17ß-Hydroxysteroid dehydrogenases types 1 and 2: Enzymatic assays based on radiometric and mass-spectrometric detection.

The 17ß-hydroxysteroid dehydrogenase type 1 (HSD17B1) has a key role in estrogen biosynthesis as it catalyzes the reduction of estrone to the most potent estrogen, estradiol. Estradiol has a high affinity for estrogen receptors and thus stimulates their transactivation, which leads to cell proliferation and numerous other effects. HSD17B2 catalyzes the oxidation of estradiol to the less potent estrone, thereby decreasing estrogen receptor activation, which results in reduction of estrogen-associated effects. HSD17B1 and HSD17B2 overexpressing E.coli homogenates or recombinant enzymes can be used for screening and development of drugs against various pathologies such as cancer, endometriosis or osteoporosis. Here we describe the preparation of HSD17B1 and HSD17B2 bacterial homogenates and purified recombinant HSD17B1 protein as enzyme sources as well as enzymatic assays based on radiometric and mass-spectrometric detection for enzyme characterization.

Analogues of Natural Chalcones as Efficient Inhibitors of AKR1C3.

Naturally occurring substances are valuable resources for drug development. In this respect, chalcones are known to be antiproliferative agents against prostate cancer cell lines through various mechanisms or targets. Based on the literature and preliminary results, we aimed to study and optimise the efficiency of a series of chalcones to inhibit androgen-converting AKR1C3, known to promote prostate cancer. A total of 12 chalcones with different substitution patterns were synthesised. Structure-activity relationships associated with these modifications on AKR1C3 inhibition were analysed by performing enzymatic assays and docking simulations. In addition, the selectivity and cytotoxicity of the compounds were assessed. In enzymatic assays, C-6' hydroxylated derivatives were more active than C-6' methoxylated derivatives. In contrast, C-4 methylation increased activity over C-4 hydroxylation. Docking results supported these findings with the most active compounds fitting nicely in the binding site and exhibiting strong interactions with key amino acid residues. The most effective inhibitors were not cytotoxic for HEK293T cells and selective for 17ß-hydroxysteroid dehydrogenases not primarily involved in steroid hormone metabolism. Nevertheless, they inhibited several enzymes of the steroid metabolism pathways. Favourable substitutions that enhanced AKR1C3 inhibition of chalcones were identified. This study paves the way to further develop compounds from this series or related flavonoids with improved inhibitory activity against AKR1C3.

Common Muscle Metabolic Signatures Highlight Arginine and Lysine Metabolism as Potential Therapeutic Targets to Combat Unhealthy Aging.

Biological aging research is expected to reveal modifiable molecular mechanisms that can be harnessed to slow or possibly reverse unhealthy trajectories. However, there is first an urgent need to define consensus molecular markers of healthy and unhealthy aging. Established aging hallmarks are all linked to metabolism, and a 'rewired' metabolic circuitry has been shown to accelerate or delay biological aging. To identify metabolic signatures distinguishing healthy from unhealthy aging trajectories, we performed nontargeted metabolomics on skeletal muscles from 2-month-old and 21-month-old mice, and after dietary and lifestyle interventions known to impact biological aging. We hypothesized that common metabolic signatures would highlight specific pathways and processes promoting healthy aging, while revealing the molecular underpinnings of unhealthy aging. Here, we report 50 metabolites that commonly distinguished aging trajectories in all cohorts, including 18 commonly reduced under unhealthy aging and 32 increased. We stratified these metabolites according to known relationships with various aging hallmarks and found the greatest associations with oxidative stress and nutrient sensing. Collectively, our data suggest interventions aimed at maintaining skeletal muscle arginine and lysine may be useful therapeutic strategies to minimize biological aging and maintain skeletal muscle health, function, and regenerative capacity in old age.

Functional characterization of two 20ß-hydroxysteroid dehydrogenase type 2 homeologs from Xenopus laevis reveals multispecificity.

