PNU 157706, a novel dual type I and II 5alpha-reductase inhibitor

PNU 157706 is a novel dual inhibitor of 5alpha-reductase (5alpha-R), the enzyme responsible for the conversion of testosterone (T) to 5alpha-dihydrotestosterone (DHT). Tested on a crude preparation of human or rat prostatic 5alpha-R, PNU 157706 caused enzyme inhibition with IC50 values of 20 and 34 nM, respectively, compared to the values of 32 and 58 nM shown by finasteride. Furthermore, PNU 157706 was highly potent in inhibiting human recombinant 5alpha-R type I and II isozymes, showing IC50 values of 3.9 and 1.8 nM and, therefore, it was several folds more potent than finasteride (IC50 values of 313 and 11.3 nM), particularly on the type I isozyme. PNU 157706 was shown to have no binding affinity for the rat prostate androgen receptor (RBA 0.009% that of DHT). In adult male rats, a single oral dose of 10 mg/kg of PNU 157706 caused a marked and longer lasting reduction of prostatic DHT than did finasteride (at 24 h inhibition by 89 and 47%, respectively). In prepubertal, T- or DHT-implanted castrated rats, PNU 157706, given orally for 7 days at the dose of 10 mg/kg/day, markedly reduced ventral prostate weight in T- but not in DHT-implanted animals, thus showing to be devoid of any anti-androgen activity. In adult rats treated orally for 28 days, PNU 157706 resulted markedly more potent (16-fold) than finasteride in reducing prostate weight, the ED50 values being 0.12 and 1.9 mg/kg/day, respectively. These results indicate that PNU 157706 is a promising, potent inhibitor of both type II and I human 5alpha-R with a very marked antiprostatic effect in the rat.     

Tetrahydronaphthalenes: influence of heterocyclic substituents on inhibition of steroid enzymes P450 arom and P450 17

In search of new leads for selective inhibition of estrogen and androgen biosynthesis, respectively, heterocyclic substituted 2-(arylmethylene)-1-tetralones (1-4, 9-17), 2-(aryl-hydroxymethyl)-1-tetralones (5-8), exo-1a,2,3,7b-tetrahydro-1H-cyclopropa[alpha] naphthalenes (18-24), and 3-alkyl substituted 4,5-dihydronaphtho[1,2-c]pyrazoles (25-27) were synthesized and tested for inhibitory activity toward four steroidogenic enzymes (P450 arom, P450 17, P450 18, and P450 scc, as well as another P450 enzyme, thromboxane A(2) (TXA(2)) synthase. The test compounds inhibited human placental P450 arom, showing a wide range of inhibitory potencies. (Z)-4-Imidazolyl compound 17 was the most potent inhibitor, with a relative potency (rp) of 110 [rp of aminoglutethimide (AG) = 1), rp of fadrozole = 359]. A competitive type of inhibition was shown by the (E)-4-imidazolyl compound 16(rp = 71). On the other hand some of these compounds inhibited rat testicular P450 17. Maximum activity was shown by the 3-pyridyl compound 20 (rp = 10, ro of ketoconazole = 1). 20 was the only compound which exhibited a marked inhibition of TXA(2) synthase (IC(50) = 14.5 microM; IC(50) of dazoxiben = 1.1 microM). Regarding selectivity toward the steroidogenic enzymes, compound 16 was relatively selective toward P450 arom, whereas compound 20 was relatively selective toward P450 17. (P450 arom: K(m) testosterone = 42 nM, K(i)16 = 33 nM, K(i)20 = 3 microM. P450 17: K(m)progesterone = 7 microM, K(i)16 = 9 microM, K(i)20 = 80 nM). 17 and 24 were not selective since they showed strong inhibition of P450 arom (K(i)17 = 26 nM, K(i)24 = 0.12 microM) and P450 17 (K(i) 17 = 0.7 microM, K(i)24 = 0.11 microM).     

