Lipid-mediated unfolding of 3β-hydroxysteroid dehydrogenase 2 is essential for steroidogenic activity

For inner mitochondrial membrane (IMM) proteins that do not undergo N-terminal cleavage, the activity may occur in the absence of a receptor present in the mitochondrial membrane. One such protein is human 3β-hydroxysteroid dehydrogenase 2 (3βHSD2), the IMM resident protein responsible for catalyzing two key steps in steroid metabolism: the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione. Conversion requires that 3βHSD2 serve as both a dehydrogenase and an isomerase. The dual functionality of 3βHSD2 results from a conformational change, but the trigger for this change remains unknown. Using fluorescence resonance energy transfer, we found that 3βHSD2 interacted strongly with a mixture of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC). 3βHSD2 became less stable when incubated with the individual lipids, as indicated by the decrease in thermal denaturation (T(m)) from 42 to 37 °C. DPPG, alone or in combination with DPPC, led to a decrease in α-helical content without an effect on the β-sheet conformation. With the exception of the 20 N-terminal amino acids, mixed vesicles protected 3βHSD2 from trypsin digestion. However, protein incubated with DPPC was only partially protected. The lipid-mediated unfolding completely supports the model in which a cavity forms between the α-helix and β-sheet. As 3βHSD2 lacks a receptor, opening the conformation may activate the protein.     

A quantitative cytochemical method for the demonstration of Β5,3β-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary

Summary A method for the quantitative measurement of ⁵, 3-hydroxysteroid dehydrogenase activity in unfixed tissue sections of rat ovary has been described. The method depends on the oxidation of dehydroepiandrosterone (DHEA) and uses nitroblue tetrazolium as the final electron acceptor. Although the dehydrogenase is not a soluble enzyme, polyvinyl alcohol is included in the reaction medium to allow the use of a high substrate concentration whilst employing a low concentration (5%) of dimethyl formamide. The enzyme is equally dependent on NAD⁺ or NADP⁺ for its activity and this activity is significantly enhanced by the presence of cyanide. The NADP⁺ dependence is not abolished by inhibiting nonspecific alkaline phosphomonoesterase. The activity of ⁵, 3-hydroxysteroid dehydrogenase is completely dependent on a functional sulphydryl group. Furthermore, the enzyme activity is totally inhibited in the presence of a steroid substrate analogue at 10^–4 M.     

Development of a sensitive and selective LC-MS/MS method for the determination of α-fluoro-β-alanine, 5-fluorouracil and capecitabine in human plasma

A sensitive and selective quantitative method to determine α-fluoro-β-alanine (FBAL), 5-fluorouracil (5-FU), and capecitabine (Cape) from a single human plasma aliquot (50 μL) has been developed and validated. First, 5-FU and Cape were extracted by liquid–liquid extraction (LLE) using a mixture of acetonitrile and ethyl acetate. This was followed by derivatization with dansyl chloride. The dansyl-derivatives from 5-FU and Cape were further purified using LLE with methyl tertiary-butyl ether (MTBE) and analyzed using a reversed-phase analytical column “Primesep D” (2.1 mm × 50 mm; 5 μm) with embedded basic ion-pairing groups. The remaining aqueous phase containing FBAL was treated with dansyl chloride and the dansyl-FBAL was purified by solid phase extraction. Ultra high pressure liquid chromatography (UPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for analysis of dansyl-FBAL. The method was validated over the concentration ranges of 10–10,000, 5–5000, and 1–1000 ng/mL for FBAL, 5-FU, and Cape, respectively. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in 1 day.     

