In Silico Workflow for the Discovery of Natural Products Activating the G Protein-Coupled Bile Acid Receptor 1

The G protein-coupled bile acid receptor (GPBAR1) has been recognized as a promising new target for the treatment of diverse diseases, including obesity, type 2 diabetes, fatty liver disease and atherosclerosis. The identification of novel and potent GPBAR1 agonists is highly relevant, as these diseases are on the rise and pharmacological unmet therapeutic needs are pervasive. Therefore, the aim of this study was to develop a proficient workflow for the in silico prediction of GPBAR1 activating compounds, primarily from natural sources. A protocol was set up, starting with a comprehensive collection of structural information of known ligands. This information was used to generate ligand-based pharmacophore models in LigandScout 4.08 Advanced. After theoretical validation, the two most promising models, namely BAMS22 and TTM8, were employed as queries for the virtual screening of natural product and synthetic small molecule databases. Virtual hits were progressed to shape matching experiments and physicochemical clustering. Out of 33 diverse virtual hits subjected to experimental testing using a reporter gene-based assay, two natural products, farnesiferol B (27) and microlobidene (28), were confirmed as GPBAR1 activators reaching more than 50% receptor activation at 20 μM with EC50s of 13.53 μM and 13.88 μM, respectively. This activity is comparable to that of the endogenous ligand lithocholic acid (1). Seven further virtual hits showed activity reaching at least 15% receptor activation either at 5 or 20 μM, including new scaffolds from natural and synthetic origin.© 2018 Kirchweger, Kratz, Ladurner, Grienke, Langer, Dirsch and Rollinge

Allspice and Clove As Source of Triterpene Acids Activating the G Protein-Coupled Bile Acid Receptor TGR5

Worldwide, metabolic diseases such as obesity and type 2 diabetes have reached epidemic proportions. A major regulator of metabolic processes that gained interest in recent years is the bile acid receptor TGR5 (Takeda G protein-coupled receptor 5). This G protein-coupled membrane receptor can be found predominantly in the intestine, where it is mainly responsible for the secretion of the incretins glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). The aim of this study was (i) to identify plant extracts with TGR5-activating potential, (ii) to narrow down their activity to the responsible constituents, and (iii) to assess whether the intestinal microbiota produces transformed metabolites with a different activity profile. Chenodeoxycholic acid (CDCA) served as positive control for both, the applied cell-based luciferase reporter gene assay for TGR5 activity and the biotransformation assay using mouse fecal slurry. The suitability of the workflow was demonstrated by the biotransformation of CDCA to lithocholic acid resulting in a distinct increase in TGR5 activity. Based on a traditional Tibetan formula, 19 plant extracts were selected and investigated for TGR5 activation. Extracts from the commonly used spices Syzygium aromaticum (SaroE, clove), Pimenta dioica (PdioE, allspice), and Kaempferia galanga (KgalE, aromatic ginger) significantly increased TGR5 activity. After biotransformation, only KgalE showed significant differences in its metabolite profile, which, however, did not alter its TGR5 activity compared to non-transformed KgalE. UHPLC-HRMS (high-resolution mass spectrometry) analysis revealed triterpene acids (TTAs) as the main constituents of the extracts SaroE and PdioE. Identification and quantification of TTAs in these two extracts as well as comparison of their TGR5 activity with reconstituted TTA mixtures allowed the attribution of the TGR5 activity to TTAs. EC50s were determined for the main TTAs, i.e., oleanolic acid (2.2 ± 1.6 μM), ursolic acid (1.1 ± 0.2 μM), as well as for the hitherto unknown TGR5 activators corosolic acid (0.5 ± 1.0 μM) and maslinic acid (3.7 ± 0.7 μM). In conclusion, extracts of clove, allspice, and aromatic ginger activate TGR5, which might play a pivotal role in their therapeutic use for the treatment of metabolic diseases. Moreover, the TGR5 activation of SaroE and PdioE could be pinpointed solely to TTAs.© 2017 Ladurner, Zehl, Grienke, Hofstadler, Faur, Pereira, Berry, Dirsch and Rollinge

