Johann Jauch

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 (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.

A new Bacillus megaterium whole-cell catalyst for the hydroxylation of the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) based on a recombinant cytochrome P450 system

The use of cytochromes P450 for the regio- and stereoselective hydroxylation of non-activated carbon atoms in biotechnological applications reflects an efficient and cost-effective alternative in comparison to classical organic chemistry. The prokaryotic cytochrome P450 CYP106A2 from Bacillus megaterium ATCC 13368 hydroxylates a variety of 3-oxo-Δ4 steroids and recently it was identified to carry out a one-step regioselective allylic hydroxylation of the diterpene abietic acid. The anti-inflammatory pentacyclic triterpene 11-Keto-β-boswellic acid (KBA) was found to be a further substrate of CYP106A2, being the first report of a pentacyclic triterpene conversion by a prokaryotic P450. The reaction products were analyzed by HPLC and the corresponding kinetic parameters were investigated. Structure determination of the main product by NMR revealed a 15α-hydroxylation of this substrate. In order to overcome the inability of a recombinant P450 whole-cell system in E. coli for the uptake of acids with terpene structure, we developed for the first time an expression system for cytochromes P450 in B. megaterium (strains MS941 and ATCC 13368). Interestingly, CYP106A2 was only successfully expressed in the plasmid-less B. megaterium strain MS941 but not in ATCC13368. This recombinant system, with the co-expressed heterologous redox chain of the P450, bovine adrenodoxin reductase (AdR), and bovine adrenodoxin (Adx), was applied for the whole-cell conversion of KBA. The formation of 15α-hydroxy-KBA was increased 15-fold in comparison with the naturally CYP106A2-expressing B. megaterium strain ATCC 13368.

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 β-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 β-boswellic acid and was without effect at higher concentrations (≥30 μM). In contrast to thrombin, β-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 β-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, β-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.

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

Are Free Radicals Involved in IspH Catalysis? An EPR and Crystallographic Investigation

The [4Fe-4S] protein IspH in the methylerythritol phosphate isoprenoid biosynthesis pathway is an important anti-infective drug target, but its mechanism of action is still the subject of debate. Here, by using electron paramagnetic resonance (EPR) spectroscopy and (2)H, (17)O, and (57)Fe isotopic labeling, we have characterized and assigned two key reaction intermediates in IspH catalysis. The results are consistent with the bioorganometallic mechanism proposed earlier, and the mechanism is proposed to have similarities to that of ferredoxin, thioredoxin reductase, in that one electron is transferred to the [4Fe-4S](2+) cluster, which then performs a formal two-electron reduction of its substrate, generating an oxidized high potential iron-sulfur protein (HiPIP)-like intermediate. The two paramagnetic reaction intermediates observed correspond to the two intermediates proposed in the bioorganometallic mechanism: the early π-complex in which the substrates 3-CH(2)OH group has rotated away from the reduced iron-sulfur cluster, and the next, η(3)-allyl complex formed after dehydroxylation. No free radical intermediates are observed, and the two paramagnetic intermediates observed do not fit in a Birch reduction-like or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy and X-ray crystallography that two substrate analogues (4 and 5) follow the same reaction mechanism.

On the interference of boswellic acids with 5-lipoxygenase: Mechanistic studies in vitro and pharmacological relevance

Boswellic acids are pharmacologically active ingredients of frankincense with anti-inflammatory properties. It was shown that in vitro 11-keto-boswellic acids inhibit 5-lipoxygenase (5-LO, EC 1.13.11.34), the key enzyme in leukotriene biosynthesis, which may account for their anti-inflammatory effectiveness. However, whether 11-keto-boswellic acids interfere with 5-LO under physiologically relevant conditions (i.e., in whole blood assays) and whether they inhibit 5-LO in vivo is unknown. Inhibition of human 5-LO by the major naturally occurring boswellic acids was analyzed in cell-free and cell-based activity assays. Moreover, interference of boswellic acids with 5-LO in neutrophil incubations in the presence of albumin and in human whole blood was assessed, and plasma leukotriene B(4) of frankincense-treated healthy volunteers was determined. Factors influencing 5-LO activity (i.e., Ca(2+), phospholipids, substrate concentration) significantly modulate the potency of 11-keto-boswellic acids to inhibit 5-LO. Moreover, 11-keto-boswellic acids efficiently suppressed 5-LO product formation in isolated neutrophils (IC(50)=2.8 to 8.8 muM) but failed to inhibit 5-LO product formation in human whole blood. In the presence of albumin (10 mg/ml), 5-LO inhibition by 11-keto-boswellic acids (up to 30 muM) in neutrophils was abolished, apparently due to strong albumin-binding (>95%) of 11-keto-boswellic acids. Finally, single dose (800 mg) oral administration of frankincense extracts to human healthy volunteers failed to suppress leukotriene B(4) plasma levels. Our data show that boswellic acids are direct 5-LO inhibitors that efficiently suppress 5-LO product synthesis in common in vitro test models, however, the pharmacological relevance of such interference in vivo seems questionable.

