inactivated at progressively slower rates, and pregnenolone no longer protects against the alkylator. The inactivation of isomerase exhibits the expected first-order kinetics (KI = 31.3 microM, k3 = 6.42 x 10(-2) min-1) at 11-30 microM secosteroid. 5-Androstene-3,17-dione protects isomerase from inactivation by 15 microM secosteroid, but the substrate steroid unexpectedly fails to slow the inactivation of isomerase by a lower concentration of alkylator (5 microM). A shift from a dehydrogenase to an isomerase conformation in response to rising secosteroid levels explains these results. Analysis of the ligand-induced conformational change along with cofactor protection data suggests that the enzyme expresses both activities at a bifunctional catalytic site. According to this model, the protein begins the reaction sequence as 3 beta-hydroxysteroid dehydrogenase. The products of the first step (principally NADH) promote a change in protein conformation that triggers the isomerase reaction.     

Mechanism-based inactivation of 17 beta,20 alpha-hydroxysteroid dehydrogenase by an acetylenic secoestradiol

14,15-Secoestra-1,3,5(10)-trien-15-yne-3,17 beta-diol (1) is a mechanism-based inactivator of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase (estradiol dehydrogenase, EC 1.1.1.62). Inactivation with alcohol 1 requires NAD-dependent enzymic oxidation and follows approximately pseudo-first-order kinetics with a limiting t1/2 of 82 min and a "Ki" of 2.0 microM at pH 9.2 and 25 degrees C. At saturating concentrations of NAD, the initial rate of inactivation is slower than in the presence of 5 microM NAD, suggesting that cofactor binding to free enzyme impedes the inactivation process. Glutathione completely protects the enzyme from inactivation at both cofactor concentrations. Inactivation with 45 microM tritiated alcohol 1 followed by dialysis and gel filtration demonstrates a covalent interaction and affords an estimated stoichiometry of 1.4 molecules of steroid per subunit (2.8 per dimer). Chemically prepared 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one (2) rapidly inactivates estradiol dehydrogenase with biphasic kinetics. From the latter phase, a Ki of 2.8 microM and a limiting t1/2 of 12 min at pH 9.2 were determined. Estradiol, NADH, and NAD all retard this latter inactivation phase. We propose that enzymatically generated ketone 2 inactivates estradiol dehydrogenase after its release from and return to the active site of free enzyme.     

Irreversible active-site-directed inhibition of Δ5-3-ketosteroid isomerase by steroidal 17-β-oxiranes. Evidence for two modes of binding in steroid-enzyme complexes

The spiro-17β-oxiranyl derivatives of d-equilenin, epiandrosterone, dehydroepiandrosterone, and Δ⁴-androsten-3,17-dione are active-site-directed irreversible inhibitors of Δ⁵-3-ketosteroid isomerase of $\text{P.}\text{testosteroni}$. The 17β-oxiranyl steroids rapidly inhibit the isomerase in a time-dependent manner which exhibits saturation kinetics. The enzyme is protected against inactivation by the competitive inhibitor 19-nortestosterone. In addition, prolonged dialysis against neutral buffer leads to no regeneration of enzyme activity. Fluorescent spectral changes associated with the incubation of the enzyme with the 17β-oxirane derived from d-equilenin indicate that there are two modes of binding for steroids to the isomerase. These results suggest that 17β-oxiranes may inhibit the enzyme by a mechanism similar to that for the previously studied spiro-3β-oxiranyl steroids.     

Linear relationships between the ligand binding energy and the activation energy of time-dependent inhibition of steroid 5alpha-reductase by delta 1-4-azasteroids

