Mansouramycins E–G, Cytotoxic Isoquinolinequinones from Marine Streptomycetes

Chemical investigation of the ethyl acetate extract from the marine-derived Streptomyces sp. isolate B1848 resulted in three new isoquinolinequinone derivatives, the mansouramycins E–G (1a–3a), in addition to the previously reported mansouramycins A (5) and D (6). Their structures were elucidated by computer-assisted interpretation of 1D and 2D NMR spectra, high-resolution mass spectrometry, and by comparison with related compounds. Cytotoxicity profiling of the mansouramycins in a panel of up to 36 tumor cell lines indicated a significant cytotoxicity and good tumor selectivity for mansouramycin F (2a), while the activity profile of E (1a) was less attractive

Untersuchungen zur Biosynthese von Indolalkaloiden aus den Basidiomyceten der Gattung Psilocybe

The goal of this doctoral thesis was the close examination of the biosynthesis of different indole alkaloides produced by different species of the fungal genus Psilocybe. This included the first characterization of the biosynthesis of psilocybin as the main psychotropic agent and the first discovery of the simultaneous production of β-carbolines. In the first step, the genome of P. cyanescens was sequenced and analyzed for characteristic genes and clusters or modifying enzymes. After the detection of the psilocybin biosynthesis cluster in Psilocybe, the cluster was expressed heterologous in E. coli and A. niger and an in vitro characterization of the psilocybin biosynthesis was done. Because of the raised pharmaceutical interest as well as some collaboration with the company Promega and the non-profit organization Usona, an expanded biocatalytic route for the in vitro production of psilocybin was established. The solution was the development of a continuous in vitro synthesis through a connection of the tryptophan synthase (TS) out of the basidiomycete P. cubensis with the biosynthesis route of psilocybin. Additionally, the substrate specifity of the TS were tested against different other halogenated indoles and for the first time different analogues of psilocybin could be produced biocatalytically with the same set of enzymes (Blei, Baldeweg et al. 2018). The generation of another derivate the 6-methylpsilocybin succeeded through an exchange with the TS of S. enterica (Fricke, Sherwood et al. 2019). Furthermore, the biosynthesis of the indole alkaloid l-Hypaphorin was characterized using the methyltransferase TrpM. Surprisingly no interference occured between the substrates of TrpM and the methyltransferase from the psilocybin biosynthesis PsiM because of strict substrate specifics. Supplemental to the already addressed indole alkaloides my work could identify further natural products out of the β-carboline class in the fruiting bodies and mycelia of different Psilocybe species. This was a big discovery in regards to the possible synergistic effects this could have on the psychoactive effect of Psilocybin through an inhibition of the monoaminoxidase enzyme (Blei, Dörner et al. 2019)

Antibacterial Activity of Endophytic Actinomycetes Isolated from the Medicinal Plant \u3cem\u3eVochysia divergens\u3c/em\u3e (Pantanal, Brazil)

Endophytic actinomycetes from medicinal plants produce a wide diversity of secondary metabolites (SM). However, to date, the knowledge about endophytes from Brazil remains scarce. Thus, we analyzed the antimicrobial potential of 10 actinomycetes isolated from the medicinal plant Vochysia divergens located in the Pantanal sul-mato-grossense, an unexplored wetland in Brazil. Strains were classified as belonging to the Aeromicrobium, Actinomadura, Microbacterium, Microbispora, Micrococcus, Sphaerisporangium, Streptomyces, and Williamsia genera, through morphological and 16S rRNA phylogenetic analyzes. A susceptibility analysis demonstrated that the strains were largely resistant to the antibiotics oxacillin and nalidixic acid. Additionally, different culture media (SG and R5A), and temperatures (28 and 36°C) were evaluated to select the best culture conditions to produce the active SM. All conditions were analyzed for active metabolites, and the best antibacterial activity was observed from metabolites produced with SG medium at 36°C. The LGMB491 (close related to Aeromicrobium ponti) extract showed the highest activity against methicillin-resistant Staphylococcus aureus (MRSA), with a MIC of 0.04 mg/mL, and it was selected for SM identification. Strain LGMB491 produced 1-acetyl-β-carboline (1), indole-3-carbaldehyde (2), 3-(hydroxyacetyl)-indole (4), brevianamide F (5), and cyclo-(L-Pro-L-Phe) (6) as major compounds with antibacterial activity. In this study, we add to the knowledge about the endophytic community from the medicinal plant V. divergens and report the isolation of rare actinomycetes that produce highly active metabolites