A Thin‐layer Chromatography Method for the Identification of Three Different Olibanum Resins (Boswellia serrata, Boswellia papyrifera and Boswellia carterii, respectively, Boswellia sacra)

INTRODUCTION Resins of the genus Boswellia are currently an interesting topic for pharmaceutical research since several pharmacological activities (e.g. anti-inflammatory, anti-microbial, anti-tumour) are reported for extracts and compounds isolated from them. Unambiguous identification of these resins, by simple and convenient analytical methods, has so far not clearly been verified. OBJECTIVE For differentiation and identification of three important Boswellia species (Boswellia serrata Roxb., Boswellia papyrifera Hochst. and Boswellia carterii Birdw., respectively Boswellia sacra Flueck.), possible even for minimally equipped laboratories, a thin-layer chromatography (TLC) method was developed, allowing unambiguous identification of the three species. METHODOLOGY Crude resin samples (commercial samples and a voucher specimen) were extracted with methanol or diethyl ether and subjected to TLC analysis (normal phase). A pentane and diethyl ether (2:1) with 1% acetic acid eluent was used. Chromatograms were analysed by UV detection (254 nm) and dyeing with anisaldehyde dyeing reagent. Significant spots were isolated and structures were assigned (mass spectrometry; nuclear magnetic resonance spectroscopy). RESULTS Incensole and incensole acetate are specific biomarkers for Boswellia papyrifera. Boswellia carterii/Boswellia sacra reveal ß-caryophyllene oxide as a significant marker compound. Boswellia serrata shows neither incensole acetate nor ß-caryophyllene oxide spots, but can be identified by a strong serratol and a sharp 3-oxo-8,24-dien-tirucallic acid spot. CONCLUSION The TLC method developed allows unambiguous identification of three different olibanum samples (Boswellia papyrifera, Boswellia serrata, Boswellia carterii/Boswellia sacra). Evidence on the specific biosynthesis routes of these Boswellia species is reported.

Tetra- and pentacyclic triterpene acids from the ancient anti-inflammatory remedy frankincense as inhibitors of microsomal prostaglandin E(2) synthase-1.

The microsomal prostaglandin E2 synthase (mPGES)-1 is the terminal enzyme in the biosynthesis of prostaglandin (PG)E2 from cyclooxygenase (COX)-derived PGH2. We previously found that mPGES-1 is inhibited by boswellic acids (IC50 = 3–30 μM), which are bioactive triterpene acids present in the anti-inflammatory remedy frankincense. Here we show that besides boswellic acids, additional known triterpene acids (i.e., tircuallic, lupeolic, and roburic acids) isolated from frankincense suppress mPGES-1 with increased potencies. In particular, 3α-acetoxy-8,24-dienetirucallic acid (6) and 3α-acetoxy-7,24-dienetirucallic acid (10) inhibited mPGES-1 activity in a cell-free assay with IC50 = 0.4 μM, each. Structure–activity relationship studies and docking simulations revealed concrete structure-related interactions with mPGES-1 and its cosubstrate glutathione. COX-1 and -2 were hardly affected by the triterpene acids (IC50 > 10 μM). Given the crucial role of mPGES-1 in inflammation and the abundance of highly active triterpene acids in frankincence extracts, our findings provide further evidence of the anti-inflammatory potential of frankincense preparations and reveal novel, potent bioactivities of tirucallic acids, roburic acids, and lupeolic acids.

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 β-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 β-BA may contribute to the observed anti-inflammatory effects of BSE during infections by suppressing LPS activity.

