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Two Bioassays for Cyanobacterial Neuro-active Metabolites

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Nogle LM, Okino T, Gerwick WH. ' Antillatoxin B, a neurotoxic lipopeptide from the marine ... Marine sponge compound Lembehyne A induces neuritogenesis ... – PowerPoint PPT presentation

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Title: Two Bioassays for Cyanobacterial Neuro-active Metabolites


1
Two Bioassays for Cyanobacterial Neuro-active
Metabolites
  • Amanda Cordes, Dr. Doug Goeger, Dr. William
    Gerwick

2
The Gerwick Group
  • Purpose Study of marine algae to discover novel
    compounds and develop biomedicinal agents
  • Focus Marine Cyanobacteria blue green algae

3
Discoveries
Applications
  • Antillatoxin
  • Kalkitoxin
  • Curacin A
  • Anticancer agents
  • Anesthetics
  • Agrichemicals

Nogle LM, Okino T, Gerwick WH. "Antillatoxin B,
a neurotoxic lipopeptide from the marine
cyanobacterium Lyngbya majuscula." Journal of
Natural Products 2001, 64983-985. Lu, W.I.,
F.W. Berman, T. Okino, F. Yokokawa, T. Shioiri,
W.H. Gerwick, and T.F. Murray (2001) Antillatoxin
is a novel marine cyanobacterial toxin that
potently activates voltage-gated sodium channels.
Proceedings of the National Academy of Sciences.
(Submitted for publication). Milligan, K. E., B
Marquez, R. T. Williamson, and W. H. Gerwick
(2000) Lyngbyabellin B, a toxic and antifungal
secondary metabolite from the marine
cyanobacterium Lyngbya majuscula. J. Natural
Products 63 1440-1443. Verdier-Pinard, P., N.
Sitachitta, J.V. Rossi, D.L. Sackett, W.H.
Gerwick and E. Hamel (1999) Biosynthesis of
radiolabeled curacin a and its rapid and
apparently irreversible binding to the colchicine
site of tubulin. Arch. Biochem. Biophys. 370
51-58.
4
Part 1. Detection and Characterization of
Cyanobacterial Neurotoxins using Zebrafish
Behavior
5
Goals
  • Determine viability of zebrafish as toxicity
    model using known neurotoxins
  • Apply model to marine cyanobacterial extracts to
    detect biological activity and characterize their
    pharmacology

6
Zebrafish(Danio rerio)
http//edtech.tph.wku.edu/jbilotta/neuro.htm
7
Experiment
  • Place fish in 100 mL of water
  • Expose fish to toxin in increasing amounts until
    response is observed
  • Isolate fish overnight to observe recovery
  • Verify response on other fish
  • In some cases, increase dose to obtain a more
    pronounced response

8
Amount of Toxin Required to Induce Response in
100 mL of Water
  • Ethanol 33 mg
  • Ouabain 3.27 mg
  • Nicotine 0.25 mg
  • Caffeine 0.68 mg

Amount of Compound (mg)
9
Responses Observed
  • Ethanol Fish at bottom, often bouncing
  • Ouabain Fish circling, may also go to bottom
  • Nicotine Fish circling beaker at surface, tilted
    upwards, quivering
  • Caffeine Fish holding at bottom

10
Results of Blind TestsOne compound per beaker,
fish introduced simultaneously
  • Ethanol, Ouabain, and Control All three systems
    were correctly identified
  • Ethanol, Ouabain, Nicotine, Caffeine, and
    Control Only Nicotine was correctly identified

11
Conclusions on Zebrafish Model
Zebrafish are not a viable model for detection
and characterization of cyanobacterial
neurotoxins
  • Fish to fish variability is high
  • Large quantities of toxin required to induce
    response