19-nor-10-azasteroids: a novel class of inhibitors for human steroid 5alpha-reductases 1 and 2

Steroid 5alpha-reductase is a system of two isozymes (5alphaR-1 and 5alphaR-2) which catalyzes the NADPH-dependent reduction of testosterone to dihydrotestosterone in many androgen sensitive tissues and which is related to several human endocrine diseases such as benign prostatic hyperplasia (BPH), prostatic cancer, acne, alopecia, pattern baldness in men and hirsutism in women. The discovery of new potent and selective 5alphaR inhibitors is thus of great interest for pharmaceutical treatment of these diseases. The synthesis of a novel class of inhibitors for human 5alphaR-1 and 5alphaR-2, having the 19-nor-10-azasteroid skeleton, is described. The inhibitory potency of the 19-nor-10-azasteroids was determined in homogenates of human hypertrophic prostates toward 5alphaR-2 and in DU-145 human prostatic adenocarcinoma cells toward 5alphaR-1, in comparison with finasteride (IC50 = 3 nM for 5alphaR-2 and approximately 42 nM for 5alphaR-1), a drug which is currently used for BPH treatment. The inhibition potency was dependent on the type of substituent at position 17 and on the presence and position of the unsaturation in the A and C rings. delta9(11)-19-Nor-10-azaandrost-4-ene-3,17-dione (or 10-azaestra-4,9(11)-diene-3,17-dione) (4a) and 19-nor-10-azaandrost-4-ene-3,17-dione (5) were weak inhibitors of 5alphaR-2 (IC50 = 4.6 and 4.4 microM, respectively) but more potent inhibitors of 5alphaR-1 (IC50 = 263 and 299 nM, respectively), whereas 19-nor-10-aza-5alpha-androstane-3,17-dione (7) was inactive for both the isoenzymes. The best result was achieved with the 9:1 mixture of delta9(11)- and delta8(9)-17beta-(N-tert-butylcarbamoyl)-19-nor-10-aza-4- androsten-3-one (10a,b) which was a good inhibitor of 5alphaR-1 and 5alphaR-2 (IC50 = 127 and 122 nM, respectively), with a potency very close to that of finasteride. The results of ab initio calculations suggest that the inhibition potency of 19-nor-10-azasteroids could be directly related to the nucleophilicity of the carbonyl group in the 3-position.     

Some 1-[(benzofuran-2-yl)methyl]imidazoles as inhibitors of 17 alpha-hydroxylase: 17, 20-lyase (P450 17) and their specificity patterns

Four 1-[(benzofuran-2-yl)methyl]imidazoles (1-4) have been evaluated as in-vitro inhibitors of human testicular and bovine adrenal microsomal 17 alpha-hydroxylase: 17,20-lyase (P450 17) as potential anti-prostatic agents. Their specificity towards other steroidogenic and liver enzymes has been compared with that of ketoconazole. All four compounds were inhibitors of the testicular enzyme (2, IC50 (concentration resulting in 50% inhibition) 0.185 microM; 4, IC50 0.18 microM) but less potent than ketoconazole (IC50 0.03 microM). Towards bovine adrenal enzyme 2 and 4 were 35- and 31-fold more potent than ketoconazole (IC50 = 39.8 microM). Compound 2 is a useful lead compound but although less potent than ketoconazole towards P450SCC and P450 11 beta, but not P450C21, at the enhanced dose required for equivalent effects in-vivo on P450 17 it is likely that cortisol and aldosterone production will be affected to a greater extent than with ketoconazole.     

Indole and benzimidazole derivatives as steroid 5alpha-reductase inhibitors in the rat prostate

A novel series of indole and benzimidazole derivatives were synthesized and evaluated for their inhibitory activity of rat prostatic 5alpha-reductase. Among these compounds, 4-?2-[1-(4,4'-dipropylbenzhydryl)indole-5-carboxamido]phenoxy?buty ric acid (15) and its benzimidazole analogue 25 showed potent inhibitory activities for rat prostatic 5alpha-reductase (IC50 values of 9.6+/-1.0 and 13+/-1.5 nM, respectively), with the potency very close to that of finasteride. Compound 30, in which the moiety between the benzene ring and amide bond was replaced by quinolin-4-one ring, showed almost equipotent activity (IC50= 19+/-6.2nM) with the correspondent amide derivative 13. This result was consistent with the previous observation that the coplanarity of this moiety might contribute to the potent inhibitory activity.     

