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.

Effects of 17ß-HSD2 inhibition in bones on osteoporosis based on an animal rat model.

Hormone replacement therapy is a viable option to protect bone from postmenopausal osteoporosis. Systemically elevated estrogen levels, however, are disadvantageous because of the risk of harmful side effects in other organs. The rationale of the study presented here is to target a key enzyme in estradiol (E2) and testosterone (T) metabolism to increase E2 levels in an organ-specific manner, thereby avoiding the disadvantages of systemically increased E2 levels. The 17ß-hydroxysteroid dehydrogenase (17ß-HSD2), which is e.g. expressed in bone, catalyzes the oxidation of E2 and T into estrone (E1) and androstenedione. We postulate that inhibiting 17ß-HSD2 should lead to elevated E2 and T levels in organs expressing the enzyme. Therefore, we can use the benefits of E2 directly, or those of T following aromatization into E2, in the bone without affecting systemic levels. We tested for the first time, the novel and potent 17ß-HSD2 inhibitor, compound 24 (C24), to explore the therapeutic potential of a 17ß-HSD2 inhibition in an ovariectomy (ovx)-induced rat model of bone loss. We tested the inhibitor alone and, together with low dose estrogen supplementation to model estrogen levels in the postmenopausal situation. Female mature Wistar-Hannover rats were treated for 8 weeks with doses of 2, 10, 50 mg C24 per kg body weight per day alone or in the presence of estradiol benzoate (E2B) supplementation to alleviate ovx-induced bone loss. Ovx placebo and sham operated animals served as negative and positive controls. The experiment was evaluated regarding aspects of efficacy and safety: Bone was analyzed to evaluate bone protective effects, and uterus for potential, unwanted E2-mediated side effects. We observed a good bioavailability of C24 as very high plasma concentrations were measured, up to a group mean of 15,412 nM for the ovx C24-high group. Histomorphometrical analyses and in vivo &ex vivo µCT revealed significant bone protective effects for the lowest inhibitor concentration used. Irrespective of the plasma concentration, no proliferative effects in the uterus could be observed. These results support our approach of intracellular targeting key enzymes of E2 and T metabolism to increase E2 and T levels in an organ specific manner.

Binding Mode Characterization and Early in Vivo Evaluation of Fragment-Like Thiols as Inhibitors of the Virulence Factor LasB from Pseudomonas aeruginosa.

The increasing emergence of antibiotic resistance necessitates the development of anti-infectives with novel modes of action. Targeting bacterial virulence is considered a promising approach to develop novel antibiotics with reduced selection pressure. The extracellular collagenase elastase (LasB) plays a pivotal role in the infection process of Pseudomonas aeruginosa and therefore represents an attractive antivirulence target. Mercaptoacetamide-based thiols have been reported to inhibit LasB as well as collagenases from clostridia and bacillus species. The present work provides an insight into the structure-activity relationship (SAR) of these fragment-like LasB inhibitors, demonstrating an inverse activity profile compared to similar inhibitors of clostridial collagenase H (ColH). An X-ray cocrystal structure is presented, revealing distinct binding of two compounds to the active site of LasB, which unexpectedly maintains an open conformation. We further demonstrate in vivo efficacy in a Galleria mellonella infection model and high selectivity of the LasB inhibitors toward human matrix metalloproteinases (MMPs).

Lead Optimization Generates CYP11B1 Inhibitors of Pyridylmethyl Isoxazole Type with Improved Pharmacological Profile for the Treatment of Cushing's Disease.

Cushing's disease, characterized by elevated plasma cortisol levels, can be controlled by inhibition of 11ß-hydroxylase (CYP11B1). The previously identified selective and potent CYP11B1 inhibitor 5-((5-methylpyridin-3-yl)methyl)-2-phenylpyridine Ref 7 (IC50= 2 nM) exhibited promutagenic potential as well as very low oral bioavailability in rats (F = 2%) and was therefore modified to overcome these drawbacks. Successful lead optimization resulted in similarly potent and selective 5-((5-methoxypyridin-3-yl)methyl)-3-phenylisoxazole 25 (IC50 = 2 nM, 14-fold selectivity over CYP11B2), exhibiting a superior pharmacological profile with no mutagenic potential. Furthermore, compound 25 inhibited rat CYP11B1 (IC50 = 2 µM) and showed a high oral bioavailability (F = 50%) and sufficient plasma concentrations in rats, providing an excellent starting point for a proof-of-principle study.

