On the Synthesis of lugdunomycins and Related Metabolites from Streptomyces

Some bacteria make very useful compounds, such as antibiotics and anti-cancer medicines. Especially the so-called streptomycetes (a kind of soil bacteria), are very good at that. But how they exactly do this on a chemical level is not always well understood. For our study we were interested in one particular compound; lugdunomycin, also produced by a streptomycete. In the lab we tried to mimic how lugdunomycin is made by the streptomycete. We predicted on the basis of our initial chemical and biological knowledge on lugdunomycin, that two other specific compounds (say building blocks) were required. We were also wondering if the bacterium had a means to couple the two compounds together like two Lego pieces, which yields lugdunomycin. To answer the main question “how does the bacteria make lugdnomycin?”, we had to make the two other compounds in the lab, by means of chemical synthesis. Synthesis is a means to make chemical compounds by means performing of chemical reactions. We succeeded to synthesise the two building blocks. After elaborate experimentation, we discovered that the two compounds react spontaneously, giving lugdunomycin, alongside with other compounds that look a lot like lugdunomycin. Eventually it was succeeded to make several milligrams of lugdunomycin. We have proven that the chemical structure of the lugdunomycin from the lab is exactly matching with lugdunomycin from the bacterium. Thereby, we have shown that our hypothesis about how the bacterium makes lugdunomycin, is likely correct. The knowledge that originated from this research is important, because it gives new insights about the ways streptomycets can produce new compounds

Halophiles and Their Biomolecules: Recent Advances and Future Applications in Biomedicine

The organisms thriving under extreme conditions better than any other organism living on Earth, fascinate by their hostile growing parameters, physiological features, and their production of valuable bioactive metabolites. This is the case of microorganisms (bacteria, archaea, and fungi) that grow optimally at high salinities and are able to produce biomolecules of pharmaceutical interest for therapeutic applications. As along as the microbiota is being approached by massive sequencing, novel insights are revealing the environmental conditions on which the compounds are produced in the microbial community without more stress than sharing the same substratum with their peers, the salt. In this review are reported the molecules described and produced by halophilic microorganisms with a spectrum of action in vitro: antimicrobial and anticancer. The action mechanisms of these molecules, the urgent need to introduce alternative lead compounds and the current aspects on the exploitation and its limitations are discussed.España, MINECO CGL2017-83385-

O-Verbrückte Angucyclinone, Verfahren zu ihrer Herstellung, sie enthaltende Arzneimittel und deren Verwendung

Es werden das verbrückte Angucyclinon Gephyromycin (1a) und Derivate dieser Verbindung nach der allgemeinen Formel 1 beschrieben sowie ein Verfahren zu ihrer Herstellung. Diese Substanzen erhöhen die intrazelluläre Calciumkonzentration in Neuronen. Weiterhin besitzen sie eine antibiotische Wirkung gegenüber Gram-positiven Bakterien

Mayamycin, a Cytotoxic Polyketide from aStreptomycesStrain Isolated from the Marine SpongeHalichondria panicea

A new benz[a]anthracene derivative called mayamycin (1) was identified in cultures of Streptomyces sp. strain HB202, which was isolated from the marine sponge Halichondria panicea and selected because of its profound antibiotic activity. The ability to produce aromatic polyketides was indicated by genetic analyses, demonstrating the presence of a type II polyketide synthase. The production of mayamycin (1) was induced by variation of the culture conditions. The structure of 1 was elucidated by HPLC-UV/MS and NMR spectroscopy. Mayamycin (1) exhibited potent cytotoxic activity against eight human cancer cell lines and showed activity against several bacteria including antibiotic-resistant strains

Selective Oxidative Dearomatization of Angular Tetracyclic Phenols by Controlled Irradiation under Air: Synthesis of an Angucyclinone-Type Double Peroxide with Anticancer Properties