The inhibition of steroid 5alpha-reductase (5AR) by Delta(1)-4-azasteroids is characterized by a two-step time-dependent kinetic mechanism where inhibitor combines with enzyme in a fast equilibrium, defined by the inhibition constant K(i), to form an initial reversible enzyme-inhibitor complex, which subsequently undergoes a time-dependent chemical rearrangement, defined by the rate constant k(3), leading to the formation of an apparently irreversible, tight-binding enzyme-inhibitor complex (Tian, G., Mook, R. A., Jr., Moss, M. L., and Frye, S. V. (1995) Biochemistry 34, 13453-13459). A detailed kinetic analysis of this process with a series of Delta(1)-4-azasteroids having different C-17 substituents was performed to understand the relationships between the rate of time-dependent inhibition and the affinity of the time-dependent inhibitors for the enzyme. A linear correlation was observed between ln(1/K(i)), which is proportional to the ligand binding energy for the formation of the enzyme-inhibitor complex, and ln(1/(k(3)/K(i))), which is proportional to the activation energy for the inhibition reaction under the second order reaction condition, which leads to the formation of the irreversible, tight-binding enzyme-inhibitor complex. The coefficient of the correlation was -0.88 +/- 0.07 for type 1 5AR and -1.0 +/- 0.2 for type 2 5AR. In comparison, there was no obvious correlation between ln(1/K(i)) and ln(1/k(3)), which is proportional to the activation energy of the second, time-dependent step of the inhibition reaction. These data are consistent with a model where ligand binding energies provided at C-17 of Delta(1)-4-azasteroids is fully expressed to lower the activation energy of k(3)/K(i) with little perturbation of the energy barrier of the second, time-dependent step.     

Modified acetylenic steroids as potent mechanism-based inhibitors of cytochrome P-450SCC

Synthesized 20-(4-tetrahydropyranyl-1-butynyloxy)-5-pregnen-3 alpha,20 beta- diol [steroid I] and 20-(3-tetrahydropyranyl-1-propargyloxy)-5-pregnen- 3 alpha,20 beta-diol [steroid III] have been found to inactivate purified adrenocortical cytochrome P-450SCC. When incubated with the enzyme under turnover conditions, steroid I inactivated cytochrome P-450SCC by about 85% in 40 min. This is in contrast to the free triol analog, steroid II which inactivated the enzyme by only 45% within the same incubation period. A comparison of steroid III with its free triol analog, steroid IV, also showed that the diol is a more effective inactivator of the enzyme than the triol. The partition ratio was calculated by two different methods. Each of the steroids I-IV bound to the enzyme with spectrophotometric dissociation constant (Ks) in the micromolar range, producing Type II low spin spectra changes during titration of the enzyme. In addition, it was found that the binding of each of the compounds to the enzyme occurred without inactivation of the enzyme and that the inactivation under turnover condition, is not as a result of conversion to the denatured P-420 species. This demonstrated that steroids I and III could correctly be designated as mechanism-based (suicide) inhibitors. The kinetic studies demonstrated that steroids with the tetrahydropyranyl substituent are more potent inhibitors of cytochrome P-450SCC as shown by an initial turnover rate of 0.06 min-1, an inactivation rate constant of 0.05 min-1, and a partition ratio of about 1.0 for steroid I. Based on our finding, possible mechanisms of inactivation of cytochrome P-450SCC by these acetylenic steroids are proposed.     

Mechanism-based inactivation of bovine adrenal cytochromes P450 C-21 and P450 17 alpha by 17 beta-substituted steroids

A series of progesterone derivatives has been studied as potential inactivators of the bovine adrenocortical cytochromes P450, P450 17 alpha, and P450 C-21. Replacement of the 21-methyl group of progesterone with a difluoromethyl group resulted in a selective inactivator of P450 C-21 in a reconstituted system. The loss of 21-hydroxylase activity caused by this compound exhibits a number of characteristics of mechanism-based inactivation including NADPH dependence, pseudo-first-order kinetics, saturability, irreversibility, and protection by substrate. In addition to the difluoro compound, 21,21-dichloroprogesterone, the acetylenic compound pregn-4-en-20-yn-3-one, and the olefinic compound pregna-4,20-dien-3-one all inactivate P450 C-21. In contrast, the only compound to inactivate the rabbit adrenal progesterone 21-hydroxylase is 21,21-dichloroprogesterone. In binding studies, the 21,21-dihalo steroids produce a greater maximal type I spectral shift of P450 C-21 than the two 17 beta-unsaturated steroids. The dihalo compounds inactivate P450 C-21 by both heme destruction and protein modification as shown by significant decreases in residual 21-hydroxylase activity and spectrally detectable P450 after incubation with P450 C-21 in a reconstituted system. Liquid chromatographic and mass spectral analyses of the organic extracts from these incubations showed that 21-pregnenoic acid is a major metabolite of the dihalo compounds with a partition ratio of 5 nmol of acid produced/nmol of P450 C-21 inactivated. This supports the hypothesis that inactivation proceeds in part through an acyl halide intermediate. In contrast, the acetylenic compound pregn-4-en-20-yn-3-one inactivates P450 C-21 mainly by protein modification, producing an NADPH-dependent irreversible type I spectral shift. The stoichiometry of inactivation is approximately 1.5 nmol of compound bound/nmol of enzyme inactivated, indicating selective modification of the enzyme at or near the substrate binding site.     

