Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G.

Age-related diseases, such as osteoarthritis, Alzheimer's disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging.

Boswellic acids from frankincense inhibit lipopolysaccharide functionality through direct molecular interference.

Lipophilic extracts of gum resins of Boswellia species (BSE) are used in folk medicine to treat various inflammatory disorders and infections. The molecular background of the beneficial pharmacological effects of such extracts is still unclear. Various boswellic acids (BAs) have been identified as abundant bioactive ingredients of BSE. Here we report the identification of defined BAs as direct inhibitors of lipopolysaccharide (LPS) functionality and LPS-induced cellular responses. In pull-down experiments, LPS could be precipitated using an immobilized BA, implying direct molecular interactions. Binding of BAs to LPS leads to an inhibition of LPS activity which was observed in vitro using a modified limulus amoebocyte lysate assay. Analysis of different BAs revealed clear structure-activity relationships with the classical ß-BA as most potent derivative (IC(50)=1.8 µM). In RAW264.7 cells, LPS-induced expression of inducible nitric oxide synthase (iNOS, EC 1.14.13.39) was selectively inhibited by those BAs that interfered with LPS activity. In contrast, interferon-γ-induced iNOS induction was not affected by BAs. We conclude that structurally defined BAs are LPS inhibiting agents and we suggest that ß-BA may contribute to the observed anti-inflammatory effects of BSE during infections by suppressing LPS activity.

Identification of human cathepsin G as a functional target of boswellic acids from the anti-inflammatory remedy frankincense.

Frankincense preparations, used in folk medicine to cure inflammatory diseases, showed anti-inflammatory effectiveness in animal models and clinical trials. Boswellic acids (BAs) constitute major pharmacological principles of frankincense, but their targets and the underlying molecular modes of action are still unclear. Using a BA-affinity Sepharose matrix, a 26-kDa protein was selectively precipitated from human neutrophils and identified as the lysosomal protease cathepsin G (catG) by mass spectrometry (MALDI-TOF) and by immunological analysis. In rigid automated molecular docking experiments BAs tightly bound to the active center of catG, occupying the same part of the binding site as the synthetic catG inhibitor JNJ-10311795 (2-[3-[methyl[1-(2-naphthoyl)piperidin-4-yl]amino]carbonyl)-2-naphthyl]-1-(1-naphthyl)-2-oxoethylphosphonic acid). BAs potently suppressed the proteolytic activity of catG (IC(50) of approximately 600 nM) in a competitive and reversible manner. Related serine proteases were significantly less sensitive against BAs (leukocyte elastase, chymotrypsin, proteinase-3) or not affected (tryptase, chymase). BAs inhibited chemoinvasion but not chemotaxis of challenged neutrophils, and they suppressed Ca(2+) mobilization in human platelets induced by isolated catG or by catG released from activated neutrophils. Finally, oral administration of defined frankincense extracts significantly reduced catG activities in human blood ex vivo vs placebo. In conclusion, we show that catG is a functional and pharmacologically relevant target of BAs, and interference with catG could explain some of the anti-inflammatory properties of frankincense.

Identification and functional analysis of cyclooxygenase-1 as a molecular target of boswellic acids.

Boswellic acids (BAs) are assumed as the anti-inflammatory principles of Boswellia species. Initially, it was found that BAs inhibit leukotriene biosynthesis and 5-lipoxygenase (EC number 1.13.11.34), whereas suppression of prostaglandin formation and inhibition of cyclooxygenases (COX, EC number 1.14.99.1) has been excluded. Recently, we demonstrated that BAs also interfere with platelet-type 12-lipoxygenase. Here, we show that BAs, preferably 3-O-acetyl-11-keto-beta-BA (AKBA), concentration-dependently inhibit COX-1 product formation in intact human platelets (IC(50)=6 microM) as well as the activity of isolated COX-1 enzyme in cell-free assays (IC(50)=32 microM). The inhibitory effect of AKBA is reversible, and increased levels of arachidonic acid (AA) as substrate for COX-1 impair the efficacy. COX-1 in platelet lysates or isolated COX-1 selectively bound to an affinity matrix composed of immobilized BAs linked via glutaric acid to sepharose and this binding was reversed by ibuprofen or AA. Automated molecular docking of BAs into X-ray structures of COX-1 yielded positive Chemscore values for BAs, indicating favorable binding to the active site of the enzyme. In contrast, COX-2 was less efficiently inhibited by BAs as compared to COX-1, and pull-down experiments as well as docking studies exclude strong affinities of BAs towards COX-2. In conclusion, BAs, in particular AKBA, directly interfere with COX-1 and may mediate their anti-inflammatory actions not only by suppression of lipoxygenases, but also by inhibiting cyclooxygenases, preferentially COX-1.

Boswellic acids stimulate arachidonic acid release and 12-lipoxygenase activity in human platelets independent of Ca2+ and differentially interact with platelet-type 12-lipoxygenase.

Boswellic acids inhibit the transformation of arachidonic acid to leukotrienes via 5-lipoxygenase but can also enhance the liberation of arachidonic acid in human leukocytes and platelets. Using human platelets, we explored the molecular mechanisms underlying the boswellic acid-induced release of arachidonic acid and the subsequent metabolism by platelet-type 12-li-poxygenase (p12-LO). Both beta-boswellic acid and 3-O-acetyl-11-keto-boswellic acid (AKBA) markedly enhanced the release of arachidonic acid via cytosolic phospholipase A2 (cPLA2), whereas for generation of 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], AKBA was less potent than beta-boswellic acid and was without effect at higher concentrations (> or =30 microM). In contrast to thrombin, beta-boswellic acid-induced release of ara-chidonic acid and formation of 12-H(P)ETE was more rapid and occurred in the absence of Ca2+. The Ca2+-independent release of arachidonic acid and 12-H(P)ETE production elicited by beta-boswellic acid was not affected by pharmacological inhibitors of signaling molecules relevant for agonist-induced arachidonic acid liberation and metabolism. It is noteworthy that in cell-free assays, beta-boswellic acid increased p12-LO catalysis approximately 2-fold in the absence but not in the presence of Ca2+, whereas AKBA inhibited p12-LO activity. No direct modulatory effects of boswellic acids on cPLA2 activity in cell-free assays were evident. Therefore, immobilized KBA (linked to Sepharose beads) selectively precipitated p12-LO from platelet lysates but failed to bind cPLA2. Taken together, we show that boswellic acids induce the release of arachidonic acid and the synthesis of 12-H(P)ETE in human platelets by unique Ca2+-independent routes, and we identified p12-LO as a selective molecular target of boswellic acids.