Andreas Koeberle

Identification of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 as functional targets of the anti-inflammatory and anti-carcinogenic garcinol

Garcinol (camboginol) from the fruit rind of Guttiferae species shows anti-carcinogenic and anti-inflammatory properties, but the underlying molecular mechanisms are unclear. Here we show that garcinol potently interferes with 5-lipoxygenase (EC 7.13.11.34) and microsomal prostaglandin (PG)E2 synthase (mPGES)-1 (EC 5.3.99.3), enzymes that play pivotal roles in inflammation and tumorigenesis. In cell-free assays, garcinol inhibited the activity of purified 5-lipoxygenase and blocked the mPGES-1-mediated conversion of PGH2 to PGE2 with IC50 values of 0.1 and 0.3 microM, respectively. Garcinol suppressed 5-lipoxygenase product formation also in intact human neutrophils and reduced PGE2 formation in interleukin-1beta-stimulated A549 human lung carcinoma cells as well as in human whole blood stimulated by lipopolysaccharide. Moreover, garcinol interfered with isolated cyclooxygenase (COX)-1 (EC 1.14.99.1, IC50 = 12 microM) and with the formation of COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and thromboxane B2 in human platelets. In contrast, neither Ca2+-ionophore (A23187)-induced arachidonic acid release in neutrophils nor COX-2 activity in A549 cells or whole blood, measured as formation of 6-keto PGF1alpha, or isolated human recombinant COX-2 were significantly affected by garcinol (< or = 30 microM). Together, the high potency of garcinol to selectively suppress PGE2 synthesis and 5-lipoxygenase product formation provides a molecular basis for the anti-inflammatory and anti-carcinogenic effects of garcinol and rationalizes its therapeutic use.

Myrtucommulone, a natural acylphloroglucinol, inhibits microsomal prostaglandin E2 synthase‐1

Background and purpose:u2002 The selective inhibition of prostaglandin (PG)E2 formation via interference with microsomal PGE2 synthase (mPGES)‐1 could have advantages in the treatment of PGE2‐associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)‐1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from Myrtus communis L. (myrtle) affected mPGES‐1.

Inhibition of microsomal prostaglandin E2 synthase-1 as a molecular basis for the anti-inflammatory actions of boswellic acids from frankincense

BACKGROUND AND PURPOSE Frankincense, the gum resin derived from Boswellia species, showed anti‐inflammatory efficacy in animal models and in pilot clinical studies. Boswellic acids (BAs) are assumed to be responsible for these effects but their anti‐inflammatory efficacy in vivo and their molecular modes of action are incompletely understood.

Arzanol, a prenylated heterodimeric phloroglucinyl pyrone, inhibits eicosanoid biosynthesis and exhibits anti-inflammatory efficacy in vivo.

Based on its capacity to inhibit in vitro HIV-1 replication in T cells and the release of pro-inflammatory cytokines in monocytes, the prenylated heterodimeric phloroglucinyl α-pyrone arzanol was identified as the major anti-inflammatory and anti-viral constituent from Helichrysum italicum. We have now investigated the activity of arzanol on the biosynthesis of pro-inflammatory eicosanoids, evaluating its anti-inflammatory efficacy in vitro and in vivo. Arzanol inhibited 5-lipoxygenase (EC 7.13.11.34) activity and related leukotriene formation in neutrophils, as well as the activity of cyclooxygenase (COX)-1 (EC 1.14.99.1) and the formation of COX-2-derived prostaglandin (PG)E(2)in vitro (IC(50)=2.3-9μM). Detailed studies revealed that arzanol primarily inhibits microsomal PGE(2) synthase (mPGES)-1 (EC 5.3.99.3, IC(50)=0.4μM) rather than COX-2. In fact, arzanol could block COX-2/mPGES-1-mediated PGE(2) biosynthesis in lipopolysaccharide-stimulated human monocytes and human whole blood, but not the concomitant COX-2-derived biosynthesis of thromboxane B(2) or of 6-keto PGF(1α), and the expression of COX-2 or mPGES-1 protein was not affected. Arzanol potently suppressed the inflammatory response of the carrageenan-induced pleurisy in rats (3.6mg/kg, i.p.), with significantly reduced levels of PGE(2) in the pleural exudates. Taken together, our data show that arzanol potently inhibits the biosynthesis of pro-inflammatory lipid mediators like PGE(2)in vitro and in vivo, providing a mechanistic rationale for the anti-inflammatory activity of H. italicum, and a rationale for further pre-clinical evaluation of this novel anti-inflammatory lead.

