18-Substituted progesterone derivatives as inhibitors of aldosterone biosynthesis

The synthesis of new progesterone derivatives substituted at the 18 methyl group is described. These compounds are designed as 18-monooxygenase, cytochrome P-450-dependent potential kcat inhibitors. Preliminary results on the in vitro biological investigation of these modified progesterones are presented.     

Inhibition of progesterone synthesis in normal and transformed human placental cells by tight binding inhibitors of 3 beta-hydroxysteroid dehydrogenase

The biosynthesis of progesterone from [3H]pregnenolone was curvilinear over a 6 h time course in human placenta cytotrophoblasts and in human placenta choriocarcinoma cells (JEG-3 cells). Mass measurements determined independently by radioimmunoassay indicate that the progesterone synthesized by cytotrophoblasts (21.0 +/- 5.20 ng/6 h/mg protein) is substantially higher than that synthesized by the JEG-3 cells (4.48 +/- 0.56 ng/6 h/mg protein). Two tight binding inhibitors of 3 beta-hydroxysteroid dehydrogenase (2 alpha-cyanoprogesterone I and cyanoketone II), and a potent inhibitor of the microsomal conversion of pregnenolone to progesterone (2 alpha-bromo-5 alpha-androstan-3-one-17 beta-acetate III) were compared as inhibitors of progesterone synthesis in the two cell-types. Compounds I and II were very potent inhibitors yielding IC50 values of between 10 and 20 nM. At higher concentrations (100 nM - 1,000 nM) compound I promoted a complete cessation of progesterone synthesis which could be reversed by washing the cells free of inhibitor. By contrast compound III was ineffectual as an inhibitor yielding an IC50 value greater than 10 microM. This 1,000-fold difference in inhibitory potency suggests that 2 alpha-cyano-substituted steroids display an unusual capacity to inhibit progesterone biosynthesis and secretion in normal and transformed human cells.     

Contrasting effects of ethylene perception and biosynthesis inhibitors on germination and seedling growth of barley (Hordeum vulgare L.)

The effects of the plant growth regulator ethylene, and of ethylene inhibitors, on barley (Hordeum vulgare L.) germination and seedling growth were investigated. Exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) at 100 microM enhanced ethylene production by barley seedlings and stimulated shoot growth, whereas both germination and seedling growth were inhibited by antagonists of ethylene perception (75 microM silver ions, 100 microM 2,5-norbornadiene (NBD)). In contrast, germination was unaffected by, and root and shoot growth of seedlings was strongly stimulated by inhibitors of ethylene biosynthesis (10 microM cobalt chloride, 10 microM aminoethoxyvinylglycine (AVG)). Since the ethylene and polyamine biosynthetic pathways are linked through S:-adenosylmethionine, this prompted further explorations into the role of polyamines in germination and seedling growth. Exogenous polyamines (putrescine, spermidine and spermine) at 1 microM concentration stimulated barley seedling growth in a similar fashion to the ethylene biosynthetic inhibitors. Both polyamines and ethylene biosynthetic inhibitors reversed the inhibitory effects of ethylene perception inhibitors on germination and seedling growth. Blocking endogenous ethylene production with aminoethoxyvinylglycine enhanced the free putrescine and spermidine content of germinating barley grains. Thus endogenous polyamines may play a complementary, growth-promotive, role to ethylene in the normal course of barley germination. Further, experiments that have been carried out using inhibitors of ethylene biosynthesis may have to be re-evaluated to take the possible effect of polyamines into account.     

The enantiomer of progesterone (ent-progesterone) is a competitive inhibitor of human cytochromes P450c17 and P450c21

Human cytochrome P450c17 (17alpha-hydroxylase, 17,20-lyase) (CYP17) and cytochrome P450c21 (21-hydroxylase) (CYP21) differ by only 14 amino acids in length and share 29% amino acid identity. Both enzymes hydroxylate progesterone at carbon atoms that lie only 2.6A apart, but CYP17 also metabolizes other steroids and demonstrates additional catalytic activities. To probe the active site topologies of these related enzymes, we synthesized the enantiomer of progesterone and determined if ent-progesterone is a substrate or inhibitor of CYP17 and CYP21. Neither enzyme metabolizes ent-progesterone; however, ent-progesterone is a potent competitive inhibitor of CYP17 (K(I)=0.2 microM). The ent-progesterone forms a type I difference spectrum with CYP17, but molecular dynamics simulations suggest different binding orientations for progesterone and its enantiomer. The ent-progesterone also inhibits CYP21, with weaker affinity than for CYP17. We conclude that CYP17 accommodates the stereochemically unnatural ent-progesterone better than CYP21. Enantiomeric steroids can be used to probe steroid binding sites, and these compounds may be effective inhibitors of steroid biosynthesis.     

