Inhibition of HIF-1α through Suppression of NF-κB Activation by Compounds Isolated from Senecio graveolens

One of the characteristics of cancer is that the lack of oxygen in the cancer cells triggers changes in their gene expression. This hypoxia activates hypoxia-inducible factor 1-alpha and this in turn sets in motion the whole family of important angiogenic genes for the tumour. Hypoxia-inducible factor 1-alpha therefore increases the density and vascular permeability within the tumours, facilitating their rapid growth and, later, the metastasis. Senecio graveolens is a South American medicinal plant commonly used for mountain sickness (lack of adaptation of the organism to hypoxia). Additionally, pharmacological studies showed that its alcoholic extracts have cytotoxic properties. This research aimed to perform a guided phytochemical study of S. graveolens to identify compounds capable of inhibiting hypoxia-inducible factor 1-alpha through suppression of nuclear factor kappa-light-chain-enhancer of activated B cell activation. The isolation led to the characterisation of phanurane (1), damsine (2), and scoparone (3), first reported in the S. graveolens species. Phanurane (1) showed inhibitory activity of hypoxia-inducible factor 1-alpha on the cancer cell lines U-373 MG (IC50 = 20.66 ± 0.04 μM), A549 (IC50 =25.80 ± 0.04 μM), Hep G2 (IC50 =29.21 ± 0.03 μM), and Caco-2 (IC50 =38.58 ± 0.02 μM). Damsine (2) hypoxia-inducible factor 1-alpha displayed inhibitory activity of hypoxiainducible factor 1-alpha on the cancer cell lines U-373 MG (IC50 =2.29 ± 0.07 μM), A549 (IC50 =4.13 ± 0.04 μM), Hep G2 (IC50 =6.40 ± 0.03 μM), and Caco-2 (IC50 =9.80 ± 0.04 μM). Finally, scoparone (3) displayed inhibitory activity of y poxiainducible factor 1-alpha on the cancer cell lines U-373 MG (IC50 =15.22±0.01μM), A549 (IC50 =17.47±0.02μM), Hep G2 (IC50=18.26±0.06μM), and Caco-2 (IC50=19.75±0.04μM). In addition, phanurane (1) displayed inhibitory activity over nuclear factor kappa-light-chain-enhancer of activated B cells on cancer cell lines U-373 MG (IC50 =7.13 ± 0.03 μM), A549 (IC50 = 8.64 ± 0.03 μM), Hep G2 (IC50 = 8.87 ± 0.04 μM), and Caco-2 (IC50 =15.11 ± 0.01 μM). Likewise, damsine (2) showed inhibitory activity over nuclear factor kappa-light-chainenhancer of activated B cells on cancer cell lines U-373 MG (IC50 =2.28 ± 0.01 μM), A549 (IC50 =3.79 ± 0.02 μM), Hep G2 (IC50 = 3.98 ± 0.05 μM), and Caco-2 (IC50 = 6.41 ± 0.02 μM). Lastly, scoparone (3) displayed inhibitory activity of nuclear factor kappa-light-chain-enhancer of activated B cells on cancer cell lines U-373 MG (IC50 = 3.62 ± 0.06 μM), A549 (IC50 = 4.48 ± 0.03 μM), Hep G2 (IC50 = 5.25 ± 0.01 μM), and Caco-2 (IC50 =11.90 ± 0.02 μM). This study corroborates the cytotoxic activity of the isolated compounds through the inhibition of hypoxia-inducible factor 1-alpha as well as its modulator nuclear factor kappa-light-chain-enhancer of activated B cellsThis work was supported by the National Herbarium of Bolivia, the Fundación de la Universidad Autónoma de Madrid (FUAM

Anti-inflammatory Potential of Macamides Isolated from Yellow Tubers of Mashua (Tropaeolum Tuberosum)

