Ulrike Grienke

Antiviral potential and molecular insight into neuraminidase inhibiting diarylheptanoids from Alpinia katsumadai.

At present, neuraminidase (NA) inhibitors are the mainstay of pharmacological strategies to fight against global pandemic influenza. In the search for new antiviral drug leads from nature, the seed extract of Alpinia katsumadai has been phytochemically investigated. Among the six isolated constituents, four diarylheptanoids showed in vitro NA inhibitory activities in low micromolar ranges against human influenza virus A/PR/8/34 of subtype H1N1. The most promising constituent, katsumadain A (4; IC(50) = 1.05 +/- 0.42 microM), also inhibited the NA of four H1N1 swine influenza viruses, with IC(50) values between 0.9 and 1.64 muM, and showed antiviral effects in plaque reduction assays. Considering the flexible loop regions of NA, extensive molecular dynamics (MD) simulations were performed to study the putative binding mechanism of the T-shaped diarylheptanoid 4. Docking results showed well-established interactions between the protein and the core of this novel NA-inhibiting natural scaffold, excellent surface complementarity to the simulated binding pocket, and concordance with experimentally derived SAR data.

Pharmacophore-based discovery of FXR-agonists. Part II: Identification of bioactive triterpenes from Ganoderma lucidum

Graphical abstract

Pharmacophore-based discovery of FXR agonists. Part I: Model development and experimental validation.

Graphical abstract

European medicinal polypores – A modern view on traditional uses

ETHNOPHARMACOLOGICAL RELEVANCE In particular five polypore species, i.e. Laetiporus sulphureus, Fomes fomentarius, Fomitopsis pinicola, Piptoporus betulinus, and Laricifomes officinalis, have been widely used in central European folk medicines for the treatment of various diseases, e.g. dysmenorrhoea, haemorrhoids, bladder disorders, pyretic diseases, treatment of coughs, cancer, and rheumatism. Prehistoric artefacts going back to over 5000 years underline the long tradition of using polypores for various applications ranging from food or tinder material to medicinal-spiritual uses as witnessed by two polypore species found among items of Ötzi, the Iceman. The present paper reviews the traditional uses, phytochemistry, and biological activity of the five mentioned polypores. MATERIALS AND METHODS All available information on the selected polypore taxa used in traditional folk medicine was collected through evaluation of literature in libraries and searches in online databases using SciFinder and Web of Knowledge. RESULTS Mycochemical studies report the presence of many primary (e.g. polysaccharides) and secondary metabolites (e.g. triterpenes). Crude extracts and isolated compounds show a wide spectrum of biological properties, such as anti-inflammatory, cytotoxic, and antimicrobial activities. CONCLUSIONS The investigated polypores possess a longstanding ethnomycological tradition in Europe. Here, we compile biological results which highlight their therapeutic value. Moreover, this work provides a solid base for further investigations on a molecular level, both compound- and target-wise.

Discovery of prenylated flavonoids with dual activity against influenza virus and Streptococcus pneumoniae

Influenza virus neuraminidase (NA) is the primary target for influenza therapeutics. Severe complications are often related to secondary pneumonia caused by Streptococcus pneumoniae (pneumococci), which also express NAs. Recently, a NA-mediated lethal synergism between influenza A viruses and pneumococci was described. Therefore, dual inhibitors of both viral and bacterial NAs are expected to be advantageous for the treatment of influenza. We investigated the traditional Chinese herbal drug sāng bái pí (mulberry root bark) as source for anti-infectives. Two prenylated flavonoid derivatives, sanggenon G (4) and sanggenol A (5) inhibited influenza A viral and pneumococcal NAs and, in contrast to the approved NA inhibitor oseltamivir, also planktonic growth and biofilm formation of pneumococci. Evaluation of 27 congeners of 5 revealed a correlation between the degree of prenylation and bioactivity. Abyssinone-V 4′-methyl ether (27) inhibited pneumococcal NA with IC50 = 2.18 μM, pneumococcal growth with MIC = 5.63 μM, and biofilm formation with MBIC = 4.21 μM, without harming lung epithelial cells. Compounds 5 and 27 also disrupt the synergism between influenza A virus and pneumococcal NA in vitro, hence functioning as dual-acting anti-infectives. The results warrant further studies on whether the observed disruption of this synergism is transferable to in vivo systems.

Accessing biological actions of Ganoderma secondary metabolites by in silico profiling

The species complex around the medicinal fungus Ganoderma lucidum Karst. (Ganodermataceae) is widely known in traditional medicines, as well as in modern applications such as functional food or nutraceuticals. A considerable number of publications reflects its abundance and variety in biological actions either provoked by primary metabolites, such as polysaccharides, or secondary metabolites, such as lanostane-type triterpenes. However, due to this remarkable amount of information, a rationalization of the individual Ganoderma constituents to biological actions on a molecular level is quite challenging. To overcome this issue, a database was generated containing meta-information, i.e., chemical structures and biological actions of hitherto identified Ganoderma constituents (279). This was followed by a computational approach subjecting this 3D multi-conformational molecular dataset to in silico parallel screening against an in-house collection of validated structure- and ligand-based 3D pharmacophore models. The predictive power of the evaluated in silico tools and hints from traditional application fields served as criteria for the model selection. Thus, the focus was laid on representative druggable targets in the field of viral infections (5) and diseases related to the metabolic syndrome (22). The results obtained from this in silico approach were compared to bioactivity data available from the literature. 89 and 197 Ganoderma compounds were predicted as ligands of at least one of the selected pharmacological targets in the antiviral and the metabolic syndrome screening, respectively. Among them only a minority of individual compounds (around 10%) has ever been investigated on these targets or for the associated biological activity. Accordingly, this study discloses putative ligand target interactions for a plethora of Ganoderma constituents in the empirically manifested field of viral diseases and metabolic syndrome which serve as a basis for future applications to access yet undiscovered biological actions of Ganoderma secondary metabolites on a molecular level.

