- Danazol Inhibits Human Adrenal 21- and 11β-Hydroxylation in vitro
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The effects of danazol on steroidogenesis in vitro in the 16-20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial 11β-hydroxylase.The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum.Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (KS) of 1 μM and with a lower affinity K'S of 10 μM.Danazol bound to mitochondrial cytochrome P-450 with a KS of 5 μM.In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant KI = 0.8 μM) and the mitrochondrial 11β-hydroxylase (KI = 3 μM).These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal in vitro.
- Barbieri, Robert L.,Osathanondh, Rapin,Canick, Jacob A.,Stillman, Robert J.,Ryan, Kenneth J.
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- Simple Route for Elaboration of the Hydroxy-ketone and Dihydroxy-acetone Side-chains of Corticosteroids from 17-Oxo-steroids
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α-Formylaminoacrylic esters, produced by the condensation of ethyl isocyanoacetate with 17-oxo-steroids have been reduced selectively to give the corresponding alcohols; the latter gave, in high yield, the hydroxyacetyl side-chain on acidic hydrolysis or the dihydroxy-acetone side-chain after appropriate oxidation and hydrolysis.
- Nedelec, Lucien,Torelli, Vesperto,Hardy, Michel
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- An efficient hemisynthesis of 20- and 21-[13C]-labeled cortexolone: A model for the study of skin sensitization to corticosteroids
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A method is described for the synthesis of isotopomers of cortexolone from the commercially available andros-4-ene-3, 17dione. The strategy is based on the use of K13CN for labeling at position 20 and of 13CH 3Mgl, generated in situ, for labeling at position 21. Because of the early introduction of the [13C] labeling, our efforts aimed at reproducible experimental procedures giving high yields with respect to the isotope containing precursors. During the development of this hemisynthesis, we noted that judicious choice of protective groups was essential as this could lead not only to mixtures or unstable intermediates but also influence considerably the output of reactions.
- Claudel, Emilie,Arbez-Gindre, Cecile,Berl, Valerie,Lepoittevin, Jean-Pierre
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- Inherent steroid 17α,20-lyase activity in defunct cytochrome P450 17A enzymes
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Cytochrome P450 (P450) 17A1 catalyzes the oxidations of progesterone and pregnenolone and is the major source of androgens. The enzyme catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction, and several mechanisms have been proposed for the latter step. Zebrafish P450 17A2 catalyzes only the 17α-hydroxylations. We previously reported high similarity of the crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1. Five residues near the heme, which differed, were changed. We also crystallized this five-residue zebrafish P450 17A1 mutant, and the active site still resembled the structure in the other proteins, with some important differences. These P450 17A1 and 17A2 mutants had catalytic profiles more similar to each other than did the wildtype proteins. Docking with these structures can explain several minor products, which require multiple enzyme conformations. The 17α-hydroperoxy (OOH) derivatives of the steroids were used as oxygen surrogates. Human P450 17A1 and zebrafish P450s 17A1 and P450 17A2 readily converted these to the lyase products in the absence of other proteins or cofactors (with catalytically competent kinetics) plus hydroxylated 17α-hydroxysteroids. The 17α-OOH results indicate that a "Compound I" (FeO3+) intermediate is capable of formation and can be used to rationalize the products. We conclude that zebrafish P450 17A2 is capable of lyase activity with the 17α-OOH steroids because it can achieve an appropriate conformation for lyase catalysis in this system that is precluded in the conventional reaction.
- Gonzalez, Eric,Johnson, Kevin M.,Pallan, Pradeep S.,Phan, Thanh T.N.,Zhang, Wei,Lei, Li,Wawrzak, Zdzislaw,Yoshimoto, Francis K.,Egli, Martin,Peter Guengerich
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- Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia
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Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone (17-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure ofWT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17-OH-progesterone).We found that the 17-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either saltwasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (Ks) for four variants were 37–13,000-fold higher than for WT P450 21A2. Cytochrome b5, which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (> 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d3- progesterone, indicating that C–H bond breaking is a ratelimiting step over a 104 -fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insigh into the effects of disease-causing mutations on this important enzyme.
- Wang, Chunxue,Pallan, Pradeep S.,Zhang, Wei,Lei, Li,Yoshimoto, Francis K.,Waterman, Michael R.,Egli, Martin,Guengerich, F. Peter
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- BIOSYNTHESIS OF ANDROGENS BY PHEOCHROMOCYTOMAS
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Homogenates from four adrenal pheochromocytomas converted 4-14C-labeled pregnenolone, 17-hydroxyprogesterone, and dehydroepiandrosterone into androstenedione and testosterone.In addition to these androgens, labeled pregnane substrates were also transformed into corticosteroids, as previously reported, and this conversion occurred in even higher yield.The formation of labeled metabolites of either pathway was greater in homogenates from intraadrenal pheochromocytomas than in those derived from an extraadrenal tumor, but less than in preparations of hyperplastic adrenal cortex.Incubations of subcellular fractions isolated from an adrenal pheochromocytoma showed that the enzyme activities involved in androgen formation from the radioactive substrates studied were associated with the microsomes and required exogenous cofactors.In contrast to adrenocortical tissue, chromaffin cell preparations uniformly failed to convert substrate cholesterol into either androgens or corticosteroids.The data available demonstrate the presence in chromaffin tissue of all of the enzyme activities required for the biosynthesis of androgens and corticosteroids except for those involved in the side-chain scission of cholesterol.