Discovery of the first small-molecule CsrA-RNA interaction inhibitors using biophysical screening technologies.

AIM: CsrA is a global post-transcriptional regulator protein affecting mRNA translation and/or stability. Widespread among bacteria, it is essential for their full virulence and thus represents a promising anti-infective drug target. Therefore, we aimed at the discovery of CsrA-RNA interaction inhibitors. Results & methodology: We followed two strategies: a screening of small molecules (A) and an RNA ligand-based approach (B). Using surface plasmon resonance-based binding and fluorescence polarization-based competition assays, (A) yielded seven small-molecule inhibitors, among them MM14 (IC50 of 4 µM). (B) resulted in RNA-based inhibitor GGARNA (IC50 of 113 µM). CONCLUSION: The first small-molecule inhibitors of the CsrA-RNA interaction were discovered exhibiting micromolar affinities. These hits represent tools to investigate the effects of CsrA-RNA interaction inhibition on bacterial virulence.

Inhibition of aldosterone synthase (CYP11B2) by torasemide prevents atrial fibrosis and atrial fibrillation in mice.

Loop diuretics are used for fluid control in patients with heart failure. Furosemide and torasemide may exert differential effects on myocardial fibrosis. Here, we studied the effects of torasemide and furosemide on atrial fibrosis and remodeling during atrial fibrillation. In primary neonatal cardiac fibroblasts, torasemide (50µM, 24h) but not furosemide (50µM, 24h) reduced the expression of connective tissue growth factor (CTGF; 65±6%) and the pro-fibrotic miR-21 (44±23%), as well as the expression of lysyl oxidase (LOX; 57±8%), a regulator of collagen crosslinking. Mineralocorticoid receptor (MR) expression and activity were not altered. Torasemide but not furosemide inhibited human aldosterone synthase (CYP11B2) activity in transfected lung fibroblasts (V79MZ cells) by 75±1.8%. The selective CYP11B2 inhibitor SL242 mimicked the torasemide effects. Mice with cardiac overexpression of Rac1 GTPase (RacET), which develop atrial fibrosis and spontaneous AF with aging, were treated long-term (8months) with torasemide (10mg/kg/day), furosemide (40mg/kg/day) or vehicle. Treatment with torasemide but not furosemide prevented atrial fibrosis in RacET as well as the up-regulation of CTGF, LOX, and miR-2, whereas MR expression and activity remained unaffected. These effects correlated with a reduced prevalence of atrial fibrillation (33% RacET+Tora vs. 80% RacET). Torasemide but not furosemide inhibits CYP11B2 activity and reduces the expression of CTGF, LOX, and miR-21. These effects are associated with prevention of atrial fibrosis and a reduced prevalence of atrial fibrillation in mice.

Composing compound libraries for hit discovery--rationality-driven preselection or random choice by structural diversity?

AIMS: In order to identify new scaffolds for drug discovery, surface plasmon resonance is frequently used to screen structurally diverse libraries. Usually, hit rates are low and identification processes are time consuming. Hence, approaches which improve hit rates and, thus, reduce the library size are required. METHODS: In this work, we studied three often used strategies for their applicability to identify inhibitors of PqsD. In two of them, target-specific aspects like inhibition of a homologous protein or predicted binding determined by virtual screening were used for compound preselection. Finally, a fragment library, covering a large chemical space, was screened and served as comparison. RESULTS & CONCLUSION: Indeed, higher hit rates were observed for methods employing preselected libraries indicating that target-oriented compound selection provides a time-effective alternative.

Design and synthesis of a library of lead-like 2,4-bisheterocyclic substituted thiophenes as selective Dyrk/Clk inhibitors.