AbstractAlthough Tropaeolum tuberosum tubers have been consumed cooked as a folk remedy for the treatment of skin, lungs, liver and kidneys diseases, these uses have very limited scientific basis. Therefore, this article develops a phytochemical analysis of the yellow tubers of T. tuberosum with the objective to assess whether the isolated compounds have anti-inflammatory potential in the CCD-1109Sk, MRC-5 and RWPE-1 cell lines. We performed an extraction of T. tuberosum tubers using different organic solvents, followed by a bioguided chromatographic separation. Four macamides were identified by LC/MS techniques, but only N-benzyllinoleamide (1) and N-benzyloleamide (2) were isolated and elucidated by NMR/MS techniques, given that they were present in a larger proportion in the tubers. The anti-inflammatory potential of macamides was evaluated by the inhibition of NF-κB and STAT3 activation. Both compounds displayed inhibition of NF-κB activation with IC50 values of 2.28±0.54 µM; 3.66±0.34 µM and 4.48±0.29 µM for compound (1) and 6.50±0.75 µM; 7.74±0.19 µM and 8.37 ±0.09 µM for compound (2) in CCD-1109Sk, MRC-5 and RWPE-1 cell lines, respectively. Moreover, both compounds inhibited the STAT3 activation with IC50 of 0.61±0.76 µM; 1.24±0.05 µM and 2.10±0.12 µM for compound (1) and 5.49±0.31 µM; 7.73 ±0.94 µM and 7.79±0.30 µM for compound (2). Therefore, isolated macamides of T. tuberosum tubers showed promising anti-inflammatory effects, suggesting a possible beneficial use to combat inflammatory processes of skin, lung and prostate

Hydroalcoholic extract of Tagetes minuta L. inhibits inflammatory bowel disease through the activity of pheophytins on the NF-κB signalling pathway

Ethno-pharmacological relevance: Species of the genus Tagetes are well known for their anti-inflammatory properties. Tagetes minuta “Huacatay” is an endemic species of South America that has been used in traditional medicine since ancient times as a remedy for stomach and intestinal discomfort. Aim of the study: The aim of this study is to investigate the anti-inflammatory activity of the aqueous and hydroalcoholic extracts of the Huacatay, identifying the compounds responsible for this activity. Materials and methods: Anti-inflammatory activity of the compounds, fractions and extracts was evaluated in Hs 746T (stomach), HIEC-6 (intestine) and THP-1 (monocytes peripheral blood) cells by measuring their inhibitory capacity against the NF-κB production. Results: Aqueous and hydroalcoholic extracts of Tagetes minuta displayed anti-inflammatory activity in vitro, the hydroalcoholic extract being the most active (IC50 between 59.72 and 66.42 μg/mL) in all cell lines. Bio-guided hydroalcoholic extract fractionation led to the isolation and characterisation of two pheophytins, pheophytin a (1) and 132-hydroxy pheophytin a (2). Both compounds inhibited the production of NF-κB with IC50 values in the low micromolar range, with an IC50 between 12.32 and 16.01 μM for compound 1 and 7.91–9.87 μM for compound 2. Conclusions: The two pheophytins isolated in this study inhibit the production of NF-κB, thus showing that the traditional anti-inflammatory use of Tagetes minuta can be proved through pharmacological assays. This contributes to understanding the anti-inflammatory activity of the Huacatay extracts and their use in the treatment of stomach and intestinal discomfortThis work was supported by the National Herbarium of Bolivia and the Fundación de la Universidad Autónoma de Madrid (FUAM

Enzymatic synthesis and molecular modelling studies of rhamnose esters using lipase from Pseudomonas stutzeri

Rhamnolipids are becoming an important class of glycolipid biosurfactants. Herein, we describe for the first time the enzymatic synthesis of rhamnose fatty acid esters by the transesterifica-tion of rhamnose with fatty acid vinyl esters, using lipase from Pseudomonas stutzeri as a biocatalyst. The use of this lipase allows excellent catalytic activity in the synthesis of 4-O-acylrhamnose (99% conversion and full regioselectivity) after 3 h of reaction using tetrahydrofuran (THF) as the reaction media and an excess of vinyl laurate as the acyl donor. The role of reaction conditions, such as temperature, the substrates molar ratio, organic reaction medium and acyl donor chain-length, was studied. Optimum conditions were found using 35 ºC, a molar ratio of 1:3 (rhamnose:acyldonor), solvents with a low logP value, and fatty acids with chain lengths from C4 to C18 as acyl donors. In hydrophilic solvents such as THF and acetone, conversions of up to 99–92% were achieved after 3 h of reaction. In a more sustainable solvent such as 2-methyl-THF (2-MeTHF), high conversions were also obtained (86%). Short and medium chain acyl donors (C4–C10) allowed maximum conversions after 3 h, and long chain acyl donors (C12–C18) required longer reactions (5 h) to get 99% conversions. Furthermore, scaled up reactions are feasible without losing catalytic action and regioselectivity. In order to explain enzyme regioselectivity and its ability to accommodate ester chains of different lengths, homology modelling, docking studies and molecular dynamic simulations were performed to explain the behaviour observe