Effects of temperature and ethanol on agonist and antagonist binding to rat heart muscarinic receptors in the absence and presence of GTP.

2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) were synthesized and screened as irreversible active-site-directed inhibitors of the delta 5-3-oxosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Both compounds were found to inhibit the purified bacterial enzyme in a time-dependent manner. In either case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond had formed between the inhibitor and the enzyme. Inactivation mediated by compounds (I) and (II) followed pseudo-first-order kinetics, and at higher inhibitor concentrations saturation was observed. The competitive inhibitor 17 beta-oestradiol offered protection against the inactivation mediated by both compounds, and initial-rate studies indicated that compounds (I) and (II) can also act as competitive inhibitors yielding Ki values identical with those generated during inactivation experiments. 2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) thus appear to be active-site-directed. To compare the reactivity of these 2-substituted progesterones with other irreversible inhibitors of the isomerase, 3 beta-spiro-oxiranyl-5 alpha-pregnan-20 beta-ol (III) was synthesized as the C21 analogue of 3 beta-spiro-oxiranyl-5 alpha-androstan-17 beta-ol, which is a potent inactivator of the isomerase [Pollack, Kayser & Bevins (1979) Biochem. Biophys. Res. Commun. 91, 783-790]. Comparison of the bimolecular rate constants for inactivation (k+3/Ki) mediated by compounds (I)-(III) indicated the following order of reactivity: (III) greater than (II) greater than (I). 2-Mercaptoethanol offers complete protection against the inactivation of the isomerase mediated by 2 alpha-cyanoprogesterone (I). Under the conditions of inactivation compound (I) appears to be completely stable, and no evidence could be obtained for enolate ion formation in the presence or absence of enzyme. It is suggested that cyanoprogesterone inactivates the isomerase after direct nucleophilic attack at the electropositive 2-position, and that tautomerization plays no role in the inactivation event. By contrast, 2-mercaptoethanol offers no protection against the inactivation mediated by 2-hydroxymethyleneprogesterone, and under the conditions of inactivation this compound appears to exist in the semi-enolized form.     

Inhibitory effect of synthetic progestins, 4-MA and cyanoketone on human placental 3 beta-hydroxysteroid dehydrogenase/5----4-ene-isomerase activity

Human placental 3 beta-hydroxysteroid dehydrogenase/5----4-ene isomerase (3 beta-HSD) purified from human placenta transforms C-21 (pregnenolone and 17 alpha-hydroxy pregnenolone) as well as C-19 (dehydroepiandrosterone and androst-5-ene-3 beta, 17 beta-diol) steroids into the corresponding 3-keto-4-ene-steroids and is thus involved in the biosynthesis of all classes of hormonal steroids. Trilostane, epostane and cyanoketone are potent inhibitors of 3 beta-HSD with Ki values of approximately 50 nM. 4-MA, a well known 5 alpha-reductase inhibitor, is also a potent inhibitor of 3 beta-HSD with a Ki value of 56 nM. Synthetic progestin compounds such as promegestone and RU2323 show relatively strong inhibitory effects with Ki values of 110 and 190 nM, respectively. Cyproterone acetate, a progestin used in the treatment of hirsutism, acne and prostate cancer as well as norgestrel and norethindrone that are widely used as oral contraceptives also inhibit 3 beta-HSD activity at Ki values of 1.5, 1.7 and 2.5 microM, respectively.     

