Design, synthesis and evaluation of side-chain hydroxylated derivatives of lithocholic acid as potent agonists of the vitamin D receptor (VDR).

A high number of biologically active and low-calcemic secosteroidal ligands of the vitamin D receptor (VDR) have been developed, some of which are already used clinically although with limited success in the treatment of hyperproliferative diseases because the required pharmaceutical dosages induce toxicity. We describe here the in silico design, synthesis, structural analysis and biological evaluation of two novel active lithocholic acid derivatives hydroxylated at the side chain as highly potent inhibitors of atopic dermatitis-relevant keratinocyte inflammation of potential therapeutic interest.

The design, synthesis, and anti-inflammatory evaluation of a drug-like library based on the natural product valerenic acid.

The plant natural product, valerenic acid (1) was chosen as a desirable scaffold for the generation of a novel screening library due to its drug-like physicochemical parameters (such as LogP, hydrogen bond donor/acceptor counts, and molecular weight). An 11-membered amide library (2-12) was subsequently generated using parallel solution-phase synthesis and Ghosez's reagent. The chemical structures of all semi-synthetic analogues (2-12) were elucidated following analysis of the NMR, MS, UV and IR data. The structures of compounds 8 and 11 were also confirmed by X-ray crystallographic analysis. All library members were evaluated for their ability to inhibit the release of IL-8 and TNF-α. Six analogues showed moderate activity in the IL-8 assay with IC50 values of 2.8-8.3µM, while none of the tested compounds showed any significant effect on inhibiting TNF-α release.

Classification complexity in myoelectric pattern recognition.

BACKGROUND: Limb prosthetics, exoskeletons, and neurorehabilitation devices can be intuitively controlled using myoelectric pattern recognition (MPR) to decode the subject's intended movement. In conventional MPR, descriptive electromyography (EMG) features representing the intended movement are fed into a classification algorithm. The separability of the different movements in the feature space significantly affects the classification complexity. Classification complexity estimating algorithms (CCEAs) were studied in this work in order to improve feature selection, predict MPR performance, and inform on faulty data acquisition. METHODS: CCEAs such as nearest neighbor separability (NNS), purity, repeatability index (RI), and separability index (SI) were evaluated based on their correlation with classification accuracy, as well as on their suitability to produce highly performing EMG feature sets. SI was evaluated using Mahalanobis distance, Bhattacharyya distance, Hellinger distance, Kullback-Leibler divergence, and a modified version of Mahalanobis distance. Three commonly used classifiers in MPR were used to compute classification accuracy (linear discriminant analysis (LDA), multi-layer perceptron (MLP), and support vector machine (SVM)). The algorithms and analytic graphical user interfaces produced in this work are freely available in BioPatRec. RESULTS: NNS and SI were found to be highly correlated with classification accuracy (correlations up to 0.98 for both algorithms) and capable of yielding highly descriptive feature sets. Additionally, the experiments revealed how the level of correlation between the inputs of the classifiers influences classification accuracy, and emphasizes the classifiers' sensitivity to such redundancy. CONCLUSIONS: This study deepens the understanding of the classification complexity in prediction of motor volition based on myoelectric information. It also provides researchers with tools to analyze myoelectric recordings in order to improve classification performance.

Assessing Specialized Metabolite Diversity in the Cosmopolitan Plant Genus Euphorbia L.

Coevolutionary theory suggests that an arms race between plants and herbivores yields increased plant specialized metabolite diversity and the geographic mosaic theory of coevolution predicts that coevolutionary interactions vary across geographic scales. Consequently, plant specialized metabolite diversity is expected to be highest in coevolutionary hotspots, geographic regions, which exhibit strong reciprocal selection on the interacting species. Despite being well-established theoretical frameworks, technical limitations have precluded rigorous hypothesis testing. Here we aim at understanding how geographic separation over evolutionary time may have impacted chemical differentiation in the cosmopolitan plant genus Euphorbia. We use a combination of state-of-the-art computational mass spectral metabolomics tools together with cell-based high-throughput immunomodulatory testing. Our results show significant differences in specialized metabolite diversity across geographically separated phylogenetic clades. Chemical structural diversity of the highly toxic Euphorbia diterpenoids is significantly reduced in species native to the Americas, compared to Afro-Eurasia. The localization of these compounds to young stems and roots suggest a possible ecological relevance in herbivory defense. This is further supported by reduced immunomodulatory activity in the American subclade as well as herbivore distribution patterns. We conclude that computational mass spectrometric metabolomics coupled with relevant ecological data provide a strong tool for exploring plant specialized metabolite diversity in a chemo-evolutionary framework.

