From Vietnamese plants to a biflavonoid that relieves inflammation by triggering the lipid mediator class switch to resolution.

Chronic inflammation results from excessive pro-inflammatory signaling and the failure to resolve the inflammatory reaction. Lipid mediators orchestrate both the initiation and resolution of inflammation. Switching from pro-inflammatory to pro-resolving lipid mediator biosynthesis is considered as efficient strategy to relieve chronic inflammation, though drug candidates exhibiting such features are unknown. Starting from a library of Vietnamese medical plant extracts, we identified isomers of the biflavanoid 8-methylsocotrin-4'-ol from Dracaena cambodiana, which limit inflammation by targeting 5-lipoxygenase and switching the lipid mediator profile from leukotrienes to specialized pro-resolving mediators (SPM). Elucidation of the absolute configurations of 8-methylsocotrin-4'-ol revealed the 2S,γS-isomer being most active, and molecular docking studies suggest that the compound binds to an allosteric site between the 5-lipoxygenase subdomains. We identified additional subordinate targets within lipid mediator biosynthesis, including microsomal prostaglandin E2 synthase-1. Leukotriene production is efficiently suppressed in activated human neutrophils, macrophages, and blood, while the induction of SPM biosynthesis is restricted to M2 macrophages. The shift from leukotrienes to SPM was also evident in mouse peritonitis in vivo and accompanied by a substantial decrease in immune cell infiltration. In summary, we disclose a promising drug candidate that combines potent 5-lipoxygenase inhibition with the favorable reprogramming of lipid mediator profiles.

Inhibitory Effects of Secondary Metabolites from the Lichen Stereocaulon evolutum on Protein Tyrosine Phosphatase 1B.

Protein tyrosine phosphatase 1B plays a significant role in type 2 diabetes mellitus and other diseases and is therefore considered a new drug target. Within this study, an acetone extract from the lichen Stereocaulon evolutum was identified to possess strong protein tyrosine phosphatase 1B inhibition in a cell-free assay (IC50 of 11.8 µg/mL). Fractionation of this bioactive extract led to the isolation of seven known molecules belonging to the depsidones and the related diphenylethers and one new natural product, i.e., 3-butyl-3,7-dihydroxy-5-methoxy-1(3H)-isobenzofurane. The isolated compounds were evaluated for their inhibition of protein tyrosine phosphatase 1B. Two depsidones, lobaric acid and norlobaric acid, and the diphenylether anhydrosakisacaulon A potently inhibited protein tyrosine phosphatase 1B with IC50 values of 12.9, 15.1, and 16.1 µM, respectively, which is in the range of the protein tyrosine phosphatase 1B inhibitory activity of the positive control ursolic acid (IC50 of 14.4 µM). Molecular simulations performed on the eight compounds showed that i) a contact between the molecule and the four main regions of the protein is required for inhibitory activity, ii) the relative rigidity of the depsidones lobaric acid and norlobaric acid and the reactivity related to hydrogen bond donors or acceptors, which interact with protein tyrosine phosphatase 1B key amino acids, are involved in the bioactivity on protein tyrosine phosphatase 1B, iii) the cycle opening observed for diphenylethers decreased the inhibition, except for anhydrosakisacaulon A where its double bond on C-8 offsets this loss of activity, iv) the function present at C-8 is a determinant for the inhibitory effect on protein tyrosine phosphatase 1B, and v) the more hydrogen bonds with Arg221 there are, the more anchorage is favored.

Finding New Molecular Targets of Familiar Natural Products Using In Silico Target Prediction.