Biological activity of pyrazole and imidazole-dehydroepiandrosterone derivatives on the activity of 17β-hydroxysteroid dehydrogenase

The enzyme type 5 17β-hydroxysteroid dehydrogenase 5 (17β-HSD5) catalyzes the transformation of androstenedione (4-dione) to testosterone (T) in the prostate. This metabolic pathway remains active in cancer patients receiving androgen deprivation therapy. Since physicians seek to develop advantageous and better new treatments to increase the average survival of these patients, we synthesized several different dehydroepiandrosterone derivatives. These compounds have a pyrazole or imidazole function at C-17 and an ester moiety at C-3 and were studied as inhibitors of 17β-HSD5. The kinetic parameters of this enzyme were determined for use in inhibition assays. Their pharmacological effect was also determined on gonadectomized hamsters treated with Δ⁴-androstenedione (4-dione) or testosterone (T) and/or the novel compounds. The results indicated that the incorporation of a heterocycle at C-17 induced strong 17β-HSD5 inhibition. These derivatives decreased flank organ diameter and prostate weight in castrated hamsters treated with T or 4-dione. Inhibition of 17β-HSD5 by these compounds could have therapeutic potential for the treatment of prostate cancer and benign prostatic hyperplasia.     

Disposition and metabolism of 2-fluoro-β-alanine conjugates of bile acids following secretion into bile

Since 2-fluoro-β-alanine (FBAL) conjugates of bile acids (BA), the primary biliary metabolites of fluoropyrimidine (FP) drugs, have been suggested to be related to the hepatotoxicity which develops in patients receiving FP chemotherapy by intrahepatic arterial infusion (Proc. Natl. Acad. Sci. USA 84, 5439–5443, 1987), it was important to determine whether they undergo enterohepatic circulation and hence accumulate in the liver and biliary system. In initial studies, sensitivity of FBAL-BA conjugates to hydrolysis by pancreatic enzymes was examined. In subsequent in vivo studies, a model FBAL-BA conjugate, FBAL-chenodeoxycholate (FBAL-CDC), was introduced into the lumen of the small intestine of anesthetized rats with biliary fistulas to quantitate the intestinal absorption, metabolism and tissue distribution of the conjugate. The results indicated that: (1) FBAL-BA conjugates were resistant to hydrolysis by pancreatic enzymes (carboxypeptidase A, carboxypeptidase B and trypsin) and by human pancreatic juice, but were completely hydrolyzed by cholylglycine hydrolase. (2) At least one-half of the administered FBAL-CDC was deconjugeted during the process of intestinal absorption, as shown by HPLC analysis of the radioactivity in portal venous blood. (3) Deconjugated FBAL or CDC was reconjugated in liver with other bile acids or amino acids (glycine and taurine), respectively, as shown by radiochromatography of bile. (4) FBAL, formed as a result of hydrolysis of FBAL-CDC, had a wide tissue distribution. In conclusion, FBAL-CDC has a rapid turnover during its enterohepatic circulation due to deconjugation in the intestine and reconjugation in the liver.     

A quantitative cytochemical method for the measurement of β-hydroxyacyl CoA dehydrogenase activity in rat heart muscle

Summary Although cytochemical methods exist for measuring dehydrogenases that act on substrates involved in the production of chemical energy from sugars, virtually no methods exist for measuring the dehydrogenases that act on fatty acids. Yet the oxidation of fatty acids accounts for over 60% of the oxidative activity of cardiac muscle. Consequently a new quantitative cytochemical method, based on a new substrate (DL-S--hydroxybutyryl-N-acetyl cysteamine), has been developed for measuring the activity of hydroxy-acyl coenzyme A dehydrogenase, which is the penultimate step of the -oxidation of fatty acids to acetyl-coenzyme A that is used in the Krebs' cycle. Menadione or phenazine methosulphate is used as the intermediate hydrogen-acceptor, with neotetrazolium chloride as the final acceptor. The medium contains nitroprusside, ostensibly to react with any cysteamine liberated by hydrolysis of the substrate. As a control, cysteamine is substituted for the substrate. The concentrations of reactants have been optimized for cardiac muscle; the reaction is linear with thickness of the sections and with time of reaction from 15 to 60 min.     

