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Species used for drug testing reveal different inhibition susceptibility for 17beta-hydroxysteroid dehydrogenase type 1.

ABSTRACT: Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1) for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17beta-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17beta-HSD types 1, 2, 4, 5 and 7 but also against 17beta-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17beta-HSDs analyzed were observed. Especially, the rodent 17beta-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17beta-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution.

SUBMITTER: Moller G

PROVIDER: S-EPMC2882332 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Species used for drug testing reveal different inhibition susceptibility for 17beta-hydroxysteroid dehydrogenase type 1.

Möller Gabriele G Husen Bettina B Kowalik Dorota D Hirvelä Leena L Plewczynski Dariusz D Rychlewski Leszek L Messinger Josef J Thole Hubert H Adamski Jerzy J

PloS one 20100608 6

Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1) for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17beta-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17beta-HSD types 1, 2, 4, 5 and 7 but also against 17b ...[more]

PMID: 20544026

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Project description:Aldo-keto reductase (AKR) 1C3 catalyzes the NADPH-dependent reduction of Delta(4)-androstene-3,17-dione to yield testosterone, reduction of estrone to yield 17beta-estradiol and reduction of progesterone to yield 20alpha-hydroxyprogesterone. In addition, it functions as a prostaglandin (PG) F synthase and reduces PGH(2) to PGF(2)alpha and PGD(2) to 11beta-PGF(2). Immunohistochemistry showed that AKR1C3 is over-expressed in invasive ductal carcinoma of the breast. Retroviral expression of AKR1C3 in MCF-7 breast carcinoma cells shows that each of the assigned reactions occur in a breast cell microenvironment. Steroid and prostaglandin conversions were monitored by radiochromatography. Prostaglandin conversion was validated by a second method using HPLC coupled to APCI-MRM/MS. The combined effect of the AKR1C3 catalyzed 17- and 20-ketosteroid reductions will be to increase the 17beta-estradiol:progesterone ratio in the breast. In addition, formation of PGF(2) epimers would activate F prostanoid receptors and deprive PPARgamma of its putative anti-proliferative PGJ(2) ligands. Thus, AKR1C3 is a source of proliferative signals and a potential therapeutic target for hormone-dependent and -independent breast cancer. Two strategies for AKR1C3 inhibition based on non-steroidal anti-inflammatory drugs were developed. The first strategy uses the Ullmann coupling reaction to generate N-phenylanthranilate derivatives that inhibit AKR1C enzymes without affecting PGH(2) synthase (PGHS) 1 or PGHS-2. The second strategy exploits the selective inhibition of AKR1C3 by indomethacin, which did not inhibit highly related AKR1C1 or AKR1C2. Using known structure-activity relationships for the inhibition of PGHS-1 and PGHS-2 by indole acetic acids we obtained N-(4-chlorobenzoyl)-melatonin as a specific AKR1C3 inhibitor (K(I)=6.0muM) that does not inhibit PGHS-1, PGHS-2, AKR1C1, or AKR1C2. Both strategies are informed by crystal structures of ternary AKR1C3.NADP(+).NSAID complexes. The identification of NSAID analogs as specific inhibitors of AKR1C3 will help validate its role in the proliferation of breast cancer cells.

Project description:Human 17beta-hydroxysteroid dehydrogenase type 1 (17?-HSD1) is a steroid-converting enzyme that has long been known to play critical roles in estradiol synthesis and more recently in dihydrotestosterone (DHT) inactivation, showing a dual function that promotes breast cancer cell proliferation. Previously, we reported the first observation of the influence of the enzyme on endogenous estrogen-responsive gene expression. Here, we demonstrate the impact of 17?-HSD1 expression on the breast cancer cell proteome and investigate its role in cell migration.17?-HSD1 was stably transfected in MCF7 cells and the proteome of the generated cells overexpressing 17?-HSD1 (MCF7-17?HSD1 cells) was compared to that of the wild type MCF7 cells. Proteomics study was performed using two-dimensional gel electrophoresis followed by mass spectrometry analysis of differentially expressed protein spots. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the transcription of individual gene. The effect of 17?-HSD1 on MCF7 cell migration was verified by a wound-healing assay.Proteomic data demonstrate that the expression of more than 59 proteins is modulated following 17?-HSD1 overexpression. 17?-HSD1 regulates the expression of important genes and proteins that are relevant to cell growth control, such as BRCA2 and CDKN1A interacting protein (BCCIP) and proliferating cell nuclear antigen (PCNA) which are down- and upregulated in MCF7-17?HSD1 cells, respectively. RT-qPCR data reveal that 17?-HSD1 increases the mRNA levels of estrogen receptors (ER) alpha and beta by 171 and 120%, respectively, while decreasing that of the androgen receptor by 64%. Interestingly, 17?-HSD1 increases the mRNA transcript (by 3.6 times) and the protein expression of the metastasis suppressor gene nm23-H1 and the expression of the two enzymes are closely correlated. We have further shown that 17?-HSD1 expression is associated with an increase of MCF7 cell migration.In addition to the regulation of important genes, we have demonstrated for the first time that 17?-HSD1 increases breast cancer cell migration, in spite of its positive regulation of the antimetastatic gene NM23. This is also correlated to its stimulation of breast cancer cell growth, further confirming its targeting in ER positive breast cancer. The novel findings in this study suggest several directions for future research on the contribution of 17?-HSD1 to breast cancer progression and related treatment.

