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Institute
The repertoire of natural products offers tremendous opportunities for chemical biology and drug discovery. Natural product-inspired synthetic molecules represent an ecologically and economically sustainable alternative to the direct utilization of natural products. De novo design with machine intelligence bridges the gap between the worlds of bioactive natural products and synthetic molecules. On employing the compound Marinopyrrole A from marine Streptomyces as a design template, the algorithm constructs innovative small molecules that can be synthesized in three steps, following the computationally suggested synthesis route. Computational activity prediction reveals cyclooxygenase (COX) as a putative target of both Marinopyrrole A and the de novo designs. The molecular designs are experimentally confirmed as selective COX-1 inhibitors with nanomolar potency. X-ray structure analysis reveals the binding of the most selective compound to COX-1. This molecular design approach provides a blueprint for natural product-inspired hit and lead identification for drug discovery with machine intelligence.
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.
Recently, we reported that in crude enzyme preparations, a monocyte-derived soluble protein (M-DSP) renders 5-lipoxygenase (5-LO) activity Ca2+-dependent. Here we provide evidence that this M-DSP is glutathione peroxidase (GPx)-1. Thus, the inhibitory effect of the M-DSP on 5-LO could be overcome by the GPx-1 inhibitor mercaptosuccinate and by the broad spectrum GPx inhibitor iodoacetate, as well as by addition of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-HPODE). Also, the chromatographic characteristics and the estimated molecular mass (80-100 kDa) of the M-DSP fit to GPx-1 (87 kDa), and GPx-1, isolated from bovine erythrocytes, mimicked the effects of the M-DSP. Intriguingly, only a trace amount of thiol (10 micro M GSH) was required for reduction of 5-LO activity by GPx-1 or the M-DSP. Moreover, the requirement of Ca2+ allowing 5-LO product synthesis in various leukocytes correlated with the respective GPx-1 activities. Mutation of the Ca2+ binding sites within the C2-like domain of 5-LO resulted in strong reduction of 5-LO activity by M-DSP and GPx-1, also in the presence of Ca2+. In summary, our data suggest that interaction of Ca2+ at the C2-like domain of 5-LO protects the enzyme against the effect of GPx-1. Apparently, in the presence of Ca2+, a low lipid hydroperoxide level is sufficient for 5-LO activation.
5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis, is expressed in a tissue- and cell differentiation-specific manner. The 5-LO core promoter required for basal promoter activity has a unique (G+C)-rich sequence that contains five tandem Sp1 consensus sequences. The mechanisms involved in the regulation of cell type-specific 5-LO expression are unknown. Here we show that 5-LO expression is regulated by DNA methylation. Treatment of the 5-LO-negative cell lines U937 and HL-60TB with the demethylating agent 5-aza-2'-deoxycytidine (AdC) up-regulated expression of 5-LO primary transcripts and mature mRNA in a similar fashion, indicating that AdC stimulates 5-LO gene transcription. Analysis of the methylation status of the 5-LO promoter revealed that the core promoter region was methylated in U937 and HL-60TB cells, whereas it was unmethylated in the 5-LO-positive parent HL-60 cell line. Reporter gene assays with 5-LO promoter constructs gave up to 68- and 655-fold repression of 5-LO promoter activity in HeLa and Mono Mac 6 cells by methylation. 1,25-dihydroxyvitamin D(3) and transforming growth factor-beta (TGFbeta), potent inducers of the 5-LO pathway in myeloid cell lines, increased 5-LO RNA expression in HL-60TB and U937 cells, but co-treatment with AdC was required to achieve 5-LO expression levels in HL-60TB cells that were comparable with wild-type HL-60 cells. In reporter gene assays, 1,25-dihydroxyvitamin D(3) and TGFbeta were unable to induce promoter activity when the 5-LO promoter constructs were methylated, which suggests that 5-LO promoter demethylation is a prerequisite for the high level induction of 5-LO gene expression by 1,25-dihydroxyvitamin D(3) and TGFbeta and that the effects of both agents on 5-LO mRNA expression are not related to DNA methylation.
Wirkungen von Heilpflanzen, Gewürzen, Tees und Lebensmitteln werden in der Naturheilkunde seit der Antike genutzt. Pharmakologisch wirksam sind in der Regel nur die sekundären Pflanzeninhaltsstoffe. Diese in den oft aus vielen Bestandteilen zusammengesetzten Naturstoffen aufzuspüren und ihren molekularbiologischen Wirkungsmechanismus im Körper aufzuklären, ist das Ziel eines Forschungsnetzwerks am Frankfurter ZAFES (Zentrum für Arzneimittelforschung, -Entwicklung und -Sicherheit). So konnten Pharmazeuten und Kliniker gemeinsam herausfinden, wie ein Bestandteil des Rotweins, das Resveratrol, vor Darmkrebs schützt. Die Inhaltsstoffe von Salbei und Rosmarin bieten vielversprechende Ausgangspunkte für neue Medikamente gegen Altersdiabetes. Weihrauch, Myrte und Johanniskraut enthalten Wirkstoffe, die Schlüsselenzyme für Entzündungsreaktionen – etwa bei rheumatischen Beschwerden – hemmen.