Natural products modulating the hERG channel: heartaches and hope

Covering: 1996–December 2016 The human Ether-à-go-go Related Gene (hERG) channel is a voltage-gated potassium channel playing an essential role in the normal electrical activity in the heart. It is involved in the repolarization and termination of action potentials in excitable cardiac cells. Mutations in the hERG gene and hERG channel blockage by small molecules are associated with increased risk of fatal arrhythmias. Several drugs have been withdrawn from the market due to hERG channel-related cardiotoxicity. Moreover, as a result of its notorious ligand promiscuity, this ion channel has emerged as an important antitarget in early drug discovery and development. Surprisingly, the hERG channel blocking profile of natural compounds present in frequently consumed botanicals (i.e. dietary supplements, spices, and herbal medicinal products) is not routinely assessed. This comprehensive review will address these issues and provide a critical compilation of hERG channel data for isolated natural products and extracts over the past two decades (1996–2016). In addition, the review will provide (i) a solid basis for the molecular understanding of the physiological functions of the hERG channel, (ii) the translational potential of in vitro/in vivo results to cardiotoxicity in humans, (iii) approaches for the identification of hERG channel blockers from natural sources, (iv) future perspectives for cardiac safety guidelines and their applications within phytopharmaceuticals and dietary supplements, and (v) novel applications of hERG channel modulation (e.g. as a drug target)

Streptomyces spp. From Ethiopia Producing Antimicrobial Compounds: Characterization via Bioassays, Genome Analyses, and Mass Spectrometry

A total of 416 actinomycete cultures were isolated from various unique environments in Ethiopia and tested for bioactivity. Six isolates with pronounced antimicrobial activity were chosen for taxonomic identification and further investigation. Morphological and cultural properties of the isolates were found to be consistent with those of the genus Streptomyces, which was further confirmed by phylogenetic analysis based on 16S rRNA gene sequences. One of the isolates, designated Streptomyces sp. Go-475, which displayed potent activity against both pathogenic yeasts and Gram-positive bacteria, was chosen for further investigation. Metabolite profiles and bioactivity of Go-475 incubated on wheat bran-based solid and soya flour-based liquid media were compared using high-resolution LC-MS. This allowed identification of several known compounds, and suggested the ability of Go-475 to produce new secondary metabolites. Major anti-bacterial compounds were purified from liquid cultures of Go-475, and their structures elucidated by NMR and HRMS as 8-O-methyltetrangomycin and 8-O-methyltetrangulol. In addition, many potentially novel metabolites were detected, the majority of which were produced in solid media-based fermentation. The genome sequence of Streptomyces sp. Go-475 was obtained using a hybrid assembly approach of high quality Illumina short read and low quality Oxford Nanopore long read data. The complete linear chromosome of 8,570,609 bp, featuring a G+C content of 71.96%, contains 7,571 predicted coding sequences, 83 t(m)RNA genes, and six rrn operons. Analysis of the genome for secondary metabolite biosynthesis gene clusters further confirmed potential of this isolate to synthesize chemically diverse natural products, and allowed to connect certain clusters with experimentally confirmed molecules.© 2018 Kibret, Guerrero-Garzón, Urban, Zehl, Wronski, Rückert, Busche, Kalinowski, Rollinger, Abate and Zotche

Drugs from nature targeting inflammation (DNTI): a successful Austrian interdisciplinary network project

Inflammation is part of numerous pathological conditions, which are lacking satisfying treatment and effective concepts of prevention. A national research network project, DNTI, involving scientists from six Austrian universities as well as several external partners aimed to identify and characterize natural products capable of combating inflammatory processes specifically in the cardiovascular system. The combined use of computational techniques with traditional knowledge, high-tech chemical analysis and synthesis, and a broad range of in vitro, cell-based, and in vivo pharmacological models led to the identification of a series of promising anti-inflammatory drug lead candidates. Mechanistic studies contributed to a better understanding of their mechanism of action and delivered new knowledge on the molecular level of inflammatory processes. Herein, the used approaches and selected highlights of the results of this interdisciplinary project are presented

Biological Activity of Flavonoids and Rare Sesquiterpene Lactones Isolated From Centaurea ragusina L.