The African clawed frog, Xenopus laevis, is a versatile model for biomedical research and is largely similar to mammals in terms of organ development, anatomy, physiology, and hormonal signaling mechanisms. Steroid hormones control a variety of processes and their levels are regulated by hydroxysteroid dehydrogenases (HSDs). The subfamily of 20ß-HSD type 2 enzymes currently comprises eight members from teleost fish and mammals. Here, we report the identification of three 20ß-HSD type 2 genes in X. tropicalis and X. laevis and the functional characterization of the two homeologs from X. laevis. X. laevis Hsd20b2.L and Hsd20b2.S showed high sequence identity with known 20ß-HSD type 2 enzymes and mapped to the two subgenomes of the allotetraploid frog genome. Both homeologs are expressed during embryonic development and in adult tissues, with strongest signals in liver, kidney, intestine, and skin. After recombinant expression in human cell lines, both enzymes co-localized with the endoplasmic reticulum and catalyzed the conversion of cortisone to 20ß-dihydrocortisone. Both Hsd20b2.L and Hsd20b2.S catalyzed the 20ß-reduction of further C21 steroids (17α-hydroxyprogesterone, progesterone, 11-deoxycortisol, 11-deoxycorticosterone), while only Hsd20b2.S was able to convert corticosterone and cortisol to their 20ß-reduced metabolites. Estrone was only a poor and androstenedione no substrate for both enzymes. Our results demonstrate multispecificity of 20ß-HSD type 2 enzymes from X. laevis similar to other teleost 20ß-HSD type 2 enzymes. X. laevis 20ß-HSD type 2 enzymes are probably involved in steroid catabolism and in the generation of pheromones for intraspecies communication. A role in oocyte maturation is unlikely.

Posterior subcapsular cataracts are a late effect after acute exposure to 0.5 Gy ionizing radiation in mice.

PURPOSE: The long-term effect of low and moderate doses of ionizing radiation on the lens is still a matter of debate and needs to be evaluated in more detail. MATERIAL AND METHODS: We conducted a detailed histological analysis of eyes from B6C3F1 mice cohorts after acute gamma irradiation (60Co source; 0.063 Gy/min) at young adult age of 10 weeks with doses of 0.063, 0.125, and 0.5 Gy. Sham irradiated (0 Gy) mice were used as controls. To test for genetic susceptibility heterozygous Ercc2 mutant mice were used and compared to wild-type mice of the same strain background. Mice of both sexes were included in all cohorts. Eyes were collected 4 h, 12, 18 and 24 months after irradiation. For a better understanding of the underlying mechanisms, metabolomics analyses were performed in lenses and plasma samples of the same mouse cohorts at 4 and 12 h as well as 12, 18 and 24 months after irradiation. For this purpose, a targeted analysis was chosen. RESULTS: This analysis revealed histological changes particularly in the posterior part of the lens that rarely can be observed by using Scheimpflug imaging, as we reported previously. We detected a significant increase of posterior subcapsular cataracts (PSCs) 18 and 24 months after irradiation with 0.5 Gy (odds ratio 9.3; 95% confidence interval 2.1-41.3) independent of sex and genotype. Doses below 0.5 Gy (i.e. 0.063 and 0.125 Gy) did not significantly increase the frequency of PSCs at any time point. In lenses, we observed a clear effect of sex and aging but not of irradiation or genotype. While metabolomics analyses of plasma from the same mice showed only a sex effect. CONCLUSIONS: This article demonstrates a significant radiation-induced increase in the incidence of PSCs, which could not be identified using Scheimpflug imaging as the only diagnostic tool.

Homology modeling meets site-directed mutagenesis: An ideal combination to elucidate the topology of 17ß-HSD2.

