Total Synthesis, Structure Reassignment, and Biological Evaluation of the Anti-Inflammatory Macrolactone 13-Hydroxy-14-deoxyoxacyclododecindione.

Herein, the first total synthesis of natural 13-hydroxy-14-deoxyoxacyclododecindione along with the revision of the proposed configuration is reported. This natural product, initially discovered in 2018, belongs to the oxacyclododecindione family, renowned for their remarkable anti-inflammatory and antifibrotic activities. The synthetic route involves an esterification/Friedel-Crafts-acylation approach and uses various triol fragments. It allows the preparation of different stereoisomers, including the (revised) natural product, two threo-derivatives, and two Z-isomers of the endocyclic C═C double bond. Furthermore, a late-stage inversion of the C-13 stereocenter could transform the originally proposed structure into the revised natural product. With this comprehensive set of compounds and the previously prepared (13R,14S,15R)-isomer, deeper insights into their structural properties and biological activities were obtained. A detailed analysis of the final macrolactones using spectroscopy (NMR, IR, UV-vis) and X-ray crystallography gave new insights such as the significance of the optical rotation for the elucidation of their configuration and the light-induced E/Z double-bond photoisomerization. The pharmacological potential of the compounds was underlined by remarkably low IC50 values in biological assays addressing the inhibition of cellular inflammatory responses.

Total Synthesis and Biological Evaluation of the Anti-Inflammatory (13R,14S,15R)-13-Hydroxy-14-deoxyoxacyclododecindione.

The first total synthesis of the natural product (13R,14S,15R)-13-hydroxy-14-deoxyoxacyclododecindione, which was isolated in 2018 as a member of the oxacyclododecindione family, is reported. A synthetic strategy through intramolecular Friedel-Crafts acylation combined with the stereoselective synthesis of a new triol key fragment allowed the preparation of the macrolactone. Due to mismatching physical data of the synthetic product, a revision of the configuration of the natural product isolated in 2018 is required. Light-induced E/Z-isomerism of the macrolactone backbone is described for the first time in the class of oxacyclododecindione-type macrolactones. The hydroxylated macrolactone prepared herein was found to show highly promising IC50 values in biological assays addressing the inhibition of inflammatory responses.

Anti-inflammatory dihydroxanthones from a Diaporthe species.

In a search for anti-inflammatory compounds from fungi inhibiting the promoter activity of the small chemokine CXCL10 (Interferon-inducible protein 10, IP-10) as a pro-inflammatory marker gene, the new dihydroxanthone methyl (1R, 2R)-1,2,8-trihydroxy-6-(hydroxymethyl)-9-oxo-2,9-dihydro-1H-xanthene-1-carboxylate (2) and the previously described dihydroxanthone AGI-B4 (1) were isolated from fermentations of a Diaporthe species. The structures of the compounds were elucidated by a combination of one- and two-dimensional NMR spectroscopy, mass spectrometry, and calculations using density functional theory (DFT). Compounds 1 and 2 inhibited the LPS/IFNγ induced CXCL10 promoter activity in transiently transfected human MonoMac6 cells in a dose-dependent manner with IC50 values of 4.1 µM (±0.2 µM) and 1.0 µM (±0.06 µM) respectively. Moreover, compounds 1 and 2 reduced mRNA levels and synthesis of pro-inflammatory mediators such as cytokines and chemokines in LPS/IFNγ stimulated MonoMac6 cells by interfering with the Stat1 and NFκB pathway.

Total synthesis and biological evaluation of seven new anti-inflammatory oxacyclododecindione-type macrolactones.

