Atanas G. Atanasov

Discovery and resupply of pharmacologically active plant-derived natural products: A review.

Medicinal plants have historically proven their value as a source of molecules with therapeutic potential, and nowadays still represent an important pool for the identification of novel drug leads. In the past decades, pharmaceutical industry focused mainly on libraries of synthetic compounds as drug discovery source. They are comparably easy to produce and resupply, and demonstrate good compatibility with established high throughput screening (HTS) platforms. However, at the same time there has been a declining trend in the number of new drugs reaching the market, raising renewed scientific interest in drug discovery from natural sources, despite of its known challenges. In this survey, a brief outline of historical development is provided together with a comprehensive overview of used approaches and recent developments relevant to plant-derived natural product drug discovery. Associated challenges and major strengths of natural product-based drug discovery are critically discussed. A snapshot of the advanced plant-derived natural products that are currently in actively recruiting clinical trials is also presented. Importantly, the transition of a natural compound from a “screening hit” through a “drug lead” to a “marketed drug” is associated with increasingly challenging demands for compound amount, which often cannot be met by re-isolation from the respective plant sources. In this regard, existing alternatives for resupply are also discussed, including different biotechnology approaches and total organic synthesis. While the intrinsic complexity of natural product-based drug discovery necessitates highly integrated interdisciplinary approaches, the reviewed scientific developments, recent technological advances, and research trends clearly indicate that natural products will be among the most important sources of new drugs also in the future.

Natural product agonists of peroxisome proliferator-activated receptor gamma (PPARγ): a review.

Graphical abstract

Hexose-6-phosphate dehydrogenase determines the reaction direction of 11β-hydroxysteroid dehydrogenase type 1 as an oxoreductase

The impact of hexose‐6‐phosphate dehydrogenase (H6PDH) on 11β‐hydroxysteroid dehydrogenase (11β‐HSD) type 1 activity was investigated upon coexpression in HEK‐293 cells. Confocal microscopy analysis indicated colocalisation of both enzymes at the lumenal side of the endoplasmic reticulum (ER) membrane. Functional analysis in intact cells revealed fivefold stimulation of 11β‐HSD1 oxoreductase activity and sixfold decrease of dehydrogenase activity upon coexpression with H6PDH, without changing kinetic parameters in cell lysates. Thus, H6PDH directly determines the reaction direction of 11β‐HSD1 in intact cells as an oxoreductase without changing intrinsic catalytic properties of 11β‐HSD1 by regenerating NADPH in the ER‐lumen.

Ethnopharmacological in vitro studies on Austria's folk medicine—An unexplored lore in vitro anti-inflammatory activities of 71 Austrian traditional herbal drugs

Ethnopharmacological relevance In Austria, like in most Western countries, knowledge about traditional medicinal plants is becoming scarce. Searching the literature concerning Austrias ethnomedicine reveals its scant scientific exploration. Aiming to substantiate the potential of medicinal plants traditionally used in Austria, 63 plant species or genera with claimed anti-inflammatory properties listed in the VOLKSMED database were assessed for their in vitro anti-inflammatory activity. Material and methods 71 herbal drugs from 63 plant species or genera were extracted using solvents of varying polarities and subsequently depleted from the bulk constituents, chlorophylls and tannins to avoid possible interferences with the assays. The obtained 257 extracts were assessed for their in vitro anti-inflammatory activity. The expression of the inflammatory mediators E-selectin and interleukin-8 (IL-8), induced by the inflammatory stimuli tumor necrosis factor alpha (TNF-α) and the bacterial product lipopolysaccharide (LPS) was measured in endothelial cells. The potential of the extracts to activate the nuclear factors PPARα and PPARγ and to inhibit TNF-α-induced activation of the nuclear factor-kappa B (NF-κB) in HEK293 cells was determined by luciferase reporter gene assays. Results In total, extracts from 67 of the 71 assessed herbal drugs revealed anti-inflammatory activity in the applied in vitro test systems. Thereby, 30 could downregulate E-selectin or IL-8 gene expression, 28 were strong activators of PPARα or PPARγ (inducing activation of more than 2-fold at a concentration of 10 µg/mL) and 21 evoked a strong inhibition of NF-κB (inhibition of more than 80% at 10 µg/mL). Conclusion Our research supports the efficacy of herbal drugs reported in Austrian folk medicine used for ailments associated with inflammatory processes. Hence, an ethnopharmacological screening approach is a useful tool for the discovery of new drug leads.