The effect of water loading on the urinary ratio of cortisone to cortisol in healthy subjects and a new approach to the evaluation of the ratio as an index for in vivo human 11β-hydroxysteroid dehydrogenase 2 activity

Factors that give rise to a large variation in the urinary ratio of free cortisone to cortisol (UFE/UFF) were investigated to accurately estimate 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity in humans in vivo. A water loading test was first carried out in two healthy subjects to examine the effect of water intake or urine volume on the urinary ratio of free cortisone to cortisol (UFE/UFF). The ratio was found to increase by water loading. We also examined urinary concentrations and amounts of cortisol, cortisone, creatinine, Na(+), K(+), and Cl(-), and urine volume, as possible factors affecting the urinary ratio (UFE/UFF), in 60 urine samples obtained from 15 healthy volunteers. Among these factors tested, the urinary concentration of cortisol was most highly correlated with the UFE/UFF ratio (r=-0.858), indicating that the in vivo activity of 11β-HSD2 (UFE/UFF) should fluctuate with the changes of the urinary concentration of cortisol. Based on the findings, we proposed a new estimation method of in vivo activity of 11β-HSD2 in humans, using the UFE/UFF ratio correlated with the urinary concentration of cortisol (UFE/UFF-cortisol concentration). Taking into consideration the intra-individual variabilities in the urinary concentration of cortisol, there were no significant within-day variations in 11β-HSD2 activity. The findings indicate that 11β-HSD2 activities can be accurately evaluated by simply measuring free cortisol and cortisone concentrations in spot urine samples. Furthermore, administrations of glycyrrhetinic acid in three healthy volunteers were performed to confirm the usefulness of the present assessment for the activity of 11β-HSD2.     

Improved histochemical method for the demonstration of the activity of α-glucan phosphorylase

Summary The activity and localization of -glucan phosphorylase in experimental canine glycogen-depleted heart tissue has been investigated with biochemical and histochemical methods using dextran as enzyme acceptor. Only linear, essentially unbranched, dextrans exhibit acceptor properties; highly branched dextrans are not suitable acceptors for the enzyme. Results of Michaelis-Menten constant measurements for the linear essentially unbranched dextran fractions used, indicate that the affinity of the enzyme for the non-reducing end group of the dextran molecule increases with increasing molecular weight of the acceptor.In the glycogen-depleted tissue of anoxic and ischaemic cardiac musculature there is a gradual inactivation of the enzyme during the ischaemic period. Shortly before total inactivation the affinity of the enzyme, especially for the lower molecular dextran fractions, is greatly reduced. Therefore, for the histochemical demonstration of phosphorylase activity in infarcted areas of the heart it is essential to use as acceptor an unbranched dextran fraction with a high average molecular weight.This investigation was partially supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Improved histochemical method for the demonstration of the activity of α-glucan phosphorylase

Summary A modified method for the histochemical demonstration of the activity of -glucan phosphorylase is described. In the histochemical system the enzyme catalyses the synthesis of glycogen by transglucosylation from -d-glucosylphosphate. The incubation medium contains dextran as glucosyl acceptor. Therefore, in contrast with the unmodified method, the new technique is able to demonstrate the activity of phosphorylase in ischaemic glycogen-depleted tissues.     

Strain-specific differences in the regulation of the hepatic cytoplasmic 17β-hydroxysteroid dehydrogenase activity of the rat

• 1.1. The effect of prepuberal gonadectomy of Sprague-Dawley/NIH/HAN rats on cytoplasmic 17β-hydroxysteroid dehydrogenase was examined on day 30, 45, 60, 75, 90 and 105 of life.
• 2.2. The activity in male rats was not significantly affected by gonadectomy, whereas the activity in females showed an age-dependent oestrogen dependency.
• 3.3. This age-dependent oestrogen dependency could also be demonstrated in 5α-dihydrotestosterone treated intact females.
• 4.4. Cytoplasmic 17β-hydroxysteroid dehydrogenase activity of female Chbb:THOM rats also showed an age-dependent oestrogen dependency, whereas the enzyme activity of male rats of this strain showed a distinct androgen dependency absent in Sprague-Dawley/NIH/HAN rats.
• 5.5. On the basis of previous investigations it is concluded that the androgen dependency of the enzyme activity of male Chbb:THOM rats has been bred into this strain in the period 1974–1977.