Natural products comprise a rich reservoir for innovative drug leads and are a constant source of bioactive compounds. To find pharmacological targets for new or already known natural products using modern computer-aided methods is a current endeavor in drug discovery. Nature's treasures, however, could be used more effectively. Yet, reliable pipelines for the large-scale target prediction of natural products are still rare. We developed an in silico workflow consisting of four independent, stand-alone target prediction tools and evaluated its performance on dihydrochalcones (DHCs)-a well-known class of natural products. Thereby, we revealed four previously unreported protein targets for DHCs, namely 5-lipoxygenase, cyclooxygenase-1, 17ß-hydroxysteroid dehydrogenase 3, and aldo-keto reductase 1C3. Moreover, we provide a thorough strategy on how to perform computational target predictions and guidance on using the respective tools.

A New Diterpene and Anti-inflammatory Sesquiterpene Lactones from Sigesbeckia orientalis.

Sigesbeckia orientalis, more commonly referred to as Herba Sigesbeckiae or Xi Xian Cao in traditional Chinese medicine and hy thiêm in traditional Vietnamese medicine, is used in China and Vietnam to treat inflammatory diseases such as arthritis, rheumatism, and joint pain. In initial investigations, the dichloromethane extract from the aerial parts of S. orientalis showed distinct inhibitory effects on the release of interleukin-8 in human neutrophils. Therefore, the purpose of the present study was the phytochemical investigation of the bioactive dichloromethane extract and the in vitro analysis of the effects of the isolated compounds on interleukin-8, interleukin-1ß, tumor necrosis factor-α, and monocyte chemoattractant protein 1 release, and surface expression of adhesion molecules (CD11a, CD11b, and CD62L) in lipopolysaccharide-stimulated human neutrophils to identify the active principle(s). The separation of the bioactive dichloromethane extract using various chromatographic techniques led to the isolation of nine compounds. Their chemical structures were elucidated from nuclear magnetic resonance and mass spectrometry data. One diterpene, 17(13 → 14)-abeo-ent-3S*,13S*,16-trihydroxystrob-8(15)-ene, was identified as a new natural product. Three germacranolide sesquiterpene lactones inhibited interleukin-8 production with IC50 values between 1.6 and 6.3 µM, respectively, and tumor necrosis factor-α production with IC50 values between 0.9 and 3.3 µM, respectively. Furthermore, they significantly inhibited interleukin-1ß and monocyte chemoattractant protein 1 production and diminished the effects of lipopolysaccharide on the surface expression of the adhesion molecules CD11a, CD11b, and CD62L. These findings support the traditional use of S. orientalis in the treatment of inflammatory diseases.

Identification and characterization of plant-derived alkaloids, corydine and corydaline, as novel mu opioid receptor agonists.

Pain remains a key therapeutic area with intensive efforts directed toward finding effective and safer analgesics in light of the ongoing opioid crisis. Amongst the neurotransmitter systems involved in pain perception and modulation, the mu-opioid receptor (MOR), a G protein-coupled receptor, represents one of the most important targets for achieving effective pain relief. Most clinically used opioid analgesics are agonists to the MOR, but they can also cause severe side effects. Medicinal plants represent important sources of new drug candidates, with morphine and its semisynthetic analogues as well-known examples as analgesic drugs. In this study, combining in silico (pharmacophore-based virtual screening and docking) and pharmacological (in vitro binding and functional assays, and behavioral tests) approaches, we report on the discovery of two naturally occurring plant alkaloids, corydine and corydaline, as new MOR agonists that produce antinociceptive effects in mice after subcutaneous administration via a MOR-dependent mechanism. Furthermore, corydine and corydaline were identified as G protein-biased agonists to the MOR without inducing ß-arrestin2 recruitment upon receptor activation. Thus, these new scaffolds represent valuable starting points for future chemical optimization towards the development of novel opioid analgesics, which may exhibit improved therapeutic profiles.

Plumericin prevents intestinal inflammation and oxidative stress in vitro and in vivo.