The 16,17-double bond is needed for irreversible inhibition of human cytochrome p45017alpha by abiraterone (17-(3-pyridyl)androsta-5, 16-dien-3beta-ol) and related steroidal inhibitors

Abiraterone (17-(3-pyridyl)androsta-5,16-dien-3beta-ol, 1) is a potent inhibitor (IC50 4 nM for hydroxylase) of human cytochrome P45017alpha. To assist in studies of the role of the 16,17-double bond in its mechanism of action, the novel 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) and 17beta-(3-pyridyl)-16,17alpha-epoxy-5alpha-androst-3beta-ol (6) were synthesized. 3beta-Acetoxyetienic acid was converted in three steps into 5 via photolysis of the thiohydroxamic ester 8. Oxidation of an appropriate 16,17-unsaturated precursor (21) with CrO3-pyridine afforded the acetate (23) of 6. Inhibition of the enzyme by 1, the similarly potent 5,6-reduced analogue 19 (IC50 5 nM), and the 4, 16-dien-3-one 26 (IC50 3 nM) and by the less potent (IC50 13 nM) 3,5, 16-triene 25 is slow to occur but is enhanced by preincubation of the inhibitor with the enzyme. Inhibition following preincubation with these compounds is not lessened by dialysis for 24 h, implying irreversible binding to the enzyme. In contrast under these conditions the still potent (IC50 27 nM) 17alpha-(4-pyridyl)androst-5-en-3beta-ol (5) showed partial reversal after 5 h of dialysis and complete reversal of inhibition after 24 h. This behavior was also shown by the less potent 16,17-reduced 3-pyridyl compounds 3 and 24. Further, in contrast to the compounds (1, 19, 25, 26) with the 16,17-double bond, the inhibition of the enzymic reaction was not enhanced by preincubation either with 5 or with the 17beta-pyridyl analogues 3, 4, and 24 which also lack this structural feature. The results show that the 16,17-double bond is necessary for irreversible binding of these pyridyl steroids to cytochrome P45017alpha. However oxidation to an epoxide is probably not involved since epoxide 6 was only a moderately potent inhibitor (IC50 260 nM).     

Endocrine properties of the testosterone 5 alpha-reductase inhibitor turosteride (FCE 26073)

Turosteride was tested in a series of studies for its effect on 5 alpha-reductase and for its possible influence on other steroidogenic enzymes and on steroid receptors. The compound was found to inhibit human and rat prostatic 5 alpha-reductases with IC50 values of 55 and 53 nM, respectively, whereas it caused a less marked inhibition of the dog enzyme (IC50 2.2 microM). Turosteride showed no relevant effect on rat adrenal C20,22-desmolase (IC50 254 microM) and human placental aromatase (IC50 > 100 microM), and only at relatively high concentrations it caused inhibition of human placental 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) (IC50 2.5 microM). Turosteride was found to be a selective 5 alpha-reductase inhibitor showing no noteworthy binding to receptors for androgens (relative binding affinity, RBA, 0.004%), estrogens (< or = 0.005%), progesterone (< 0.005%), glucocorticoids (< 0.01%) and mineralocorticoids (< 0.03%). Its biochemical profile was similar to that of finasteride, whereas 4-MA (17 beta-N,N-diethyl-carbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one) was confirmed to be a non-selective 5 alpha-reductase inhibitor, showing a degree of binding affinity to the androgen receptor (RBA 0.1%) and a marked inhibition of 3 beta-HSD-I (IC50 32 nM). When given orally in immature castrated rats together with subcutaneous testosterone propionate (TP) for 7 consecutive days, turosteride reduced the ventral prostate and seminal vesicle growth promoting effect of TP, with IC50 values of approximately 5 and 6.7 mg/kg/day, whereas levator ani weight was unchanged. In comparison, 4-MA was approx. 3-fold less potent than turosteride in reducing the prostate and seminal vesicle weights and caused a marked reduction of levator ani weight, thus showing its unselectivity.     