Through variation of our previously published total synthesis of two highly active anti-inflammatory macrolactones from the oxacyclododecindione family (J. Tauber, M. Rohr, T. Walter, G. Erkel and T. Opatz, Org. Biomol. Chem., 2015, 13, 7813-7821), seven new representatives of this compound class were prepared. Substitution of the 14-hydroxy group in oxacyclododecindione with a methyl substituent provided a readily accessible non-natural analogue which has similar pharmacological properties to the scarcely available natural product. Since the producible amount of substance is therefore no longer restricted by low fermentation yields, extensive in vivo studies become possible for the first time. Based on this finding, further investigations on structure-activity relationships were undertaken by variation of the halogen atom, which showed that exchange or omission of the chloro substituent led to significantly lower binding affinities. Furthermore, it was found that elongation of the crucial and characteristic aliphatic side chain at C-10 also increased the IC50 value in the biological assays of interest.

Anti-inflammatory tetraquinane diterpenoids from a Crinipellis species.

The small pro-inflammatory 10kDa chemokine CXCL10 (Interferon-inducible protein 10, IP-10) plays an important role in mediating immune responses through the activation and recruitment of leukocytes such as T cells, eosinophils, monocytes and NK cells to the sites of inflammation. Elevated levels of CXCL10 have been associated with chronic inflammatory and infectious diseases and therefore CXCL10 represents an attractive target for the development of new anti-inflammatory drugs. In a search for anti-inflammatory compounds from fungi inhibiting the inducible CXCL10 promoter activity, four new tetraquinane diterpenoids, crinipellin E (1), crinipellin F (2), crinipellin G (3) and crinipellin H (4) were isolated from fermentations of a Crinipellis species. The structures of the compounds were elucidated by a combination of one- and two-dimensional NMR spectroscopy and mass spectrometry. Compounds 1, 2, and 3 inhibited the LPS/IFN-γ induced CXCL10 promoter activity in transiently transfected human MonoMac6 cells in a dose-dependent manner with IC50 values of 15µM, 1.5µM, and 3.15µM respectively, whereas compound 4 was devoid of any biological activity. Moreover, compounds 1, 2 and 3 reduced mRNA levels and synthesis of pro-inflammatory mediators such as cytokines and chemokines in LPS/IFN-γ stimulated MonoMac6 cells.

A surprising switch in absolute configuration of anti-inflammatory macrolactones.

Oxacyclododecindione-type macrolactones exhibit highly potent anti-inflammatory activities even at nanomolar concentration. After the determination of the relative configuration of the stereocenters at C14 and C15 by total synthesis of 4-dechloro-14-deoxyoxacyclododecindione and 14-deoxyoxacyclododecindione, the absolute configuration has now been assigned by X-ray crystallography. Surprisingly, the absolute configuration is (14S,15R) which differs for C15 from that of the well-known derivatives of (S)-curvularin. The biological activities of both enantiomers of 14-deoxyoxacyclododecindione, obtained by racemic synthesis and optical resolution, were investigated and the ring conformation of the natural product was compared to that of (S)-curvularin and (R)-dehydrocurvularin.

Total synthesis of two potent anti-inflammatory macrolactones of the oxacyclododecindione type.

An esterification/Friedel-Crafts-cyclization approach permitted the first successful synthetic entry into the oxacyclododecindione subclass of the dihydroxyphenylacetic acid lactone-type natural products. This route allowed the preparation of two highly active anti-inflammatory fungal secondary metabolites 14-deoxyoxacyclododecindione and 14-deoxy-4-dechlorooxacyclododecindione as well as their 14-desmethyl analogues.

4-Dechloro-14-deoxy-oxacyclododecindione and 14-deoxy-oxacylododecindione, two inhibitors of inducible connective tissue growth factor expression from the imperfect fungus Exserohilum rostratum.