β-Carboline Alkaloids From the Deep-Sea Fungus Trichoderma sp. MCCC 3A01244 as a New Type of Anti-pulmonary Fibrosis Agent That Inhibits TGF-β/Smad Signaling Pathway

Pulmonary fibrosis is a scarring disease of lung tissue, which seriously threatens human health. Treatment options are currently limited, and effective strategies are still lacking. In the present study, 25 compounds were isolated from the deep-sea fungus Trichoderma sp. MCCC 3A01244. Among them, two β-carboline alkaloids, trichocarbolines A (1) and C (4) are new compounds. The chemical structures of these compounds were elucidated based on their HRESIMS, 1D and 2D NMR spectra, optical rotation calculation, and comparisons with data reported in the literature. Trichocarboline B [(+)- and (–)-enantiomers] had previously been synthesized, and this is its first report as a natural product. Their anti-pulmonary fibrosis (PF) activity and cytotoxicity were investigated. Compounds 1, 11, and 13 strongly inhibited TGF-β1-induced total collagen accumulation and showed low cytotoxicity against the HFL1 cell line. Further studies revealed compound 1 inhibited extracellular matrix (ECM) deposition by downregulating the expression of protein fibronectin (FN), proliferating cell nuclear antigen (PCNA), and α-smooth muscle actin (α-SMA). Mechanistic study revealed that compound 1 decreased pulmonary fibrosis by inhibiting the TGF-β/Smad signaling pathway. As a newly identified β-carboline alkaloid, compound 1 may be used as a lead compound for developing more efficient anti-pulmonary fibrosis agents

Implementation of the novel high-throughput Fungal Isolation one step Device method FIND and secondary metabolite extraction from the isolate <em>Heydenia</em> cf. <em>alpina</em>

Fungi are an important source of bioactive metabolites. The cultivation of source organisms is a crucial step in natural product isolation. With standard isolation methods for fungi it is difficult to isolate rare und underexamined fungi from environmental samples. The Fungal one-step IsolatioN Device (FIND) technology allows the isolation of rare fungi from terrestrial and marine samples. The FIND comprises a multi-chambered micro agar plate, where initially only one fungal part (e.g., hyphal cell, mycelial fragment or spore) is located in each chamber. After inoculation the device is placed back into the original natural environment of sample collection, to ensure favourable growth conditions. A comprehensive analysis of state-of-the-art isolation techniques for filamentous fungi emphasises the advantages of the FIND method compared to the standard isolation methods. With the FIND, it was possible to isolate 12 rare and underexamined axenic fungal cultures directly from their sample sources, i.e. terrestrial soil, sea sediment and seawater, without any purification or passaging steps. The taxonomy of nine of these strains could be determined to species level by DNA sequencing. One of the isolated fungi, i.e. Heydenia cf. alpina (strain 824) was never before investigated chemically and cultures of our strain yielded three novel compounds, namely heydenoic acid A (1), heydenoic acid B (2) and (3E,6E)-4,6,8-trimethyldodeca-3,6-diendioic acid (8). The first two compounds belong to the sesquiterpene family of bergamotenes. Using the latest approaches, i.e. HRMS based dereplication (GNPS) and deep learning NMR guided structure elucidation (SMART), fascinating insights into the metabolome of Heydenia cf. alpina were displayed, after challenging cultures of this strain with varying (partially extreme) conditions (OSMAC).Pilze bilden eine der wichtigsten Quellen für bioaktive Sekundärmetabolite. Die Kultivierung von Quellorganismen spielt eine entscheidende Rolle bei der Isolierung von Naturstoffen. Mit herkömmlichen Methoden ist es jedoch diffizil seltene und wenig erforschte Pilze aus Umweltproben zu isolieren. Die Fungal one-step IsolatioN Device (FIND) Technologie ermöglicht die Isolierung von seltenen Pilzen aus marinen und terrestrischen Proben. Das FIND beinhaltet Mikroagarplatten in getrennten Kammern, welche zuvor mit einem einzigen Pilzbestandteil (z.B. Hyphenzelle, Myzelfragment oder Spore) beladen wurden. Nach der Inokulation wird die Vorrichtung in die Umgebung der Probenentnahme zurückgeführt um optimale Wachstumsbedingungen zu garantieren. Eine vollständige Revision der gängigsten Isolierungsmethoden für filamentöse Pilze stellt die Vorteile der FIND Methode deutlich heraus. Mithilfe der FIND Methode wurden 12 seltene und wenig erforschte Stämme in Reinkultur direkt aus der Probe isoliert, welche aus Bodenproben, Meeressediment oder Meerwasser bestand, ohne die Notwendigkeit zusätzlicher Aufreinigungsschritte. Die Taxonomie von 9 der 12 Stämme konnte mittels DNA-Sequenzierung bis auf Spezieslevel bestimmt werden. Einer der isolierten Stämme, Heydenia cf. alpina (Stamm 824), wurde bisher noch nicht auf seine Chemie hin untersucht und Kulturen unseres Isolats produzierten drei neuartige Naturstoffe, nämlich heydenoic acid A (1), heydenoic acid B (2) und (3E,6E)-4,6,8-trimethyldodeca-3,6-diendioic acid (8). Die ersten beiden gehören zur Naturstoffklasse der Bergamotensesquiterpene. Durch den Einsatz modernster Analysetechniken, z.B. HRMS basierte Dereplizierung (GNPS) und deep learning NMR vermittelte Strukturaufklärung (SMART), konnten faszinierende Einblicke in das Metabolom von Heydenia cf. alpina gewonnen werden, nachdem der Stamm unterschiedlichen, teils extremen Wachstumsbedingungen ausgesetzt wurde (OSMAC)