Inhibition of 11β-hydroxysteroid dehydrogenase type 2 by dithiocarbamates

Dithiocarbamates (DTCs), important therapeutic and industrial chemicals released in high quantities into the environment, exhibit complex chemical and biological activities. Here, we demonstrate an effect of DTCs on glucocorticoid action due to inhibition of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) type 2, converting cortisol to cortisone in the kidney, but not 11 beta-HSD1, catalyzing the reverse reaction in liver and adipose tissue. Thus, DTCs may locally increase active glucocorticoid concentrations. Preincubation with the DTC thiram abolished 11 beta-HSD2 activity, suggesting irreversible enzyme inhibition. The sulfhydryl protecting reagent dithiothreitol blocked thiram-induced inhibition and NAD+ partially protected 11 beta-HSD2 activity, indicating that DTCs act at the cofactor-binding site. A 3D-model of 11 beta-HSD2 identified Cys90 in the NAD(+)-binding site as a likely target of DTCs, which was supported by a 99% reduced activity of mutant Cys90 to serine. The interference of DTCs with glucocorticoid-mediated responses suggests a cautious approach in the use of DTCs in therapeutic applications and in exposure to sources of DTCs such as cosmetics and agricultural products by pregnant women and others.

Organotins Disrupt the 11β-Hydroxysteroid Dehydrogenase Type 2-Dependent Local Inactivation of Glucocorticoids

Organotins, important environmental pollutants widely used in agricultural and industrial applications, accumulate in the food chain and induce imposex in several marine species as well as neurotoxic and immunotoxic effects in higher animals. Reduced birth weight and thymus involution, observed upon exposure to organotins, can also be caused by excessive glucocorticoid levels. We now demonstrate that organotins efficiently inhibit 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), converting active 11β-hydroxyglucocorticoids into inactive 11-ketoglucocorticoids, but not 11β-HSD1, which catalyzes the reverse reaction. Di- and tributyltin as well as di- and triphenyltin inhibited recombinant and endogenous 11β-HSD2 in lysates and intact cells with IC50 values between 500 nM and 3 μM. Dithiothreitol protected 11β-HSD2 from organotin-dependent inhibition, indicating that organotins act by binding to one or more cysteines. Mutational analysis and 3-D structural modeling revealed several important interactions of cysteines in 11β-HSD2. Cys90, Cys228, and Cys264 were essential for enzymatic stability and catalytic activity, suggesting that disruption of such interactions by organotins leads to inhibition of 11β-HSD2. Enhanced glucocorticoid concentrations due to disruption of 11β-HSD2 function may contribute to the observed organotin-dependent toxicity in some glucocorticoid-sensitive tissues such as thymus and placenta.

Why is 11β-hydroxysteroid dehydrogenase type 1 facing the endoplasmic reticulum lumen?: Physiological relevance of the membrane topology of 11β-HSD1

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is essential for the local activation of glucocorticoid receptors (GR). Unlike unliganded cytoplasmic GR, 11beta-HSD1 is an endoplasmic reticulum (ER)-membrane protein with lumenal orientation. Cortisone might gain direct access to 11beta-HSD1 by free diffusion across membranes, indirectly via intracellular binding proteins or, alternatively, by insertion into membranes. Membranous cortisol, formed by 11beta-HSD1 at the ER-lumenal side, might then activate cytoplasmic GR or bind to ER-lumenal secretory proteins. Compartmentalization of 11beta-HSD1 is important for its regulation by hexose-6-phosphate dehydrogenase (H6PDH), which regenerates cofactor NADPH in the ER lumen and stimulates oxoreductase activity. ER-lumenal orientation of 11beta-HSD1 is also essential for the metabolism of the alternative substrate 7-ketocholesterol (7KC), a major cholesterol oxidation product found in atherosclerotic plaques and taken up from processed cholesterol-rich food. An 11beta-HSD1 mutant adopting cytoplasmic orientation efficiently catalyzed the oxoreduction of cortisone but not 7KC, indicating access to cortisone from both sides of the ER-membrane but to 7KC only from the lumenal side. These aspects may be relevant for understanding the physiological role of 11beta-HSD1 and for developing therapeutic interventions to control glucocorticoid reactivation.