Structures of Fluoro, Amino and Thiol Inhibitors Bound to the [Fe4S4] Protein IspH

Isoprenoids derive from two universal precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP).[1] In mammals, these key intermediates are crucial for cell survival, e.g. in cholesterol biosynthesis, and are generated via the mevalonate pathway, while in most bacteria as well as in malaria parasites such as Plasmodium falciparum, the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway is used.[2] The DXP pathway is absent in humans and is, therefore, considered to be an important drug target against many infectious diseases.[3] Scheme 1 shows the last step of the DXP pathway, the conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP, 1) to a mixture of IPP (2) and DMAPP (3). This reaction is catalyzed by HMBPP reductase (IspH), a monomeric enzyme that consists of three domains binding to a redox-active [Fe4S4] cluster in its central cavity (Figure 1a).[4] The cluster is linked to the protein by the side-chains of three cysteine residues, and one of the iron atoms possesses an unoccupied coordination site that has not been found to bind to any amino acid residue. Mechanistic studies with wild type Escherichia coli IspH as well as IspH mutants have revealed two different conformations of 1 inside the active site that are adopted in the catalytic cycle (Figure 1b and c): one in which O1 binds to the 4th iron atom, and a second in which it undergoes numerous hydrogen bond interactions with its diphosphate group and protein residues.[5] Figure 1 Crystal structure of IspH in complex with 1. a) Top and side view of the overall structure of E. coli IspH. Active site of b) wild type IspH forming an alkoxide complex with 1 and c) the IspH E126Q mutant bound to 1 in the cyclic conformation revealing ... Scheme 1 Reductive dehydration catalyzed by IspH. The proposed mechanism for the IspH reaction is shown in Scheme 2 and involves four intermediate states that have been identified by crystallography, Mosbauer and electron paramagnetic resonance (EPR) spectroscopy.[5–6] The detailed structure of IspH in the absence of exogenous ligands is not known (state 0) but binding of 1 to oxidized IspH leads to formation of an alkoxide complex with weak pi interactions (state I; spin S=0). One-electron reduction of the cluster results in [Fe4S4]+ with spin S=1/2, and correlates with a rotation of the ligands hydroxymethyl group away from the cluster to form a cyclic conformation (state II) which has essential impact on the stereochemical course of the IspH reaction.[7] The transfer of two electrons from the cofactor to the substrate produces a HiPIP-type [Fe4S4]3+ cluster and leads to C-O bond cleavage and water release. The allyl anion (state III) then abstracts a proton from the diphosphate group, either at the ligands C2 or C4 atom, to form IPP and DMAPP, respectively. Scheme 2 Proposed mechanism of IspH catalysis. Besides the intensive investigation of the IspH reaction mechanism, a remarkable effort was put into the design and characterization of inhibitors.[8] Recently, synthesis and spectroscopic studies of three substrate analogs with the hydroxyl group in HMBPP replaced by fluoro (4)[9], amino (5)[10], or thiol (6) groups have been reported. Compound 4 is slowly converted by IspH, whereas 5 and 6 inhibit the enzyme. In order to analyze the structure-function relationship of these derivatives we synthesized 4[11], 5[12], and 6 (see SI), performed co-crystallization with E. coli IspH and determined the crystal structure of the complexes. The X-ray structure of IspH in complex with the fluoro analog 4 was determined to 1.8 A resolution [Rfree = 23.2%, Figure 2a, Protein Data Bank (PDB)[13] ID 4H4C] and reveals that 4 binds to the active site of IspH in a similar way as the substrate 1.[14] However, the C-F bond is rotated by 106° compared to the C-O bond in the IspH:1 complex (Figure 2b), the fluorine atom is thus located inside a hydrophobic pocket stabilized by van der Waals interactions with His74Cδ (3.6 A), Ala73C (3.9 A), and Ala73Cβ (3.9 A). This unique conformation allows water molecules to occupy positions W1 and W2.[14] Although it displays an unusual orientation, 4 is converted to 2 or 3 by IspH, but with a decreased rate (kcat = 28 min−1) compared to 1 (kcat = 604 min−1). The differences in these reaction rates are likely due, at least in part, to the increased bond energies of C-F versus C-O.[15] Furthermore, the lack of a direct interaction with the apical iron atom leads to the high Km value of 4 (Km = 104 μM) compared to 1 (Km = 20 μM). Figure 2 Complex structure of IspH bound to the fluorinated derivative 4. a) Active site of IspH showing the bound ligand and two water molecules. A 2FO-FC omit electron density map (blue mesh, contoured at 1.0 σ) is shown for the [Fe4S4] cluster, the ... Recent inhibition studies have shown that the amino and thiol substrate analogs 5 and 6 exhibit potent inhibition of IspH with IC50 values of 0.15 μM and 0.21 μM, respectively.