Design,synthesis and biological evaluation of novel HIV-1 protease inhibitors with pentacyclic triterpenoids as P2-ligands

The design, synthesis and SAR study of a new series of HIV-1 protease inhibitors with pentacyclic triterpenoids as P2 ligands and phenylsulfonamide as P2′ ligands were discussed. These compounds exhibited micromolar inhibitory potency, among which compound T1c displayed HIV-1 protease inhibition with IC₅₀ values of 0.12?μM, which was 67 times the inhibitory activity of its raw material Ursolic acid (8.0?μM).     

C6-(N,N-butyl-methyl-heptanamide) derivatives of estrone and estradiol as inhibitors of type 1 17beta-hydroxysteroid dehydrogenase: Chemical synthesis and biological evaluation

A series of estrone and estradiol derivatives having an N-butyl,methyl heptanamide side chain at C6-position were synthesized, tested as inhibitors of type 1 17beta-HSD and assessed for their possible estrogenic activity. A better type 1 17beta-HSD inhibition was obtained for the 6beta-side chain orientation over 6alpha; the C17-alcohols are more potent inhibitors than the corresponding ketones; introducing a 2-methoxy group decreased the inhibitory potency; and the replacement of a C-S bond by a C-C bond in the C6beta-side chain is not detrimental to inhibition. Interestingly, the new inhibitors were also found less estrogenic than the lead compound in two breast cancer cell lines, T-47D and MCF-7.     

Chemical synthesis of 16beta-propylaminoacyl derivatives of estradiol and their inhibitory potency on type 1 17beta-hydroxysteroid dehydrogenase and binding affinity on steroid receptors

The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are members of a family of enzymes that catalyze the interconversion of weakly active sexual hormones (ketosteroids) and potent hormones (17beta-hydroxysteroids). Among the known isoforms of 17beta-HSD, the type 1 catalyzes the NAD(P)H-mediated reduction of estrone (E(1)) to estradiol (E(2)), a predominant mitogen for the breast cancer cells. Therefore, the inhibition of this particular enzyme is a logical approach to reduce the concentration of estradiol in breast tumors. To develop inhibitors of type 1 17beta-HSD activity, we hypothesized that molecules containing both hydrophobic and hydrophilic components should be interesting candidates for interacting with both the steroid binding domain and some amino acid residues of the cofactor binding domain of the enzyme. Firstly, a conveniently protected 16beta-(3-aminopropyl)-E(2) derivative was synthesized from commercially available E(1). Then, a representative of all class of NHBoc-protected amino acids (basic, acid, aromatic, aliphatic, hydroxylated) were coupled using standard procedures to the amino group of the precursor. Finally, cleavage of all protecting groups was performed in a single step to generate a series of 16beta-propylaminoacyl derivatives of E(2). The enzymatic screening revealed that none of the novel compounds can inhibit the reductive activity of type 1 17beta-HSD. On the other hand, all of these E(2) derivatives did not show any significant binding affinity on four steroid receptors including the estrogen receptor. Additional efforts aimed at improving the inhibitory potency of these steroidal derivatives on type 1 17beta-HSD without providing estrogenic activities is under investigation using a combinatorial chemistry approach.     

Some coumarins and triphenylethene derivatives as inhibitors of human testes microsomal 17beta-hydroxysteroid dehydrogenase (17beta-HSD type 3): further studies with tamoxifen on the rat testes microsomal enzyme

The 7-hydroxycoumarins, umbelliferone and 4-methylumbelliferone (IC50 = 1.4 and 1.9 microM, respectively) were potent inhibitors of human testes microsomal 17beta-HSD (type 3) enzyme whereas 7-methoxycoumarin, 4-hydroxycoumarin and 7-ethoxycoumarin had little or no inhibitory activity. Analogues of the weak inhibitory triphenylethenes tamoxifen and clomiphene but lacking the basic substituent, were weak inhibitors of the human microsomal enzyme. Inhibitory activity was improved by replacement of the triphenylethene structure with a triphenylmethyl (17, 52.6% inhibition) or phenylpropyl (16, 94.8%, IC50 = 42.1 microM) skeleton. Further studies on tamoxifen using rat testes microsomal 17beta-HSD showed that the inhibition was time-dependent and irreversible but not specifically mechanism-based.     