Potent and selective aromatase inhibitor: in vitro and in vivo studies with s-triazine derivative SEF19

We found a potent aromatase inhibitor through the screening of agents for estrogen-dependent breast cancer. SEF19 (2-(imidazol-1-yl)-4,6-dimorphorino-1,3,5-triazine) decreased 50% of human placental aromatase activity in vitro at the concentration of 5.3 nM. In order to clarify the selectivity of SEF19 for enzyme inhibition, we determined the effect of SEF19 on the activities of four steroidogenic cytochrome P450 enzymes in porcine adrenal gland, P450SCC(side-chain cleavage of cholesterol), P450(11 beta) (11 beta-hydroxylase), P450(17 alpha)(17 alpha-hydroxylase/C17,20 lyase) and P450C21 (21-hydroxylase). SEF19 failed to inhibit the activities of porcine adrenal P450SCC, P450(17 alpha) and P450C21 up to the concentration of 100 microM and showed some inhibition on P450(11 beta) activity at 100 microM, while SEF19 completely nullified the aromatase activity at 1 microM. We also determined the potency of SEF19 for the suppression of aromatase activity in vivo. SEF19 suppressed dose-dependently the uterine hypertrophy of immature rats caused by administration of androstenedione (30 mg/kg, s.c.). The ED50 of SEF19 for the suppression of uterine hypertrophy was 0.8 mumol/kg. These results suggest that SEF19 may serve as a potent and selective agent for the treatment of estrogen-dependent breast cancer.     

N-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide: a potent and selective dopamine D4 ligand

A series of new 1-aryl-4-alkylpiperazines containing a terminal benzamide fragment or a tetralin-1-yl nucleus on the alkyl chain were synthesized and tested for binding at cloned human dopamine D4 and D2 receptor subtypes. A SAFIR (structure-affinity relationship) study on this series is herein discussed. The most relevant D4 receptor affinities were displayed by N-[omega-[4-arylpiperazin-1-yl]alkyl]-methoxybenzamides (compounds 5, 16-20), their IC50 values ranging between 0.057 and 7.8 nM. Among these, N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (17) emerged since it exhibited very high affinity for dopamine D4 receptor (IC50 = 0.057 nM) with selectivity of >10 000 for the D4 versus the D2 receptor; compound 17 was also selective versus serotonin 5-HT1A and adrenergic alpha1 receptors.     

Effect of 17 alpha hydroxy-20 beta dihydroprogesterone and progesterone on maturation and ovulation in vivo and on the plasma level of gonadotropic hormone t-GtH in rainbow trout Salmo gairdneri

Ovulation occurred 5 to 9 days after injection of 2 mg/kg 17 alpha-hydroxy-20 beta dihydroprogesterone (17 alpha-20 beta Pg) in rainbow trout with oocytes in which the germinal vesicle was subperipheral. Whereas oocyte maturation was initiated by 17 alpha-20 beta Pg without an immediate increase in the concentration of t-GtH, in controls an increased level was found to coincide with initiation of the process and the concentration of t-GtH was higher at all stages. Progesterone treatment was less effective.     