Inflammatory bowel diseases (IBDs) are characterized by an inflammatory and oxidative stress condition in the intestinal tissue. In this study, we evaluated the effect of plumericin, one of the main bioactive components of Himatanthus sucuuba (Woodson) bark, on intestinal inflammation and oxidative stress, both in vitro and in vivo. The effect of plumericin (0.5-2 µM) in vitro was evaluated in rat intestinal epithelial cells (IEC-6) treated with lipopolysaccharides from E. coli (10 µg/mL) plus interferon-γ (10 U/mL). Moreover, a 2,4,6-dinitrobenzene sulfonic acid (DNBS)-induced colitis model was used to evaluate the anti-inflammatory and antioxidant activity of plumericin (3 mg/kg) in vivo. The results showed that plumericin significantly reduces intestinal inflammatory factors such as tumor necrosis factor-α, cyclooxygenase-2 and inducible nitric oxide synthase expression, and nitrotyrosine formation. Plumericin also inhibited nuclear factor-κB translocation, reactive oxygen species (ROS) release, and inflammasome activation. Moreover, plumericin activated the nuclear factor erythroid-derived 2 pathway in IEC-6. Using the DNBS-induced colitis model, a significant reduction in the weight loss and in the development of the macroscopic and histologic signs of colon injury, together with a reduced inflammatory and oxidative stress state, were observed in plumericin-treated mice. These results indicate that plumericin exerts a strong anti-inflammatory and antioxidant activity. Thus, it might be a candidate for the development of a new pharmacologic approach for IBDs treatment.

From bench to counter: Discovery and validation of a peony extract as tyrosinase inhibiting cosmeceutical.

The anti-melanogenic principle of peony (Paeonia officinalis subsp. officinalis) seeds was elucidated via activity-guided isolation. Resveratroloside (trans-resveratrol-4'-O-beta-d-glucopyranoside) was found to be the main metabolite of P. officinalis subsp. officinalis seeds and its tyrosinase inhibiting activity was confirmed via an enzymatic assay. Furthermore, the in vitro activity and the therapeutic window were studied employing the murine melanoma cell line B16F10. The results from the conducted stability assay and the high content of resveratroloside in the seeds (i.e. 10.4% dw) motivated us to push the extract forward to an in vivo tolerance assay. A clinical study with forty Caucasian participants proofed a good skin-tolerance with high moisture effect and reduction of pores.

Melodamide A from Melodorum fruticosum - Quantification using HPLC and one-step-isolation by centrifugal partition chromatography.

Melodamide A, a phenolic amide from the leaves of Melodorum fruticosum Lour., has previously shown pronounced anti-inflammatory activity. In order to rapidly isolate larger quantities for biological testing, a fast, one-step isolation method by centrifugal partition chromatography was developed within this study. Fractionation of the dichloromethane extract was performed with a two-phase solvent system consisting of n-hexane, ethyl acetate, methanol, and water (3:7:5:5, v/v), leading to the isolation of melodamide A with a purity of >90% and a yield of 6.7 w% within 32 min. The developed method can also be used in dual mode for the enrichment of further constituents like flavonoids or chalcones. In order to support the centrifugal partition chromatography method development, additionally, a high-performance liquid chromatography method was established and validated to determine quantities of melodamide A in plant material and crude extracts. Analysis of M. fruticosum leaves and a dichloromethane extract obtained from this plant material showed a total melodamide A content of 0.19 ± 0.008 and 8.9 ± 0.249 w%, respectively.

Ginkgolic Acid is a Multi-Target Inhibitor of Key Enzymes in Pro-Inflammatory Lipid Mediator Biosynthesis.