Photoaffinity modification of delta 5-3-ketosteroid isomerase by light-activatable steroid ketones covalently coupled to agarose beads

In order to identify the minor site(s) of photoattachment of unsaturated steroid ketones to delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni, we have developed a solid-state photoaffinity labeling technique. Two solid-state reagents, O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-19-nortestosterone and O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadien-3-one, have been synthesized. Under anaerobic conditions, isomerase bound to these resins is photoinactivated by UV light (lambda greater than 290 nm) whereas isomerase bound to O-carboxymethylagarose-ethylenediamine-deoxycholate or isomerase in the presence of O-carboxymethylagarose-ethylenediamine-acetate is almost completely stable to irradiation under the same conditions. Photoinactivation under anaerobic condition promoted by the resin-bound steroid ketones results from a reaction at the active site since the competitive inhibitor, sodium cholate, which does not absorb light above 290 nm, provides protection toward photoinactivation. Preliminary analysis of isomerase that has been photolyzed in the presence of O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadiene-3-one has established that the enzyme is converted to at least two different forms. One form binds more tightly to the resin than does the native enzyme. This form can be eluted by a sodium dodecyl sulfate containing buffer. The second form is not eluted by this buffer but can be released from the resin by cleavage of the ester bond linking the steroid to the derivatized agarose. We presume that the latter form is covalently coupled to the resin-linked steroid. In the presence of oxygen, additional nonspecific inactivation reactions occur, but these can be suppressed by the singlet oxygen trap, L-histidine. The application of solid-state photoaffinity reagents to some areas of receptor isolation and characterization is discussed.     

Structural requirements for the action of steroids as quenchers of albumin fluorescence.

1. Androgens, corticoids, gestagens, estrogens and related steroids are effective quenchers of the intrinsic fluorescence of bovine serum albumin. The quenching effect involves the formation of a steroid albumin complex which formation constant (Kf) and free energy of formation (delta G 0) can be determined by fluorescence titration. The fluorimetrically determined delta G 0 values range from -6.5 to -7.5 kcal/mol. 2. 5 alpha-Androstane and 5 alpha-pregnane are effective quenchers of albumin fluorescence, in accord with the essentially hydrophobic nature of the steroid-albumin interaction. Introduction of hydroxy or oxo groups in 5 alpha-androstane decreases the fluorescence quenching action, but the effect of each group declines when other polar groups are present in the steroid molecule. Similar effects occur with 5 alpha-pregnane except that 20-hydroxy (or oxo) duo-polar derivatives are more effective than the parent hydrocarbon. 3. Comparison of delta G 0 values for steroids differing in a single grouping shows that the steroid-albumin interaction is increased by (a) the benzenoid A-ring; (b) sulfate or carboxylate ions in the vicinity of C-3; (c) the 3-oxo group in place of the 3 alpha-hydroxyl (with 5 beta-pregnane derivatives; not with 5 alpha-androstane derivatives); (d) 17 beta-acetyl or 17 beta-hydroxyethyl residues; (e) acetylated or propionated 17 beta-hydroxy groups; (f) acetylated or methylated hydroxy groups at the C-3 of estrogens; (g) delta 5 and delta 6 double bonds; and (h) the 19 beta-methyl group. The maximal variation of delta G 0 determined by affinity-enhancing groups is -0.8 kcal/mol. Conversely, the steroid-albumin interaction is decreased by introduction of (i) oxygen atoms at C-3, C-6, C-11, C-16, and C-17; (j) 17 alpha-ethynyl and 17 alpha-acetoxyl residues; (k) benzoylated or hexahydro-benzoylated beta-hydroxy groups at C-17; (l) acetylated and benzoylated hydroxy groups at C-3; and delta 1 (conjugated) double bond. Oxo groups at C-3, C-6, C-16 and the 16 alpha, 17 alpha-epoxy group are more effective than the corresponding alpha-hydroxyl in decreasing affinity, while at C-11 and C-17, the alpha-hydroxyl is more effective than the beta-hydroxyl and the oxo group. The effect of substituents is influenced by the whole molecular structure, particularly, by the stereostructure at the A/B juncture, and the presence of an oxo group at C-17. 4. The stereospecific effect of substituents at different positions in the steroid molecule suggests that with non-aromatic, A/B trans (planar) steroids, binding to albumin primarily involves the (alpha) rear surface of the B-, C- and D-ring, and possibly, the 17 beta-side chain. With estrogens and A/B cis (dihedral) steroids, the benzenoid A-ring and electron attracting groups at C-3, respectively, may participate in binding.     