The Dyrk family of protein kinases is implicated in the pathogenesis of several diseases, including cancer and neurodegeneration. Pharmacological inhibitors were mainly described for Dyrk1A so far, but in fewer cases for Dyrk1B, Dyrk2 or other isoforms. Herein, we report the development and optimization of 2,4-bisheterocyclic substituted thiophenes as a novel class of Dyrk inhibitors. The optimized hit compounds displayed favorable pharmacokinetic properties and high ligand efficiencies, and inhibited Dyrk1B in intact cells. In a larger selectivity screen, only Clk1 and Clk4 were identified as additional targets of compound 48, but no other kinases frequently reported as off-targets. Interestingly, Dyrk1A is implicated in the regulation of alternative splicing, a function shared with Clk1/Clk4; thus, some of the dual inhibitors might be useful as efficient splicing modulators. A further compound (29) inhibited Dyrk1A and 1B with an IC50 of 130 nM, showing a moderate selectivity over Dyrk2. Since penetration of the central nervous system (CNS) seems possible based on the physicochemical properties, this compound might serve as a lead for the development of potential therapeutic agents against glioblastoma. Furthermore, an inhibitor selective for Dyrk2 (24) was also identified, which might be are suitable as a pharmacological tool to dissect Dyrk2 isoform-mediated functions.

Synthesis and biological evaluation of phenyl substituted 1H-1,2,4-triazoles as non-steroidal inhibitors of 17ß-hydroxysteroid dehydrogenase type 2.

A series of disubstituted-1H-1,2,4-triazole derivatives was synthesized with the aim of developing new non-steroidal inhibitors of 17ß-hydroxysteroid dehydrogenase type 2 (17ßHSD2) - a novel and attractive target for the treatment of osteoporosis. 17ßHSD2 catalyzes the oxidation of the highly active estrogen 17ß-estradiol (E2) and androgen testosterone (T) into the weak estrone and androstenedione, respectively. Inhibition of this enzyme will locally in the bone lead to an increase in E2 and T levels, two key players in the maintenance of the balance between bone resorption and bone formation. In this study, a new class of 17ßHSD2 inhibitors with a 1H-1,2,4-triazole scaffold was identified; the three best compounds 8b, 8f, and 13a showed moderate 17ßHSD2 inhibitory activity and a good selectivity toward 17ßHSD1. They could be a useful tool to map the unexplored enzyme active site.

Facile synthesis of chrysin-derivatives with promising activities as aromatase inhibitors.

Flavones such as chrysin show structural similarities to androgens, the substrates of human aromatase, which converts androgens to estrogens. Aromatase is a key target in the treatment of hormone-dependent tumors, including breast cancer. Flavone-based aromatase inhibitors are of growing interest, and chrysin in particular provides a (natural) lead structure. This paper reports multicomponent synthesis as a means for facile modification of the chrysin core structure in order to add functional elements. A Mannich-type reaction was used to synthesize a range of mono- and disubstituted chrysin derivatives, some of which are more effective aromatase inhibitors than the benchmark compound, aminoglutethimide. Similarly, the reaction of chrysin with various isonitriles and acetylene dicarboxylates results in a new class of flavone derivatives, tricyclic pyrano-flavones which also inhibit human aromatase. Multicomponent reactions involving flavones therefore enable the synthesis of a variety of derivatives, some of which may be useful as anticancer agents.

Selective aldosterone synthase inhibitors reduce aldosterone formation in vitro and in vivo.

Aldosterone plays a crucial role in salt and water homeostasis but in case of pathologically increased plasma aldosterone levels it is also involved in the development and the progression of severe cardiovascular diseases like heart failure and myocardial fibrosis. For the treatment of these diseases we propose inhibition of the aldosterone forming enzyme CYP11B2 as a new pharmacological strategy. We recently developed in vitro highly potent and selective inhibitors of human CYP11B2, but the evidence of their in vivo activity is still missing. For this purpose, rat aldosterone synthase gene was cloned and expressed in V79MZ cells to establish a new screening assay for the identification of "rat-active" substances. Compound 7 from the class of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones showed a moderate inhibitory effect (65% at 2 microM) on rat CYP11B2 in vitro. Furthermore, it diminished the conversion of deoxycorticosterone to aldosterone in rat adrenals and significantly reduced plasma aldosterone levels in vivo.

Development of a biological screening system for the evaluation of highly active and selective 17beta-HSD1-inhibitors as potential therapeutic agents.