Structure–activity relationships of lanostane-type triterpenoids from Ganoderma lingzhi as α-glucosidase inhibitors

A series of lanostane-type triterpenoids, identified as ganoderma alcohols and ganoderma acids, were isolated from the fruiting body of Ganoderma lingzhi. Some of these compounds were confirmed as active inhibitors of the in vitro human recombinant aldose reductase. This paper aims to explain the structural requirement for α-glucosidase inhibition. Our structure–activity studies of ganoderma alcohols showed that the OH substituent at C-3 and the double-bond moiety at C-24 and C-25 are necessary to increase α-glucosidase inhibitory activity. The structure–activity relationships of ganoderma acids revealed that the OH substituent at C-11 is an important feature and that the carboxylic group in the side chain is essential for the recognition of α-glucosidase inhibitory activity. Moreover, the double-bond moiety at C-20 and C-22 in the side chain and the OH substituent at C-3 of ganoderma acids improve α-glucosidase inhibitory activity.These results provide an approach with which to consider the structural requirements of lanostane-type triterpenoids from G. lingzhi. An understanding of these requirements is considered necessary in order to improve a new type of α-glucosidase inhibitor.     

Structure-activity relationships of ganoderma acids from Ganoderma lucidum as aldose reductase inhibitors

A series of lanostane-type triterpenoids, known as ganoderma acids were isolated from the fruiting body of Ganoderma lucidum. Some of these compounds were identified as active inhibitors of the in vitro human recombinant aldose reductase. To clarify the structural requirement for inhibition, some structure–activity relationships were determined. Our structure–activity studies of ganoderma acids revealed that the OH substituent at C-11 is an important feature and the carboxylic group in the side chain is essential for the recognition of aldose reductase inhibitory activity. Moreover, double bond moiety at C-20 and C-22 in the side chain contributes to improving aldose reductase inhibitory activity. In the case of ganoderic acid C2, all of OH substituent at C-3, C-7 and C-15 is important for potent aldose reductase inhibition. These results provide an approach to understanding the structural requirements of ganoderma acids from G. lucidum for aldose reductase inhibitor. This understanding is necessary to design a new-type of aldose reductase inhibitor.     

Impairment of the antiproliferative effect of glucocorticosteroids by 11beta-hydroxysteroid dehydrogenase type 2 overexpression in MCF-7 breast-cancer cells.

To verify the relevance of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) activity in controlling breast-cancer cell growth, we have evaluated the correlation of 11beta-HSD2 expression and antiproliferative effects of glucocorticosteroids (GCs) on breast cancer cell proliferation. We cloned human 11beta-HSD2 cDNA into the expression vector pBK-CMV. The interspersing lac promoter region was deleted, achieving differential translational efficiency. The constructs were stably transfected into wild-type MCF-7 breast-cancer cells possessing almost no oxidative and no reductive 11beta-HSD activity. Low (times 7) and high (times 718) 11beta-HSD2 overexpression was achieved. We compared growth behavior of transfected cells In the presence of GCs to MCF-7 cells transfected with pBK-CMV alone (internal control). The antiproliferative effects of GCs were reversed and total cell growth boosted by overexpression of 11beta-HSD2; about 50 % of the increase in cell proliferation was attained by low 11beta-HSD2 overexpression, while high enzyme overexpression led to an increase in cell growth of about 120 %. Using direct evidence, this study shows 11beta-HSD2 to impair antiproliferative glucocorticosteroid effects, thus acting as an enzymatic shield aggravating breast-cancer cell growth. These results indicate a possible therapeutic role for 11beta-HSD inhibitors in the treatment of breast cancer.     

Discovery of novel inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 by docking and pharmacophore modeling

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is a potential target for treatment of diabetes and metabolic syndrome. Docking and pharmacophore modeling have been used to discover novel inhibitors of 11beta-HSD1. Several compounds, with large structural diversity and good potency against 11beta-HSD1, have been found and their potency was determined by the enzyme assay. New scaffolds of 11beta-HSD1 inhibitors are also reported.     