- Carballeira, Andres,Brown, John W.,Fishman, Lawrence M.
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- Human cytochrome P450 21A2, the major steroid 21-hydroxylase: Structure of the enzyme?progesterone substrate complex and rate-limiting C-H bond cleavage
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Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ～95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613-10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C-H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 107 M-1 s-1) is only ～2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 107 M-1 s-1) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.
- Pallan, Pradeep S.,Wang, Chunxue,Lei, Li,Yoshimoto, Francis K.,Auchus, Richard J.,Waterman, Michael R.,Guengerich, F. Peter,Egli, Martin
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- Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution
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A current challenge in synthetic organic chemistry is the development of methods that allow the regio- and stereoselective oxidative C - H activation of natural or synthetic compounds with formation of the corresponding alcohols. Cytochrome P450 enzymes enable C - H activation at non-activated positions, but the simultaneous control of both regio- and stereoselectivity is problematic. Here, we demonstrate that directed evolution using iterative saturation mutagenesis provides a means to solve synthetic problems of this kind. Using P450 BM3(F87A) as the starting enzyme and testosterone as the substrate, which results in a 1:1 mixture of the 2β- and 15β-alcohols, mutants were obtained that are 96 - 97% selective for either of the two regioisomers, each with complete diastereoselectivity. The mutants can be used for selective oxidative hydroxylation of other steroids without performing additional mutagenesis experiments. Molecular dynamics simulations and docking experiments shed light on the origin of regio- and stereoselectivity.
- Kille, Sabrina,Zilly, Felipe E.,Acevedo, Juan P.,Reetz, Manfred T.
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scheme or table
p. 738 - 743
(2012/02/15)
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- Inhibition of angiogenesis
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Angiogenesis in mammals is inhibited by administration of the active agents (1) heparin or a heparin fragment which is a hexasaccharide or larger or analogous compounds and (2) a steroid having 17α- and 21-hydroxy groups, 3- and 20-one groups, and in the 16-position hydrogen hydroxy or a methyl group, and non-toxic physiologically acceptable carboxylates, acetals, ketals and phosphates thereof.
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- Total Synthesis of ( +/-)-Cortisone. Double-Hydroxylation Reaction for Corticoid Synthesis
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Total syntheses of (+/-)-cortisone and (+/-)-adrenosterone have been achieved in 18 steps with the aid of metal-assisted new synthetic sequences in particular, ene reaction and homoenolate chemistry.A novel double-hydroxylation reaction of enol silyl ethers leading to a single step construction of the dihydroxyacetone side chain in corticoids has been developed and applied to the synthesis of cortisone, cortexolone, and 16α-methylcortexolone.
- Horiguchi, Yoshiaki,Nakamura, Eiichi,Kuwajima, Isao
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p. 6257 - 6265
(2007/10/02)
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- SYNTHESIS OF 9α-HYDROXYSTEROIDS BY A RHODOCOCCUS SP.
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9α-Hydroxylation of Δ5-3β-hydroxysteroids (of androstane, pregnane, 24-nor- and 21,24-bisnorcholane groups) was carried out by a Rhodococcus sp., isolated from a petroleum-containing soil sample.A large number of the investigated steroids was transformed into 9α-hydroxy-Δ4-3-ketones in satisfactory yields (50 - 90percent) at high initial concentrations of the substrates (0.5 - 5.0 g/L).The influence of some structural features of the steroid molecule on the progress and effectiveness of the microbial transformation was also shown.
- Datcheva, Violeta K.,Voishvillo, Natalia E.,Kamernitskii, Aleksei V.,Vlahov, Radoslav J.,Reshetova, Irina G.
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p. 271 - 286
(2007/10/02)
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- Process and intermediates for the preparation of 17 alphahydroxyprogesterones and corticoids from an enol steroid
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This invention discloses an improved process for the production of corticoids from 17α-hydroxy steroids utilizing peroxymonosulfate.
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- New Preparation and Controlled Alkaline Hydrolysis of 21-Bromo-20-ketopregnenes. A Facile Synthesis of Deoxycorticoids
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Syntheses of deoxycorticoids 7b, 8b, and 9b are described.Treatment of 20-oxo steroid 1 with 3 mol equiv of CuBr2 in MeOH in the presence or absence of pyridine gave the 21-bromide 4a or the 17α-methoxide 2 in high yields, respectively.When 6 mol equiv of the brominating reagent was used in the absence of pyridine, the 21-bromo-17α-methoxide 5a was formed. 17α-hydroxy 20-ones 3 could be similarly converted to the 21-bromides 6a and 6b.Oxidation of 4a, 5a, and 6a with CrO3 and subsequent isomerization of a double bond at C-5 with acid gave the corresponding 4-ene-3-ones 7a, 8a, and 9a, of which 7a and 9a were efficiently hydrolized to 7b and 9b under controlled conditions with a K2CO3-H2O-acetone system.On the other hand, 8a was converted to 8b by reaction with NaOCH3 in MeOH.
- Numazawa, Mitsuteru,Nagaoka, Masao
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