Connective tissue growth factor (CTGF/CCN2), a member of the CCN superfamily of secreted cysteine-rich glycoproteins, is a central mediator of tissue remodeling and fibrosis. CTGF is suggested to be an important down-stream effector of transforming growth factor-beta (TGF-ß) signaling and has therefore reached considerable pathophysiological relevance because of its involvement in the pathogenesis of fibrotic diseases, atherosclerosis, skin scarring, and other conditions with excess production of connective tissue. In a search for inhibitors of inducible CTGF expression from fungi, two new macrocyclic lactones, namely 4-dechloro-14-deoxy-oxacyclododecindione (1) and 14-deoxy-oxacylododecindione, (2) along with the previously described congener oxacyclododecindione (3) were isolated from fermentations of the imperfect fungus Exserohilum rostratum. The structure of the compounds were elucidated by a combination of one- and two-dimensional NMR spectroscopy and mass spectrometry. Compounds 1 and 2 turned out to inhibit TGF-ß induced CTGF promoter activity in transiently transfected HepG2 cells in a dose-dependent manner with IC50 values of 1.8 µM and 336 nM, respectively, and also antagonized TGF-ß induced cellular effects including CTGF mRNA levels, CTGF protein expression and tube formation.

The fungal lactone oxacyclododecindione is a potential new therapeutic substance in the treatment of lupus-associated kidney disease.

Recently oxacyclododecindione (Oxa), a macrocyclic lactone isolated from the imperfect fungus Exserohilum rostratum, has been described as a potent transcription inhibitor of inducible proinflammatory and profibrotic genes in cell culture models. As kidney disease in systemic lupus erythematosus is characterized by aberrant expression of inflammatory mediators and infiltration of immune cells, we investigated the effect of Oxa in MRL-Fas(lpr) mice, a model of systemic lupus erythematosus. These mice develop a spontaneous T-cell and macrophage-dependent autoimmune disease including severe glomerulonephritis that shares features with human lupus. Comparable to the results of in vitro models, we found a reduced expression of the cytokines IFN-γ, IL-6, and TNF-α and the chemokines CCL2, RANTES, and CSF-1 on mRNA and protein level in the kidney of Oxa-treated MRL-Fas(lpr) mice. Accordingly, Oxa treatment reduced the infiltration of immune cells and the frequency of activated proinflammatory T cells in the kidney. Moreover, kidney disease, measured by histopathology, IgG and collagen deposition, and proteinuria, was ameliorated in Oxa-treated MRL-Fas(lpr) mice compared with the control group. Thus, Oxa is a new effective anti-inflammatory compound, which may serve as base structure for the development of new therapeutics for the treatment of chronic inflammatory and/or fibrotic diseases.

Total synthesis and biological evaluation of the natural product (-)-cyclonerodiol, a new inhibitor of IL-4 signaling.

In a screening program of natural compounds from fungi, the known cyclopentanoid sesquiterpene (-)-cyclonerodiol was identified as a specific inhibitor of the IL-4 induced STAT6 signaling pathway (IC50 = 9.7 µM) which is required for the differentiation of naive CD4 T cells to T helper type 2 (Th2) lymphocytes. As many allergic conditions, including allergic asthma and atopic diseases, are driven by an excessive Th2 response, STAT6 is a promising target for the development of new therapeutics. The compound was synthesized in six steps from (-)-linalool using an epoxide radical cyclization as the key step.

Anti-inflammatory drimane sesquiterpene lactones from an Aspergillus species.

IFN-γ inducible protein 10 (IP-10, CXCL10) is a 10 kDa chemokine, which is secreted from various cell types after exposure to pro-inflammatory stimuli. This chemokine is a ligand for the CXCR3 receptor and regulates immune responses by activating and recruiting leukocytes such as T cells, eosinophils, monocytes, and NK cells to sites of inflammation. Altered expression of CXCL10 has been associated with chronic inflammatory and infectious diseases and therefore CXCL10 represents a promising target for the development of new anti-inflammatory drugs. In a search for inhibitors of CXCL10 promoter activity, three structurally related drimane sesquiterpene lactones (compounds 1-3) were isolated from fermentations of an Aspergillus species. Compounds 1 and 2 inhibited the IFN-γ/TNF-α/IL-1ß induced CXCL10 promoter activity in transiently transfected human DLD-1 colon carcinoma cells in a dose-dependent manner with IC50 values of 12.4 µM for 1 and 55 µM for 2, whereas 3 was devoid of any biological activity. Moreover, compounds 1 and 2 reduced CXCL10 mRNA levels and synthesis in IFN-γ/TNF-α/IL-1ß stimulated DLD-1 cells.