Monitoring system for the totally implantable ventricular assist system by use of sensors for virtual reality

The aim of this study was to compare the clinical efficacy and safety of low dose cyproterone acetate-estrogen combination (Diane) and the 5 alpha-reductase inhibitor finasteride in the treatment of hirsutism. Fourty-two women with hirsutism were included in the study. Twenty-one patients treated with cyproterone acetate (CPA) 2 mg and ethinyl estradiol (E) 35 micrograms daily on days 5-25 of the menstrual cycle, 21 with finasteride 5 mg daily. Hirsutism score, hormone levels, multiscreen blood chemistry and side effects were evaluated at three-monthly intervals for 9 months. A significant decrease in hirsutism score as compared to baseline was observed after 9 months with either CPA + E (Diane) (mean +/- SE, 15.81 +/- 1.19 vs 8.38 +/- 1.21) or finasteride treatment (17.81 +/- 1.05 vs 10.86 +/- 0.91) (p < 0.0005). The reductions in hirsutism scores (mean% +/- SE) were 14.23 +/- 2.29 vs 19.77 +/- 2.22 (p < 0.05) at 3, 40.23 +/- 4.58 vs 29.49 +/- 2.69 (p < 0.02) at 6 and 50.99 +/- 4.13 vs 39.87 +/- 3.30 (p < 0.02) at 9 months in CPA + E and finasteride groups, respectively. No significant changes were observed in hormone levels during finasteride treatment. Serum free testosterone significantly decreased at the third month of treatment, and remained suppressed for the duration of treatment in CPA + E group. DHEAS levels also decreased significantly after 6 and 9 months of therapy with CPA + E. SHBG significantly increased during CPA + E treatment. We conclude that both drugs are effective and well tolerated, but CPA + E appears to be more effective than 5 alpha-reductase inhibitor finasteride in long-term treatment of hirsute women. Diane is also a cost-effective drug.     

(1-Benzylindole-3-yl)alkanoic acids; novel nonsteroidal inhibitors of steroid 5 alpha-reductase (I)

A novel series of indole-3-alkanoic acids with varied N-benzyl substituents were synthesized as nonsteroidal inhibitors of steroid 5 alpha-reductase. The structure-activity relationships in this series were studied and the optimum carboxylic acid side chain was butyric acid. Furthermore, compounds with a diaryl substituent at the 1-position of the indole ring displayed strong inhibitory activities in vitro. Amongst these derivatives, 4-[1-(6,6-dimethyl-6H-dibenzo[b,d]pyran-3-yl)methylindol-3-yl]b uty ric acid (FR119680) displayed very high inhibitory activity in vitro against rat prostatic 5 alpha-reductase (IC50 = 5.0 nM) and good in vivo activity in the castrated young rat model.     

Reproductive steroids during maturation in a primitive teleost, the Pacific herring (Clupea harengus pallasi)

Concentrations of reproductive steroids were measured in the plasma of captive Pacific herring, Clupea harengus pallasi, (1) prior to ovulation and milt production, (2) during the periovulatory and newly milt-producing period, (3) during a "ripe" holding period after ovulation and during milt production, and (4) before and after spawning. 17alpha,20beta-Dihydroxyprogesterone (17,20beta-P), despite being present only in low concentrations in the unconjugated (free) form (<10 ng/ml), is likely to be the maturation inducing steroid in females and was associated with the initiation of milt production in males since its levels are elevated coincidentally with these events. Glucuronated 17,20beta-P, free 17alpha-hydroxyprogesterone (17-P), and free and glucuronated 3alpha, 17alpha-dihydroxy-5beta-pregnan-20-one (3alpha,17-P-5beta) were present in high concentrations (140-250 ng/ml) in periovulatory females and newly milt-producing males. This steroid pattern suggests that the low levels of 17,20beta-P are due to glucuronation and competitive conversion of its precursor, 17-P, to free and glucuronated 3alpha, 17-P-5beta. Glucuronated testosterone was the principal steroid in preovulatory and premilt-producing fish (200-350 ng/ml), coincident with similar levels of glucuronated 11-ketotestosterone in males. After ovulation females did not spawn synchronously until 2 months later, which may be partially due to reduced environmental cues in the captive situation, while male fish released milt sporadically throughout the ripe holding period. Steroidal indicators of readiness to spawn in females or males were not detected. Rather, levels of all steroids gradually decreased in ripe holding fish (<30 ng/ml) to reach even lower levels (<1 ng/ml) after spawning. We suggest that "runniness" of gametes is a distinctive characteristic of females that are ready to spawn, but that this may result from relaxation of sphincter muscles rather than being an additional maturational step.     

