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Bioactive compounds from cultured (mainly marine) micro-organisms Translational Signaling Group (S. Døskeland), Department of biomedicine, University of Bergen Major contributors: Lars Herfindal, Silje Oftedal, Lene Myhren, ….. Origin of micro-organisms: Supported from: Univ. Helsinki: Kaarina Sivonen, J. Jokela, M.Wahlsten Univ. Bergen: Gjert Knutsen, Siv Prestegard Univ. Porto: Vitor Vasconçelos The NIVA collection: B. Edvardsen, O. Skulberg SINTEF / NTNU: H. Sletta, S. Zotchev Bioprospecting – for what? 1. Compounds that kill or incapacitate cancer cells without harming the patient 2. Compounds preventing pathological blood platelet aggregation without causing bleeding 3. Compounds that block or stimulate major cell processes (like key signal pathways) by binding precise molecular targets (can serve as cell biology reagents and drug scaffolds) Requires optimism: A looong way from a hit on screening, via pure compound, extensive validation on cell lines, validation in small animal models, toxicity studies in rodents and non-rodents, phase I/II clinical trials, negotiations with “Big Pharma”, phase III/IV trials, approval of drug, fame + money Primary bioactivity screens: Viable AML cells 1) Induction of leukemia (AML) cell death *AML hits adults; is most frequent leukemia; > 50% mortality *AML is suited for early phase clinical testing; we have in vivo mouse models for AML and access to a biobank of viable patient AML cells. Assay is by tetrazolium-based colorimetry + microscopic evaluation of cell death: Apoptotic AML cells 2a) Induction of primary hepatocyte death •Helps eliminate anti-cancer candidates attacking normal cells Normal Apoptotic Necrotic 2b) Induction of cytoskeletal abnormality (helps identify useful cell biology reagents) control Effect of 1h incubation with various benthic cyanobacterial extracts Bioprospecting - why search cultured micro-organisms? 1. Provides stable source of bioactive compound 2. Less risk of contamination (by compounds from other organisms in the environment of the original isolate) 3. Allows cloning of genes responsible for biosynthesis of compound of interest (useful for transgene production by microbes or for isolating enzymes that can be used for critical steroselective biochemical synthesis steps) 4. Allows metabolic labeling of compound (like 15N, 13C for MR and/or MS analyses) 5. Environmentally sustainable (no need to go back to e.g. coral reef to obtain high amounts of biomass) BUT: 6. Will production of bioactive substance persist upon long term culture? Microbes can retain bioactive compound production during long periods of culture: •A cyanobacterium collected in 1956 still produced same cytotoxins in 2007 (collaboration with NIVA; Oftedal et al. J Industr. Microbiol Biotechnol 2010) •A diatom produced similar amount of bioactive compound ”B59a,b” in 2010 and 2000: 2000 : 2010: *An example of retained bioactivity production in nature: Cyanobacterium of same species collected (by NIVA) from same location in 1982 and 1998 had unaltered toxin profile BUT: Promising anti-leukemic activities in cyanobacterial cultures that were “lost” Involvement of caspases Synergy with daunorubicin Bioprospecting - why search cultured micro-organisms? 1. Provides stable source of bioactive compound 2. Less risk of contamination (by compounds from other organisms in the environment of the original isolate) 3. Allows cloning of genes responsible for biosynthesis of compound of interest (useful for transgene production by microbes or for isolating enzymes that can be used for critical steroselective biochemical synthesis steps) 4. Allows metabolic labeling of compound (like 15N, 13C for MR and/or MS analyses) 5. Environmentally sustainable (no need to go back to e.g. coral reef to obtain high amounts of biomass) BUT: 6. Will production of bioactive substance persist upon long term culture? 7. Micro-organisms are extremely diverse, numerous and nearly ubiquitous (everywhere). So – where start “digging” or “diving” for the biological gold? Where search micro-organisms with novel bioactive compounds? Marine environment has highest microbial biodiversity, and is least exploited But: Culturing of marine microbes in general more demanding Interesting extreme habitats may be hard to come by (deep mid-ocean vents, oil-containing subsea reservoirs) ------- Where in the sea expect microbes producing biological weapons against eukaryotes, including cancer cells? Benthic biotopes – exposed to fierce competition and eukaryotic grazers/predators Bottom-dwelling organisms may be hard to mass-culture in suspension, but