Identification of Human Cathepsin G As a Functional Target of Boswellic Acids from the Anti-Inflammatory Remedy Frankincense

Abstract

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.

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

“…Pull-down assays using immobilized BAs were previously applied to demonstrate a direct interference of BAs with COX-1, 12S-LOX and cathepsin G (Poeckel et al, 2006b;Siemoneit et al, 2008;Tausch et al, 2009), supporting the suitability of this methodology for target identification. The SPR-based ligand-analyte studies revealed KD values of 5.2-23 mM for 3-Ooxaloyl-KBA, and these values fit the IC50 (5 mM) of 3-O-oxaloyl-KBA in the mPGES1 cell-free assay, suggesting specific binding to mPGES1 and hence a direct relation between enzyme binding and interference with mPGES1 activity.…”

“…Therefore, we conclude that impaired PGE2 biosynthesis is the result of the selective inhibition of the transformation of PGH2 to PGE2 by interference of BAs with mPGES1, rather than with COX-2 or other distal events (such as AA release). In particular b-BA, the major BA present in frankincense that reaches the highest plasma levels (up to 10.1 mM) among the BAs in treated humans (Buchele and Simmet, 2003;Tausch et al, 2009), suppressed PGE2 formation in human whole blood, again without significant reduction of 6-keto PGF1a or TXB2 levels. Interference with COX-1 was excluded as 12-HHT formation in whole blood was unaffected by b-BA, although BAs (in particular AKBA and KBA) may inhibit COX-1 in cell-free and cell-based models at higher concentrations .…”

“…However, compared with indomethacin, b-BA was less potent in reducing PGE2 levels, but still efficiently suppressed exudate formation and infiltration of inflammatory cells. It is possible that other antiinflammatory features of b-BA, such as inhibition of cathepsin G (Tausch et al, 2009), may contribute to the overall anti-inflammatory effects. The inhibition of cathepsin G may also explain the slight but still significant reduction of oedema formation and cell infiltration upon pretreatment with Ab-BA, which was the least potent inhibitor of mPGES1 and failed to suppress PGE2 formation in whole blood.…”

“…Most studies have focused on AKBA or KBA as the pharmacologically active principles and numerous targets (5-LOX, 12S-LOX, COX-1, HLE, IkB kinases and topoisomerases) have been proposed, but the interference with these targets in vivo has been largely neglected and thus, the pharmacological relevance is still unclear. Recently, we presented the identification of cathepsin G as a pharmacologically relevant target of all major BAs (Tausch et al, 2009). PGE2 is a key player in inflammation and pain, and mPGES1 is regarded as a potential target for the development of antiinflammatory therapeutics (Samuelsson et al, 2007).…”

“…However, in vivo studies confirming the pharmacological relevance of these proposed target interactions are still missing, and the efficacy of defined BAs in in vivo models of inflammation remains to be assessed. Recently, we showed that the serine protease cathepsin G is a high affinity and pharmacologically relevant target of BAs (Tausch et al, 2009).…”