Characteristics of estrogen-2/4-hydroxylase of porcine ovarian follicles: influence of steroidal and non-steroidal agents on the activity of the enzyme in vitro

The conversion of [3H]estradiol to 2-hydroxyestradiol (2-OH-E2) by homogenates of porcine ovarian follicles was assayed in vitro in the presence and absence of 10 and 100 microM concentrations of the following potential substrates or inhibitors of estrogen-2/4-hydroxylase (E-2/4-H): (1) estrogens; estrone (E1), estriol (E3) and 17 alpha-estradiol (17 alpha-E2), (2) catecholestrogens; 2-hydroxyestradiol (2-OH-E2), 4-hydroxyestradiol (4-OH-E2) and 2-hydroxyestrone (2-OH-E1); (3) 2-methoxyestradiol (2-MeO-E2); (4) halogenated estrogens; 2-bromoestradiol, (2-Bromo-E2) 4-bromoestradiol and 2,4-dibromoestradiol; (5) androgens; testosterone (T), dihydrotestosterone (DHT) and androstenedione; (6) progesterone; (7) epinephrine; (8) inhibitors of steroid aromatase; aminoglutethimide and 4-hydroxyandrostenedione and (9) SKF 525A, an inhibitor of cytochrome P-450. Progesterone and 2-Bromo-E2 were the two most effective inhibitors (2-OH-E2 formation = 4 and 5% of control at 100 microM and 29.6 and 17.4% at 10 microM of progesterone and 2-Bromo-E2, respectively). 2-MeO-E2 at 100 microM was nearly as effective as progesterone in inhibiting E-2/4-H activity but only caused about 50% inhibition at 10 microM. The three catecholestrogens reduced 2-OH-E2 formation to about the same degree (21-23% of control at 100 microM). The 2,4-dibromo-E2 was equipotent with the catecholestrogens while 4-bromo-E2 was about half as effective. The phenolic estrogens, potential substrates for the enzyme, reduced 2-OH-E2 formation to different degrees, with E3 being the most effective. Among the androgens, DHT was almost as effective an inhibitor as the catecholestrogens, T was about half as effective while androstenedione had no effect. Epinephrine and the two inhibitors of aromatase did not inhibit E-2/4-H activity. SKF 525A inhibited E-2/4-H activity but with a potency only about 1/10th that reported for liver.     

Product inhibition of steroid-17 alpha-monooxygenase by endogenous 17 alpha-hydroxyprogesterone in microsomes and isolated Leydig cells from rat testis

Use of integrated rate equations for analysis of progesterone metabolism by isolated Leydig cells and microsomes from rat testis in presence of several progesterone concentrations within several periods reveals competitive product inhibition by endogenously formed 17 alpha-hydroxyprogesterone (Kpm = 0.1 microM) of steroid-17 alpha-monooxygenase activity (Ksm = 0.8 microM). The discrepancy between this very low interaction constant of endogenous 17 alpha-hydroxyprogesterone with the steroid-17 alpha-monooxygenase, and the respective values (from the literature) for exogenous 17 alpha-hydroxyprogesterone which are about 50-fold higher, may be explained by accumulation of endogenous 17 alpha-hydroxyprogesterone at the catalytic site of the steroid-17 alpha-monooxygenase. This mechanism may be important for intratesticular regulation of androgen biosynthesis from precursor steroids.     