Differential regulation of the oxidative 11beta-hydroxysteroid dehydrogenase activity in testis and liver

11Beta-hydroxysteroid dehydrogenase (11beta-HSD) Type I enzyme is found in testis and liver. In Leydig cell cultures, 11beta-HSD activity is reported to be primarily oxidative while another report concluded that is primarily reductive. Hepatic 11beta-HSD preferentially catalyzes reduction and the reaction direction is unaffected by the external factors. Recent analysis of testicular 11beta-HSD revealed two kinetically distinct components. In the present study, various steroid hormones or glycyrrhizic acid (GCA), given for 1 week, or thyroxine given for 5 weeks to normal intact rats had different effects on the 11beta-HSD oxidative activity in testis and liver. Deoxycorticosterone, dexamethasone, progesterone, thyroxine, and clomiphene citrate increased testicular 11beta-HSD oxidative activity, but decreased hepatic enzyme activity except for deoxycorticosterone (unchanged). Corticosterone and testosterone decreased 11beta-HSD oxidative activity in testis but not that of liver (which was unchanged). Estradiol, GCA and adrenalectomy lowered oxidative activity of 11beta-HSD in testis and liver, but the degrees of reduction were different. The in vivo effects of glucocorticoids too were different, even in the same organ. Dexamethasone, a pure glucocorticoid, has greater affinity for glucocorticoid receptors (GR) than corticosterone. The direct effects of dexamethasone via GR in increasing testicular 11beta-HSD oxidative activity may override its indirect effects. Possibly, the reverse occurs with corticosterone treatment, as it has both glucocorticoid and mineralocorticoid effects. Because both organs have Type I isoenzyme, the difference in 11beta-HSD oxidative activities of these two organs could be attributable to the presence of an additional isozyme in testis or differences in tissue-specific regulatory mechanisms.     

Effect of immunosuppressive and other drugs on the cortisol-cortisone shuttle in human kidney and liver.

BACKGROUND: Impaired 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) has been suggested in patients with hypertension or renal disease, where it may contribute to sodium retention and hypertension. 11beta-HSD1, which is expressed predominantly in liver and adipose tissue, influences glucose homeostasis and fat distribution by altering intracellular cortisol (F) concentrations. We tested immunosuppressive drugs that cause hypertension, and substances that interfere with steroidogenesis or influence glucose homeostasis for their ability to influence the inhibition of 11beta-HSD isozymes. METHODS: For inhibition experiments, we used microsomes prepared from unaffected parts of human liver segments and resected human kidney cortex because of hepatocarcinoma or renal cell cancer. The inhibitory potency of several compounds was evaluated in concentrations from 10(-9)-10(-5) mol/l. RESULTS: Only sirolimus, but not cyclosporine A, tacrolimus, mycophenolate mofetil, or azathioprine showed a slight inhibition of 11beta-HSD2 activity. None of the drugs that inhibit steroidogenesis (suramine, mitotane, etomidate, and aminogluthethimide) or steroid metabolism (rifampicine) influenced 11beta-HSDs, nor did ginsenoides Re, Rc, and Rb1. Among sulfonylureas, only gliclazide decreased significantly 11beta-HSD1 activity. CONCLUSIONS: Increased blood pressure due to immunosuppressive drugs is probably not caused by direct inhibition of 11beta-HSD2. An additional glucose lowering effect of sulfonylurea gliclazide may be due to its ability to inhibit 11beta-HSD1.     

Novel enzymological profiles of human 11beta-hydroxysteroid dehydrogenase type 1

The human enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the reversible oxidoreduction of 11beta-OH/11-oxo groups of glucocorticoid hormones. Besides this important endocrinological property, the type 1 isozyme (11beta-HSD1) mediates reductive phase I reactions of several carbonyl group bearing xenobiotics, including drugs, insecticides and carcinogens. The aim of this study was to explore novel substrate specificities of human 11beta-HSD1, using heterologously expressed protein in the yeast system Pichia pastoris. In addition to established phase I xenobiotic substrates, it is now demonstrated that transformed yeast strains catalyze the reduction of ketoprofen to its hydroxy metabolite, and the oxidation of the prodrug DFU-lactol to the pharmacologically active lactone compound. Purified recombinant 11beta-HSD1 mediated oxidative reactions, however, the labile reductive activity component could not be maintained. In conclusion, evidence is provided that human 11beta-HSD1 in vitro is involved in phase I reactions of anti-inflammatory non-steroidal drugs like ketoprofen and DFU-lactol.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of 4-hydroxyphenyl ketones as potent and specific inhibitors of the type 3 of 17beta-hydroxysteroid dehydrogenase (17beta-HSD3)