Green tea epigallocatechin-3-gallate inhibits microsomal prostaglandin E2 synthase-1

Prostaglandin (PG)E(2) is a critical lipid mediator connecting chronic inflammation to cancer. The anti-carcinogenic epigallocatechin-3-gallate (EGCG) from green tea (Camellia sinensis) suppresses cellular PGE(2) biosynthesis, but the underlying molecular mechanisms are unclear. Here, we investigated the interference of EGCG with enzymes involved in PGE(2) biosynthesis, namely cytosolic phospholipase (cPL)A(2), cyclooxygenase (COX)-1 and -2, and microsomal prostaglandin E(2) synthase-1 (mPGES-1). EGCG failed to significantly inhibit isolated COX-2 and cPLA(2) up to 30 microM and moderately blocked isolated COX-1 (IC(50)>30 microM). However, EGCG efficiently inhibited the transformation of PGH(2) to PGE(2) catalyzed by mPGES-1 (IC(50)=1.8 microM). In lipopolysaccharide-stimulated human whole blood, EGCG significantly inhibited PGE(2) generation, whereas the concomitant synthesis of other prostanoids (i.e., 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-keto PGF(1alpha)) was not suppressed. Conclusively, mPGES-1 is a molecular target of EGCG, and inhibition of mPGES-1 is seemingly the predominant mechanism underlying suppression of cellular PGE(2) biosynthesis by EGCG.

Total Synthesis of Myrtucommulone A

reported that 1 is highly active against Gram-positive bacteria. Three years later Lounasmaa and co-workers isolated myrtucommulone A from other members of the myrtacea family. After that, interest in myrtle died down until 2002 when Appendino and co-workers re-examined extracts of this Mediterranean shrub and described additional myrtucommulones and their anti-oxidative properties. Shaheen et al. recently isolated the myrtucommulones C to E and other natural products from Myrtus communis. Quinn and coworkers examined extracts from Corymbia scabrida and could identify 1 and the myrtucommulones F to I. We became interested in the myrtucommulones when it was reported that these compounds show very significant antiinflammatory activity as well as highly selective apoptosisinducing activity. For detailed studies of the pharmacological activities of these compounds it seemed reasonable to develop a synthetic strategy leading to myrtucommulone A (1) and the other myrtucommulones. Here, we report on our total synthesis of myrtucommulone A (1), myrtucommulone F (13), myrtucommulone C (16), and three analogues thereof. Based on the constitutional symmetry of 1 the retrosynthetic disconnection shown in Scheme 1 seems reasonable. It should be possible to synthesize 1 from isobutyryl phloroglucinol (2), isobutyraldehyd (3), and syncarpic acid (4) in one step. Isobutyryl phloroglucinol (2) is readily available through Friedel–Crafts acylation of phloroglucinol (5) in 70–80% yield (Scheme 2). Syncarpic acid (4) is described in the

MK-886, an inhibitor of the 5-lipoxygenase-activating protein, inhibits cyclooxygenase-1 activity and suppresses platelet aggregation.

MK-886, an inhibitor of the 5-lipoxygenase-activating protein (FLAP), potently suppresses leukotriene biosynthesis in intact cells and is frequently used to define a role of the 5-lipoxygenase (EC 1.13.11.34) pathway in cellular or animal models of inflammation, allergy, cancer, and cardiovascular disease. Here we show that MK-886 also interferes with the activities of cyclooxygenases (COX, EC 1.14.99.1). MK-886 inhibited isolated COX-1 (IC(50)=8 microM) and blocked the formation of the COX-1-derived products 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid (12-HHT) and thromboxane B(2) in washed human platelets in response to collagen as well as from exogenous arachidonic acid (IC(50)=13-15 microM). Isolated COX-2 was less affected (IC(50)=58 microM), and in A549 cells, MK-886 (33 microM) failed to suppress COX-2-dependent 6-keto-prostaglandin (PG)F(1alpha) formation. The distinct susceptibility of MK-886 towards COX-1 and -2 is apparent in automated molecular docking studies that indicate a preferred binding of MK-886 to COX-1 into the active site. MK-886 (10 microM) inhibited COX-1-mediated platelet aggregation induced by collagen or arachidonic acid whereas thrombin- or U-46619-induced (COX-independent) aggregation was not affected. Since leukotrienes and prostaglandins share (patho)physiological properties in the development and regulation of carcinogenesis, inflammation, and vascular functions, caution should be used when interpreting data where MK-886 is used as tool to determine the involvement of FLAP and/or the 5-lipoxygenase pathway in respective experimental models.