     

TTC-based screening assay for ω-transaminases: a rapid method to detect reduction of 2-hydroxy ketones

A rapid TTC-based screening assay for ω-transaminases was developed to determine the conversion of substrates with a 2-hydroxy ketone motif. Oxidation of the compounds in the presence of 2,3,5-triphenyltetrazolium chloride (TTC) results in a reduction of the colourless TTC to a red-coloured 1,3,5-triphenylformazan. The enzymatic reductive amination of a wide range of various aliphatic, aliphatic-aromatic and aromatic-aromatic 2-hydroxy ketones can be determined by the decrease of the red colouration due to substrate consumption. The conversion can be quantified spectrophotometrically at 510 nm based on reactions, e.g. with crude cell extracts in 96-well plates. Since the assay is independent of the choice of diverse amine donors a panel of ω-transaminases was screened to detect conversion of 2-hydroxy ketones with three different amine donors: l-alanine, (S)-α-methylbenzylamine and benzylamine. The results could be validated using HPLC and GC analyses, showing a deviation of only 5-10%. Using this approach enzymes were identified demonstrating high conversions of acetoin and phenylacetylcarbinol to the corresponding amines. Among these enzymes three novel wild-type ω-transaminases have been identified.     

3β-Hydroxy steroid dehydrogenase activity in the mitochondria of rat adrenal homogenates

The activity of 3beta-hydroxy steroid dehydrogenase (EC 1.1.1.51) in the mitochondrial fraction of rat adrenal homogenates was approx. 31% of the total activity recovered after differential centrifugation and washing of the particulate fractions. Some 45% of the total activity was found in the microsomal fraction. The activity was assayed by a radioisotopic method devised in this laboratory for the purpose of studying small quantities of tissue and cell fractions. Satisfactory separation of the two fractions was demonstrated by electron microscopy of the pellets and by comparative recoveries of RNA, steroid 21-hydroxylase and cytochrome c oxidase in the various compartments. Analyses of the kinetics of the enzyme activity in the two fractions revealed no significant differences in apparent K(m) for pregnenolone, dehydroepiandrosterone or NAD(+), but demonstrated a distinct difference in the K(m) for NADP(+). pH optima and susceptibility to cyanoketone inhibition were similar in both fractions.     

Engineering a hydroxysteroid dehydrogenase to improve its soluble expression for the asymmetric reduction of cortisone to 11β-hydrocortisone

11β-Hydrocortisone (11β-HC) is an important anti-inflammatory drug and intermediate for the synthesis of other steroids. One of the methods for the synthesis of 11β-HC is the asymmetric reduction of cortisone catalyzed by a highly regioselective and stereoselective 11β-hydroxysteroid dehydrogenase (11β-HSDH). However, this process has been prohibited by the poor soluble expression of the membrane-anchoring protein 11β-HSDH in prokaryotes. To overcome this obstacle, a mutant III-1G1 (Phe80Leu/Thr105Ser/Ala260Thr/Tyr274Stop) truncated at position 274 with improved yield of soluble protein was stepwise obtained from the 11β-HSDH from guinea pig by random mutagenesis combining with structural complementation assay and C-terminal truncating library screening. The improved 11β-HSDH mutant and glucose dehydrogenase (GDH) from Bacillus subtilis were coexpressed in Escherichia coli. The resulting whole-cell biocatalyst catalyzed the reduction of cortisone to 11β-HC with 98 % conversion in 20 h, laying foundation for the development of an asymmetric reduction process for the production of 11β-HC.     

11β-Hydroxysteroid dehydrogenase 1 contributes to the pro-inflammatory response of keratinocytes

The endogenous glucocorticoid, cortisol, is released from the adrenal gland in response to various stress stimuli. Extra-adrenal cortisol production has recently been reported to occur in various tissues. Skin is known to synthesize cortisol through a de novo pathway and through an activating enzyme. The enzyme that catalyzes the intracellular conversion of hormonally-inactive cortisone into active cortisol is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). We recently reported that 11β-HSD1 is expressed in normal human epidermal keratinocytes (NHEKs) and negatively regulates proliferation of NHEKs. In this study, we investigated the role of 11β-HSD1 in skin inflammation. Expression of 11β-HSD1 was induced by UV-B irradiation and in response to the pro-inflammatory cytokines, IL-1β and TNFα. Increased cortisol concentrations in culture media also increased in response to these stimuli. To investigate the function of increased 11β-HSD1 in response to pro-inflammatory cytokines, we knocked down 11β-HSD1 by transfecting siRNA. Production of IL-6 and IL-8 in response to IL-1β or TNFα stimulation was attenuated in NHEKs transfected with si11β-HSD1 compared with control cells. In addition, IL-1β-induced IL-6 production was enhanced in cultures containing 1 × 10⁻¹³ M cortisol, whereas 1 × 10⁻⁵ M cortisol attenuated production of IL-6. Thus, cortisol showed immunostimulatory and immunosuppressive activities depending on its concentration. Our results indicate that 11β-HSD1 expression is increased by various stimuli. Thus, regulation of cytosolic cortisol concentrations by 11β-HSD1 appears to modulate expression of inflammatory cytokines in NHEKs.     