Synthesis of carbolines and analogues via rhodhium-catalyzed [2+2+2] cycloaddition with alkynyl-ynamides : application to the total synthesis of alkaloids

The main research theme of this dissertation is the synthesis of g- and b-carbolines using a metal-catalyzed [2+2+2] cycloaddition strategy of tethered alkynyl-ynamides (diynes) with nitriles. g- and b-carbolines form the core of a large group of natural product and represent important targets for organic chemists. Many of these carbolines showed pharmacological effects ranging from anti-tumor to anxiolytic and anti-HIV activity. A model study with N-Ethynyl-N-tosyl-2-(2-phenylethynyl)aniline and methyl cyanoformate showed that rhodium-based catalysts promote efficiently the reaction. A further optimization showed that the regioselectivity of the reaction can be tuned by the choice of the solvent or by the catalytic system. Application to a larger scope of diynes showed that the regioselectivity strongly depends on the type of substitution of the alkynyl moieties, giving regioselectivities in the range g:b = 1/0 to g:b = 0/1. This [2+2+2] cycloaddition approach for the synthesis of the g- and b-carboline cores was successfully applied to the first total synthesis of Isoperlolyrine and the total synthesis of Perlolyrine. Extension of this strategy to heterocumulenes as cycloaddition partners allowed the synthesis of a g-carbolinone, a thiopyrano[3,4-b]indol-3-imine and thiopyranothiones.Diese Dissertation berichtet über der Synthese von g- and b-Carbolinen durch Metall-katalysierte [2+2+2]-Cycloadditionen von Alkynyl-Ynamiden (Diinen) mit Nitrilen. Die g- und b-Carboline sind die Grundgerüste vieler Naturstoffe, die wichtige Zielverbindungen in der organischen Chemie sind. Viele Carboline zeigen starke pharmakologische Wirkungen, insbesondere anxiolytische, Antitumor- und anti-HIV-Aktivitäten. Eine Modellstudie mit N-Ethinyl-N-tosyl-2-(2-phenylethinyl)anilin und Cyanameisensäuremethylester zeigte, daß Rhodium-basierte Katalysatoren die Reaktion effizient ermöglichen. Eine weitere Optimierung zeigte, daß die Regioselektivität von dem Lösungsmittel und dem Katalysator abhängig ist. Anwendung der Reaktion auf andere Diine zeigte auch eine Abhängigkeit der Regioselektiviät des Cyanideinbaus von den Substituenten an den Alkinen, es wurden Regioselektivitäten von g:b = 1/0 bis g:b = 0/1 gefunden. Diese Cycloadditionsmethode wurde zur Totalsynthese von Perlolyrin und Isoperlolyin erfolgreich angewendet. In Erweiterung der Methode konnten Heterokumulene als Cycloadditionenspartner erfolgreich zur Synthese von Verbindungen mit einem g-Carbolinon-, Thiopyrano[3,4-b]indol-3-imin- und Indolothiopyranothion-Gerüst genutzt werden.248 S