Synthesis and biochemical studies of 17-substituted androst-3-enes and 3,4-epoxyandrostanes as aromatase inhibitors

A series of 5alpha-androst-3-enes and 3alpha,4alpha-epoxy-5alpha-androstanes were synthesized and tested for their abilities to inhibit aromatase in human placental microsomes. In these series the original C-17 carbonyl group was replaced by hydroxyl, acetyl and hydroxyimine groups. Inhibition kinetic analysis on the most potent steroid of these series revealed that it inhibits the enzyme in a competitive manner (IC(50)=6.5 microM). The achieved data pointed out the importance of the C-17 carbonyl group in the D-ring of the studied steroids as a structural feature required to reach maximum aromatase inhibitory activity. Further, at least one carbonyl group (C-3 or C-17) seems to be essential to effective aromatase inhibition.     

Steroid-transforming enzymes in fungi

Fungal species are a very important source of many different enzymes, and the ability of fungi to transform steroids has been used for several decades in the production of compounds with a sterane skeleton. Here, we review the characterised and/or purified enzymes for steroid transformations, dividing them into two groups: (i) enzymes of the ergosterol biosynthetic pathway, including data for, e.g. ERG11 (14α-demethylase), ERG6 (C-24 methyltransferase), ERG5 (C-22 desaturase) and ERG4 (C-24 reductase); and (ii) the other steroid-transforming enzymes, including different hydroxylases (7α-, 11α-, 11β-, 14α-hydroxylase), oxidoreductases (5α-reductase, 3β-hydroxysteroid dehydrogenase/isomerase, 17β-hydroxysteroid dehydrogenase, C-1/C-2 dehydrogenase) and C-17-C-20 lyase. The substrate specificities of these enzymes, their cellular localisation, their association with protein super-families, and their potential applications are discussed. Article from a special issue on steroids and microorganisms.     

Inhibition of rat ovarian 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase and 17,20 lyase by progestins and danazol

The site of action of synthetic progestins or danazol in the treatment of endometriosis is considered to be mainly the hypothalamo-pituitary level, but the direct action to the uterine endometrium and the ovary is also suggested. We investigated the effect of these synthetic steroids to rat ovarian steroidogenic enzymes. The effect of norethisterone, levonorgestrel, danazol, gestrinone, desogestrel and 3-keto-desogestrel was studied in vitro. The sources of the enzymes were prepared from ovaries of immature rats treated either with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) for 3 beta-hydroxy steroid dehydrogenase (3 beta-HSD), or with PMS for 17 alpha-hydroxylase and 17,20 lyase. The substrates used were pregnenolone (P5) for 3 beta-HSD, progesterone (P4) for 17 alpha-hydroxylase, and 17 alpha-hydroxy-progesterone (17 alpha-OH-P4) for 17,20 lyase. The substrates were incubated with the enzyme sources and coenzymes, and the products formed were measured. All the steroids inhibited 3 beta-HSD, and the inhibition by gestrinone (Ki = 3.0 microM) and 3-keto-desogestrel (17.5 microM) was particularly marked. Only desogestrel (Ki = 30.3 microM) and danazol (168 microM) inhibited 17 alpha-hydroxylase. All the steroids inhibited 17,20 lyase, and the inhibition by desogestrel (Ki = 0.70 microM), danazol (0.80 microM), and gestrinone (30 microM) was particularly marked.     