culturing may be the only way to produce enough biomass from isolates from small benthic biotopes, like a small rock in the tidal zone. Micro-organisms extracted •Cyanobacteria •Microalgae •Diatoms •Green-algae •Actinomycetes •Many were marine, non-planctonic (benthic) •All could be grown in batch culture Cytotoxic cyanobacteria. Photo: Matti Wahlsten, Univ Helsinki (0.1 to 100 litres) Basic questions: •Are some groups of micro-organisms inherently more likely than others to express ”bioactivity aginst erukaryotic processes”? •Does microbial habitat predict probability of expressing interesting bioactivity? Diatom cultures. Photo: Siv Prestegard, Univ. of Bergen Primary hepatocytes (from rat liver) A: Aqueous extract B: Methanol extract AML cells (IPC-81) * * ** * * * ** * * * * C: * Organic extract Most toxin activity in Benthic Anabaena*cyanobacteria Note different no sequence Some micro-organisms are more prone to express ”interesting compounds” Fraction of tested isolates with high content of modulators of either: AML/hepatocyte cell death/deformation, blood platelet aggregation, inhibition of thrombin-induced aggregation, and OATP1B1/3 mediated transport. Hit rate n=10 n>80 n>100 n=19 * *Higher for benthic Anabaena B59 (KA-1) - a novel substance with unusual biological properties from the marine diatom Craspedostauros britannicus Isolated near Bergen by Siv Prestegard / Gjert Knutsen) – Benthic Blood platelets are extremely B59 (KA-1) sensitive - via PI3 pathway perturbation? Inhibition of aggregation Platelet aggregation B59/KA-1 inhibits PI3-kinase product accumulation in thrombin-stimulated HL60 leukemia cells (A,B) and thrombinactivated platelets (C). Affects also platelet tyrosine phosphorylation (D). B59/KA-1 inhibits platelet activation and reverses platelet aggregation like the PI3kinase inhibitor Wortmannin The intact shape change response to thrombin peptide indicates that the platelets are viable PI3-kinase is pivotal in survival signalling • B59 induces rapid cell death Morphological alterations of HL-60 AML leukemia cells already within 5-10 minutes of B59 exposure Autophagic (mitophagic) vesicles observed after prolonged exposure to low B59 concentration Control, HL-60 Control, NRK B59 > loss of mitochondrial membrane potential B59 > disintegration of the actin cytoskeleton + B59 • One microbial strategy is to ”starve” predators and competitors by blocking important surface transport proteins •We identified, purified and structure-elucidated (w. Sivonen, Helsinki) a cyanobacterial cyclic peptide (Ncp-M1) that selectively blocks the important human drug transporters OATP1B1 and OATP1B3. (Incidentally these transporters also carry the hepatotoxins microcystin and nodularin, so Ncp-M1 protects hepatocytes against them) Surface Nucleus Untreated Microycystin Microcystin + Ncp-M1 Herfindal, Myhren et al. Mol Pharm, 2011 Analogs of Ncp-M1 are being synthesized in collaboration with chemists in Tromsø and Bergen O Gly I Other types of bioactivity Gly II HN O NH NH O OH •Detergent-like activity •Anabaenolysin from a cyanobacterium perforates membranes containing cholesterol •Mitochondrial membrane contain small amounts of cholesterol Liposome leakage H N HO O O O Illustration of usefulness of working with cultured organisms for structure elucidation by metabolic labeling: Metabolic labeling of cyanobacteria with 15N revealed that anabaenolysin contains four nitrogen atoms Cell surface becomes leaky (with enhanced import of small peptide toxins and to some degree cDNA) BUT: mitochondria are not targeted by anabaenolysins Isolated mitochondria have normal morphology and intact membrane Collaborators Norway Department of Biomedicine, University of Bergen Leader: Prof. Stein Ove Døskeland Post. Doc. Lars Herfindal PhD. Linn Oftedal MSc. Lene Myhren Assoc. Prof. Frode Selheim Ing. Nina Lied Larsen Department of Biology University of Bergen Leader: Prof. Gjert Knutsen Post. Doc. Siv Prestegård Finland Department of Food and Environmental Sciences University of Helsinki Leader: Acad. Prof. Kaarina Sivonen Post. Doc. Jouni Jokela Tech. Matti Wahlsten Department of Biotechnology, NTNU Leader: Prof. Sergey Zotchev Dr. Espen Fjærvik Department of Industrial Biotechnology, SINTEF Leader: Dr. Trond Ellingsen, Marbio, University of Tromsø Leader: Jeanette H Andersen Post. Doc. Maria Perander NIVA Leader: Prof. Olav Skulberg Prof. Bente Edvardsen (UiO) Portugal Department of Zoology and Anthropology University of Porto Leader: Prof. Vitor Vasconçelos Post. Doc. Rosario Màrtens Republic of South Africa Department of Organic Chemistry University of the Free State, Bloemfontain Prof. Andrew Marston