Enzyme-inhibitory secondary metabolites and their exudation in the marine-derived fungus <em>Epicoccum nigrum</em> link

The kingdom of fungi harbors a huge diversity of life forms. The Ascomycota in particular are a prolific source of medicinal drugs and new lead structures with potent bioactivities. During this work, two marine-derived strains of Epicoccum nigrum – strains 749 and 800 – were assessed for their production of bioactive secondary metabolites, which could serve as lead structures for medicinal use. In this way, 12 compounds with different scaffolds were discovered and identified. Three of these structures are new chemical entities, 5´-6´-dihydroxyacetosellin, 1F4-1-6 and epicaronic acid. Pronounced inhibitory activities against human serine- and cysteine-proteases were revealed for epipyrones. Furthermore, epipyrones were shown to be the first inhibitors of human cerebroside sulfotransferase (hCST). Through the use of trace metals, the biosynthesis of the two main secondary metabolites – epipyrones and acetosellin – could be controlled. The optimized production of acetosellin enabled studies on its unusual biosynthetic origin. Thus, the assembly from two separate polyketide-derived chains was shown using 13C-labeled sodium acetate. Furthermore, the excretion of epipyrones and acetosellin into guttation droplets was studied in dependence of the trace metal content in the medium. Thereby, the eligibility of guttation droplets for the study of the secondary metabolism in fungi was to be studied. Surprisingly, the concentration of epipyrones was strongly elevated in the guttation fluid under optimal production conditions (in comparison to the culture extracts). In contrary, acetosellin was mainly found in the culture extracts. The underlying mechanisms of this observation could be elucidated in future studies

Gênero Microbispora : reclassificação filogenética, bioprospecção e identificação de metabólitos secundários