Metabolism of 7 beta-hydroxycholesterol in astrocyte primary cultures and derived spontaneously transformed cell lines: correlation between the esterification on C-3 -OH by naturally occurring fatty acids and cytotoxicity

The lethal effect of 7 beta-hydrocholesterol (7 beta-OHC) on spontaneously transformed cell lines, derived from neonatal rat astrocyte primary cultures and the extent of 7 beta-OHC esterification by naturally occurring fatty acids on C-3 -OH (metabolite) was investigated. The extent of cellular death and metabolite biosynthesis matched with the 7 beta-OHC concentrations. Incubation of the cells with 10 microM 7 beta-OHC in the presence of either lipoproteins depleted fetal calf serum or with increasing serum concentrations revealed proportionality between the degree of cellular cytotoxicity and metabolite levels. The use of tetracaine or progesterone as acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors indicated that ACAT was involved in metabolite production; the inhibition of metabolite biosynthesis slowed down 7 beta-OHC lethal effect. Incubation of the cells with 1 mM db-cAMP, prior 7 beta-OHC treatment, enhanced both metabolite production and cellular death. These findings support the view that the metabolite is directly implicated in the cytotoxic action.     

Inhibition of hamster adrenal 11 beta/19-hydroxylase activity

Increased mineralocorticoid activity has been associated with elevated urinary levels of 19-nordeoxycorticosterone in several forms of experimental and human hypertension. Biosynthesis of 19-norsteroids involves hydroxylation of the C-19 methyl group. We synthesized the 4-hydroxy analogs of deoxycorticosterone, deoxycorticosterone acetate, progesterone, and androstenedione and evaluated them as inhibitors of deoxycorticosterone 11 beta/19-hydroxylase using hamster adrenal mitochondrial preparations. These 4-hydroxy analogs were inhibitors of this P 450 hydroxylase, with approximately 10 times weaker affinity than their respective natural substrates. 4-Hydroxydeoxycorticosterone was the most potent inhibitor evaluated in this study. The half-maximal inhibitory concentration of deoxycorticosterone hydroxylation was 5 microM, 15 microM, more than 50 microM, and 14 microM, respectively, for the above compounds.     

Design, synthesis and structure-activity relationships of new phosphinate inhibitors of MurD

A series of new phosphinate compounds were designed and synthesized as inhibitors of the d-glutamic acid-adding enzyme (MurD) involved in peptidoglycan biosynthesis. They were tested against the MurD enzyme from Escherichia coli, allowing initial structure-activity relationships to be deduced. Two compounds had IC(50) values near 100 microM and constitute a promising starting point for further development.     

Effect of steroid biosynthesis modifiers on progesterone biotransformation by recombinant yeasts expressing cytochrome P450cl7

Using recombinant microorganisms S. cerevisiae GRF18/YEp 5117α, expressing bovine adrenocortical cytochrome P450cl7, we have studied the effect of various modifiers of steroid biosynthesis on the relationship between reactions of the 17α-hydroxylation and 20α-reduction of progesterone. Dexamethasone and metyrapone had no effect on the reaction of progesterone 17α-hydroxylation and 20α-reduction of 17α-hydroxyprogesterone. Mifepriston and danazol did not covalently modify amino acid residues of the cytochrome P450cl7 or its heme group under the conditions of progesterone biotransformation by recombinant yeasts. Ketokonazole, mifepriston and danazol were found to be low-affinity competitive inhibitors, but the 20-dihydroderivatives of progesterone were mixed type inhibitors of the cytochrome P450cl7. All modifiers used did not affect the functional properties of the yeast analog of 20α-hydroxysteroid dehydrogenase. Based on the effect on catalytic parameters of the cytochrome P450cl7, the all modifiers used can be arranged in the following order: 20β-dihydroprogesterone (maximal effect) > mifepriston = ketokonazole > 20α-dihydroprogesterone > danazol > dexamethasone, metyrapone (without effect).     

An in vitro method to determine the selective inhibition of estrogen biosynthesis by aromatase inhibitors