We report the synthesis and biochemical evaluation of a number of 4-hydroxyphenyl ketones as potential inhibitors of the enzyme 17beta-hydroxysteroid dehydrogenase (17beta-HSD). In particular, we evaluated compounds against the catalysis of the conversion of androstenedione (AD) to testosterone (T) [17beta-HSD type 3 (17beta-HSD3)], furthermore, in an effort to determine the specificity of our compounds, we evaluated the ability of the compounds to inhibit the catalysis of the conversion of estrone (E1) to estradiol (E2) [17beta-HSD type 1 (17beta-HSD1)] as well as the conversion of dehydroepiandrosterone (DHEA) to AD [by 3beta-hydroxysteroid dehydrogenase (3beta-HSD)]. The results of our study suggest that the synthesised compounds are, in general, able to inhibit 17beta-HSD3 whilst being weak inhibitors of 17beta-HSD1. Against 3beta-HSD, we discovered that all of the synthesised compounds were weak inhibitors (all were found to possess less than 50% inhibition at [I]=500 microM). More specifically, we discovered that 1-(4-hydroxy-phenyl)-nonan-1-one (15) was the most potent against 17beta-HSD3 (IC(50)=2.9 microM) whilst possessing poor inhibitory activity against 17beta-HSD1 ( approximately 36% inhibitory activity against this reaction at [I]=100 microM) and less than 10% inhibition for the conversion of DHEA to AD. We have therefore provided good lead compounds in the design and synthesis of novel non-steroidal inhibitors of 17beta-HSD3.     

Type 2 11beta-hydroxysteroid dehydrogenase activity in human ovarian cancer

In the ovary cortisol-cortisone inter-conversion is catalyzed by the enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD). Its role in carcinomas of human ovary is unknown. The majority of ovarian cancers are derived from ovarian surface epithelium and the inflammation caused by successive ovulation seems to a play a role in the development of cancer. Cortisol is known to act as anti-inflammatory agent and its metabolism by type 1 and type 11beta-HSD may control the inflammatory action by cortisol in ovary. We undertook this study to investigate type 2 11beta-HSD activity which functions exclusively oxidative direction, in normal ovarian tissue compared to ovarian epithelial cancer. Ovarian tissue was obtained from patients undergoing hysterectomy for both benign and malignant disease. Tissue was placed immediately on dry ice and subsequently transferred to a freezer where they were maintained at -70 degrees C. NAD dependent 11beta-HSD activity was then determined in this tissue. T-test was performed to determine statistical significance. Mean type 2 enzyme activity was 0.87 +/- 1.65 pmol/min g tissue in normal ovarian tissue versus a mean enzyme activity of 2.96 +/- 1.37 pmol/mim g tissue in from cancer specimens. This difference was statistically significant with a p-value of 0.03. Type 2 1beta-HSD activity in ovarian cancer specimens was significantly higher than enzyme activity measured in normal post-menopausal ovarian tissue. Decreased cortisol levels due type 2 1beta-HSD activity may play a role neoplastic transformation as well as tumor proliferation in ovarian cancer by eliminating anti-inflammatory action of cortisol.     