Project description:BackgroundWe have recently discovered that human type 12 17beta-HSD (h17beta-HSD12), a homolog of type 3 17beta-HSD, is a new estrogen-specific 17beta-hydroxysteroid dehydrogenase involved in the production of estradiol (E2). To further characterize this estradiol-producing enzyme, we have isolated the corresponding cDNA in the cynomolgus monkey (Macaca fascicularis), characterized its enzymatic activities and performed cellular localization using in situ hybridization.ResultsUsing HEK-293 cells stably expressing Macaca fascicularis type 12 17beta-HSD (mf17beta-HSD12), we have found that the mf17beta-HSD12 catalyzes efficiently and selectively the transformation of El into E2, in analogy with the h17beta-HSD12. We have also quantified the mf17beta-HSD12 mRNA expression levels in a series of Macaca fascicularis tissues using Quantitative RealTime PCR. The Macaca fascicularis 17beta-HSD12 mRNA is widely expressed with the highest levels tissues found in the cerebellum, spleen and adrenal with moderate level observed in all the other examined, namely the testis, ovary, cerebral cortex, liver, heart, prostate, mammary gland, myometrium, endometrium, skin, muscle and pancreas. To gain knowledge about the cellular localization of the mf17beta-HSD12 mRNA expression, we performed in situ hybridization using a 35S-labeled cRNA probe. Strong labeling was observed in epithelial cells and stromal cells of the mammary gland. In the uterus, the labeling is detected in epithelial cells and stromal cells of the endometrium.ConclusionThese results strongly suggest that the Macaca fascicularis 17beta-HSD12 is an essential partner of aromatase in the biosynthesis of estradiol (E2). It strongly suggests that in the estradiol biosynthesis pathway, the step of 17-ketoreduction comes after the step of the aromatization (the aromatization of 4-androstendione to estrone followed by the conversion of estrone into estradiol by estrogen specific l7beta-HSDs) which is in contrast with the hypothesis suggesting that 4-androstenedione is converted to testosterone followed by the aromatization of testosterone.

Project description:BACKGROUND: The Abeta-binding alcohol dehydrogenase/17beta-hydroxysteroid dehydrogenase type 10 (ABAD/HSD10) is an enzyme involved in pivotal metabolic processes and in the mitochondrial dysfunction seen in the Alzheimer's disease. Here we use comparative genomic analyses to study the evolution of the HADH2 gene encoding ABAD/HSD10 across several eukaryotic species. RESULTS: Both vertebrate and nematode HADH2 genes showed a six-exon/five-intron organization while those of the insects had a reduced and varied number of exons (two to three). Eutherian mammal HADH2 genes revealed some highly conserved noncoding regions, which may indicate the presence of functional elements, namely in the upstream region about 1 kb of the transcription start site and in the first part of intron 1. These regions were also conserved between Tetraodon and Fugu fishes. We identified a conserved alternative splicing event between human and dog, which have a nine amino acid deletion, causing the removal of the strand betaF. This strand is one of the seven strands that compose the core beta-sheet of the Rossman fold dinucleotide-binding motif characteristic of the short chain dehydrogenase/reductase (SDR) family members. However, the fact that the substrate binding cleft residues are retained and the existence of a shared variant between human and dog suggest that it might be functional. Molecular adaptation analyses across eutherian mammal orthologues revealed the existence of sites under positive selection, some of which being localized in the substrate-binding cleft and in the insertion 1 region on loop D (an important region for the Abeta-binding to the enzyme). Interestingly, a higher than expected number of nonsynonymous substitutions were observed between human/chimpanzee and orangutan, with six out of the seven amino acid replacements being under molecular adaptation (including three in loop D and one in the substrate binding loop). CONCLUSION: Our study revealed that HADH2 genes maintained a reasonable conserved organization across a large evolutionary distance. The conserved noncoding regions identified among mammals and between pufferfishes, the evidence of an alternative splicing variant conserved between human and dog, and the detection of positive selection across eutherian mammals, may be of importance for further research on ABAD/HSD10 function and its implication in the Alzheimer's disease.

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Species used for drug testing reveal different inhibition susceptibility for 17beta-hydroxysteroid dehydrogenase type 1.

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Species used for drug testing reveal different inhibition susceptibility for 17beta-hydroxysteroid dehydrogenase type 1.

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