17beta-Hydroxysteroid dehydrogenase type 1 (17beta-HSD1) catalyses the intracellular conversion of oestrone (E1) to oestradiol (E2). E2 is known to be involved in the development and progression of breast cancer and endometriosis. Since 17beta-HSD1 is overexpressed in these oestrogen-dependent diseases, inhibition of this enzyme may be a more target-directed therapeutical approach compared to established medical treatments. For the identification of highly active and selective 17beta-HSD1-inhibitors that are suitable for application as potential therapeutics, there is a need for an appropriate, efficient and reliable screening system. Here, we report the development and application of our screening system using our in house library of potential 17beta-HSD1-inhibitors. Four potent and selective compounds with a good first pharmacokinetic profile were identified.

Effects of Leuzea carthamoides on human breast adenocarcinoma MCF-7 cells determined by gene expression profiling and functional assays.

Products derived from roots of Leuzea carthamoides (Maral root) are being promoted as dietary supplements with anti-aging, adaptogenic and anabolic activity, without much scientific evidence. We investigated the effects of a lipophilic Leuzea root extract and the major phytoecdysteroid, 20-hydroxyecdysone, in human breast adenocarcinoma MCF-7 cells. Cell proliferation was inhibited by the extract (IC50 = 30 microg/mL) but not by 20-hydroxyecdysone. Genome-wide expression profiling using Affymetrix HG U133 Plus 2.0 microarrays was carried out to analyse effects at the transcriptional level. 241 genes appeared to be differentially expressed after Leuzea treatment, more than after treatment with either 17beta-estradiol or tamoxifen. Transcripts linked to cell cycle regulation and DNA replication were highly over-represented and regulated in an anti-proliferative manner. Genes involved in apoptosis were regulated in a pro-apoptotic manner. Expression levels of several oxidoreductase transcripts were strongly induced, most prominent CYP1A1, known to be regulated via the aryl hydrocarbon receptor pathway. An XRE-dependent reporter gene assay confirmed the AhR-agonistic activity of the Leuzea root extract, whereas 20-hydroxyecdysone was not active. Leuzea extract also inhibited 5alpha-reductase, type II. While the extract significantly modulates cellular activities, the phytoecdysteroids, are most likely not the active principles of L. carthamoides.

Structural modifications of salicylates: inhibitors of human CD81-receptor HCV-E2 interaction.

Starting point of the present paper was the result of a virtual screening using the open conformation of the large extracellular loop (LEL) of the CD81-receptor (crystal structure: PDB-ID: 1G8Q). After benzyl salicylate had been experimentally validated to be a moderate inhibitor of the CD81-LEL-HCV-E2 interaction, further optimization was performed and heterocyclic-substituted benzyl salicylate derivatives were synthesized. The compounds were tested for their ability to inhibit the interaction of a fluorescence-labeled antibody to CD81-LEL using HUH7.5 cells. No compound showed an increase concerning the inhibition of the protein-protein interaction compared to benzyl salicylate.

Identification of terfenadine as an inhibitor of human CD81-receptor HCV-E2 interaction: synthesis and structure optimization.

Terfenadine (4-[4-(hydroxydiphenylmethyl)-1-piperidyl]-1-(4-tert-butylphenyl)-butan-1-ol) was identified in a biological screening to be a moderate inhibitor (27% inhibition) of the CD81-LEL-HCV-E2 interaction. To increase the observed biological activity, 63 terfenadine derivates were synthesized via microwave assisted nucleophilic substitution. The prepared compounds were tested for their inhibitory potency by means ofa fluorescence labeled antibody assay using HUH7.5 cells. Distinct structure-activity relationships could be derived. Optimization was successful, leading to 3g, identified as the most potent compound (69 % inhibition). Experiments with viral particles revealed that there might be additional HCV infection reducing mechanisms.

Development and evaluation of a pharmacophore model for inhibitors of aldosterone synthase (CYP11B2).

Recently, we proposed inhibition of aldosterone synthase (CYP11B2) as a novel strategy for the treatment of congestive heart failure and myocardial fibrosis and synthesized a large number of inhibitors. In this work, a pharmacophore model for CYP11B2 inhibitors was developed by superimposition of active and non-active compounds. This model was confirmed by the synthesis of two pyridyl substituted acenaphthene derivatives (A,B). This new class of compounds as well as the pharmacophore could be helpful for the discovery of novel inhibitors.