17ß-Hydroxysteroid dehydrogenase type 2 (17ß-HSD2) catalyzes the conversion of highly active estrogens and androgens into their less active forms using NAD+ as cofactor. Substrate and cofactor specificities of 17ß-HSD2 have been reported and potent 17ß-HSD2 inhibitors have been discovered in a ligand-based approach. However, the molecular basis and the amino acids involved in the enzymatic functionality are poorly understood, as no crystal structure of the membrane-associated 17ß-HSD2 exists. The functional properties of only few amino acids are known. The lack of topological information impedes structure-based drug design studies and limits the design of biochemical experiments. The aim of this work was the determination of the 17ß-HSD2 topology. For this, the first homology model of 17ß-HSD2 in complex with NAD+ and 17ß-estradiol was built, using a multi-fragment "patchwork" approach. To confirm the quality of the model, fifteen selected amino acids were exchanged one by one using site directed mutagenesis. The mutants' functional behavior demonstrated that the generated model was of very good quality and allowed the identification of several key amino acids involved in either ligand or internal structure stabilization. The final model is an optimal basis for further experiments like, for example, lead optimization.

Finding New Molecular Targets of Familiar Natural Products Using In Silico Target Prediction.

Natural products comprise a rich reservoir for innovative drug leads and are a constant source of bioactive compounds. To find pharmacological targets for new or already known natural products using modern computer-aided methods is a current endeavor in drug discovery. Nature's treasures, however, could be used more effectively. Yet, reliable pipelines for the large-scale target prediction of natural products are still rare. We developed an in silico workflow consisting of four independent, stand-alone target prediction tools and evaluated its performance on dihydrochalcones (DHCs)-a well-known class of natural products. Thereby, we revealed four previously unreported protein targets for DHCs, namely 5-lipoxygenase, cyclooxygenase-1, 17ß-hydroxysteroid dehydrogenase 3, and aldo-keto reductase 1C3. Moreover, we provide a thorough strategy on how to perform computational target predictions and guidance on using the respective tools.

Dual Inhibitory Action of a Novel AKR1C3 Inhibitor on Both Full-Length AR and the Variant AR-V7 in Enzalutamide Resistant Metastatic Castration Resistant Prostate Cancer.

The expanded use of second-generation antiandrogens revolutionized the treatment landscape of progressed prostate cancer. However, resistances to these novel drugs are already the next obstacle to be solved. Various previous studies depicted an involvement of the enzyme AKR1C3 in the process of castration resistance as well as in the resistance to 2nd generation antiandrogens like enzalutamide. In our study, we examined the potential of natural AKR1C3 inhibitors in various prostate cancer cell lines and a three-dimensional co-culture spheroid model consisting of cancer cells and cancer-associated fibroblasts (CAFs) mimicking enzalutamide resistant prostate cancer. One of our compounds, named MF-15, expressed strong antineoplastic effects especially in cell culture models with significant enzalutamide resistance. Furthermore, MF-15 exhibited a strong effect on androgen receptor (AR) signaling, including significant inhibition of AR activity, downregulation of androgen-regulated genes, lower prostate specific antigen (PSA) production, and decreased AR and AKR1C3 expression, indicating a bi-functional effect. Even more important, we demonstrated a persisting inhibition of AR activity in the presence of AR-V7 and further showed that MF-15 non-competitively binds within the DNA binding domain of the AR. The data suggest MF-15 as useful drug to overcome enzalutamide resistance.

Lipidomic Phenotyping Reveals Extensive Lipid Remodeling during Adipogenesis in Human Adipocytes.

Differentiation of preadipocytes into mature adipocytes is a highly complex cellular process. At lipidome level, the adipogenesis remains poorly characterized. To investigate the lipidomic changes during human adipogenesis, we used the LipidyzerTM assay, which quantified 743 lipid species from 11 classes. The undifferentiated human SGBS cell strain showed a heterogeneous lipid class composition with the most abundant classes, phosphatidylethanolamines (PE), phosphatidylcholines (PC), and sphingomyelins (SM). The differentiation process was accompanied by increased ceramide concentrations. After completion of differentiation around day 4, massive lipid remodeling occurred during maturation, characterized by substantial synthesis of diacylglycerols (DAG), lysophosphatidylethanolamines (LPE), PC, PE, SM, and triacylglycerols (TAG). Lipid species composition became more homogeneous during differentiation to highly concentrated saturated and monounsaturated long-chain fatty acids (LCFA), with the four most abundant being C16:0, C16:1, C18:0, and C18:1. Simultaneously, the amount of polyunsaturated and very long-chain fatty acids (VLCFA) markedly decreased. High negative correlation coefficients between PE and PC species containing VLCFA and TAG species as well as between ceramides and SM imply that PE, PC, and ceramides might have served as additional sources for TAG and SM synthesis, respectively. These results highlight the enormous remodeling at the lipid level over several lipid classes during adipogenesis.