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from mitochondria and kinetic profiles, biophysical characterization of the purified mitochondrial and microsomal enzymes

In human placenta, 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric Mr = 41,000), gel filtration at constant specific enzyme activity (Mr = 77,000), and a single NH2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone (Km = 1.6 microM, Vmax = 48.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.4 microM, Vmax = 48.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.3 microM, Vmax = 914.2 nmol/min/mg) and 5-androstene-3,17-dione (Km = 27.6 microM, Vmax = 888.4 nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and androstenedione, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD+) reduction (mean Km +/- SD using C-19 and C-21 dehydrogenase substrates = 26.4 +/- 0.8 microM, mean Vmax = 73.2 +/- 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37 degrees C), stability in storage and solution, effects of divalent cations, and the single NH2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles.     

Effect of the progestogens, gestodene, 3-keto desogestrel, levonorgestrel, norethisterone and norgestimate on the oxidation of ethinyloestradiol and other substrates by human liver microsomes

A number of different progestogens, levonorgestrel (LNG), norethisterone (NET), gestodene (GSD), desogestrel (DG) and norgestimate (NORG) are used in combination with the oestrogen ethinyloestradiol (EE2) in oral contraceptive steroid preparations. All the progestogens are acetylenic steroids and previous studies have indicated the potential of acetylenic steroids to cause mechanism-based or "suicide" inactivation of cytochrome P-450. We have compared the effects of the different progestogens on EE2 2-hydroxylation (a reaction catalyzed by enzymes from the P-450IIC, P-450IIIA and P-450IIE gene families) and also the oxidative metabolism of other drug substrates (cyclosporin, diazepam, tolbutamide) by human liver microsomes. On coincubation with EE2 as substrate, GSD, 3-keto desogestrel (3-KD, the active metabolite of desogestrel) and LNG produced some concentration-dependent inhibition of EE2 2-hydroxylation (maximum 32% inhibition at 100 microM 3-keto desogestrel). Ki values determined for GSD and 3-KD were 98.5 +/- 12.3 and 93.2 +/- 10.3 microM (mean +/- SD; n = 4), respectively. Preincubation of progestogens in a small volume (50 microliters) incubation for 30 min in the presence of an NADPH-generating system enhanced the inhibitory potential of all the steroids (at 100 microM, inhibition was for GSD 39%, 3-KD 46%, LNG 46%, NET 51% and NORG 43%). Inhibitory effects were therefore comparable and also similar to the macrolide antibiotic troleandomycin. The most marked inhibition seen was of diazepam N-demethylation and hydroxylation by GSD (71 and 57%, respectively) and 3-KD (62 and 50%, respectively). In preincubation studies involving cyclosporin as the substrate, the order of inhibitory potency was GSD greater than 3-KD greater than NET greater than LNG for production of both metabolite M17 and M21. The results of the study indicate that all the progestogens in common use have the propensity to inhibit a number of oxidative pathways but there is little evidence for one progestogen being more markedly inhibitory than others.     

Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes: potent inhibition by imidazole-type antimycotic drugs and lack of inhibition by steroid 5 alpha-reductase inhibitors.

5 alpha-Dihydrotestosterone, the principal androgen mediating prostate growth and function in the rat, is formed from testosterone by steroid 5 alpha-reductase. The inactivation of 5 alpha-dihydrotestosterone involves reversible reduction to 5 alpha-androstane-3 beta,17 beta-diol by 3 beta-hydroxysteroid oxidoreductase followed by 6 alpha-, 7 alpha-, or 7 beta-hydroxylation. 5 alpha-Androstane-3 beta,17 beta-diol hydroxylation represents the ultimate inactivation step of dihydrotestosterone in rat prostate and is apparently catalyzed by a single, high-affinity (Km approximately 0.5 microM) microsomal cytochrome P450 enzyme. The present studies were designed to determine if 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes is inhibited by agents that are known inhibitors of androgen-metabolizing enzymes. Inhibitors of steroid 5 alpha-reductase (4-azasteroid analogs; 10 microM) or inhibitors of 3 beta-hydroxysteroid oxidoreductase (trilostane, azastene, and cyanoketone; 10 microM) had no appreciable effect on the 6 alpha-, 7 alpha-, or 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol (10 microM) by rat prostate microsomes. Imidazole-type antimycotic drugs (ketoconazole, clotrimazole, and miconazole; 0.1-10 microM) all markedly inhibited 5 alpha-androstane-3 beta,17 beta-diol hydroxylation in a concentration-dependent manner, whereas triazole-type antimycotic drugs (fluconazole and itraconazole; 0.1-10 microM) had no inhibitory effect. The rank order of inhibitory potency of the imidazole-type antimycotic drugs was miconazole greater than clotrimazole greater than ketoconazole. In the case of clotrimazole, the inhibition was shown to be competitive in nature, with a Ki of 0.03 microM. The imidazole-type antimycotic drugs inhibited all three pathways of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation to the same extent, which provides further evidence that, in rat prostate microsomes, a single cytochrome P450 enzyme catalyzes the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol. These studies demonstrate that certain imidazole-type compounds are potent, competitive inhibitors of 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat prostate microsomes, which is consistent with the effect of these antimycotic drugs on cytochrome P450 enzymes involved in the metabolism of other androgens and steroids.     