The adrenocortical tumor cell line NCI-H295R as an in vitro screening system for the evaluation of CYP11B2 (aldosterone synthase) and CYP11B1 (steroid-11beta-hydroxylase) inhibitors.

Aldosterone plays a key role in salt and water homeostasis but is also involved in the development and progression of congestive heart failure and myocardial fibrosis. As a new pharmacological strategy for the treatment of these diseases, we propose the inhibition of the key enzyme of mineralcorticoid formation, CYP11B2 (aldosterone synthase). For studies of the effects of CYP11B2 inhibitors on the adrenal cortex, we selected the NCI-H295R cell line which expresses most of the key enzymes necessary for steroidogenesis. To evaluate this cell line as a test system for effects and side effects of CYP inhibitors, we established assays using radiolabeled substrates of CYP11B2 and CYP11B1 and subsequently tested a series of CYP11B2 inhibitors including the CYP19 inhibitor fadrozole. Fadrozole and compounds 6, 9 and 10 were more potent towards CYP11B2 compared to CYP11B1 with IC(50) values in the nanomolar range. To analyze their overall effect, the formation of steroids in the cell culture supernatant was monitored. All compounds led to a concentration-dependent reduction of the aldosterone secretion but also reduced the formation of cortisol and androgens. In conclusion, the H295R cell line is a suitable tool for the prediction of overall side effects of CYP11B2 inhibitors on steroidogenesis.

A method for screening enzyme inhibitors using size exclusion chromatography and ESI-LC-MS/MS.

A pilot study was performed for the development of a method to screen compound libraries using an electrospray mass spectrometer interfaced with liquid chromatography (LC). The mixture of compounds was obtained by combining low-molecular weight inhibitors of carboxypeptidase A (CPA), a representative zinc-containing proteolytic enzyme. After the incubation of the mixture with CPA, the enzyme-bound compounds were separated by size exclusion chromatography (SEC) from unbound compounds. The separation of compounds was affected by LC. Three compounds were identified, which represent the tight binding inhibitors of the library. These compounds were quantitated using an automatic switching valve to avoid the interference of buffer salts with the detection of analytes. The quantitated amounts of the compounds were found to be in good accordance with the K(i) values.

Synthesis of hydroxy derivatives of highly potent non-steroidal CYP 17 inhibitors as potential metabolites and evaluation of their activity by a non cellular assay using recombinant human enzyme.

Inhibition of CYP 17 is a promising strategy for the treatment of prostate cancer. Recently two non-steroidal compounds with high in vitro activity were synthesized in our group (BW19 and BW95). However, after a few hours they showed in vivo a strong decrease in their activity. This might be due to a fast biodegradation. Potential hydroxy and epoxy metabolites were synthesized and their inhibitory activities were tested by a new non-cellular assay using recombinant enzyme. As source, membrane fractions of E. coli pJL17/OR coexpressing human CYP 17 and rat NADPH-P450-reductase were, used. Showing a high and constant CYP 17 activity and a fast and easy isolation procedure the new method was advantageous compared with the microsomal assay. Interestingly, all the new synthesized hydroxy and epoxy compounds except one showed a lower inhibition of CYP 17 than the parent compounds. Thus, the loss of in vivo activity may be partly explained.

2- and 3-[(aryl)(azolyl)methyl]indoles as potential non-steroidal aromatase inhibitors.

The present study was designed to follow our pharmacomodulation work in the field of non-steroidal aromatase inhibitors. All target compounds 12a-h and 28a-h were tested in vitro for human placental aromatase inhibition, using testosterone or androstenedione as the substrate for the aromatase enzyme and the IC50 and relative potency to aminoglutethimide data are included. A SAR study indicated that 3-[(4-fluorophenyl)(1H-imidazol-1-yl)methyl]-1-ethyl-2-methyl-1H-indole (28 g) was a highly potent and selective aromatase inhibitor with IC50 value of 0.025 microM. 28 g was also a weak inhibitor of androstenedione synthesis.