We demonstrated previously that 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, can be phosphorylated by p38 MAPK-regulated MAPKAP kinases (MKs). Here we show that mutation of Ser-271 to Ala in 5-LO abolished MK2 catalyzed phosphorylation and clearly reduced phosphorylation by kinases prepared from stimulated polymorphonuclear leukocytes and Mono Mac 6 cells. Compared with heat shock protein 27 (Hsp-27), 5-LO was a weak substrate for MK2. However, the addition of unsaturated fatty acids (i.e. arachidonate 1-50 microm) up-regulated phosphorylation of 5-LO, but not of Hsp-27, by active MK2 in vitro, resulting in a similar phosphorylation as for Hsp-27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca(2+)/calmodulin-dependent kinase II), but these activities were not increased by fatty acids. HeLa cells expressing wild type 5-LO or S271A-5-LO, showed prominent 5-LO activity when incubated with Ca(2+)-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower as compared with wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus that does not prominently increase intracellular Ca(2+) and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation.
Extracts of frankincense, the gum resin of Boswellia species, have been extensively used in traditional folk medicine since ancient times and are still of great interest as promising anti-inflammatory remedies in Western countries. Despite their common therapeutic use and the intensive pharmacological research including studies on active ingredients, modes of action, bioavailability, pharmacokinetics, and clinical efficacy, frankincense preparations are available as nutraceuticals but have not yet approved as a drug on the market. A major issue of commercially available frankincense nutraceuticals is the striking differences in their composition and quality, especially related to the content of boswellic acids (BAs) as active ingredients, mainly due to the use of material from divergent Boswellia species but also because of different work-up and extraction procedures. Here, we assessed three frequently used frankincense-based preparations for their BA content and the interference with prominent pro-inflammatory actions and targets that have been proposed, that is, 5-lipoxygenase and leukotriene formation in human neutrophils, microsomal prostaglandin E2 synthase-1, and inflammatory cytokine secretion in human blood monocytes. Our data reveal striking differences in the pharmacological efficiencies of these preparations in inflammation-related bioassays which obviously correlate with the amounts of BAs they contain. In summary, high-quality frankincense extracts display powerful anti-inflammatory effectiveness against multiple targets which can be traced back to BAs as bioactive ingredients.
5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.
A series of derivatives of the potent dual soluble epoxide hydrolase (sEH)/5-lipoxygenase-activating protein (FLAP) inhibitor diflapolin was designed, synthesized, and characterized by 1H NMR, 13C NMR, and elemental analysis. These novel compounds were biologically evaluated for their inhibitory activity against sEH and FLAP. Molecular modeling tools were applied to analyze structure–activity relationships (SAR) on both targets. Results show that even small modifications on the lead compound diflapolin markedly influence the inhibitory potential, especially on FLAP, suggesting very narrow SAR.
The miRNA biogenesis is tightly regulated to avoid dysfunction and consequent disease development. Here, we describe modulation of miRNA processing as a novel noncanonical function of the 5-lipoxygenase (5-LO) enzyme in monocytic cells. In differentiated Mono Mac 6 (MM6) cells, we found an in situ interaction of 5-LO with Dicer, a key enzyme in miRNA biogenesis. RNA sequencing of small noncoding RNAs revealed a functional impact, knockout of 5-LO altered the expression profile of several miRNAs. Effects of 5-LO could be observed at two levels. qPCR analyses thus indicated that (a) 5-LO promotes the transcription of the evolutionarily conserved miR-99b/let-7e/miR-125a cluster and (b) the 5-LO-Dicer interaction downregulates the processing of pre-let-7e, resulting in an increase in miR-125a and miR-99b levels by 5-LO without concomitant changes in let-7e levels in differentiated MM6 cells. Our observations suggest that 5-LO regulates the miRNA profile by modulating the Dicer-mediated processing of distinct pre-miRNAs. 5-LO inhibits the formation of let-7e which is a well-known inducer of cell differentiation, but promotes the generation of miR-99b and miR-125a known to induce cell proliferation and the maintenance of leukemic stem cell functions.
Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol.
The enzyme acetyl-CoA carboxylase (ACC) plays a crucial role in fatty acid metabolism. In recent years, ACC has been recognized as a promising drug target for treating different diseases. However, the role of ACC in vascular endothelial cells (ECs) has been neglected so far. To characterize the role of ACC, we used the ACC inhibitor, soraphen A, as a chemical tool, and also a gene silencing approach. We found that ACC1 was the predominant isoform in human umbilical vein ECs as well as in human microvascular ECs and that soraphen A reduced the levels of malonyl-CoA. We revealed that ACC inhibition shifted the lipid composition of EC membranes. Accordingly, membrane fluidity, filopodia formation, and migratory capacity were reduced. The antimigratory action of soraphen A depended on an increase in the cellular proportion of PUFAs and, most importantly, on a decreased level of phosphatidylglycerol. Our study provides a causal link between ACC, membrane lipid composition, and cell migration in ECs. Soraphen A represents a useful chemical tool to investigate the role of fatty acid metabolism in ECs and ACC inhibition offers a new and valuable therapeutic perspective for the treatment of EC migration-related diseases.