Download Bioactive compounds from cultured (mainly marine) micro

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