Introduction: Lipid mediators (LMs) comprise bioactive metabolites of polyunsaturated fatty acids, including pro-inflammatory prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs), as well as specialized pro-resolving mediators (SPMs). They are essentially biosynthesized via cyclooxygenase (COX) and lipoxygenase (LO) pathways in complex networks and regulate the progression as well as the resolution of inflammatory disorders including inflammation-triggered cancer. Ginkgolic acid (GA) is a phenolic acid contained in Ginkgo biloba L. with neuroprotective, antimicrobial, and antitumoral properties. Although LMs regulate microbial infections and tumor progression, whether GA affects LM biosynthesis is unknown and was investigated here in detail. Methods: Pharmacophore-based virtual screening was performed along with docking simulations. Activity assays were conducted for isolated human recombinant 5-LO, cytosolic phospholipase (PLA)2α, COX-2, and ovine COX-1. The activity of human mPGES-1 and thromboxane A2 synthase (TXAS) was determined in crude cellular fractions. Cellular LM formation was studied using human monocytes, neutrophils, platelets, and M1- and M2-like macrophages. LMs were identified after (ultra)high-performance liquid chromatography by UV detection or ESI-tandem mass spectrometry. Results: GA was identified as virtual hit in an mPGES-1 pharmacophore-based virtual screening. Cell-free assays revealed potent suppression of mPGES-1 activity (IC50 = 0.7 µM) that is fully reversible and essentially independent of the substrate concentration. Moreover, cell-free assays revealed COX-1 and TXAS as additional targets of GA with lower affinity (IC50 = 8.1 and 5.2 µM). Notably, 5-LO, the key enzyme in LT biosynthesis, was potently inhibited by GA (IC50 = 0.2 µM) in a reversible and substrate-independent manner. Docking simulations support the molecular interaction of GA with mPGES-1 and 5-LO and suggest concrete binding sites. Interestingly, interference of GA with mPGES-1, COX-1, TXAS, and 5-LO was evident also in intact cells with IC50 values of 2.1-3.8 µM; no radical scavenging or cytotoxic properties were obvious. Analysis of LM profiles from bacteria-stimulated human M1- and M2-like macrophages confirmed the multi-target features of GA and revealed LM redirection towards the formation of 12-/15-LO products including SPM. Conclusions: We reveal GA as potent multi-target inhibitor of key enzymes in the biosynthesis of pro-inflammatory LMs that contribute to the complex pharmacological and toxicological properties of GA.

Eupatoriopicrin Inhibits Pro-inflammatory Functions of Neutrophils via Suppression of IL-8 and TNF-alpha Production and p38 and ERK 1/2 MAP Kinases.

During chronic inflammation, neutrophils acting locally as effector cells not only activate antibacterial defense but also promote the inflammatory response. Interleukin 8 (IL-8), the main cytokine produced by activated neutrophils, positively correlates with the severity of respiratory tract diseases. By screening European plants traditionally used for treating respiratory tract diseases, we found that extracts of aerial parts of Eupatorium cannabinum inhibit IL-8 release from neutrophils. Using bioassay-guided fractionation, we identified five sesquiterpene lactones, eupatoriopicrin (1), 5'-deoxyeupatoriopicrin (2), hiyodorilactone A (3), 3-hydroxy-5'- O-acetyleupatoriopicrin = hiyodorilactone D (4), and hiyodorilactone B (5), that efficiently (IC50 < 1 µM) inhibited IL-8 and TNF-α release in lipopolysaccharide (LPS)-stimulated human neutrophils. Moreover, all these sesquiterpene lactones suppressed the adhesion of human neutrophils to an endothelial monolayer by downregulating the expression of the ß2 integrin CD11b/CD18 on the neutrophil surface. Furthermore, eupatoriopicrin efficiently suppressed LPS-induced phosphorylation of p38 MAPK and ERK and attenuated neutrophil infiltration in the thioglycolate-induced peritonitis model in mice. Altogether, these results demonstrate the potential of the sesquiterpene lactone eupatoriopicrin as a lead substance for targeting inflammation.

Mushroom Tyrosinase-Based Enzyme Inhibition Assays Are Not Suitable for Bioactivity-Guided Fractionation of Extracts.