Unlocking the full potential of open innovation in the life sciences through a classification system.

A common understanding of expectations and requirements is critical for boosting research-driven business opportunities in open innovation (OI) settings. Transparent communication requires common definitions and standards for OI to align the expectations of both parties. Here, we suggest a five-level classification system for OI models, reflecting the degree of openness. The aim of this classification system is to reduce contract negotiation complexity and times between two parties looking to engage in OI. Systematizing definitions and contractual terms for OI in the life sciences helps to reduce entry barriers and boosts collaborative value generation. By providing a contractual framework with predefined rules, science will be allowed to move more freely, thus maximizing the potential of OI.

Evolutionary prediction of medicinal properties in the genus Euphorbia L.

The current decrease of new drugs brought to the market has fostered renewed interest in plant-based drug discovery. Given the alarming rate of biodiversity loss, systematic methodologies in finding new plant-derived drugs are urgently needed. Medicinal uses of plants were proposed as proxy for bioactivity, and phylogenetic patterns in medicinal plant uses have suggested that phylogeny can be used as predictive tool. However, the common practice of grouping medicinal plant uses into standardised categories may restrict the relevance of phylogenetic predictions. Standardised categories are mostly associated to systems of the human body and only poorly reflect biological responses to the treatment. Here we show that medicinal plant uses interpreted from a perspective of a biological response can reveal different phylogenetic patterns of presumed underlying bioactivity compared to standardised methods of medicinal plant use classification. In the cosmopolitan and pharmaceutically highly relevant genus Euphorbia L., identifying plant uses modulating the inflammatory response highlighted a greater phylogenetic diversity and number of potentially promising species than standardised categories. Our interpretation of medicinal plant uses may therefore allow for a more targeted approach for future phylogeny-guided drug discovery at an early screening stage, which will likely result in higher discovery rates of novel chemistry with functional biological activity.

Global medicinal uses of Euphorbia L. (Euphorbiaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Euphorbia (spurges, Euphorbiaceae) is the third largest genus of flowering plants, with almost 2000 species. Its exceptional diversity of growth forms and near-cosmopolitan distribution have attracted human interest since ancient times. For instance in Australia, topical application of latex of Euphorbia peplus L. is used as a home treatment for skin cancer and actinic keratosis. Its use in Australian folk medicine has inspired the release of the drug Picato® (ingenol mebutate), and further fostered interest in natural products and medicinal uses of Euphorbia in recent years. AIM OF THE STUDY: To provide an indicative overview of medicinal uses of the genus Euphorbia driven by the recent interest in biologically active natural products from Euphorbia in drug discovery. We assess documented medicinal knowledge and value of the genus Euphorbia and the taxonomic distribution of this value. MATERIALS AND METHODS: We undertook an extensive survey of over 260 multidisciplinary publications on the online repository JSTOR using the search term "Euphorbia medicinal". RESULTS: Medicinal uses were identified for >5% of the species in the genus, including descriptions of treatments for a variety of diseases. The most-cited medicinal uses around the world were treatments for digestive system disorders, skin ailments and, especially in the Southern hemisphere, infections. Consensus ratios indicated that the most-valued medicinal uses of Euphorbia species are in the treatment of digestive and respiratory complaints, inflammation and injuries, especially by members of Euphorbia subg. Chamaesyce. CONCLUSIONS: The present study gives a first indicative overview of Euphorbia species used for health and wellbeing around the world. The exceptional diversity of the genus Euphorbia is not only represented by its growth forms but also by its diverse medicinal uses. Our results highlight the importance of research into medicinal uses of Euphorbia species and their importance as a source of natural products. Furthermore the medicinally highly valuable subgenus Chamaesyce was identified as chemically under-investigated, emphasizing the need for further studies investigating the chemical diversity to which the high medicinal value of Euphorbia subg. Chamaesyce can be attributed.

Reverse pharmacology and the de-orphanization of 7TM receptors.

Approximately 800 genes coding for seven-transmembrane, G-protein-coupled receptors have so far been recognized. In spite of this, many of these receptors are defined by their sequence only, and are therefore classified as orphan receptors. Without knowing what their endogenous ligands are, we lack the information needed to understand their physiological role and hence cannot make use of them as drug targets. In this communication, we discuss different strategies, as well as difficulties in the deorphanizing process.:

Lysophosphatidic acid binds to and activates GPR92, a G protein-coupled receptor highly expressed in gastrointestinal lymphocytes.