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HEK293 cells were stably transfected with rat neuronal nicotinic alpha4 and beta2 subunits. Binding of tritiated cytisine and nicotine to cell homogenates revealed the presence of a single class of high-affinity sites (dissociation constants 0.1 nM and 0.4 nM, respectively). Activation of nicotinic receptors was studied using whole-cell patch clamp methods, and acetylcholine, nicotine, dimethylphenylpiperazinium, and cytisine all produced a conductance increase. Responses desensitized to prolonged applications, at both positive and negative membrane potentials. The conductance was strongly rectifying, and outward currents were essentially absent. Responses were maximal at about 2 mM external calcium ion concentration and were reduced by about one-half at either nominally 0 or 10 mM external calcium. Di-hydro-beta-erythroidine blocked physiological responses to acetylcholine and nicotine (IC50, 2.5 nM), and reduced cytisine binding in a competitive manner (Ki 20 nM). Physostigmine enhanced the response to low concentrations of acetylcholine or nicotine. The anesthetic steroid (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile blocked responses to acetylcholine (IC50, 1.3 microM), but had no effect on cytisine binding at a concentration of 30 microM. The binding properties of the receptors are those expected for rat neuronal nicotinic receptors composed of alpha4 and beta2 subunits. The pharmacological properties indicate that the responsiveness of the receptors may be allosterically enhanced or inhibited.     

Study of 3 alpha, 20 beta-hydroxysteroid dehydrogenase with an enzyme-generated affinity alkylator: dual enzyme activity at a single active site

The substrate 17 beta-[(1S)-1-hydroxy-2-propynyl]-androst-4-en-3-one (beta-HPA) and its enzyme-generated alkylating product 17 beta-(1-oxo-2-propynyl)androst-4-en-3-one (OPA) were synthesized to investigate the relationship between the 3 alpha and 20 beta activities observed in commercially available cortisone reductase (EC 1.1.1.53) from Streptomyces hydrogenans. beta-HPA, a substrate [apparent Km = 145 microM; Vmax = 63 nmol (min microgram)-1], when enzymatically oxidized by cortisone reductase of OPA, inactivates simultaneously the 3 alpha and 20 beta activities in a time-dependent and irreversible manner following pseudo-first-order kinetics. OPA alone, an affinity alkylating steroid (KI = 40.5 microM; k3 = 1.8 X 10(-2) S-1), simultaneously inactivates 3 alpha and 20 beta activities in a time-dependent and irreversible manner. At pH 7, the t 1/2 of enzyme inactivation for beta-HPA (10 h) or OPA (41 min) is slower than at pH 9.2 (beta-HPA, 16 min, and OPA, 3.3 min). Substrates (progesterone, 20 beta-hydroxypregn-4-en-3-one, and 5 alpha-dihydrotestosterone), but not all steroids (20 al]ha-delta 4-pregn-4-en-3-one and 17 beta-estradiol), protect against loss of both enzyme activities by beta-HPA and OPA. The alpha isomer of HPA is not enzymatically oxidized and therefore does not cause inactivation of either 3 alpha or 20 alpha activity. Thus, beta-HPA functions as a substrate for the enzymatic generation of a powerful affinity alkylator of cortisone reductase. Second, the identical change in both the 3 alpha and 20 beta activities in all experimental conditions clearly results from dual enzyme activity at a single enzyme active site.     