The endemic Croatian species Centaurea ragusina L., like other species from the genus Centaurea, has been traditionally used in Croatia as an antibacterial agent and for the treatment of gastrointestinal and urogenital disorders. In several chromatographic steps, three flavonoids and three sesquiterpene lactones (STLs) were isolated and identified from the most active fractions of the ethanol extract. Two STLs, one for which we created the trivial name ragusinin, and hemistepsin A are here reported for the first time as constituents of the genus Centaurea. All six compounds were screened for their effect on several tumor and one normal cell lines. Among them, ragusinin showed the best bioactivity and high specificity to affect tumor murine SCCVII, human HeLa and Caco-2 cell lines, but not the viability of normal V79 fibroblasts. Due to these characteristics the action of ragusinin was investigated in more detail. Since DNA is the primary target for many drugs with antibacterial and anticancer activity, we studied its interaction with ragusinin. Rather moderate binding affinity to DNA excluded it as the primary target of ragusinin. Due to the possibility of STL interaction with glutathione (GSH), the ubiquitous peptide that traps reactive compounds and other xenobiotics to prevent damage to vital proteins and nucleic acids, its role in deactivation of ragusinin was evaluated. Addition of the GSH precursor N-acetyl-cysteine potentiated the viability of HeLa cells, while the addition of GSH inhibitor L-buthionine sulfoximine decreased it. Moreover, pre-treatment of HeLa cells with the inhibitor of glutathione-S-transferase decreased their viability indicating the detoxifying role of GSH in ragusinin treated cells. Cell death, derived by an accumulation of cells in a G2 phase of the cell cylce, was shown to be independent of poly (ADP-ribose) polymerase and caspase-3 cleavage pointing toward an alternative cell death pathway.© 2018 Grienke, Radić Brkanac, Vujčić, Urban, Ivanković, Stojković, Rollinger, Kralj, Brozovic and Radić Stojkovi

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

Discovery and Characterization of Diazenylaryl Sulfonic Acids as Inhibitors of Viral and Bacterial Neuraminidases

Viral neuraminidases are an established drug target to combat influenza. Severe complications observed in influenza patients are primarily caused by secondary infections with e.g., Streptococcus pneumoniae. These bacteria engage in a lethal synergism with influenza A viruses (IAVs) and also express neuraminidases. Therefore, inhibitors with dual activity on viral and bacterial neuraminidases are expected to be advantageous for the treatment of influenza infections. Here we report on the discovery and characterization of diazenylaryl sulfonic acids as dual inhibitors of viral and Streptococcus pneumoniae neuraminidase. The initial hit came from a virtual screening campaign for inhibitors of viral neuraminidases. For the most active compound, 7-[2-[4-[2-[4-[2-(2-hydroxy-3,6-disulfo-1-naphthalenyl)diazenyl]-2-methylphenyl]diazenyl]-2-methylphenyl]diazenyl]-1,3-naphthalenedisulfonic acid (NSC65847; 1), the Ki-values measured in a fluorescence-based assay were lower than 1.5 μM for both viral and pneumococcal neuraminidases. The compound also inhibited N1 virus variants containing neuraminidase inhibitor resistance-conferring substitutions. Via enzyme kinetics and nonlinear regression modeling, 1 was suggested to impair the viral neuraminidases and pneumococcal neuraminidase with a mixed-type inhibition mode. Given its antiviral and antipneumococcal activity, 1 was identified as a starting point for the development of novel, dual-acting anti-infectives.© 2017 Hoffmann, Richter, von Grafenstein, Walther, Xu, Schumann, Grienke, Mair, Kramer, Rollinger, Liedl, Schmidtke and Kirchmai

Dual Acting Neuraminidase Inhibitors Open New Opportunities to Disrupt the Lethal Synergism between Streptococcus pneumoniae and Influenza Virus

Secondary infections with Streptococcus pneumoniae cause severe pneumonia and enhance lethality during influenza epidemics and pandemics. Structural and functional similarities with viral neuraminidase (NA) suggest that the highly prevalent pneumococcal NAs, NanA and NanB, might contribute to this lethal synergism by supporting viral replication and that dual acting NA inhibitors (NAIs) will disrupt it. To verify this hypothesis, NanA and NanB were expressed in E. coli. After confirming their activity in enzyme assays, in vitro models with influenza virus A/Jena/8178/09 (Jena/8178) and the recombinant NanA or NanB (rNanA and rNanB) were established in A549 and MDCK cells to mimic the role of these pneumococcal NAs during co-infection. Studies on the influence of both NAs on viral receptor expression, spread, and yield revealed a distinct effect of NanA and NanB on viral replication in these in vitro models. Both enzymes were able to support Jena/8178 replication at certain concentrations. This synergism was disrupted by the NAIs oseltamivir, DANA, katsumadain A, and artocarpin exerting an inhibitory effect on viral NA and NanA. Interestingly, katsumadain A and artocarpin inhibited rNanA and rNanB similarly. Zanamivir did not show activity. These results demonstrate a key role of pneumococcal NAs in the lethal synergism with influenza viruses and reveal opportunities for its effective disruption