Inhibition of TGF-ß signaling by the fungal lactones (S)-curvularin, dehydrocurvularin, oxacyclododecindione and galiellalactone.

TGF-ß is a multifunctional cytokine that regulates cell proliferation, differentiation, apoptosis and extracellular matrix production. Deregulation of TGF-ß production or signaling plays a pivotal role in a variety of pathological processes such as cancer, metastasis, angiogenesis and fibrosis. Therefore, TGF-ß inhibitors should be promising therapeutic agents for the suppression of cancer progression and metastasis as well as fibrotic disorders. In a screening program of natural compounds from fungi inhibiting the TGF-ß dependent expression of a reporter gene in HepG2 cells, we found that the fungal lactones (S)-curvularin, dehydrocurvularin, oxacyclododecindione and galiellalactone inhibited the binding of the activated Smad2/3 transcription factors to the DNA and antagonized the cellular effects of TGF-ß including reporter gene activation and expression of TGF-ß induced genes in HepG2 and MDA-MB-231 cells. The most active compound oxacyclododecindione inhibited TGF-ß dependent reporter activity with IC50-values of 190-217 nM. In an in vitro angiogenesis assay, the fungal lactones strongly decreased the formation of capillary-like tubules of MDA-MB-231 cells on Matrigel.

SF002-96-1, a new drimane sesquiterpene lactone from an Aspergillus species, inhibits survivin expression.

Survivin, a member of the IAP (inhibitor of apoptosis) gene family, is overexpressed in virtually all human cancers and is functionally involved in the inhibition of apoptosis, regulation of cell proliferation, metastasis and resistance to therapy. Because of its upregulation in malignancy, survivin has currently attracting considerable interest as a new target for anticancer therapy. In a screening of approximately 200 strains of imperfect fungi for the production of inhibitors of survivin promoter activity, a new drimane sesquiterpene lactone, SF002-96-1, was isolated from fermentations of an Aspergillus species. The compound inhibited survivin promoter activity in transiently transfected Colo 320 cells in a dose dependent manner with IC50 values of 3.42 µM (1.3 µg/mL). Moreover, it also reduced mRNA levels and protein synthesis of survivin and triggered apoptosis.

Structure elucidation and biological activities of perylenequinones from an Alternaria species.

The KEAP1-Nrf2/ARE pathway is a pivotal cytoprotective regulator against oxidative stress which plays an important role in the development of many inflammatory diseases and cancer. Activation of the Nrf2 transcription factor by oxidative stress or electrophiles regulates antioxidant response element (ARE)-dependent transcription of antioxidative, detoxifying, and anti-inflammatory proteins. Therefore, modulators of the KEAP1-Nrf2/ARE pathway have received considerable interest as therapeutics to protect against diseases where oxidative stress constitutes the underlying pathophysiology. In a search for fungal secondary metabolites affecting the Nrf2/ARE-dependent expression of a luciferase reporter gene in BEAS-2B cells, three new perylenequinones, compounds 1, 2, and 3, together with altertoxin-I (ATX-I), were isolated from fermentations of an Alternaria species. The structures of the compounds were elucidated by a combination of one- and two-dimensional NMR spectroscopy and mass spectrometry. Compound 1 and ATX-I exhibited strong cytotoxic effects with LC50-values of 3.8 µM and 6.43 µM, respectively, whereas compound 3 showed no cytotoxic effects up to 100 µM on BEAS-2B cells. ATX-I induced ARE-dependent luciferase expression approximately fivefold and compound 1 approximately 2.6-fold at a concentration of 3 µM in transiently transfected BEAS-2B cells. In addition, compound 1 and ATX-I exhibited strong oxidative effects, whereas compound 3 did not show significant oxidative properties. For compound 1 and ATX-I, a strong upregulation of heme oxygenase-1 could be observed on mRNA and protein level in treated BEAS-2B cells. Moreover, compound 3 significantly decreased sod3 mRNA levels after induction of oxidative stress with benzoquinone.

