Elke H. Heiss

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

Chronic Treatment with Resveratrol Induces Redox Stress- and Ataxia Telangiectasia-mutated (ATM)-dependent Senescence in p53-positive Cancer Cells

The induction of senescence, an irreversible growth arrest, in cancer cells is regarded as a mean to halt tumor progression. The phytoalexin resveratrol (RV) is known to possess a variety of cancer-preventive, -therapeutic, and -chemosensitizing properties. We report here that chronic treatment with RV in a subapoptotic concentration induces senescence-like growth arrest in tumor cells. In contrast to the widely accepted antioxidant property of RV, we demonstrate that one causative stimulus for senescence induction by chronic RV is an increased level of reactive oxygen species (ROS). The ROS formed upon RV exposure include hydrogen peroxide and superoxide and originate largely from mitochondria. Consistently, co-incubation with the antioxidant N-acetyl cysteine interfered with RV-mediated reactivation of the senescence program. Molecular mediators on the way from increased ROS levels to the observed growth arrest include p38 MAPK, p53, and p21. Moreover, we provide evidence that RV-initiated replication stress, apparent by activation of the ataxia telangiectasia-mutated kinase pathway, is associated with increased ROS levels and senescence induction. This is the first report linking cell cycle effects with a pro-oxidant and pro-senescent effect of RV in cancer cells.

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.

NADPH oxidases 1 and 4 mediate cellular senescence induced by resveratrol in human endothelial cells.

Resveratrol is believed to be partially responsible for the French paradox--the low risk of cardiovascular disease despite a high-fat diet in the French population. Recently, resveratrol has also been discussed as a life-span booster in several organisms. Age-related diseases are associated on the cellular level with senescence. We, therefore, hypothesized that resveratrol is vasoprotective by counteracting endothelial cell senescence. Surprisingly, we observed that chronic treatment with resveratrol (10 microM) was prosenescent in primary human endothelial cells. Resveratrol induced elevated reactive oxygen species (ROS) levels that were associated with and causally linked to an accumulation of cells in the S phase of the cell cycle, as measured by flow cytometry. We further show that cell accumulation in S phase leads to increased ROS and finally senescence. Using an siRNA approach, we clearly identified two NADPH oxidases, Nox1 and Nox4, as major targets of resveratrol and primary sources of ROS that act upstream of the observed S-phase accumulation.

Active NF-E2-related Factor (Nrf2) Contributes to Keep Endothelial NO Synthase (eNOS) in the Coupled State ROLE OF REACTIVE OXYGEN SPECIES (ROS), eNOS, AND HEME OXYGENASE (HO-1) LEVELS

The aim of our study was to examine in detail the impact of NF-E2-related factor (Nrf2) activation on endothelial cell function with focus on redox homeostasis and the endothelial nitric oxide synthase (eNOS) system. We administered 2-cyano-3,12-dioxooleana-1,9-dien-28-oic imidazolide (CDDO-IM), a known activator of Nrf2, to primary human umbilical vein endothelial cells. Activation of Nrf2 by CDDO-IM increased the amount of bioavailable nitric oxide (NO), a major contributor to vascular homeostasis, in naive and stressed cells. Concomitantly, intracellular reactive oxygen species were dose-and time-dependently reduced. In apparent contrast to elevated NO levels, eNOS protein expression was transiently decreased in an Nrf2-dependent manner. Employing pharmacological inhibitors as well as a small interfering RNA approach, we identified de novo protein synthesis of heme oxygenase 1 (HO-1) and its enzymatic activity as cause for the observed reduction of eNOS. We hypothesize that under redox stress, when the availability of tetrahydrobiopterin, a pivotal stoichiometric cofactor for eNOS, is limited, activation of Nrf2 leads (a) to transient reduction of eNOS protein levels and (b) to an antioxidant defense in human umbilical vein endothelial cells. Both activities ensure that a stoichiometric ratio of eNOS and tetrahydrobiopterin is sustained and that the risk of eNOS uncoupling is reduced. Our study is the first to provide a causal link between Nrf2 activation and eNOS expression and NO levels, respectively.

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.

Effect of resveratrol on endothelial cell function: Molecular mechanisms

The polyphenolic natural product resveratrol (RV), best known for its occurrence in grape skin and red wine, is considered a candidate drug for prevention and treatment of cardiovascular diseases. This review aims to summarize the molecular effects of RV on endothelial cells, which line the inner walls of blood vessels and play a key role in the development of those diseases. We describe how RV enhances endothelial nitric oxide production, improves endothelial redox balance and inhibits endothelial activation in response to pro‐inflammatory and metabolic insults. Furthermore, we summarize effects of RV on endothelial senescence, apoptosis, endothelin‐1 release, and endothelial progenitor cell function. As many of RVs actions seem to be mediated by SIRT1, different mechanistic possibilities how RV may lead to SIRT1 activation are discussed.