Antipneumococcal activity of neuraminidase inhibiting artocarpin

Streptococcus (S.) pneumoniae is a major cause of secondary bacterial pneumonia during influenza epidemics. Neuraminidase (NA) is a virulence factor of both pneumococci and influenza viruses. Bacterial neuraminidases (NAs) are structurally related to viral NA and susceptible to oseltamivir, an inhibitor designed to target viral NA. This prompted us to evaluate the antipneumococcal potential of two NA inhibiting natural compounds, the diarylheptanoid katsumadain A and the isoprenylated flavone artocarpin. Chemiluminescence, fluorescence-, and hemagglutination-based enzyme assays were applied to determine the inhibitory efficiency (IC(50) value) of the tested compounds towards pneumococcal NAs. The mechanism of inhibition was studied via enzyme kinetics with recombinant NanA NA. Unlike oseltamivir, which competes with the natural substrate of NA, artocarpin exhibits a mixed-type inhibition with a Ki value of 9.70 μM. Remarkably, artocarpin was the only NA inhibitor (NAI) for which an inhibitory effect on pneumococcal growth (MIC: 0.99-5.75 μM) and biofilm formation (MBIC: 1.15-2.97 μM) was observable. In addition, we discovered that the bactericidal effect of artocarpin can reduce the viability of pneumococci by a factor of >1000, without obvious harm to lung epithelial cells. This renders artocarpin a promising natural product for further investigations.

Applications of integrated data mining methods to exploring natural product space for acetylcholinesterase inhibitors.

Nature, especially the plant kingdom, is a rich source for novel bioactive compounds that can be used as lead compounds for drug development. In order to exploit this resource, the two neural network-based virtual screening techniques novelty detection with self-organizing maps (SOMs) and counterpropagation neural network were evaluated as tools for efficient lead structure discovery. As application scenario, significant descriptors for acetylcholinesterase (AChE) inhibitors were determined and used for model building, theoretical model validation, and virtual screening. Top-ranked virtual hits from both approaches were docked into the AChE binding site to approve the initial hits. Finally, in vitro testing of selected compounds led to the identification of forsythoside A and (+)-sesamolin as novel AChE inhibitors.

Human Ether-à-go-go Related Gene (hERG) Channel Blocking Aporphine Alkaloids from Lotus Leaves and Their Quantitative Analysis in Dietary Weight Loss Supplements

Blockage of the human ether-à-go-go related gene (hERG) channel can result in life-threatening ventricular tachyarrhythmia. In an in vitro screening of herbal materials for hERG blockers using an automated two-microelectrode voltage clamp assay on Xenopus oocytes, an alkaloid fraction of Nelumbo nucifera Gaertn. (lotus) leaves induced ∼50% of hERG current inhibition at 100 μg/mL. Chromatographic separation resulted in the isolation and identification of (-)-asimilobine, 1, nuciferine, 2, O-nornuciferine, 3, N-nornuciferine, 4, and liensinine, 5. In agreement with in silico predicted ligand-target interactions, 2, 3, and 4 revealed distinct in vitro hERG blockages measured in HEK293 cells with IC50 values of 2.89, 7.91, and 9.75 μM, respectively. Because lotus leaf dietary weight loss supplements are becoming increasingly popular, the identified hERG-blocking alkaloids were quantitated in five commercially available products. Results showed pronounced differences in the content of hERG-blocking alkaloids ranging up to 992 μg (2) in the daily recommended dose.

Computer-guided approach to access the anti-influenza activity of licorice constituents.

Neuraminidase (NA), a key enzyme in viral replication, is the first-line drug target to combat influenza. On the basis of a shape-focused virtual screening, the roots of Glycyrrhiza glabra (licorice) were identified as plant species with an accumulation of constituents that show 3D similarities to known influenza NA inhibitors (NAIs). Phytochemical investigation revealed 12 constituents identified as (E)-1-[2,4-dihydroxy-3-(3-methyl-2-butenyl)phenyl]-3-(8-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-yl)-2-propen-1-one (1), 3,4-dihydro-8,8-dimethyl-2H,8H-benzo[1,2-b:3,4-b′]dipyran-3-ol (2), biochanin B (3), glabrol (4), glabrone (5), hispaglabridin B (6), licoflavone B (7), licorice glycoside B (8), licorice glycoside E (9), liquiritigenin (10), liquiritin (11), and prunin (12). Eleven of these constituents showed significant influenza virus NA inhibition in a chemiluminescence (CL)-based assay. Additional tests, including (i) a cell-based cytopathic effect inhibition assay (general antiviral activity), (ii) the evaluation of cytotoxicity, (iii) the inhibition of the NA of Clostridium perfringens (CL- and fluorescence (FL)-based assay), and (iv) the determination of self-fluorescence and quenching, provided further perspective on their anti-influenza virus potential, revealing possible assay interference problems and false-positive results. Compounds 1, 3, 5, and 6 showed antiviral activity, most likely caused by the inhibition of NA. Of these, compounds 1, 3, and 6 were highly ranked in shape-focused virtual screening.