Hydroxybenzothiazoles as New Nonsteroidal Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 1 (17β-HSD1)

17β-estradiol (E2), the most potent estrogen in humans, known to be involved in the development and progession of estrogen-dependent diseases (EDD) like breast cancer and endometriosis. 17β-HSD1, which catalyses the reduction of the weak estrogen estrone (E1) to E2, is often overexpressed in breast cancer and endometriotic tissues. An inhibition of 17β-HSD1 could selectively reduce the local E2-level thus allowing for a novel, targeted approach in the treatment of EDD. Continuing our search for new nonsteroidal 17β-HSD1 inhibitors, a novel pharmacophore model was derived from crystallographic data and used for the virtual screening of a small library of compounds. Subsequent experimental verification of the virtual hits led to the identification of the moderately active compound 5. Rigidification and further structure modifications resulted in the discovery of a novel class of 17β-HSD1 inhibitors bearing a benzothiazole-scaffold linked to a phenyl ring via keto- or amide-bridge. Their putative binding modes were investigated by correlating their biological data with features of the pharmacophore model. The most active keto-derivative 6 shows IC50-values in the nanomolar range for the transformation of E1 to E2 by 17β-HSD1, reasonable selectivity against 17β-HSD2 but pronounced affinity to the estrogen receptors (ERs). On the other hand, the best amide-derivative 21 shows only medium 17β-HSD1 inhibitory activity at the target enzyme as well as fair selectivity against 17β-HSD2 and ERs. The compounds 6 and 21 can be regarded as first benzothiazole-type 17β-HSD1 inhibitors for the development of potential therapeutics

Insights in 17β-HSD1 Enzyme Kinetics and Ligand Binding by Dynamic Motion Investigation

BACKGROUND: Bisubstrate enzymes, such as 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), exist in solution as an ensemble of conformations. 17beta-HSD1 catalyzes the last step of the biosynthesis of estradiol and, thus, it is a potentially attractive target for breast cancer treatment. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the conformational transitions of its catalytic cycle, a structural analysis of all available crystal structures was performed and representative conformations were assigned to each step of the putative kinetic mechanism. To cover most of the conformational space, all-atom molecular dynamic simulations were performed using the four crystallographic structures best describing apoform, opened, occluded and closed state of 17beta-HSD1 as starting structures. With three of them, binary and ternary complexes were built with NADPH and NADPH-estrone, respectively, while two were investigated as apoform. Free energy calculations were performed in order to judge more accurately which of the MD complexes describes a specific kinetic step. CONCLUSIONS/SIGNIFICANCE: Remarkably, the analysis of the eight long range trajectories resulting from this multi-trajectory/-complex approach revealed an essential role played by the backbone and side chain motions, especially of the betaF alphaG'-loop, in cofactor and substrate binding. Thus, a selected-fit mechanism is suggested for 17beta-HSD1, where ligand-binding induced concerted motions of the FG-segment and the C-terminal part guide the enzyme along its preferred catalytic pathway. Overall, we could assign different enzyme conformations to the five steps of the random bi-bi kinetic cycle of 17beta-HSD1 and we could postulate a preferred pathway for it. This study lays the basis for more-targeted biochemical studies on 17beta-HSD1, as well as for the design of specific inhibitors of this enzyme. Moreover, it provides a useful guideline for other enzymes, also characterized by a rigid core and a flexible region directing their catalysis

Gene expression in macrophage-rich inflammatory cell infiltrates in human atherosclerotic lesions as studied by laser microdissection and DNA array

Objective- Inflammatory cells play an important role in atherogenesis. However, more information is needed about their gene expression profiles in human lesions. Methods and Results— We used laser microdissection (LMD) to isolate macrophage-rich shoulder areas from human lesions. Gene expression profiles in isolated cells were analyzed by cDNA array and compared with expression patterns in normal intima and THP-1 macrophages. Upregulation of 72 genes was detected with LMD and included 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, interferon regulatory factor-5 (IRF-5), colony stimulating factor (CSF) receptors, CD11a/CD18 integrins, interleukin receptors, CD43, calmodulin, nitric oxide synthase (NOS), and extracellular superoxide dismutase (SOD). Several of these changes were also present in PMA-stimulated THP-1 macrophages in vitro. On the other hand, expression of several genes, such as VEGF, tissue factor pathway inhibitor 2, and apolipoproteins C-I and C-II, decreased. Conclusions— Overexpression of HMG-CoA reductase in macrophage-rich lesion areas may explain some beneficial effects of statins, which can also modulate increased expression of CD11a/CD18 and CD43 found in microdissected cells. We also found increased expression of CSF receptors, IRF-5, and interleukin receptors, which could become useful therapeutic targets for the treatment of atherosclerotic diseases