Padé-Laplace method for the analysis of time-resolved fluorescence decay curves

The interpretation of fluorescence intensity decay times in terms of protein structure and dynamics depends on the accuracy and sensitivity of the methods used for data analysis. The are many methods available for the analysis of fluorescence decay data, but justification for choosing any one of them is unclear. In this paper we generalize the recently proposed Padé-Laplace method [45] to include deconvolution with respect to the instrument response function. In this form the method can be readily applied to the analysis of time-correlated single photon counting data. By extensive simulations we have shown that the Padé-Laplace method provides more accurate results than the standard least squares method with iterative reconvolution under the condition of closely spaced lifetimes. The application of the Padé-Laplace method to several experimental data sets yielded results consistent with those obtained by use of the least squares analysis. Offprint requests to: F. G. Prendergast     

S-Adenosylhomocysteine hydrolase of the protozoan parasite Trichomonas vaginalis: potent inhibitory activity of 9-(2-deoxy-2-fluoro-β,D-arabinofuranosyl)adenine

In the present study, we carried out a structure-activity analysis in Trichomonas vaginalis of a series of adenosine and uridine analogues. The most potent compounds were found to be 2' and 3' modified adenosine analogues some of which are potent inhibitors of S-adenosylhomocysteine hydrolase. The 9-(2-deoxy-2-fluoro-β,D-arabinofuranosyl)adenine compound was more potent than metronidazole, a current FDA approved and commonly prescribed drug for treatment of trichomoniasis. Its IC(50) was 0.09 μM compared to 0.72 μM for metronidazole.     

Characterization of activity and binding mode of glycyrrhetinic acid derivatives inhibiting 11β-hydroxysteroid dehydrogenase type 2

Modulation of intracellular glucocorticoid availability is considered as a promising strategy to treat glucocorticoid-dependent diseases. 18β-Glycyrrhetinic acid (GA), the biologically active triterpenoid metabolite of glycyrrhizin, which is contained in the roots and rhizomes of licorice (Glycyrrhiza spp.), represents a well-known but non-selective inhibitor of 11β-hydroxysteroid dehydrogenases (11β-HSDs). However, to assess the physiological functions of the respective enzymes and for potential therapeutic applications selective inhibitors are needed. In the present study, we applied bioassays and 3D-structure modeling to characterize nine 11β-HSD1 and fifteen 11β-HSD2 inhibiting GA derivatives. Comparison of the GA derivatives in assays using cell lysates revealed that modifications at the 3-hydroxyl and/or the carboxyl led to highly selective and potent 11β-HSD2 inhibitors. The data generated significantly extends our knowledge on structure-activity relationship of GA derivatives as 11β-HSD inhibitors. Using recombinant enzymes we found also potent inhibition of mouse 11β-HSD2, despite significant species-specific differences. The selected GA derivatives potently inhibited 11β-HSD2 in intact SW-620 colon cancer cells, although the rank order of inhibitory potential differed from that obtained in cell lysates. The biological activity of compounds was further demonstrated in glucocorticoid receptor (GR) transactivation assays in cells coexpressing GR and 11β-HSD1 or 11β-HSD2. 3D-structure modeling provides an explanation for the differences in the selectivity and activity of the GA derivatives investigated. The most potent and selective 11β-HSD2 inhibitors should prove useful as mechanistic tools for further anti-inflammatory and anti-cancer in vitro and in vivo studies. Article from the Special issue on Targeted Inhibitors.