Tyrosinase (Tyr) catalyzes the rate-limiting step of melanogenesis in human skin and is thus the main target for treating pigmentation disorders today. This has led to an increased research interest in Tyr inhibitors during the last decades, with a frequent focus on polyphenols. In the early stages of drug discovery, it is typical to avoid the high costs of human Tyr by using the more economic mushroom tyrosinase (mh-Tyr). Since some polyphenols are accepted as substrates by mh-Tyr, the present study aimed to more generally investigate this enzyme's specificity toward polyphenols and to discuss its significance in the context of bioactivity-guided fractionation. Mh-Tyr substrates can change the sample color during an inhibition assay, leading to unreliable inhibition constants or to the discontinuation of a bioactivity-guided fractionation campaign. A data set of 56 natural products was investigated and classified into assay interferers (AIs) and noninterferers, using a spectrophotometric and an LC-ESIHRMS assay. Based on these experimental findings, structure-activity relationships defining AIs were deduced and implemented into an in silico tool that will allow for rapid prescreening in the future. We anticipate that these results will aid in the search for new Tyr inhibitors and contribute to the understanding of this enzyme, as well as its optimal use in pharmacological research.

HPTLC Autography Based Screening and Isolation of Mushroom Tyrosinase Inhibitors of European Plant Species.

In the course of this project, 133 plants were evaluated on their ability to inhibit tyrosinase, a key enzyme in melanogenesis. The screening was performed by means of a HPTLC autographic assay, resulting in the selection of three plants, Asplenium trichomanes, Pinus uncinata, and Scutellaria altissima, with promising tyrosinase inhibiting activities. With the aid of the HPTLC assay, it was not only possible to select the most interesting plant extracts, but also to monitor the activity-guided fractionation which, in a relatively short time period, led to the isolation of active principles. Benzoic acid, roseoside, and dihydrovomifoliol-O-ß-d-glucopyranoside could be identified as tyrosinase inhibitors present in P. uncinata. Globularin turned out to be the active principle of S. altissima, and 4-ethenylphenyl 6-O-(6-deoxy-α-l-mannopyranosyl)-ß-d-glucopyranoside was detected as tyrosinase inhibitor of A. trichomanes. The pure compounds were tested also in a 96 well-plate assay in order to determine their IC50 values. The lowest IC50 value (42 µm) could be obtained for globularin, whereas the other compounds, e. g., benzoic acid exhibited a rather high IC50 value (IC50 =552 µm). This stood in clear contrast to the autographic assay, but is has to be taken into account that the outcome of the autography assay is not only depending on the IC50 value of a compound, but also on the content of the respective constituent in the extract.

Inhibition of Pro-Inflammatory Functions of Human Neutrophils by Constituents of Melodorum fruticosum Leaves.

In an initial screening, the dichloromethane extract from the leaves of Melodorum fruticosum showed distinct inhibitory effects on the release of interleukin-8 (IL-8) in human neutrophils. Therefore, the aim of the present study was the phytochemical and pharmacological investigation of this extract, to better understand which compounds might be responsible for the anti-inflammatory effect. Phytochemical analysis led to the isolation of 12 known compounds and two new natural products, 5-hydroxy-6-(2-hydroxybenzyl)-4',7-dimethoxyflavanone (13) and 2',4'-dihydroxy-3'-(2-hydroxybenzyl)-4,6'-dimethoxychalcone (14). The influence of the isolated compounds on the production and release of the pro-inflammatory factors IL-8, tumor necrosis factor alpha (TNF-α), reactive oxygen species (ROS), and adhesion molecules (CD62L and CD11b) in human neutrophils was evaluated. Three constituents, melodamide A, 2',4'-dihydroxy-4,6'-dimethoxychalcone, and 2',6'-dihydroxy-4'-methoxychalcone, showed significant inhibition of IL-8 release (IC50 =6.6, 8.6, and 11.6 µm, respectively) and TNF-α production (IC50 =4.5, 13.3, and 6.2 µm, respectively).

Endogenous metabolites of vitamin E limit inflammation by targeting 5-lipoxygenase.