Potency and selectivity of aromatase inhibition are parameters which ultimately influence the therapeutic efficacy of aromatase inhibitors. This report describes an in vitro model which allows an assessment of the selectivity with which aromatase inhibitors inhibit estrogen biosynthesis. Estrogen production was stimulated by incubating adult female hamster ovarian tissue with ovine LH. The production rates of estrogens (E), testosterone (T) and progesterone (P) were determined using radioimmunoassays to measure the amount of these steroids released into the incubation medium over a 4-hour incubation period. The selectivity of aromatase inhibition was assessed by determining the IC50S with which each inhibitor inhibited the production of E (end product), T (immediate precursor of E) and P (early precursor of E). Selectivity was studied for each of the 4 aromatase inhibitors, CGS 16949A (a new non-steroidal compound), 4-OH-androstenedione, aminoglutethimide and testolactone. CGS 16949A was the most potent of the four, followed by 4-OH-androstenedione, aminoglutethimide and testolactone. As far as selectivity was concerned, both CGS 16949A and 4-OH-androstenedione selectively inhibited aromatase judging from the IC50s for E and P production (CGS 16949A: IC50 for E & P = 0.03 & 160 microM, resp.; 4-OH-androstenedione: IC50 for E & P = 0.88 & greater than or equal to 330 microM, resp.). Aminoglutethimide was the least selective inhibitor of aromatase (IC50 for E & P = 13 & 60 microM, resp.). For testolactone, the least potent of the four (IC50 for E = 130 microM), no conclusive data were obtained concerning the selectivity of aromatase inhibition. Thus a simple, effective and reproducible method is described for assessing the selectivity with which aromatase inhibitors inhibit aromatase.     

Design and synthesis of lignostilbene-alpha,beta-dioxygenase inhibitors

Lignostilbene-alpha,beta-dioxygenase cleaves the olefinic double bond of phenolic stilbenes by a mechanism similar to that of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis. Several analogues of stilbene were designed and synthesized, and their efficacy as inhibitors of lignostilbene-alpha,beta-dioxygenase was examined. The compound (Z)-1-(4-hydroxyphenyl)-1-fluoro-2-phenylethene (2) was found to be a potent inhibitor of this enzyme with an IC(50) of 3 microM.     

Biosynthesis of paf-acether. XI. Regulation of acetyltransferase by enzyme-substrate imbalance

Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase is the key enzyme in paf-acether (paf) biosynthesis, since it yields the active mediator from its nonacetylated precursor, lyso-paf. In microsomal fractions obtained from the ionophore A23187-stimulated human polymorphonuclear neutrophils, the optimal conditions allowing the full acetylation of lyso-paf were: 2-2.5 mg.ml-1 bovine serum albumin, 40 microM lyso-paf, 200 microM acetyl-CoA and acetyltransferase of high specific activity, at least 18 nmol.min-1.mg protein- -1. The reaction frequently stopped before the substrate was consumed due to spontaneous decay of the enzyme activity at 37 degrees C and inhibition of the enzyme by the paf formed in the reaction. However, low concentrations of acetyltransferase substrates (lyso-paf or lysophosphatidylcholine) and the antioxidant dithiothreitol, but not the inhibitors of proteinases or phosphatases, protected the enzyme against decay. In contrast, high concentrations of those lyso substrates inhibited the enzyme activity in the assay. This inhibition as well as that due to paf was overcome by raising the concentration of the enzyme contained in the microsomal fraction or the bovine serum albumin in the assay. These results suggest that the biosynthesis of paf in cell-free assay and most probably in intact cells might be controlled to a larger extent by the acetyltransferase concentration rather than by that of its substrates.     

Design and synthesis of pyrimido[4,5-b][1,4]benzothiazine derivatives, as potent 15-lipoxygenase inhibitors

A group of 2-substituted pyrimido[4,5-b][1,4]benzothiazines were designed, synthesized, and evaluated as potential inhibitors of 15-lipoxygenase (15-LO). Compounds 4d and 4e showed the best IC50 of 15-LO inhibition (IC50=18 and 34 microM, respectively). All compounds were docked into 15-LO. As a result the sulfur atom was oriented toward the iron atom of the active site of 15-LO. We suggest the interaction of the iron atom is essential for the activity of the inhibitors.     

Effect of genistein, tyrphostin and herbimycin on prolactin-stimulated progesterone production by porcine theca and luteal cells.

The potential involvement of protein tyrosine kinases (PTK) in the mechanism of prolactin (Prl) action on ovarian cell steroidogenesis has not been elucidated and information about research on this subject is scarce. In this preliminary study pharmacological intervention was used to provide support for a possible involvement of tyrosine kinases in prolactin induction of progesterone secretion by porcine thecal and luteal cells. Material used in this experiment were cultures of porcine follicular theca interna and early corpus luteum cells. The former were isolated from, proestrous preovulatory follicles and the latter were obtained by enzymatic dispersion of luteal tissue. Three of tyrosine kinase inhibitors, genistein, herbimycin and tyrphostin, were applied. They act through different mechanisms, partially blocking Prl-stimulated progesterone secretion. Herbimycin at a dose of 3 microM inhibited Prl-stimulated progesterone secretion beneath the control level in theca and by 70% in luteal cells. Genistein at a dose of 45 microM inhibited Prl-stimulated progesterone secretion beneath the control level in theca and down to the control in luteal cells. On the other hand, tyrphostin at a dose of 100 microM only slightly suppressed Prl-stimulated progesterone secretion by thecal and luteal cells (33% and 40% respectively). This investigation is the first search for evidence of involvement of tyrosine kinases in Prl-stimulated progesterone production by ovarian cells in the pig.     