Activated AMPK boosts the Nrf2/HO-1 signaling axis—A role for the unfolded protein response

In light of the emerging interplay between redox and metabolic signaling pathways we investigated the potential cross talk between nuclear factor E2-related factor 2 (Nrf2) and AMP-activated kinase (AMPK), central regulators of the cellular redox and energy balance, respectively. Making use of xanthohumol (XN) as an activator of both the AMPK and the Nrf2 signaling pathway we show that AMPK exerts a positive influence on Nrf2/heme oxygenase (HO)-1 signaling in mouse embryonic fibroblasts. Genetic ablation and pharmacological inhibition of AMPK blunts Nrf2-dependent HO-1 expression by XN already at the mRNA level. XN leads to AMPK activation via interference with mitochondrial function and activation of liver kinase B1 as upstream AMPK kinase. The subsequent AMPK-mediated enhancement of the Nrf2/HO-1 response does not depend on inhibition of the mammalian target of rapamycin, inhibition of glycogen synthase kinase 3β, or altered abundance of Nrf2 (total and nuclear). However, reduced endoplasmic reticulum stress was identified and elaborated as a step in the AMPK-augmented Nrf2/HO-1 response. Overall, we shed more light on the hitherto incompletely understood cross talk between the LKB1/AMPK and the Nrf2/HO-1 axis revealing for the first time involvement of the unfolded protein response as an additional player and suggesting tight cooperation between signaling pathways controlling cellular redox, energy, or protein homeostasis.

Computer-Aided Discovery, Validation, and Mechanistic Characterization of Novel Neolignan Activators of Peroxisome Proliferator-Activated Receptor gamma

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are used for the treatment of type 2 diabetes and metabolic syndrome. However, the currently used PPARγ agonists display serious side effects, which has led to a great interest in the discovery of novel ligands with favorable properties. The aim of our study was to identify new PPARγ agonists by a PPARγ pharmacophore–based virtual screening of 3D natural product libraries. This in silico approach led to the identification of several neolignans predicted to bind the receptor ligand binding domain (LBD). To confirm this prediction, the neolignans dieugenol, tetrahydrodieugenol, and magnolol were isolated from the respective natural source or synthesized and subsequently tested for PPARγ receptor binding. The neolignans bound to the PPARγ LBD with EC50 values in the nanomolar range, exhibiting a binding pattern highly similar to the clinically used agonist pioglitazone. In intact cells, dieugenol and tetrahydrodieugenol selectively activated human PPARγ-mediated, but not human PPARα- or -β/δ-mediated luciferase reporter expression, with a pattern suggesting partial PPARγ agonism. The coactivator recruitment study also demonstrated partial agonism of the tested neolignans. Dieugenol, tetrahydrodieugenol, and magnolol but not the structurally related eugenol induced 3T3-L1 preadipocyte differentiation, confirming effectiveness in a cell model with endogenous PPARγ expression. In conclusion, we identified neolignans as novel ligands for PPARγ, which exhibited interesting activation profiles, recommending them as potential pharmaceutical leads or dietary supplements.

Honokiol: A non-adipogenic PPARγ agonist from nature☆

Background Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are clinically used to counteract hyperglycemia. However, so far experienced unwanted side effects, such as weight gain, promote the search for new PPARγ activators. Methods We used a combination of in silico, in vitro, cell-based and in vivo models to identify and validate natural products as promising leads for partial novel PPARγ agonists. Results The natural product honokiol from the traditional Chinese herbal drug Magnolia bark was in silico predicted to bind into the PPARγ ligand binding pocket as dimer. Honokiol indeed directly bound to purified PPARγ ligand-binding domain (LBD) and acted as partial agonist in a PPARγ-mediated luciferase reporter assay. Honokiol was then directly compared to the clinically used full agonist pioglitazone with regard to stimulation of glucose uptake in adipocytes as well as adipogenic differentiation in 3T3-L1 pre-adipocytes and mouse embryonic fibroblasts. While honokiol stimulated basal glucose uptake to a similar extent as pioglitazone, it did not induce adipogenesis in contrast to pioglitazone. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain. Conclusion We identified honokiol as a partial non-adipogenic PPARγ agonist in vitro which prevented hyperglycemia and weight gain in vivo. General significance This observed activity profile suggests honokiol as promising new pharmaceutical lead or dietary supplement to combat metabolic disease, and provides a molecular explanation for the use of Magnolia in traditional medicine.