Molecular genetics of naringenin biosynthesis, a typical plant secondary metabolite produced by Streptomyces clavuligerus

Background: Some types of flavonoid intermediates seemed to be restricted to plants. Naringenin is a typical plant metabolite, that has never been reported to be produced in prokariotes. Naringenin is formed by the action of a chalcone synthase using as starter 4-coumaroyl-CoA, which in dicotyledonous plants derives from phenylalanine by the action of a phenylalanine ammonia lyase. Results: A compound produced by Streptomyces clavuligerus has been identified by LC-MS and NMR as naringenin and coelutes in HPLC with a naringenin standard. Genome mining of S. clavuligerus revealed the presence of a gene for a chalcone synthase (ncs), side by side to a gene encoding a P450 cytochrome (ncyP) and separated from a gene encoding a Pal/Tal ammonia lyase (tal). Deletion of any of these genes results in naringenin non producer mutants. Complementation with the deleted gene restores naringenin production in the transformants. Furthermore, naringenin production increases in cultures supplemented with phenylalanine or tyrosine. Conclusion: This is the first time that naringenin is reported to be produced naturally in a prokariote. Interestingly three non-clustered genes are involved in naringenin production, which is unusual for secondary metabolites. A tentative pathway for naringenin biosynthesis has been proposedThis work was supported by Grant BIO2012-34723 from the Spanish Ministry of Economy and Competitivity. R. Álvarez-Álvarez received a FPU fellowship from the Spanish Ministry of Education, Culture and Sport

Synthesis and biological screening of a library of macamides as TNF-α inhibitors

Thirty-five macamide analogues were synthesised by modifying the initial molecular structure. The resulting structures were confirmed using NMR and MS. Cytotoxicity and the anti-inflammatory activity of these synthetic macamides were evaluated in the THP-1 cell line. Preliminary biological evaluation indicated that most of these synthetic macamides did not present cytotoxicity (MTT assay) in the tested cell line with respect to the control (actinomycin D). Regarding the anti-inflammatory activity, several analogues had a greater potential for inhibition of TNF-α than natural macamides. Synthetic macamide 4a was the most active (IC50 = 0.009 ± 0.001 μM) compared to the C87 (control). Through looking at the link between the chemical structure and the activity, our study proves that changes made to natural macamides at the level of the alkyl chain, the benzyl position, the amide bond, and the addition of two methyl groups to the aromatic ring (meta position) lead us to obtaining new macamides with greater anti-inflammatory activityThis work was supported by the Fundación de la Universidad Autónoma de Madrid (FUAM

Structural and bioactivity characterization of filipin derivatives from engineered streptomyces filipinensis strains reveals clues for reduced haemolytic action

The rise in the number of immunocompromised patients has led to an increased incidence of fungal infections, with high rates of morbidity and mortality. Furthermore, misuse of antifungals has boosted the number of resistant strains to these agents; thus, there is urgent need for new drugs against these infections. Here, the in vitro antifungal activity of filipin III metabolic intermediates has been characterized against a battery of opportunistic pathogenic fungi—Candida albicans, Candida glabrata, Candida krusei, Cryptococcus neoformans, Trichosporon cutaneum, Trichosporon asahii, Aspergillus nidulans, Aspergillus niger, and Aspergillus fumigatus—using the Clinical and Laboratory Standards Institute broth microdilution method. Structural characterization of these compounds was undertaken by mass spectrometry (MS) and nuclear magnetic resonance (NMR) following HPLC purification. Complete NMR assignments were obtained for the first time for filipins I and II. In vitro haemolytic assays revealed that the haemolytic action of these compounds relies largely on the presence of a hydroxyl function at C26, since derivatives lacking such moiety show remarkably reduced activity. Two of these derivatives, 1′-hydroxyfilipin I and filipin I, show decreased toxicity towards cholesterol-containing membranes while retaining potent antifungal activity, and could constitute excellent leads for the development of efficient pharmaceuticals, particularly against CryptococcosisThis research was funded by the Spanish Ministerio de Economía, Industria y Competitividad (grants BIO2013-42983-P and PCIN-2016-190 to J.F.A.), an FPU contract of the Ministerio de Educación, Cultura y Deporte (FPU13/01537 to A.P.), and contracts from the Junta de Castilla y León (to E.G.B.) and from the youth employment initiative (to E.J.), both co-financed by the European Social Fun

Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives

Background: Streptomyces filipinensis is the industrial producer of filipin, a pentaene macrolide, archetype of non-glycosylated polyenes, and widely used for the detection and the quantitation of cholesterol in biological membranes and as a tool for the diagnosis of Niemann-Pick type C disease. Genetic manipulations of polyene biosynthetic pathways have proven useful for the discovery of products with improved properties. Here, we describe the late biosynthetic steps for filipin III biosynthesis and strategies for the generation of bioactive filipin III derivatives at high yield. Results: A region of 13,778 base pairs of DNA from the S. filipinensis genome was isolated, sequenced, and characterized. Nine complete genes and two truncated ORFs were located. Disruption of genes proved that this genomic region is part of the biosynthetic cluster for the 28-membered ring of the polyene macrolide filipin. This set of genes includes two cytochrome P450 monooxygenase encoding genes, filC and filD, which are proposed to catalyse specific hydroxylations of the macrolide ring at C26 and C1' respectively. Gene deletion and complementation experiments provided evidence for their role during filipin III biosynthesis. Filipin III derivatives were accumulated by the recombinant mutants at high yield. These have been characterized by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification thus revealing the post-polyketide steps during polyene biosynthesis. Two alternative routes lead to the formation of filipin III from the initial product of polyketide synthase chain assembly and cyclization filipin I, one trough filipin II, and the other one trough 1'-hydroxyfilipin I, all filipin III intermediates being biologically active. Moreover, minimal inhibitory concentration values against Candida utilis and Saccharomyces cerevisiae were obtained for all filipin derivatives, finding that 1'-hydroxyfilipin and especially filipin II show remarkably enhanced antifungal bioactivity. Complete nuclear magnetic resonance assignments have been obtained for the first time for 1'-hydroxyfilipin I. Conclusions: This report reveals the existence of two alternative routes for filipin III formation and opens new possibilities for the generation of biologically active filipin derivatives at high yield and with improved propertiesThis work was supported by the Spanish Ministerio de Economía y Competitividad (Grants BIO2010-19911 and BIO2013-42983-P to JFA), F.P.U. fellowships of the Ministerio de Educación, Cultura y Deporte (AP2005-3644 to JSA, AP2007-02055 to TDP, FPU13/01537 to AP), a contract from the Junta de Castilla y León cofinanced by the European Social Fund (to EGB), and a fellowship from the Portuguese Fundação para a Ciência e a Tecnologia (SFRH/BD/64006/2009 to CMV

Molecular genetics of naringenin biosynthesis, a typical plant secondary metabolite produced by Streptomyces clavuligerus

Background: Some types of flavonoid intermediates seemed to be restricted to plants. Naringenin is a typical plant metabolite, that has never been reported to be produced in prokariotes. Naringenin is formed by the action of a chal cone synthase using as starter 4-coumaroyl-CoA, which in dicotyledonous plants derives from phenylalanine by the action of a phenylalanine ammonia lyase. Results: A compound produced by Streptomyces clavuligerus has been identified by LC–MS and NMR as naringenin and coelutes in HPLC with a naringenin standard. Genome mining of S. clavuligerus revealed the presence of a gene for a chalcone synthase (ncs), side by side to a gene encoding a P450 cytochrome (ncyP) and separated from a gene encoding a Pal/Tal ammonia lyase (tal). Deletion of any of these genes results in naringenin non producer mutants. Complementation with the deleted gene restores naringenin production in the transformants. Furthermore, narin genin production increases in cultures supplemented with phenylalanine or tyrosine. Conclusion: This is the first time that naringenin is reported to be produced naturally in a prokariote. Interestingly three non-clustered genes are involved in naringenin production, which is unusual for secondary metabolites. A ten tative pathway for naringenin biosynthesis has been proposed