Kinetic characterization of the carbon monoxide-acetyl-CoA (carbonyl group) exchange activity of the acetyl-CoA synthesizing CO dehydrogenase from Clostridium thermoaceticum

CO dehydrogenase from Clostridium thermoaceticum is a nickel-containing enzyme that catalyzes both the reversible conversion of CO2 to CO (for incorporation into the carbonyl group of acetate) and the synthesis of acetyl-CoA from methyl corrinoid, CO, and CoASH. The latter activity is conveniently assayed by monitoring the exchange of [1-14C]acetyl-CoA (carbonyl group) with 12CO. Kinetic parameters for the highly oxygen sensitive exchange activity have been determined: Km (acetyl-CoA) = 600 microM; Vmax = 440 min-1. In addition, coenzyme A analogues have been tested as inhibitors of the exchange to probe the active site of the enzyme; each has no effect on the CO2 in equilibrium CO activity of CO dehydrogenase. Coenzyme A, the substrate for acetate biosynthesis, is a potent competitive inhibitor, KI = 7 microM. Comparison of this value with that for desulfo-CoA (KI = 6000 microM) suggests that a key mode of binding is through the sulfur atom, possibly to a metal site on the enzyme. The relatively high affinity of the enzyme for CoASH relative to acetyl-CoA is consistent with its proposed operation in the acetogenic direction. The differential sensitivity to oxygen and storage of the two activities of CO dehydrogenase as well as the contrasting effect of coenzyme A inhibitors suggests that acetate assemblage occurs at a site distinct from that for CO dehydrogenation.     

Inhibition of the severe acute respiratory syndrome 3CL protease by peptidomimetic alpha,beta-unsaturated esters

The proteolytic processing of polyproteins by the 3CL protease of severe acute respiratory syndrome coronavirus is essential for the viral propagation. A series of tripeptide alpha,beta-unsaturated esters and ketomethylene isosteres, including AG7088, are synthesized and assayed to target the 3CL protease. Though AG7088 is inactive (IC50 > 100 microM), the ketomethylene isosteres and tripeptide alpha,beta-unsaturated esters containing both P1 and P2 phenylalanine residues show modest inhibitory activity (IC50 = 11-39 microM). The Phe-Phe dipeptide inhibitors 18a-e are designed on the basis of computer modeling of the enzyme-inhibitor complex. The most potent inhibitor 18c with an inhibition constant of 0.52 microM is obtained by condensation of the Phe-Phe dipeptide alpha,beta-unsaturated ester with 4-(dimethylamino)cinnamic acid. The cell-based assays also indicate that 18c is a nontoxic anti-SARS agent with an EC50 value of 0.18 microM.     

N-benzylideneaniline and N-benzylaniline are potent inhibitors of lignostilbene-alpha,beta-dioxygenase, a key enzyme in oxidative cleavage of the central double bond of lignostilbene

Lignostilbene-alpha,beta-dioxygenase (LSD, EC 1.13.11.43) is involved in oxidative cleavage of the central double bond of lignostilbene to form the corresponding aldehydes by a mechanism similar to those of 9-cis-epoxycarotenoid dioxygenase and beta-carotene 15,15'-dioxygenase, key enzymes in abscisic acid biosynthesis and vitamin A biosynthesis, respectively. In this study, several N-benzylideneanilines and amine were synthesized and examined for their efficacy as inhibitors of LSD. N-(4-Hydroxybenzylidene)-3-methoxyaniline was found to be a potent inhibitor with IC50 = 0.3 microM and N-(4-hydroxybenzyl)-3-methoxyaniline was also active with IC50 = 10 microM. The information obtained from the structure-activity relationships study here can aid in discovering inhibitors of both abscisic acid and vitamin A biosynthesis.