Synthesis and structure-activity relationships of 6-phenylaliphatic-substituted C19 steroids having a 1,4-diene, 4,6-diene, or 1,4,6-triene structure as aromatase inhibitors.

A series of 6alpha- and 6beta-phenylaliphatic-substituted androsta-1,4-diene-3,17-diones [9b-f and 10b-f; (CH2)nPh, n = 1-5] and their 4,6-diene and 1,4,6-triene analogs (11b-f and 12b-f) along with their respective phenyl analogs 9a-12a were synthesized and tested as aromatase inhibitors. All of the steroids examined were very powerful competitive inhibitors of aromatase in human placental microsomes with apparent Ki values ranging from 8.5 to 80 nM. The inhibitory activities of the benzyl- and phenethyl-4,6-dienes 11b and 11c (Ki, 9.0 and 10 nM) as well as the 6-phenethyl-1,4,6-triene 12c (Ki, 8.5 nM) were extremely high among them. All of the phenylaliphatic steroids, except for the 6beta-phenethyl compound 10c, and the 6-phenyl-4,6-diene 11a had higher affinity for aromatase than the corresponding parent 1,4-diene, 4,6-diene, and 1,4,6-triene steroids 9g, 11g, and 12g. All of the 6alpha-substituted 1,4-dienes (9a-9g) and the 6-substituted 1,4,6-trienes (12a-12g) caused a time-dependent inactivation of aromatase. On the other hand, only the 6beta-substituted 1,4-dienes (10a-10d) having no or less than four carbon atoms between the steroid nucleus and the phenyl group also caused a time-dependent inactivation of aromatase. Their inactivation rates (k(inact) 0.076-0.156 min(-1)) were higher than the respective parent steroids, 9g and 12g. In contrast, in the 4,6-diene series, only the 6-phenpropyl steroids 11d inactivated aromatase in a time-dependent manner with 0.155 min(-1) of k(inact) value. The inactivation was prevented by the substrate androstenedione, and no significant effect of L-cysteine on the inactivation was observed in each case. These results indicate that length and/or stereochemistry of the C-6 substituent of steroids 9-12 as well as a terminal phenyl group incorporated in the C-6 substituent play a critical role not only in tight binding to the active site of aromatase but also in the cause of a time-dependent inactivation of the enzyme.     

Affinity labeling of rat glutathione S-transferase isozyme 1-1 by 17beta -iodoacetoxy-estradiol-3-sulfate

