The anti-inflammatory drug licofelone [ϭML3000; 2-[6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] acetic acid], currently undergoing phase III trials for osteoarthritis, inhibits the prostaglandin (PG) and leukotriene biosynthetic pathway. Licofelone was reported to suppress the formation of PGE 2 in various cell-based test systems, but the underlying molecular mechanisms are not entirely clear. Here, we examined the direct interference of licofelone with enzymes participating in PGE 2 biosynthesis, that is, cyclooxygenase (COX)-1 and COX-2 as well as microsomal PGE 2 synthase (mPGES)-1. Licofelone concentration-dependently inhibited isolated COX-1 (IC 50 ϭ 0.8 M), whereas isolated COX-2 was less affected (IC 50 Ͼ 30 M). However, licofelone efficiently blocked the conversion of PGH 2 to PGE 2 mediated by mPGES-1 (IC 50 ϭ 6 M) derived from microsomes of interleukin-1-treated A549 cells, being about equipotent to 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid (MK-886), a well recognized mPGES-1 inhibitor. In intact interleukin-1-treated A549 cells, licofelone potently (IC 50 Ͻ 1 M) blocked formation of PGE 2 in response to calcimycin (A23187) plus exogenous arachidonic acid, but the concomitant generation of 6-keto PGF 1␣ , used as a biomarker for COX-2 activity, was not inhibited. We conclude that licofelone suppresses inflammatory PGE 2 formation preferentially by inhibiting mPGES-1 at concentrations that do not affect COX-2, implying an attractive and thus far unique molecular pharmacological dynamics as inhibitor of COX-1, the 5-lipoxygenase pathway, and of mPGES-1.Prostaglandins (PGs) and leukotrienes are powerful bioactive lipid mediators that are involved not only in numerous homeostatic biological functions but also in inflammation (Funk, 2001). The biosynthesis of PGs is initialized by COX isoenzymes, namely, COX-1, a constitutively expressed enzyme in numerous cell types thought to provide PGs mainly for physiological functions; and COX-2, an inducible isoform in inflammatory cells, primarily producing PGs relevant for inflammation, fever, and pain (Hawkey, 1999). After conversion of arachidonic acid to PGH 2 by COX enzymes, PGH 2 is subsequently isomerized by three different PGE 2 synthases to PGE 2 . Whereas the cytosolic PGE 2 synthase (cPGES) and the membrane-bound PGE 2 synthase (mPGES)-2 are constitutive enzymes, the mPGES-1 is an inducible isoform (Samuelsson et al., 2007). Cotransfection experiments of COX-1/2 with PGES isoenzymes imply that select molecular interactions between COX and PGES isoenzymes cause preferential functional coupling (Murakami et al., 2000;Samuelsson et al., 2007). Thus, cPGES uses PGH 2 produced by COX-1, whereas mPGES-1 receives PGH 2 from COX-2. PGE 2 plays a major role in the pathophysiology of inflammation, pain, and pyresis, but it also regulates physiological functions in the gastrointestinal tract, the kidney, and in the immune and nervous system (Smith, 1989). The nonsteroidal anti-inflamm...
BACKGROUND AND PURPOSEFrankincense, 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. EXPERIMENTAL APPROACHA protein fishing approach using immobilized BA and surface plasmon resonance (SPR) spectroscopy were used to reveal microsomal prostaglandin E2 synthase-1 (mPGES1) as a BA-interacting protein. Cell-free and cell-based assays were applied to confirm the functional interference of BAs with mPGES1. Carrageenan-induced mouse paw oedema and rat pleurisy models were utilized to demonstrate the efficacy of defined BAs in vivo. KEY RESULTSHuman mPGES1 from A549 cells or in vitro-translated human enzyme selectively bound to BA affinity matrices and SPR spectroscopy confirmed these interactions. BAs reversibly suppressed the transformation of prostaglandin (PG)H2 to PGE2 mediated by mPGES1 (IC50 = 3-10 mM). Also, in intact A549 cells, BAs selectively inhibited PGE2 generation and, in human whole blood, b-BA reduced lipopolysaccharide-induced PGE2 biosynthesis without affecting formation of the COX-derived metabolites 6-keto PGF1a and thromboxane B2. Intraperitoneal or oral administration of b-BA (1 mg·kg -1 ) suppressed rat pleurisy, accompanied by impaired levels of PGE2 and b-BA (1 mg·kg -1 , given i.p.) also reduced mouse paw oedema, both induced by carrageenan. CONCLUSIONS AND IMPLICATIONSSuppression of PGE2 formation by BAs via interference with mPGES1 contribute to the anti-inflammatory effectiveness of BAs and of frankincense, and may constitute a biochemical basis for their anti-inflammatory properties.
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 (IC50 of ∼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 Ca2+ 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.
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