Here, the ligand binding, activation, and tissue distribution of the orphan G protein-coupled receptor (GPCR) GPR92 were studied. GPR92 binds and is activated by compounds based on the lysophosphatidic acid (LPA) backbone. The binding of LPA to GPR92 was of high affinity (K(D) = 6.4 +/- 0.9 nM) and led to an increase in both phosphoinositide hydrolysis and cAMP production. GPR92 is atypical in that it has a low sequence homology with the classic LPA(1-3) receptors (21-22%). Expression of GPR92 is mainly found in heart, placenta, spleen, brain, lung, and gut. Notably, GPR92 is highly expressed in the lymphocyte compartment of the gastrointestinal tract. It is the most abundant GPCR activated by LPA found in the small intestinal intraepithelial CD8+ cytotoxic T cells.

Identification of a free fatty acid receptor, FFA2R, expressed on leukocytes and activated by short-chain fatty acids.

Short-chain fatty acids (SCFAs) have long been known to exert cellular effects on blood leukocytes. Acetate, propionate, and butyrate represent the most capable SCFA, inducing calcium mobilization which subsequently regulates leukocyte function in the immune system. We have cloned the previously described putative orphan G-protein coupled receptor, GPR43, and have functionally identified SCFA as the activating ligands. Acetate and propionate were found to be the two most potent ligands, although butyrate, formate, and valerate (in this order of potency) also were able to induce receptor activation. Both the human and mouse receptor homologues were found to share the same pattern of ligand activation. This finding, together with a high degree of amino acid sequence similarity between the mouse and human homologues, indicates an evolutionary conserved function. Upon ligand stimulation, the receptor mobilized intracellular calcium in both a recombinant system as well as in human granulocytes. We found the human gene to be predominantly expressed in peripheral blood leukocytes and, to a lesser extent, in spleen. We suggest the designation FFA(2)R to this second receptor activated by free fatty acids. The first-described FFAR, now named FFA(1)R, is activated by medium- to long-chain free fatty acids.

Progress in methodology. Improved reporter gene assays used to identify ligands acting on orphan seven-transmembrane receptors.

Seven-transmembrane G-protein-coupled receptors play a central role in physiology by facilitating cell communication through recognition of a wide range of ligands. Even more important, they represent important drug targets. Unfortunately, for many of these receptors the endogenous ligands, and hence their functions, remain to be identified. These receptors are referred to as "orphan" receptors. A pre-requisite for the identification of ligands activating orphan receptors is powerful assay systems. Until now, reporter gene assays have not been in common use in this process. Here, we summarize our development of improved reporter gene assays. We optimized reporter gene assays in respect of (i) the promoter region of the construct, (ii) the reporter enzyme used, (iii) and the assay procedure. Furthermore, an unique fluorescence-based clone selection step was introduced, allowing rapid selection of the most sensitive reporter cell clones when establishing stable reporter cell lines. Mathematical formulae are provided to enable a simple and reliable comparison between different cell lines, when tested with a compound of interest. The resulting reporter cell lines responded in a very sensitive way to the stimulation of various test receptors. The reporter system was termed HighTRACE (high-throughput reporter assay with clone election). Its high assay quality makes it suitable as a primary screening tool. Ligands for two recently unknown 7TM receptors were identified using the HighTRACE system i.e., two cell surface free fatty acid receptors, GPR40 (FFA1R) and GPR43 (FFA2R). The identification was accomplished using a reverse pharmacology approach.

A human cell surface receptor activated by free fatty acids and thiazolidinedione drugs.

Fatty acids, which are essential nutritional components, are also involved in cardiovascular and metabolic diseases. Here we report a human cell surface receptor that we name free fatty acid receptor (FFAR), because it is specifically activated by medium to long-chain free fatty acids. The receptor belongs to the class of seven-transmembrane, G-protein coupled receptors (GPCRs) and also mediates responses to antidiabetic drugs of the thiazolidinedione type. It is expressed in skeletal muscle, heart, liver, and pancreatic beta-cells. Stimulation of FFAR increases the intracellular calcium concentration in cells expressing the receptor in a native (pancreatic beta-cell line) or in a recombinant form. In view of the nature of the activating substances, their physiological role in the body, and the tissue distribution of FFAR we suggest the term "nutrient sensing receptor" for receptors acting at the interface between dietary components and signalling molecules.