The macrocyclic lactone oxacyclododecindione reduces fibrosis progression.

Background: Renal fibrosis is one of the most important triggers of chronic kidney disease (CKD), and only a very limited number of therapeutic options are available to stop fibrosis progression. As fibrosis is characterized by inflammation, myofibroblast activation, and extracellular matrix (ECM) deposition, a drug that can address all these processes might be an interesting therapeutic option. Methods: We tested in vivo in an ischemia-reperfusion (I/R) model in C57BL/6 mice and in kidney tubular epithelial cells (TEC) (HK2 cell line and primary cells) whether the natural product oxacyclododecindione (Oxa) reduces fibrosis progression in kidney disease. This was evaluated by Western blot, mRNA expression, and mass spectrometry secretome analyses, as well as by immunohistochemistry. Results: Indeed, Oxa blocked the expression of epithelial-mesenchymal transition marker proteins and reduced renal damage, immune cell infiltration, and collagen expression and deposition, both in vivo and in vitro. Remarkably, the beneficial effects of Oxa were also detected when the natural product was administered at a time point of established fibrotic changes, a situation close to the clinical situation. Initial in vitro experiments demonstrated that a synthetic Oxa derivative possesses similar features. Conclusion: Although open questions such as possible side effects need to be investigated, our results indicate that the combination of anti-inflammatory and anti-fibrotic effects of Oxa make the substance a promising candidate for a new therapeutic approach in fibrosis treatment, and thus in the prevention of kidney disease progression.

The anti-inflammatory fungal compound (S)-curvularin reduces proinflammatory gene expression in an in vivo model of rheumatoid arthritis.

In previous studies, we identified the fungal macrocyclic lactone (S)-curvularin (SC) as an anti-inflammatory agent using a screening system detecting inhibitors of the Janus kinase/signal transducer and activator of transcription pathway. The objective of the present study was to investigate whether SC is able to decrease proinflammatory gene expression in an in vivo model of a chronic inflammatory disease. Therefore, the effects of SC and dexamethasone were compared in the model of collagen-induced arthritis (CIA) in mice. Total genomic microarray analyses were performed to identify SC target genes. In addition, in human C28/I2 chondrocytes and MonoMac6 monocytes, the effect of SC on proinflammatory gene expression was tested at the mRNA and protein level. In the CIA model, SC markedly reduced the expression of a number of proinflammatory cytokines and chemokines involved in the pathogenesis of CIA as well as human rheumatoid arthritis (RA). In almost all cases, the effects of SC were comparable with those of dexamethasone. In microarray analyses, we identified additional new therapeutic targets of SC. Some of them, such as S100A8, myeloperoxidase, or cathelicidin, an antimicrobial peptide, are known to be implicated in pathophysiological processes in RA. Similar anti-inflammatory effects of SC were also observed in human C28/I2 chondrocyte cells, which are resistant to glucocorticoid treatment. These data indicate that SC and glucocorticoid effects are mediated via independent signal transduction pathways. In summary, we demonstrate that SC is a new effective anti-inflammatory compound that may serve as a lead compound for the development of new drugs for the therapy of chronic inflammatory diseases.

Inhibition of TGF-ß signaling, vasculogenic mimicry and proinflammatory gene expression by isoxanthohumol.