Angular tetracyclic p-peroxyquinols, p-quinols and a pentacyclic double peroxide, showing anticancer properties, were Selective oxidative dearomatization of angular tetracyclic phenols by controlled irradiation under air: synthesis of an angucyclinone‐type double peroxide with anticancer properties María J. Cabrera-Afonso,a Silvia R. Lucena,b Ángeles Juarranz,b Antonio Urbano,*a,c M. Carmen Carreño*a,c aDepartamento de Química Orgánica, Universidad Autónoma de Madrid (UAM), Cantoblanco, 28049-Madrid, Spain. bDepartamento de Biología, UAM, Cantoblanco, 28049-Madrid, Spain. cInstitute for Advanced Research in Chemical Sciences (IAdChem), UAM, Cantoblanco, 28049-Madrid, Spain. Supporting Information Placeholder synthesized from the corresponding phenols by an environmentally friendly solvent- and wavelength-controlled irradiation under air in the absence of an external photosensitizerWe thank MINECO (Grants CTQ2017-83309-P, CTQ2014-53894R and FIS PI15/00974) for financial suppor

Functional and structural analysis of the angucyclinone ketoreductase SimC7

The natural product simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The biosynthetic simocyclinone (sim) gene cluster has been sequenced and a hypothetical biosynthetic pathway has been proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase working in trans with two monofunctional enzymes. SimC7, which belongs to the short-chain dehydrogenase/reductase (SDR) superfamily of proteins, was proposed to supply the dehydratase activity missing from two modules of the polyketide synthase. In this study, I report the structure and function of recombinant S. antibioticus SimC7. Because the natural simocyclinone producer is genetically intractable, the ~72-kb sim cluster was isolated on a single phage artificial chromosome (PAC) clone for heterologous production in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in production of a novel simocyclinone, 7-oxo-SD8, which carried a normal tetraene linker but was altered in the angucyclinone. I demonstrate that SimC7 is an NAD(P)H-dependent polyketide ketoreductase that catalyses the reduction of the C-7 carbonyl of the angucyclinone and the resulting hydroxyl group is essential for antibiotic activity. SimC7 shares little sequence similarity with characterized ketoreductases, suggesting it might have a distinct catalytic mechanism. To investigate this possibility, I determined the structures of SimC7 alone, in complex with NADP+, and in complex with NADP+ and the substrate 7-oxo-SD8. These structures show that SimC7 is distinct from previously characterized polyketide ketoreductases, lacking the conserved catalytic triad (Ser-Tyr-Lys), including the active site tyrosine that acts as central acid-base catalyst in canonical SDR proteins. Taken together with functional analyses of active site mutants, my data suggest SimC7 catalyses a substrate-assisted, two-step reaction for reduction of the C-7 carbonyl group involving intramolecular transfer of a substrate-derived proton to generate a phenolate intermediate

Structural and functional Studies of angucycline tailoring enzymes

Polyketides are a diverse group of natural products produced in many bacteria, fungi and plants. These metabolites have diverse biological activities and several members of this group are in clinical use as antibiotics, anticancer agents, antifungals and immunosuppressants. The different polyketides are produced by polyketide synthases, which catalyze the condensation of extender units into various polyketide scaffolds. After the biosynthesis of the polyketide backbone, more versatility is created to the molecule by tailoring enzymes catalyzing for instance hydroxylations, methylations and glycosylations. Flavoprotein monooxygenases (FPMO) and short-chain alcohol dehydrogenases/reductases (SDR) are two enzyme families that catalyze unusual tailoring reactions in the biosynthesis of natural products. In the experimental section, functions of homologous FPMO and SDR tailoring enzymes from five different angucycline pathways were studied in vitro. The results revealed how different angucyclinones are produced from a common intermediate and that FPMO JadH and SDR LanV are responsible for the divergence of jadomycins and landomycins, respectively, from other angucyclines. Structural studies of these tailoring enzymes revealed differences between homologous enzymes and enabled the use of structure-based protein engineering. Mutagenesis experiments gave important information about the enzymes behind the evolution of distinct angucycline metabolites. These experiments revealed a correlation between the substrate inhibition and bi-functionality in JadH homologue PgaE. In the case of LanV, analysis of mutagenesis results revealed that the difference between the stereospecificities of LanV and its homologues CabV and UrdMred is unexpectedly related to the conformation of the substrate rather than to the structure of the enzyme. Altogether, the results presented here have improved our knowledge about different steps of angucycline biosynthesis and the reaction mechanisms used by the tailoring enzymes behind these steps. This information can hopefully be used to modify these enzymes to produce novel metabolites, which have new biological targets or possess novel modes-of-action. The understanding of these unusual enzyme mechanisms is also interesting to enzymologists outside the field of natural product research.Siirretty Doriast