Systemic vitamin E metabolites have been proposed as signaling molecules, but their physiological role is unknown. Here we show, by library screening of potential human vitamin E metabolites, that long-chain ω-carboxylates are potent allosteric inhibitors of 5-lipoxygenase, a key enzyme in the biosynthesis of chemoattractant and vasoactive leukotrienes. 13-((2R)-6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-2,6,10-trimethyltridecanoic acid (α-T-13'-COOH) can be synthesized from α-tocopherol in a human liver-on-chip, and is detected in human and mouse plasma at concentrations (8-49 nM) that inhibit 5-lipoxygenase in human leukocytes. α-T-13'-COOH accumulates in immune cells and inflamed murine exudates, selectively inhibits the biosynthesis of 5-lipoxygenase-derived lipid mediators in vitro and in vivo, and efficiently suppresses inflammation and bronchial hyper-reactivity in mouse models of peritonitis and asthma. Together, our data suggest that the immune regulatory and anti-inflammatory functions of α-tocopherol depend on its endogenous metabolite α-T-13'-COOH, potentially through inhibiting 5-lipoxygenase in immune cells.

Novel Natural Products for Healthy Ageing from the Mediterranean Diet and Food Plants of Other Global Sources-The MediHealth Project.

There is a rapid increase in the percentage of elderly people in Europe. Consequently, the prevalence of age-related diseases will also significantly increase. Therefore, the main goal of MediHealth, an international research project, is to introduce a novel approach for the discovery of active agents of food plants from the Mediterranean diet and other global sources that promote healthy ageing. To achieve this goal, a series of plants from the Mediterranean diet and food plants from other origins are carefully selected and subjected to in silico, cell-based, in vivo (fly and mouse models), and metabolism analyses. Advanced analytical techniques complement the bio-evaluation process for the efficient isolation and identification of the bioactive plant constituents. Furthermore, pharmacological profiling of bioactive natural products, as well as the identification and synthesis of their metabolites, is carried out. Finally, optimization studies are performed in order to proceed to the development of innovative nutraceuticals, dietary supplements or herbal medicinal products. The project is based on an exchange of researchers between nine universities and four companies from European and non-European countries, exploiting the existing complementary multidisciplinary expertise. Herein, the unique and novel approach of this interdisciplinary project is presented.

Vascular smooth muscle cell proliferation as a therapeutic target. Part 2: Natural products inhibiting proliferation.

Many natural products have been so far tested regarding their potency to inhibit vascular smooth muscle cell proliferation, a process involved in atherosclerosis, pulmonary hypertension and restenosis. Compounds studied in vitro and in vivo as VSMC proliferation inhibitors include, for example indirubin-3'-monoxime, resveratrol, hyperoside, plumericin, pelargonidin, zerumbone and apamin. Moreover, taxol and rapamycin, the most prominent compounds applied in drug-eluting stents to counteract restenosis, are natural products. Numerous studies show that natural products have proven to yield effective inhibitors of vascular smooth muscle cell proliferation and ongoing research effort might result in the discovery of further clinically relevant compounds.

Vascular smooth muscle cell proliferation as a therapeutic target. Part 1: molecular targets and pathways.

Cardiovascular diseases are a major cause of human death worldwide. Excessive proliferation of vascular smooth muscle cells contributes to the etiology of such diseases, including atherosclerosis, restenosis, and pulmonary hypertension. The control of vascular cell proliferation is complex and encompasses interactions of many regulatory molecules and signaling pathways. Herein, we recapitulated the importance of signaling cascades relevant for the regulation of vascular cell proliferation. Detailed understanding of the mechanism underlying this process is essential for the identification of new lead compounds (e.g., natural products) for vascular therapies.

Identification of the NADPH Oxidase 4 Inhibiting Principle of Lycopus europaeus.