TGF-ß is a multifunctional cytokine that regulates cell proliferation, differentiation, apoptosis and extracellular matrix production. Deregulation of TGF-ß production or signaling has been associated with a variety of pathological processes such as cancer, metastasis, angiogenesis and fibrosis. Therefore, TGF-ß signaling has emerged as an attractive target for the development of new cancer therapeutics. In a screening program of natural compounds from fungi inhibiting the TGF-ß dependent expression of a reporter gene in HepG2 cells, we found that the flavone isoxanthohumol inhibited the binding of the activated Smad2/3 transcription factors to the DNA and antagonized the cellular effects of TGF-ß including reporter gene activation and expression of TGF-ß induced genes in HepG2 and MDA-MB-231 cells. In an in vitro angiogenesis assay, isoxanthohumol (56 µM) strongly decreased the formation of capillary-like tubules of MDA-MB-231 cells on Matrigel. In addition, we found that isoxanthohumol blocked IFN-γ, IL-4 and IL-6 dependent Jak/Stat signaling and strongly inhibited the induction of pro-inflammatory genes in MonoMac6 cells at the transcriptional level after LPS/TPA treatment.

Induction of tolerogenic lung CD4+ T cells by local treatment with a pSTAT-3 and pSTAT-5 inhibitor ameliorated experimental allergic asthma.

Signal transducer and activator of transcription (STAT)-3 inhibitors play an important role in regulating immune responses. Galiellalactone (GL) is a fungal secondary metabolite known to interfere with the binding of phosphorylated signal transducer and activator of transcription (pSTAT)-3 as well of pSTAT-6 dimers to their target DNA in vitro. Intra nasal delivery of 50 µg GL into the lung of naive Balb/c mice induced FoxP3 expression locally and IL-10 production and IL-12p40 in RNA expression in the airways in vivo. In a murine model of allergic asthma, GL significantly suppressed the cardinal features of asthma, such as airway hyperresponsiveness, eosinophilia and mucus production, after sensitization and subsequent challenge with ovalbumin (OVA). These changes resulted in induction of IL-12p70 and IL-10 production by lung CD11c(+) dendritic cells (DCs) accompanied by an increase of IL-3 receptor α chain and indoleamine-2,3-dioxygenase expression in these cells. Furthermore, GL inhibited IL-4 production in T-bet-deficient CD4(+) T cells and down-regulated the suppressor of cytokine signaling-3 (SOCS-3), also in the absence of STAT-3 in T cells, in the lung in a murine model of asthma. In addition, we found reduced amounts of pSTAT-5 in the lung of GL-treated mice that correlated with decreased release of IL-2 by lung OVA-specific CD4(+) T cells after treatment with GL in vitro also in the absence of T-bet. Thus, GL treatment in vivo and in vitro emerges as a novel therapeutic approach for allergic asthma by modulating lung DC phenotype and function resulting in a protective response via CD4(+)FoxP3(+) regulatory T cells locally.

Production and secretion of functional SARS-CoV-2 spike protein in Chlamydomonas reinhardtii.

The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1,273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to higher eukaryotes. Here we report the production of full-length SARS-CoV-2 spike protein - lacking the C-terminal membrane anchor - as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries.

Drug Candidates for Autoimmune Diseases.

Most of the immunosuppressive drugs used in the clinic to prevent organ rejection or to treat autoimmune disorders were originally isolated from fungi or bacteria. Therefore, in addition to plants, these are valuable sources for identification of new potent drugs. Many side effects of established drugs limit their usage and make the identification of new immunosuppressants necessary. In this review, we present a comprehensive overview of natural products with potent anti-inflammatory activities that have been tested successfully in different models of chronic inflammatory autoimmune diseases. Some of these candidates already have passed first clinical trials. The anti-inflammatory potency of these natural products was often comparable to those of established drugs, and they could be used at least in addition to standard therapy to reduce their dose to minimize unwanted side effects. A frequent mode of action is the inhibition of classical inflammatory signaling pathways, such as NF-κB, in combination with downregulation of oxidative stress. A drawback for the therapeutic use of those natural products is their moderate bioavailability, which can be optimized by chemical modifications and, in addition, further safety studies are necessary. Altogether, very interesting candidate compounds exist which have the potential to serve as starting points for the development of new immunosuppressive drugs.