Identification of steroidal derivatives inhibiting the transformations of allopregnanolone and estradiol by 17β-hydroxysteroid dehydrogenase type 10

17β-Hydroxysteroid dehydrogenase type 10 (17β-HSD10) is a mitochondrial enzyme known for its potential role in Alzheimer’s Disease (AD). 17β-HSD10, by its oxidative activity, could decrease the concentration of two important neurosteroids, allopregnanolone (ALLOP) and 17β-estradiol (E2), respectively preventing their neurogenesis and neuroprotective effects. Since the inhibition of 17β-HSD10 could lead to a new treatment for AD, we developed two biological assays using labeled ALLOP or E2 as substrates to measure the inhibitory activity of compounds against pure 17β-HSD10 protein. After the optimization of different parameters (time, concentration of enzyme, substrate and cofactor), analogs of the first reported steroidal inhibitor of 17β-HSD10 in intact cells were screened to determine their inhibitory potency for the ALLOP or the E2 oxidation. One compound, androstane derivative 5, possesses the best dual inhibition against both transformations (ALLOP, IC₅₀?=?235?μM and E2, IC₅₀?=?610?μM). Some compounds are dual inhibitors to a lesser extent, and others seem selective for one of the transformations in particular. By developing two reliable assays and by identifying a first generation of steroidal inhibitors of pure 17β-HSD10, this preliminary study opens the door to new and more potent inhibitors.     

Human 11beta-hydroxysteroid dehydrogenase type 1 is enzymatically active in its nonglycosylated form

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a microsomal enzyme responsible for the reversible interconversion of active 11beta-hydroxyglucocorticoids into inactive 11-ketosteroids and by this mechanism regulates access of glucocorticoids to the glucocorticoid receptor. The enzyme has also been proven to participate in xenobiotic carbonyl compound detoxification. 11beta-HSD 1 is anchored within the membranes of the endoplasmic reticulum (ER) by its N-terminus, whereby its active site protrudes into the lumen of the ER. In the primary structure of 11beta-HSD 1 three Asn-X-Ser glycosylation motifs have been identified. However, the importance of N-linked glycosylation of 11beta-HSD 1 for catalytic activity has been controversely discussed. To clarify if glycosylation is essential for enzyme activity, we performed deglycosylation experiments of native 11beta-HSD 1 from human liver as well as site-directed mutagenesis to remove potential glycosylation sites upon overexpression in Pichia pastoris. The altered proteins were examined regarding their catalytic activity towards their physiological glucocorticoid substrates. The molecular size of the various 11beta-HSD 1 forms was analyzed by immunoblotting with a polyclonal antibody raised against 11beta-HSD 1 protein from human liver. By stepwise enzymatic deglycosylation of native 11beta-HSD 1 we could demonstrate that all potential glycosylation sites carry N-linked oligosaccharide residues under physiological conditions. Interestingly, complete deglycosylation did not affect enzyme activity, neither in the reductive (cortisone) nor in the oxidative (cortisol) direction. Upon overexpression in the yeast P. pastoris, 11beta-HSD 1 did not undergo glycosylation, but, in spite of this, yielded a fully active enzyme. Our results conclusively demonstrate that 11beta-HSD 1 does not need to be glycosylated to perform its physiological role as glucocorticoid oxidoreductase.     

Spironolactone-related inhibitors of type II 17beta-hydroxysteroid dehydrogenase: chemical synthesis, receptor binding affinities, and proliferative/antiproliferative activities.

The family of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyzes the formation and inactivation of testosterone (T), dihydrotestosterone (DHT), and estradiol (E2), thus playing a crucial role in the regulation of active steroid hormones in target tissues. Among the five known 17beta-HSD enzymes, type II catalyzes the oxidation of E2 into estrone (E1), T into androstenedione, DHT into androstanedione, and 20alpha-dihydroprogesterone into progesterone. Specific inhibitors are thus an interesting means to study the regulation and to probe the structure of type II 17beta-HSD. In this context, we have efficiently synthesized a series of 7alpha-thioalkyl and 7alpha-thioaryl derivatives of spironolactone that inhibit type II 17beta-HSD. These new C19-steroidal inhibitors possess two important pharmacophores, namely 17-spiro-gamma-lactone and a bulky side-chain at the 7alpha-position. It was found that a para-substituted benzylthio group at the 7alpha-position enhances the inhibitory potency of spironolactone derivatives on type II 17beta-HSD. In fact, the compound with a para-hydroxy-benzylthio group showed an IC50 value of 0.5 microM against type II 17beta-HSD, whereas the compound with a para-[2-(1-piperidinyl)-ethoxy]-benzylthio group inhibited this enzyme with an IC50 value of 0.7 microM. The latter inhibitor is more selective than the former because it did not show any inhibitory potency against P450 aromatase as well as any affinity towards four steroid receptors (AR, PR, GR, ER). As a result, this inhibitor did not show any proliferative effect on androgen-sensitive Shionogi cells and estrogen-sensitive ZR-75-1 cells. These findings contribute to a better knowledge of the structure of type II 17beta-HSD and offer an interesting tool to study the regulation of this enzyme in several biological systems.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of phenyl alkyl imidazole-based compounds as potent inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