CHAPTER 1:Microsomal Prostaglandin E2 Synthase-1

The prostanoids and leukotrienes (LTs) formed from arachidonic acid (AA) via the cyclooxygenase (COX)-1/2 and 5-lipoxygenase (5-LO) pathway, respectively, mediate inflammatory responses, chronic tissue remodelling, cancer, asthma and autoimmune disorders, but also possess homeostatic functions in the gastrointestinal tract, uterus, brain, kidney, vasculature and host defence. Based on the manifold functions of these eicosanoids, the clinical use of non-steroidal anti-inflammatory drugs (NSAIDs), a class of drugs that block formation of all prostanoids, is hampered by severe side-effects including gastrointestinal injury, renal irritations and cardiovascular risks. Therefore, anti-inflammatory agents interfering with eicosanoid biosynthesis require a well-balanced pharmacological profile to minimize these on-target side-effects. Current anti-inflammatory research aims at identifying compounds that can suppress the massive formation of pro-inflammatory prostaglandin (PG)E2 without affecting homeostatic PGE2 and PGI2 synthesis. The inducible microsomal prostaglandin E2 synthase-1 (mPGES-1) is one promising target enzyme. We will give an overview about the structure, regulation and function of mPGES-1 and then present novel inhibitors of mPGES-1 that may possess a promising pharmacological profile.

Raman spectroscopic profiling of intracellular lipid compositions of macrophages induced in the vicinity of cancer cells (Conference Presentation)

Lipidomics is a vast field of intracellular pathways of lipids and their biochemical functions. In analogy to genomics and proteomics it contributes to the overall comprehension of system biology. The field elucidates the role of lipids as a subset of the major biological components. Within this family of molecules, often referred to as metabolic lipidome, lipid mediators (LMs) are currently under detailed investigations. Being part of lipid signaling events, which are unique in a sense that they are produced “on demand” at the site of action, LMs fulfill important roles in receptor and enzyme regulated processes. Furthermore, LMs along with phospholipids (PL) are known to have pro- and anti-tumoral properties, and cancer cells exhibit aberrant LM and PL profiles. Typical cells that produce a broad variety of LMs are monocytes and macrophages, which also use these chemical mediators to influence the communication between monocytes and macrophages with cancer cells. As lipidomics research involves the identification and quantification of the thousands of cellular lipid molecular species and their interactions with other lipids, proteins, and other metabolites, comparably fast analytical techniques that detect the overall lipid composition of individual cells are highly advantageous. Several types of analytical methodologies are applied for characterization of the lipidome of cells. By far most commonly used is mass spectrometry in combination with separation techniques that can provide a profile of the variety of lipids present, as well as their identification. Similar information can be obtained utilizing NMR spectroscopy. Meanwhile also well established for profiling biological samples is Raman spectroscopy. As Raman micro-spectroscopy can be used to image individual cells and depict subcellular components based on their spectroscopic fingerprints, it appears as an ideal label-free technique to investigate intracellular alterations noninvasively. minute spectral changes, due to compositional alterations can be reproducibly detected. In this context Raman micro-spectroscopy has for instance been applied to typing of bacteria or the differentiation between cancerous and normal cells. Raman spectroscopy can provide an OMIC-like view of the chemical status of individual cells and metabolism and has been suggested for lipidomic profiling.(1,2) The obtained data sets of were subjected to common statistical data evaluation, such as hierarchical cluster (HCA) and principal component analysis (PCA), in order to relate spectroscopic alterations to the compositional changes associated with the presence of a cancerous environment. Here we present first results obtained from M1 and M2 macrophages cocultured in vitro with cancer cells in order to evaluate the potential of Raman spectroscopy for lipid profiling.nnAcknowledgements: nFinancial support from the Carl Zeiss Foundation is highly acknowledged. nnReferencesn1. Huang W, Spiers A. Consideration of Future Requirements for Raman Microbiology as an Examplar for the Ab Initio Development of Informatics Frameworks for Emergent OMICS Technologies OMICS: A Journal of Integrative Biology 2006;10:238-41.n2. Wu H, Volponi J, Oliver A, Parikh A, Simmons B, Singh S. In vivo lipidomics using single-cell Raman spectroscopy. Proc Natl Acad Sci USA 2011;108:3809-14.