Rat liver glutathione S-transferase, isozyme 1-1, catalyzes the glutathione-dependent isomerization of Delta(5)-androstene-3,17-dione and also binds steroid sulfates at a nonsubstrate inhibitory steroid site. 17beta-Iodoacetoxy-estradiol-3-sulfate, a reactive steroid analogue, produces a time-dependent inactivation of this glutathione S-transferase to a limit of 60% residual activity. The rate constant for inactivation (k(obs)) exhibits a nonlinear dependence on reagent concentration with K(I) = 71 microm and k(max) = 0.0133 min(-1). Complete protection against inactivation is provided by 17beta-estradiol-3,17-disulfate, whereas Delta5-androstene-3,17-dione and S-methylglutathione have little effect on k(obs). These results indicate that 17beta-iodoacetoxy-estradiol-3-sulfate reacts as an affinity label of the nonsubstrate steroid site rather than of the substrate sites occupied by Delta5-androstene-3,17-dione or glutathione. Loss of activity occurs concomitant with incorporation of about 1 mol 14C-labeled reagent/mol enzyme dimer when the enzyme is maximally inactivated. Isolation of the labeled peptide from the chymotryptic digest shows that Cys(17) is the only enzymic amino acid modified. Covalent modification of Cys(17) by 17beta-iodoacetoxy-estradiol-3-sulfate on subunit A prevents reaction of the steroid analogue with subunit B. These results and examination of the crystal structure of the enzyme suggest that the interaction between the two subunits of glutathione S-transferase 1-1, and the electrostatic attraction between the 3-sulfate of the reagent and Arg(14) of subunit B, are important in binding steroid sulfates at the nonsubstrate steroid binding site and in determining the specificity of this affinity label.     

In vitro inhibition by ketoconazole of human testicular steroid oxidoreductases

An oral antimycotic agent, ketoconazole has been demonstrated to be an inhibitor of cytochrome P-450-dependent monooxygenases. To investigate its effect on steroid oxidoreductases, in vitro studies were carried out using subcellular fractions of human testes. Ketoconazole competitively inhibited activities of 3 beta-hydroxy-5-ene-steroid oxidoreductase/isomerase and NADH-linked 20 alpha-hydroxysteroid oxidoreductase for steroid substrate and the Ki values were 2.9 and 0.9 microM, respectively. In contrast, ketoconazole inhibited neither 17 beta-hydroxysteroid oxidoreductase nor NADPH-linked 20 alpha-hydroxysteroid oxidoreductase, indicating that the two 20 alpha-hydroxysteroid oxidoreductases are distinct. Further, ketoconazole inhibited non-competitively the above enzyme activities for the corresponding cofactors of NAD and NADH. From the binding mode of ketoconazole to cytochrome P-450 and the present findings, it appears likely that the agent binds to a site which is different from that of steroids or pyridine nucleotides.     

Active-site directed inactivation of rat ovarian 20 alpha-hydroxysteroid dehydrogenase.

Rat ovarian 20 alpha-hydroxysteroid dehydrogenase plays a pivotal role in leuteolysis and parturition by catalysing the reduction of progesterone to give the progestationally inactive steroid 20 alpha-hydroxyprogesterone. Putative mechanism based inhibitors of this enzyme were synthesized as potential progestational maintaining agents, including the epimeric allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol and the related vinyl ketone 1-(3 beta-hydroxy-5 alpha-androstan-17 beta-yl)-2-propen-1-one. The vinyl ketone inactivates rat ovarian 20 alpha-hydroxysteroid dehydrogenase, semi-purified by poly(L-lysine)-agarose column chromatography, in a rapid time-dependent manner. Analysis of the pseudo-first-order inactivation plots gave a Ki of 2.0 microM for the inhibitor and a t1/2 for the enzyme of 20 s at saturation. These data indicate that the vinyl ketone is a potent and efficient inactivator of the ovarian dehydrogenase. Neither dialysis in the presence or absence of a competing nucleophile nor gel filtration reserves the inactivation, suggesting that a stable covalent bond is formed between the enzyme and steroid ligand. Both substrates (20 alpha-hydroxyprogesterone and NADP+) protect the enzyme from inactivation; moreover, initial velocity measurements in the presence of saturating concentrations of both substrates indicate that the vinyl ketone can behave as a competitive inhibitor, yielding a Ki value identical with that obtained in the inactivation experiments. Our results imply that the vinyl ketone is an active-site directed alkylating agent. By contrast the allylic alcohol pair 3 beta-hydroxy-alpha-vinyl-5 alpha-androstane-17 beta-methanol are neither substrates nor inhibitors of the ovarian enzyme and appear to be excluded from the catalytic site. The rapid inactivation observed with the vinyl ketone suggests that this compound may be useful as a progestational maintaining agent.