[10] Additionally, Mosbauer spectroscopy has suggested that both ligands interact with the [Fe4S4] cluster. However, it is not immediately obvious that 5 binds to the 4th iron atom via its amino group, or whether it forms an alternative complex that allows a water molecule to coordinate to the 4th iron atom as previously observed with an acetylene inhibitor.[8c] The structure of 5 in complex with IspH was determined to 1.35 A resolution (Rfree = 21.0% Figure 3a, PDB[13] ID 4H4D) and clearly shows two ligand conformations within the same crystal[16]: (i) a ligand-cluster complex in which the amino group coordinates to the apical iron atom and (ii) a conformation in which the amino group is rotated by approximately 74° in the opposite direction to that observed with 4. The amino-iron complex is similar to that seen with the alkoxide-iron complex formed by 1 (Figure 3b), indicating that the affinity of the free amino group with the [Fe4S2]2+ cluster is comparable to that of the hydroxyl group. The second conformation observed in the crystal structure is stabilized by hydrogen bonding of the amino (or ammonium) group to one of the diphosphate oxygen atoms (2.8 A), Glu126Oe (2.9 A) and Thr167Oγ (3.1 A), and a water molecule in the W1 position (3.1 A). Figure 3 X-ray structure of IspH in complex with the amino derivative 5. a) Active site with the ligand in two orientations. The electron density map is displayed according to Figure 2a. b) Superposition of the IspH:5 complex structures with the alkoxide complex ... The amino-iron complex is in good agreement with the Mosbauer spectroscopic data indicating a tetrahedral (3S/N) coordination sphere at the apical iron atom.[10] Thus, it seems likely that the alternative conformation may have arisen due to cluster reduction in the X-ray beam – a result that would be of particular interest in the context of inhibition of the IspH reaction which is, of course, carried out under reducing conditions.[17] However, unlike with 1, no turnover was observed with 5. One possible explanation for this is the formation of a stable ammonium-carboxylate ion pair that prevents the release of ammonia (Scheme 4). Scheme 4 Mechanism of IspH catalysis with 1 and inhibition by 5. a) Dehydroxylation of 1, facilitated by an acidic proton donor (Glu126OH or the diphosphate) represented by X-OH. b) Corresponding hypothetical reaction with 5. c) Formation of a stable ammonium-carboxylate ... Next, we determined the crystal structure of IspH bound to 6 to 1.7 A resolution (Rfree = 21.3%, Figure 4a, PDB[13] ID 4H4E), which displays complex formation between the unique iron atom of the cluster and the thiol group of 6. No solvent molecule is located in the active site, since the increased size of the bridging group leads to movement of the hydrocarbon chain of 6 towards the diphosphate moiety. Furthermore, the hydrogen bond network is modified, preventing the stabilization of the water molecules in the position as found in the IspH:1 complex (Figure 4b). The coordination by the ligands thiol group generates a [Fe4S4] cluster that is coordinated by four sulfur ligands, similar to that found in most proteins catalyzing electron transfer reactions.[18] Figure 4 IspH bound to the thiol derivative 6. a) Active site with thiol-iron complex. The electron density map is displayed according to Figure 2a. b) Structural superposition of IspH:6 with the alkoxide complex in the IspH:1 structure and the cyclic ligand orientation ... In conclusion, the results presented here provide new insights into the mechanism of the IspH reaction with the artificial substrate 4, which involves a previously unknown intermediate that is not bound to the [Fe4S4] cluster. With the amino (5) and thiol (6) species, we find in both cases that the ligands bind to the 4th iron atom. However, it still remains to be determined whether IspH inhibition is due to bonding of these ligands to the oxidized protein, the reduced protein, or both. With 5, we see evidence for a second conformation in which the side-chain is not interacting with the cluster and propose formation of a non-reactive ion-pair complex with Glu126Oe and the diphosphate group (possibly in both oxidized and reduced IspH). As expected, the IspH:6 complex reveals that the ligands sulfur atom binds to the 4th iron atom, and there is no alternative conformation, most likely due to the fact that the SH group is a poor base/hydrogen bond acceptor. The structures of these substrate analogs thus provide new mechanistic insights, as well as suggest novel strategies for the development of antibiotic and antimalarial drugs.