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 target the human immune system-modulating antimicrobial peptide LL-37.

The antimicrobial peptide LL-37 is the sole member of the human cathelicidin family with immune system-modulating properties and roles in autoimmune disease development. Small molecules able to interact with LL-37 and to modulate its functions have not been described yet. Boswellic acids (BAs) are pentacyclic triterpene acids that are bioactive principles of frankincense extracts used as anti-inflammatory remedies. Although various anti-inflammatory modes of action have been proposed for BAs, the pharmacological profile of these compounds is still incompletely understood. Here, we describe the identification of human LL-37 as functional target of BAs. In unbiased target fishing experiments using immobilized BAs as bait and human neutrophils as target source, LL-37 was identified as binding partner assisted by MALDI-TOF mass spectrometry. Thermal stability experiments using circular dichroism spectroscopy confirm direct interaction between BAs and LL-37. Of interest, this binding of BAs resulted in an inhibition of the functionality of LL-37. Thus, the LPS-neutralizing properties of isolated LL-37 were inhibited by 3-O-acetyl-ß-BA (Aß-BA) and 3-O-acetyl-11-keto-ß-BA (AKß-BA) in a cell-free limulus amoebocyte lysate assay with EC50=0.2 and 0.8 µM, respectively. Also, LL-37 activity was inhibited by these BAs in LL-37-enriched supernatants of stimulated neutrophils or human plasma derived from stimulated human whole blood. Together, we reveal BAs as inhibitors of LL-37, which might be a relevant mechanism underlying the anti-inflammatory properties of BAs and suggests BAs as suitable chemical tools or potential agents for intervention with LL-37 and related disorders.

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.

The MAP kinase JNK-1 of Caenorhabditis elegans: location, activation, and influences over temperature-dependent insulin-like signaling, stress responses, and fitness.

The mitogen-activated protein kinase (MAPK) pathways and insulin-like signaling play pivotal roles in cellular stress response. Using an anti-phospho-SAPK/JNK antibody and a daf-16::GFP-based reporter assay, the present study shows in Caenorhabditis elegans that ambient temperature (1-37 degrees C) specifically influences the activation (phosphorylation) of the MAP kinase JNK-1 as well as the nuclear translocation of DAF-16, the main downstream target of insulin-like signaling. Activated JNK-1 was detected only in neuronal cells, and JNK-1 was found to be controlled by the MAPK JKK-1 under heat stress. Comparative analyses on the wildtype and a jnk-1 deletion mutant revealed a promoting influence of JNK-1 on both nuclear DAF-16 translocations and DAF-16 target gene (superoxide dismutase 3, sod-3) expressions within peripheral, non-neuronal tissue. Consequently, the mutant exhibited a reduced thermal tolerance and reproductive fitness at higher temperatures. These results provide evidence of indirect interactions between neuronal MAPK and peripheral insulin-like signaling in response to environmental stimuli (temperature, H2O2).

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.

Interference of alpha-alkyl-substituted pirinixic acid derivatives with neutrophil functions and signalling pathways.

Pirinixic acid (Wy-14,643) is an agonist of the peroxisome proliferator-activated receptor (PPAR) subtype alpha exhibiting beneficial effects in various inflammation-related processes in a slow, long-termed fashion. We recently showed that alpha-substituted pirinixic acid derivatives are agonists of PPAR alpha and act as dual inhibitors of 5-lipoxygenase (5-LO, EC 1.13.11.34) and the microsomal prostaglandin E(2) synthase-1 (EC 5.3.99.3). Here, we explored short-term effects of alpha-substituted pirinixic acid derivatives on typical neutrophil functions evoked by the agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP) including leukotriene formation, generation of reactive oxygen species, and release of human leukocyte elastase (EC 3.4.21.37), and we investigated the modulation of related signalling pathways. Pirinixic acid derivatives that are substituted with alkyl residues in alpha-position of the carboxylic group and with a 6-aminoquinoline residue at the pyrimidine moiety cause inhibition of leukotriene formation, reactive oxygen species formation, and leukocyte elastase release in response to fMLP. In parallel, Ca(2+) mobilisation and the phosphorylation (activation) of p38 mitogen-activated protein kinase was significantly reduced, whereas phosphorylation of the extracellular signal-regulated kinase-2 was unaffected. Pirinixic acid itself was not or only marginally active in all these assays. Conclusively, targeted structural modification of pirinixic acid leads to bioactive compounds that display immediate anti-inflammatory properties in human neutrophils with potential therapeutic value.