Substrate multispecificity among 20ß-hydroxysteroid dehydrogenase type 2 members.

Steroids regulate many physiological processes. Hydroxysteroid dehydrogenases (HSDs) modulate the levels of steroids in pre- and post-receptor metabolism. The subfamily of 20ß-HSD type 2 currently comprises six members from six different species. The zebrafish ortholog converts cortisone to 20ß-dihydrocortisone and is involved in the catabolism of the stress hormone cortisol. Here, we elucidated the substrate preferences of all 20ß-HSD type 2 enzymes towards a selected panel of steroids. For quantification of the substrates and their respective 20ß-reduced products, we first developed and validated a liquid chromatography-mass spectrometry based method. Applying this method to activity assays with recombinantly expressed enzymes, our findings indicate that the 20ß-HSD type 2 enzymes catalyze the 20ß-reduction of a plethora of steroids of the glucocorticoid biosynthesis pathway. The observed multispecificity among the homologous 20ß-HSD type 2 enzymes implies different physiological roles in different species.

Engineering aldo-keto reductase 1B10 to mimic the distinct 1B15 topology and specificity towards inhibitors and substrates, including retinoids and steroids.

The aldo-keto reductase (AKR) superfamily comprises NAD(P)H-dependent enzymes that catalyze the reduction of a variety of carbonyl compounds. AKRs are classified in families and subfamilies. Humans exhibit three members of the AKR1B subfamily: AKR1B1 (aldose reductase, participates in diabetes complications), AKR1B10 (overexpressed in several cancer types), and the recently described AKR1B15. AKR1B10 and AKR1B15 share 92% sequence identity, as well as the capability of being active towards retinaldehyde. However, AKR1B10 and AKR1B15 exhibit strong differences in substrate specificity and inhibitor selectivity. Remarkably, their substrate-binding sites are the most divergent parts between them. Out of 27 residue substitutions, six are changes to Phe residues in AKR1B15. To investigate the participation of these structural changes, especially the Phe substitutions, in the functional features of each enzyme, we prepared two AKR1B10 mutants. The AKR1B10 m mutant carries a segment of six AKR1B15 residues (299-304, including three Phe residues) in the respective AKR1B10 region. An additional substitution (Val48Phe) was incorporated in the second mutant, AKR1B10mF48. This resulted in structures with smaller and more hydrophobic binding pockets, more similar to that of AKR1B15. In general, the AKR1B10 mutants mirrored well the specific functional features of AKR1B15, i.e., the different preferences towards the retinaldehyde isomers, the much higher activity with steroids and ketones, and the unique behavior with inhibitors. It can be concluded that the Phe residues of loop C (299-304) contouring the substrate-binding site, in addition to Phe at position 48, strongly contribute to a narrower and more hydrophobic site in AKR1B15, which would account for its functional uniqueness. In addition, we have investigated the AKR1B10 and AKR1B15 activity toward steroids. While AKR1B10 only exhibits residual activity, AKR1B15 is an efficient 17-ketosteroid reductase. Finally, the functional role of AKR1B15 in steroid and retinaldehyde metabolism is discussed.

Initial characterization of human DHRS1 (SDR19C1), a member of the short-chain dehydrogenase/reductase superfamily.