Inhibition studies of dehydroepiandrosterone 7 alpha- and 7 beta- hydroxylation in mouse liver microsomes

Hydroxylations of dehydroepiandrosterone (DHEA) at the 7 alpha- and 7 beta- positions have been reported in numerous murine tissues and organs, including liver, and the responsible cytochrome P450 (P450) species await identification. Using thin layer chromatography and gas chromatography-mass spectrometry, we report identification of 7 alpha-hydroxy-DHEA and 7 beta-hydroxy-DHEA metabolites produced in mouse liver microsome digests and kinetic studies of their production with apparent KM values of 3.19 +/- 0.292 microM and 2.82 +/- 0.241 microM for 7 alpha- and 7 beta-hydroxylation, respectively. Investigation of P450 inhibitor and of steroid hormone effects on both 7 alpha- and 7 beta-hydroxylation of DHEA showed that, 1) different P450s were involved in 7 alpha- and 7 beta-hydroxylation of DHEA because metyrapone inhibited solely 7 alpha-hydroxylation, 2) P450 2D6, 2B1, and 2B11 were not responsible for 7 alpha- and 7 beta-hydroxylation of DHEA because respective specific inhibitors quinidine and chloramphenicol triggered no inhibition, 3) aside from P450 7b, P450 1A1, and 1A2 may be responsible for a fraction of DHEA 7 alpha- and 7 beta-hydroxylation because alpha-naphthoflavone and furafylline, which inhibit specifically P450 1A1 and 1A2, decreased the 7 alpha- and 7 beta-hydroxylation partly, 4) comparison of these findings with those obtained with brain microsomes suggested that tissue-specific P450 species are responsible for the 7 alpha- and 7 beta-hydroxylation of DHEA, 5) 7 alpha-hydroxylation of DHEA may be shared with other 3 beta-hydroxysteroids, such as 3 beta-hydroxy-5 alpha-androstan-17-one, 5-androstene-3 beta,17 beta-diol and pregnenolone, which acted in a noncompetitive manner. Taken together, these findings will be of use for identification of the P450 species responsible for 7 alpha- and 7 beta-hydroxylation of DHEA and for studies of their activities in liver.     

The molecular basis of isolated 17,20 lyase deficiency

Human P450c17 catalyzes the 17alpha-hydroxylation of pregnenolone to 17OH pregnenolone and of progesterone to 17alpha-OH progesterone; the same P450c17 enzyme also catalyzes 17,20 lyase activity on the same active site, converting 17OH-pregnenolone to DHEA. Rodent and porcine P450c17 also catalyze 17,20 lyase activity with delta4 substrates, converting 17OH-progesterone to delta4 androstenedione, but human P450c17 catalyzes this reaction very inefficiently, so that virtually all human C19 sex steroids are made via 17OH pregnenolone and DHEA. P450c17 is encoded by a single gene and a single species of mRNA. Many mutations of this gene have been described, but until recently all of these either entirely eliminated both 17alpha-hydroxylase and 17,20 lyase activity, or affected each activity equivalently. We have identified and characterized the first patients with P450c17 mutations that selectively ablate 17,20 lyase activity while retaining 17alpha-hydroxylase activity. Through a combination of enyzmologic experiments in transfected mammalian cells and in genetically manipulated yeast, plus a computer model of human P450c17, we have proven that the responsible mutations, R347H and R358Q lie in the redox-partner binding site of P450c17. This site, through which P450c17 interacts with P450 oxidoreductase to receive the electrons needed for catalysis, can be allosterically influenced by cytochrome b5. These two mutations have contributed substantially to our understanding of the mechanisms by which 17alpha-hydroxylase and 17,20 lyase activities are regulated independently, and thus have contributed to the study of regulated 17,20 lyase activity in adrenarche, aging, and the polycystic ovary syndrome.     