The cytochrome P450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitors of the two components of P450(17alpha), i.e., 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results suggest that the imidazole-based compounds are highly potent inhibitors of both components, with N-7-phenyl heptyl imidazole (21) (IC(50)=0.32 microM against 17alpha-OHase and IC(50)=0.10 microM against lyase) and N-8-phenyl octyl imidazole (23) (IC(50)=0.25 microM against 17alpha-OHase and IC(50)=0.21 microM against lyase) being the two most potent compounds within the current study, in comparison to ketoconazole (KTZ) (IC(50)=3.76 microM against 17alpha-OHase and IC(50)=1.66 microM against lyase). Furthermore, consideration of the inhibitory activity against the two components show that the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha). Structure-activity relationship determination of the range of compounds synthesised suggests that logP (log of the partition coefficient) is a key physicochemical factor in determining the overall inhibitory activity. In an effort to determine the viability of these compounds becoming potential drug candidates as well as to show specificity of these compounds, we undertook the biochemical evaluation of the synthesised compounds against two isozymes of 17beta-hydroxysteroid dehydrogenase [namely type 1 (17beta-HSD1) and type 3 (17beta-HSD3)] and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Consideration of the inhibitory activity possessed by the compounds considered within the current study against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 shows that there is no clear structure-activity relationship and that the compounds appear to possess similar inhibitory activity against both 3beta-HSD and 17beta-HSD3 whilst against 17beta-HSD1, the compounds appear to possess poor inhibitory activity at [I]=100 microM. Indeed, two of the most potent inhibitors of P450(17alpha), (compounds 21 and 23), were found to possess relatively good levels of inhibition against the three enzymes-compound 21 was found to possess approximately 32%, approximately 21% and approximately 37% inhibition whilst compound 23 was found to possess approximately 38%, approximately 30% and approximately 28% inhibition against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 respectively. We therefore concluded that the azole-based compounds synthesised within the current study are not suitable for further consideration as potential drug candidates due to their lack of specificity.     

Chronic corticosterone treatment impairs Leydig cell 11beta-hydroxysteroid dehydrogenase activity and LH-stimulated testosterone production.

The effects of excess corticosterone on luteinizing hormone (LH)-stimulated Leydig cell testosterone production and activity of 11beta-HSD was studied. Adult male rats (200-250 g body weight) were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days. Another set of rats was treated with corticosterone (dose as above) plus LH (ovine LH 100 microg/kg body weight, s.c., daily) for 15 days. Corticosterone administration significantly increased serum and testicular interstitial fluid (TIF) corticosterone but decreased testosterone levels. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone. The oxidative activity of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) was significantly decreased in Leydig cells of rats treated with corticosterone alone and in combination with LH. The direct effect of corticosterone on Leydig cell steroidogenic potency was also studied in vitro. Addition of corticosterone to Leydig cell culture showed a dose dependent effect on LH-stimulated testosterone production. Corticosterone at 50 and 100 ng/ml did not alter LH-stimulated testosterone production, but at high doses (200-400 ng/ml), decreased basal and LH-stimulated testosterone production. Basal and LH-stimulated cAMP production was not altered by corticosterone in vitro. It is concluded from the present study that elevated levels of corticosterone decreased the oxidative activity of 11beta-HSD and thus resulting in impaired Leydig cell steroidogenesis and the inhibitory effects of corticosterone on testosterone production appear to be mediated through inhibition of LH signal transduction at post-cAMP level.