NADPH oxidase 4 (Nox4) has recently been implicated as driving force in cellular senescence. Thus, there is growing interest to develop Nox4 inhibitors, which might be valuable agents for cosmeceutical applications. Alpine plants represent a valuable source for the identification of novel bioactive natural products with anti-ageing effects, especially substances that protect plants against UV radiation, which is also known to contribute to the ageing of human skin. Therefore, the aim of this study was to identify novel Nox4 inhibitors from alpine plants. Within an initial screening of extracts of alpine plants on their ability to inhibit Nox4 activity in HEK cells, the methanolic extract of the subaerial parts of Lycopus europaeus showed a strong inhibition of Nox4 (81% chemiluminescence quenching) and a simultaneously high cell viability (91% vitality). Rosmarinic acid was isolated and identified as the major compound in this bioactive extract. It showed a dose dependent inhibitory activity on Nox4 with an IC50 of 1 µM. Moreover, it also showed a significant inhibitory activity on Nox2 in the low micromolar range, whereas no inhibition of Nox5 was detected. Further investigations confirmed that the observed effects of rosmarinic acid on Nox2 and Nox4 are real inhibitory activities, and not due to ROS scavenging effects. Therefore, L. europaeus, which we demonstrated to be a good source of rosmarinic acid, has great potential for usage in cosmeceutical products with anti-ageing activity.

Constituents of Mediterranean Spices Counteracting Vascular Smooth Muscle Cell Proliferation: Identification and Characterization of Rosmarinic Acid Methyl Ester as a Novel Inhibitor.

SCOPE: Aberrant vascular smooth muscle cell (VSMC) proliferation is involved in atherosclerotic plaque formation and restenosis. Mediterranean spices have been reported to confer cardioprotection, but their direct influence on VSMCs has largely not been investigated. This study aims at examining rosmarinic acid (RA) and 11 related constituents for inhibition of VSMC proliferation in vitro, and at characterizing the most promising compound for their mode of action and influence on neointima formation in vivo. METHODS AND RESULTS: RA, rosmarinic acid methyl ester (RAME), and caffeic acid methyl ester inhibit VSMC proliferation in a resazurin conversion assay with IC50 s of 5.79, 3.12, and 6.78 µm, respectively. RAME significantly reduced neointima formation in vivo in a mouse femoral artery cuff model. Accordingly, RAME leads to an accumulation of VSMCs in the G0 /G1 cell-cycle phase, as indicated by blunted retinoblastoma protein phosphorylation upon mitogen stimulation and inhibition of cyclin-dependent kinase 2 in vitro. CONCLUSION: RAME represses PDGF-induced VSMC proliferation in vitro and reduces neointima formation in vivo. These results recommend RAME as an interesting compound with VSMC-inhibiting potential. Future metabolism and pharmacokinetics studies might help to further evaluate the potential relevance of RAME and other spice-derived polyphenolics for vasoprotection.

A combinatorial approach for the discovery of cytochrome P450 2D6 inhibitors from nature.

The human cytochrome P450 2D6 (CYP2D6) enzyme is part of phase-I metabolism and metabolizes at least 20% of all clinically relevant drugs. Therefore, it is an important target for drug-drug interaction (DDI) studies. High-throughput screening (HTS) assays are commonly used tools to examine DDI, but show certain drawbacks with regard to their applicability to natural products. We propose an in silico - in vitro workflow for the reliable identification of natural products with CYP2D6 inhibitory potential. In order to identify candidates from natural product-based databases that share similar structural features with established inhibitors, a pharmacophore model was applied. The virtual hits were tested for the inhibition of recombinant human CYP2D6 in a bioluminescence-based assay. By controlling for unspecific interferences of the test compounds with the detection reaction, the number of false positives were reduced. The success rate of the reported workflow was 76%, as most of the candidates identified in the in silico approach were able to inhibit CYP2D6 activity. In summary, the workflow presented here is a suitable and cost-efficient strategy for the discovery of new CYP2D6 inhibitors with natural product libraries.