Many enzymes from the short-chain dehydrogenase/reductase superfamily (SDR) have already been well characterized, particularly those that participate in crucial biochemical reactions in the human body (e.g. 11ß-hydroxysteroid dehydrogenase 1, 17ß-hydroxysteroid dehydrogenase 1 or carbonyl reductase 1). Several other SDR enzymes are completely or almost completely uncharacterized, such as DHRS1 (also known as SDR19C1). Based on our in silico and experimental approaches, DHRS1 is described as a likely monotopic protein that interacts with the membrane of the endoplasmic reticulum. The highest expression level of DHRS1 protein was observed in human liver and adrenals. The recombinant form of DHRS1 was purified using the detergent n-dodecyl-ß-D-maltoside, and DHRS1 was proven to be an NADPH-dependent reductase that is able to catalyse the in vitro reductive conversion of some steroids (estrone, androstene-3,17-dione and cortisone), as well as other endogenous substances and xenobiotics. The expression pattern and enzyme activities fit to a role in steroid and/or xenobiotic metabolism; however, more research is needed to fully clarify the exact biological function of DHRS1.

Three-Dimensional Quantitative Co-Mapping of Pulmonary Morphology and Nanoparticle Distribution with Cellular Resolution in Nondissected Murine Lungs.

Deciphering biodistribution, biokinetics, and biological effects of nanoparticles (NPs) in entire organs with cellular resolution remains largely elusive due to the lack of effective imaging tools. Here, light sheet fluorescence microscopy in combination with optical tissue clearing was validated for concomitant three-dimensional mapping of lung morphology and NP biodistribution with cellular resolution in nondissected ex vivo murine lungs. Tissue autofluorescence allowed for label-free, quantitative morphometry of the entire bronchial tree, acinar structure, and blood vessels. Co-registration of fluorescent NPs with lung morphology revealed significant differences in pulmonary NP distribution depending on the means of application (intratracheal instillation and ventilator-assisted aerosol inhalation under anesthetized conditions). Inhalation exhibited a more homogeneous NP distribution in conducting airways and acini indicated by a central-to-peripheral (C/P) NP deposition ratio of unity (0.98 ± 0.13) as compared to a 2-fold enhanced central deposition (C/P = 1.98 ± 0.37) for instillation. After inhalation most NPs were observed in the proximal part of the acini as predicted by computational fluid dynamics simulations. At cellular resolution patchy NP deposition was visualized in bronchioles and acini, but more pronounced for instillation. Excellent linearity of the fluorescence intensity-dose response curve allowed for accurate NP dosimetry and revealed ca. 5% of the inhaled aerosol was deposited in the lungs. This single-modality imaging technique allows for quantitative co-registration of tissue architecture and NP biodistribution, which could accelerate elucidation of NP biokinetics and bioactivity within intact tissues, facilitating both nanotoxicology studies and the development of nanomedicines.

Structure-based design and profiling of novel 17ß-HSD14 inhibitors.

The human enzyme 17ß-hydroxysteroid dehydrogenase 14 (17ß-HSD14) oxidizes the hydroxyl group at position 17 of estradiol and 5-androstenediol using NAD+ as cofactor. However, the physiological role of the enzyme remains unclear. We recently described the first class of nonsteroidal inhibitors for this enzyme with compound 1 showing a high 17ß-HSD14 inhibitory activity. Its crystal structure was used as starting point for a structure-based optimization in this study. The goal was to develop a promising chemical probe to further investigate the enzyme. The newly designed compounds revealed mostly very high inhibition of the enzyme and for seven of them the crystal structures of the corresponding inhibitor-enzyme complexes were resolved. The crystal structures disclosed that a small change in the substitution pattern of the compounds resulted in an alternative binding mode for one inhibitor. The profiling of a set of the most potent inhibitors identified 13 (Ki = 9 nM) with a good selectivity profile toward three 17ß-HSDs and the estrogen receptor alpha. This inhibitor displayed no cytotoxicity, good solubility, and auspicious predicted bioavailability. Overall, 13 is a highly interesting 17ß-HSD14 inhibitor, which might be used as chemical probe for further investigation of the target enzyme.

High-throughput extraction and quantification method for targeted metabolomics in murine tissues.