Thromboxane A2 stimulated signal transduction in vascular smooth muscle

Thromboxane A2 (TXA2) is a potent, labile vasoconstrictor which stimulates vessel contraction through vascular smooth muscle TXA2 receptors differing from those in platelets. We studied TXA2-stimulated events in cultured adult rat aortic smooth muscle cells. The stable TXA2 mimetic (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619) competed for TXA2 agonist binding to vascular smooth muscle cells with an IC50 of 10 +/- 1 nM. In fura-2-loaded cells, U46619 increased free cytosolic Ca++ concentration with an EC50 of 49 +/- 14 nM. The increase in free cytosolic Ca++ was rapid, transient and independent of extracellular Ca++ or Ca++ antagonists and thus was due to release from intracellular stores. U46619-mediated Ca++ release was temporally associated with phosphorylation of myosin light chains, increased accumulation of 1,4,5-inositol trisphosphate (EC50 = 32 +/- 4 nM) and cytoplasmic acidification from pH 7.06 +/- 0.01 to 7.00 +/- 0.02 (P = .02). Ca++ release was 53% attenuated by the phospholipase C inhibitor, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H- pyrrole-2,5-dione. In rat aortic rings U46619 caused TXA2 receptor-mediated contractions (EC50 of 28 +/- 2 nM) which were not attenuated by removal of extracellular Ca++ from the superfusion buffer. Together, these results suggest that agonist occupation of TXA2 receptors produces vascular smooth muscle contraction through initial activation of phospholipase C with production of 1,4,5-inositol phosphate, release of intracellular calcium stores and phosphorylation of myosin light chains associated with cellular acidification, presumably via activation of Ca++ ATPase.     

17 alpha-alkyl- or 17 alpha-substituted benzyl-17 beta-estradiols: a new family of estrone-sulfatase inhibitors

A series of 17 alpha-derivatives of 17 beta-estradiol was synthesized and tested for their ability to inhibit the estrone-sulfatase activity transforming estrone sulfate to estrone. A strong inhibitory activity was obtained when an alkyl side chain or a substituted benzyl was introduced at position 17 alpha of estradiol. The 17 alpha-(3'-bromobenzyl)-estradiol (26) and 17 alpha-(4'-t-butylbenzyl)-estradiol (30) were the most potent estrone-sulfatase inhibitors obtained in our study with IC50 values of 24 and 28 nM, respectively. They also represent a new family of estrone-sulfatase inhibitors. These compounds are about 300-fold more effective in interacting with the enzyme than the substrate estrone sulfate itself.     

Interactions of 16alpha,17alpha-cyclohexane derivatives of progesterone with the progesterone receptor from rat uterus

Pregna-D'-pentaranes, 16alpha,17alpha-cyclohexanoprogesterone and 6alpha-methyl-16alpha,17alpha-cyclohexanoprogestero ne, were found to specifically interact with the progesterone receptor of soluble fraction from rat uterus. The formation of complexes between 3H-labeled derivatives of these steroids and the protein was complete within 1 to 3 h at 0-4 degreesC. The dissociation of these complexes was a two-phase process, the contribution of the fast dissociating complexes decreasing with increasing preincubation time. The dissociation constant (k-1) values for progesterone, 16alpha, 17alpha-cyclohexanoprogesterone, and 6alpha-methyl-16alpha, 17alpha-cyclohexanoprogesterone complexes with the protein after 1 h preincubation were 6.5 +/- 0.8, 8.8 +/- 5.5, and (16.6 +/- 5.6).10(-4) sec(-1) for the fast phase and 5.1 +/- 0.5, 3.5 +/- 0.8, and (2.8 +/- 0.6).10(-5) sec(-1) for slow phase, respectively. The equilibrium Kd values were 11.7 +/- 2.1, 19.0 +/- 2.0, and 66.1 +/- 14.6 nM for progesterone, 16alpha,17alpha-cyclohexanoprogesterone, and 6alpha-methyl-16alpha,17alpha-cyclohexanoprogestero ne, respectively. The steroids mutually inhibited the binding of their 3H-labeled derivatives to the protein, the inhibition being of competitive type. In the case of [3H]6alpha-methyl-16alpha, 17alpha-cyclohexanoprogesterone, the inhibitory efficacy of progesterone declined with an increase of its concentration; this points to possible heterogeneity of binding sites for the 3H-labeled ligand. The comparison of the results with those obtained by us earlier (Biochemistry (Moscow), 1996, 61, 1034-1041) suggests the existence of significant species differences in progesterone receptor structure within or near the region that interacts with the D-ring of a hormone molecule.