Orientadora : Profª Drª Chirlei GlienkeCo-orientadora : Drª Josiane Gomes Figueiredo e Dr. Jurgen RohrTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 27/08/2015Inclui referênciasÁrea de concentraçãoResumo: A ioprospecção de microrganismos hoje é a fonte mais promissora para obtenção de novos compostos bioativos. Actinomicetos do solo pertencentes ao gênero Streptomyces, produzem uma vasta gama de compostos amplamente utilizados pela industría farmacêutica e agronômica. Porém, devido ao grande número de pesquisas envolvendo estes microrganismos a probablidade da descoberta de novos compostos a partir de linhagens isoladas do solo torna-se cada vez menos provável, levando a exploração de actinomicetos de outras fontes naturais, como por exemplo, isolados de plantas medicinais. Neste contexto, o presente trabalho objetivou o isolamento, bioprospecção, e a identificação de actinomicetos endofíticos da planta medicinal Vochysia divergens. O trabalho foi dividido em 3 capítulos voltados a bioprospecção e avaliação da atividade biológica dos isolados endofíticos - principalmente pertencentes ao gênero Microbispora - seguido de identificação dos compostos produzidos pela linhagem mais promissora e posterior estudo filogenético dos isolados pertencentes ao gênero Microbispora. O primeiro capítulo, intitulado "Antitumor, antioxidant and antibacterial activities of secondary metabolites extracted by endophytic actinomycetes isolated from Vochysia divergens", explorou o isolamento e biodiversidade de actinomicetos isolados da planta Vochysia divergens, bem como a capacidade de seis isolados em produzir compostos com atividade antibacteriana, antioxidante e antitumoral. O segundo capítulo, "Microbispora sp. LGMB259 Endophytic Actinomycete Isolated from Vochysia divergens (Pantanal, Brazil) Producing ?- Carbolines and Indoles with Biological Activity" objetivou o isolamento e identificação de compostos produzidos pela linhagem de Microbispora sp. LGMB259. No trabalho foram identificados sete compostos, quatro ?-carbolinas e três indóis, sendo que o composto 1-vinil- ?-carbolina-3-ácido carboxílico foi responsável pela atividade biológica desta linhagem, o qual apresentou atividade antibacteriana, antifúngica e citotóxica. O último capítulo, intitulado "Multilocus Sequence Analysis of the Genus Microbispora" baseou-se em análise multigênica utilizando os genes 16S rRNA, 23S rRNA, gyrB e rpoB para o estudo filogenético dentro do gênero Microbispora. As análises realizadas sugerem que as espécies M. amethystogenes, M. chromogenes, M. karnatakensis, M. parva, M. aerata, M. thermodiastatica e M. thermorosea são espécies distintas da espécie M. rosea; diferentemente do proposto na literatura por Miyadoh et al. (1990). M. aerata e M. thermodiastica compartilharam elevada similaridade genética e provavelmente pertencem a mesma espécie, assim como M. indica e M. rosea. As linhagens endofíticas pertencentes aos clusters Microbispora sp. 1, Microbispora sp. 2 e Microbispora sp. 3 são diferentes das espécies de Microbispora previamente descritas, sendo que mais estudos são necessários para a descrição das mesmas como novas espécies. Foi proposto também a análise concatenada dos genes gyrB-rpoB como uma alternativa à técnica de hibridização de DNA para a identificação de espécies dentro do gênero Microbispora, e o valor de 98,0% como o valor de homologia mínimo para classificar os isolados de Microbispora como pertencentes a mesma espécie. Palavras-chave: Vochysia divergens - Gênero Microbispora - metabólitos secundários - ?- carbolinas - reclassificação filogenéticaAbstract: The Bioprospecting of Microorganisms is the most promising source for new bioactive compounds. Actinomycetes - mainly that belonging to the genus Streptomyces - isolated from soil produced a wide range of bioactive compounds widely used by the pharmaceutical and agronomic industries. However, due to the large number of research involving these microorganisms the probablidade of discovery novel compounds isolated from soil strains is increasingly less, leading to exploration of actinomycetes isolated from others natural sources such as, endophytic isolated by medicial plants. In this context, our objectives were the isolation, bioprospecting, and the identification of endophytic actinomycetes from medicinal plant Vochysia divergens. The work was divided into three chapters and includes bioprospecting and avaliation of biological activity of endophytic isolates - mainly belonging to the genus Microbispora - followed by identification of compounds produced by the most promising strain and subsequent phylogenetic study of isolates belonging to Microbispora genus. The first chapter, entitled "Antitumor, antioxidant and antibacterial activities of secondary metabolites extracted by endophytic actinomycetes isolated from Vochysia divergens" is about the isolation and biodiversity of actinomycetes from the medicinal plant Vochysia divergens, and the ability of six isolates to produce compounds with antibacterial, antioxidant and antitumor activities. The second chapter, "Microbispora sp. LGMB259 Endophytic actinomycete Isolated from Vochysia divergens (Pantanal, Brazil) Producing ?-Carbolines and Indoles with Biological Activity" aimed to isolate and identify compounds produced by the strain Microbispora sp. LGMB259. We identified seven compounds produced by this strain - four ?-carbolines and three indoles- wherein the compound 1-vinyl-?-carboline-3-carboxylic acid was responsible for the biological activities of this strain, which showed antibacterial, antifungal and cytotoxic activities. The last chapter, entitled "Multilocus Sequence Analysis of the Genus Microbispora" was based on multigene analysis using the 16S rRNA, 23S rRNA, gyrB and rpoB for phylogenetic studies in the Microbispora genus. The analyzes suggest that the species M. amethystogenes, M. chromogenes, M. karnatakensis, M. parva, M. aerata, M. thermodiastatica and M. thermorosea are distinct from M. rosea; different by proposed for Miyadoh et al. (1990). However M. aerata and M. thermodiastatica probably are the same species, as well M. indica and M. rosea. Endophytic isolates belongs to the clusters Microbispora sp. 1, Microbispora sp. 2 and Microbispora sp. 3 are different from the Microbispora species previous described and futures studies are required to describe these species. It is also proposed the concatenated analyses of gyrB-rpoB genes as a useful alternative to DNA-DNA hybridization for the identification and phylogenetic analysis in the Microbispora genus, and values less than 98,0% to characterizate and determine relationship at species level. Key-words: Vochysia divergens - Microbispora Genus - secondary metabolites - ?- carbolines - phylogenetic reclassificatio