INTRODUCTION: Global metabolomics analyses using body fluids provide valuable results for the understanding and prediction of diseases. However, the mechanism of a disease is often tissue-based and it is advantageous to analyze metabolomic changes directly in the tissue. Metabolomics from tissue samples faces many challenges like tissue collection, homogenization, and metabolite extraction. OBJECTIVES: We aimed to establish a metabolite extraction protocol optimized for tissue metabolite quantification by the targeted metabolomics AbsoluteIDQ™ p180 Kit (Biocrates). The extraction method should be non-selective, applicable to different kinds and amounts of tissues, monophasic, reproducible, and amenable to high throughput. METHODS: We quantified metabolites in samples of eleven murine tissues after extraction with three solvents (methanol, phosphate buffer, ethanol/phosphate buffer mixture) in two tissue to solvent ratios and analyzed the extraction yield, ionization efficiency, and reproducibility. RESULTS: We found methanol and ethanol/phosphate buffer to be superior to phosphate buffer in regard to extraction yield, reproducibility, and ionization efficiency for all metabolites measured. Phosphate buffer, however, outperformed both organic solvents for amino acids and biogenic amines but yielded unsatisfactory results for lipids. The observed matrix effects of tissue extracts were smaller or in a similar range compared to those of human plasma. CONCLUSION: We provide for each murine tissue type an optimized high-throughput metabolite extraction protocol, which yields the best results for extraction, reproducibility, and quantification of metabolites in the p180 kit. Although the performance of the extraction protocol was monitored by the p180 kit, the protocol can be applicable to other targeted metabolomics assays.

Long-Term Stability of Human Plasma Metabolites during Storage at -80 °C.

Prolonged storage of biospecimen can lead to artificially altered metabolite concentrations and thus bias data analysis in metabolomics experiments. To elucidate the potential impact of long-term storage on the metabolite profile, a pooled human plasma sample was aliquoted and stored at -80 °C. During a time period of five years, 1012 of the aliquots were measured with the Biocrates AbsoluteIDQ p180 targeted-metabolomics assay at 193 time points. Modeling the concentration courses over time revealed that 55 out of 111 metabolites remained stable. The statistically significantly changed metabolites showed on average an increase or decrease of +13.7% or -14.5%, respectively. In detail, increased concentration levels were observed for amino acids (mean: + 15.4%), the sum of hexoses (+7.9%), butyrylcarnitine (+9.4%), and some phospholipids mostly with chain lengths exceeding 40 carbon atoms (mean: +18.0%). Lipids tended to exhibit decreased concentration levels with the following mean concentration changes: acylcarnitines, -12.1%; lysophosphatidylcholines, -15.1%; diacyl-phosphatidylcholines, -17.0%; acyl-alkyl-phosphatidylcholines, -13.3%; sphingomyelins, -14.8%. We conclude that storage of plasma samples at -80 °C for up to five years can lead to altered concentration levels of amino acids, acylcarnitines, glycerophospholipids, sphingomyelins, and the sum of hexoses. These alterations must be considered when analyzing metabolomics data from long-term epidemiological studies.

Endocrinology Meets Metabolomics: Achievements, Pitfalls, and Challenges.

The metabolome, although very dynamic, is sufficiently stable to provide specific quantitative traits related to health and disease. Metabolomics requires balanced use of state-of-the-art study design, chemical analytics, biostatistics, and bioinformatics to deliver meaningful answers to contemporary questions in human disease research. The technology is now frequently employed for biomarker discovery and for elucidating the mechanisms underlying endocrine-related diseases. Metabolomics has also enriched genome-wide association studies (GWAS) in this area by providing functional data. The contributions of rare genetic variants to metabolome variance and to the human phenotype have been underestimated until now.

Characterization of AKR1B16, a novel mouse aldo-keto reductase.