Investigations of Novel Mechanisms of Action for Anti-Bacterial and Anti-Cancer Agent Development

The development of drugs and therapeutic agents for combating infections and human malignancies continues to be a forefront area in both academic and industrial research. This is driven by the rapid emergence of multi-drug resistant bacterial strains and accumulating mutations in cancer targets that is quickly rendering our current arsenal of drugs ineffective for these therapies. Unless new drugs with novel mechanisms of action are identified and developed at a faster pace, we face a losing battle in managing these diseases. The first part of this work concerns with the natural product Simocyclinone D8 (SD8). Simocyclinone D8 is an angucyclinone antibiotic that inhibits DNA gyrase with a novel mechanism of action that has been termed competitive inhibition. Simocyclinone D8 was found to inhibit the growth of both Gram-(+ve) and Gram-(–ve) organisms and also inhibit a fluoroquinolone resistant mutant of DNA gyrase. Inspired by the structure and novel mechanism of action that SD8 displays, we synthesized analogues based on the co-crystal structure of SD8 with DNA gyrase. These compounds were found to inhibit DNA gyrase, albeit by a different mechanism of action than that of SD8. We also conducted studies towards the total chemical synthesis of SD8 and made three out of the four fragments in SD8 in decent yields. The second part of this work is focused on the development of a substrate-competitive covalent inhibitor for protein kinase B (AKT). AKT is a valid target for cancer research with two compounds currently in late stage clinical trials. Developing substrate- competitive inhibitors for kinases is a novel approach in targeting them, with very few examples in the literature. This mechanism has been postulated to overcome common resistance mutations that cancer targets harbor. A major drawback in this approach is the low binding affinity for peptide substrates by kinases. We circumvented this problem of affinity by utilizing a covalent mode of binding and synthesized a potent non-peptide active-site directed irreversible compound that inhibits AKT. Further studies on this compound are underway and are expected to yield a compound that can be used as a therapeutic agent or as a probe for AKT

Antibiotic R2, a new angucyclinone compound from Streptosporangium sp. Sg3

Antibiotic R2, a new angucyclinone compound from Streptosporangium sp. Sg

SEQUENCING AND ANALYSIS OF PUTATIVE 3D-4H RING CYCLASE GENE lndF OF STREPTOMYCES GLOBISPORUS 1912 LANDOMYCIN E BIOSYNTHETIC GENE CLUSTER

DNA fragment of landomycin E biosynthesis gene cluster 700 bp in size has been completely sequenced. 3?-end of lndE (oxygenase) was identified, 5?-end of lndA (ketosynthase) and entire ORF for previously not sequenced lanF homologue, lndF. Analysis of lndF putative translation product revealed that it is highly conservative in comparison with other known cyclases from antibiotic biosynthesis gene clusters. Unlike urdamycin, jadomycin biosynthetic clusters, lndF and lndA are uncoupled, as well as genes lanF and lanA. Genes lndF and lndA are not preceded by direct or inverted repeats, putative sites for binding of transcriptional activator LndI. In contrast, lanF is flanked at it's 5?-end by three direct repeats, possible target for regulatory protein LanI.L. Castro is acknowledged for help in sequencing. This work was supported by INTAS grant YSF 00-208 to B.O