Aldo-keto reductases (AKRs) are distributed in three families and multiple subfamilies in mammals. The mouse Akr1b3 gene is clearly orthologous to human AKR1B1, both coding for aldose reductase, and their gene products show similar tissue distribution, regulation by osmotic stress and kinetic properties. In contrast, no unambiguous orthologs of human AKR1B10 and AKR1B15.1 have been identified in rodents. Although two more AKRs, AKR1B7 and AKR1B8, have been identified and characterized in mouse, none of them seems to exhibit properties similar to the human AKRs. Recently, a novel mouse AKR gene, Akr1b16, was annotated and the respective gene product, AKR1B16 (sharing 83% and 80% amino acid sequence identity with AKR1B10 and AKR1B15.1, respectively), was expressed as insoluble and inactive protein in a bacterial expression system. Here we describe the expression and purification of a soluble and enzymatically active AKR1B16 from E. coli using three chaperone systems. A structural model of AKR1B16 allowed the estimation of its active-site pocket volume, which was much wider (402 Å3) than those of AKR1B10 (279 Å3) and AKR1B15.1 (60 Å3). AKR1B16 reduced aliphatic and aromatic carbonyl compounds, using NADPH as a cofactor, with moderate or low activity (highest kcat values around 5 min-1). The best substrate for the enzyme was pyridine-3-aldehyde. AKR1B16 showed poor inhibition with classical AKR inhibitors, tolrestat being the most potent. Kinetics and inhibition properties resemble those of rat AKR1B17 but differ from those of the human enzymes. In addition, AKR1B16 catalyzed the oxidation of 17ß-hydroxysteroids in a NADP+-dependent manner. These results, together with a phylogenetic analysis, suggest that mouse AKR1B16 is an ortholog of rat AKR1B17, but not of human AKR1B10 or AKR1B15.1. These human enzymes have no counterpart in the murine species, which is evidenced by forming a separate cluster in the phylogenetic tree and by their unique activity with retinaldehyde.

First Structure-Activity Relationship of 17ß-Hydroxysteroid Dehydrogenase Type 14 Nonsteroidal Inhibitors and Crystal Structures in Complex with the Enzyme.

17ß-HSD14 belongs to the SDR family and oxidizes the hydroxyl group at position 17 of estradiol and 5-androstenediol using NAD+ as cofactor. The goal of this study was to identify and optimize 17ß-HSD14 nonsteroidal inhibitors as well as to disclose their structure-activity relationship. In a first screen, a library of 17ß-HSD1 and 17ß-HSD2 inhibitors, selected with respect to scaffold diversity, was tested for 17ß-HSD14 inhibition. The most interesting hit was taken as starting point for chemical modification applying a ligand-based approach. The designed compounds were synthesized and tested for 17ß-HSD14 inhibitory activity. The two best inhibitors identified in this study have a very high affinity to the enzyme with a Ki equal to 7 nM. The strong affinity of these inhibitors to the enzyme active site could be explained by crystallographic structure analysis, which highlighted the role of an extended H-bonding network in the stabilization process. The selectivity of the most potent compounds with respect to 17ß-HSD1 and 17ß-HSD2 is also addressed.

Removing the bottlenecks of cell culture metabolomics: fast normalization procedure, correlation of metabolites to cell number, and impact of the cell harvesting method.

INTRODUCTION: Although cultured cells are nowadays regularly analyzed by metabolomics technologies, some issues in study setup and data processing are still not resolved to complete satisfaction: a suitable harvesting method for adherent cells, a fast and robust method for data normalization, and the proof that metabolite levels can be normalized to cell number. OBJECTIVES: We intended to develop a fast method for normalization of cell culture metabolomics samples, to analyze how metabolite levels correlate with cell numbers, and to elucidate the impact of the kind of harvesting on measured metabolite profiles. METHODS: We cultured four different human cell lines and used them to develop a fluorescence-based method for DNA quantification. Further, we assessed the correlation between metabolite levels and cell numbers and focused on the impact of the harvesting method (scraping or trypsinization) on the metabolite profile. RESULTS: We developed a fast, sensitive and robust fluorescence-based method for DNA quantification showing excellent linear correlation between fluorescence intensities and cell numbers for all cell lines. Furthermore, 82-97 % of the measured intracellular metabolites displayed linear correlation between metabolite concentrations and cell numbers. We observed differences in amino acids, biogenic amines, and lipid levels between trypsinized and scraped cells. CONCLUSION: We offer a fast, robust, and validated normalization method for cell culture metabolomics samples and demonstrate the eligibility of the normalization of metabolomics data to the cell number. We show a cell line and metabolite-specific impact of the harvesting method on metabolite concentrations.