12
Part 2. Ability of Cyanobacterial Metabolites to
Induce Neuritogenesis
13
First Defining Some Terms
  • Neuro 2a Neuroblastoma Cells A mouse cancer cell
    line deriving from neurons
  • Neuron Cell with capability of transmitting
    electric signals, found in nervous system
  • Neurite Long, branching outgrowth from a neuron
  • Differentiate Cells mature, adopt distinctive
    functions, less likely to divide

http//cancerweb.ncl.ac.uk
14
Neuro 2a Cells with Neurites
http//users.jagunet.com/meledy/cell2.jpg
15
Background
  • Marine sponge compound Lembehyne A induces
    neuritogenesis
  • Both Lactacystin and 8-Bromo-Cyclic AMP
    (8-Br-cAMP) also induce neuritogenesis

Aoki, S., Matsui, K., Takata, T., Hong, W., and
Kobayashi, M. (2001) Lembehyne A, a Spongean
Polyacetylene, Induces Neuronal Differentiation
in Neuroblastoma Cell. Biochem. Biophys Res
Commun. 289, 558-563. Fenteany, G., and
Schreiber, S. (1998) Lactacystin, Proteasome
Function, and Cell Fate. J Biol. Chem. 273,
8545-8548.
16
Experiment
  • Neuro 2a Cells are cultured in 60 mm dishes
  • Cells then exposed to novel marine extracts,
    observed in 24 hr. increments
  • Neurite outgrowth compared against untreated
    control cells and ones treated with Lactacystin
    and with 8-Br-cAMP, known outgrowth promoters

17
Neurite Outgrowth Controls
Treated
Control
18
Screening for Pure Cyanobacterial Natural
Products that Induce Neurite Outgrowth
  • Based on of cells with outgrowths after 24 hours

19
Inactive Compounds
  • Octadec-5-yne-7Z,9Z,12Z-trienoic Acid
  • 10 ug/mL 0.65
  • 3 ug/mL 0.86
  • Malhamensilipin A
  • 10 ug/mL 2.2
  • 3 ug/mL 2.
  • Avrainvilleol
  • 10 ug/mL 2.2
  • 3 ug/mL 4.2

20
Contd
  • Gloiosiphone A Dimethyl Ether
  • 10 ug/mL 2.6
  • 3 ug/mL 3.8
  • Pacifenol
  • 10 ug/mL 0.68
  • 3 ug/mL 3.3
  • Dilophic Acid
  • 10 ug/mL 1.2
  • 3 ug/mL 2.8

21
Contd
  • Cymathere Lactone
  • 10 ug/mL 1.5
  • 3 ug/mL 2.4
  • Malyngolide
  • 10 ug/mL 0.59
  • 3 ug/mL 2.8
  • Spiro-bis-pinnaketal
  • 10 ug/mL 1.6
  • 3 ug/mL 2.4

22
Contd
  • Palisadin A
  • 10 ug/mL 2.4
  • 3 ug/mL 2.9
  • Carmabin A
  • 10 ug/mL 0
  • 3 ug/mL 2.8
  • Martensia Indole
  • 10 ug/mL 0
  • 3 ug/mL 1.2

23
Toxic Compounds
  • Hormothamnione
  • 10 ug/mL toxic
  • 3 ug/mL 1.1
  • Malyngamide F Acetate
  • 10 ug/mL toxic
  • 3 ug/mL toxic
  • Ptilodene Methyl Ester
  • 10 ug/mL toxic
  • 3 ug/mL 1.8

24
Contd
  • Cymopol
  • 10 ug/mL toxic
  • 3 ug/mL 1.5

25
Active Compounds
  • Allolaurinterol
  • 10ug/mL 3.3
  • 3ug/mL 6.2

26
Contd
  • Methyl 12S-HETE
  • 10ug/mL 2.3
  • 3ug/mL 5.4

27
Contd
  • Sarcolactone A
  • 10ug/mL 3.7
  • 3ug/mL 5.6

28
Contd
  • Sarcolactone B
  • 10ug/mL 3.0
  • 3ug/mL 4.2

29
Contd
  • Ecklonialactone B
  • 10ug/mL 4.7
  • 3ug/mL 4.2

30
Contd
  • Constanolactone A
  • 10ug/mL 2.1
  • 3ug/mL 4.7

31
Contd
  • Lyngbya chlorohydrin (Higa)
  • 10ug/mL 1.0
  • 3ug/mL 8.1

32
Current and Future Plans
  • Continue screening pure compounds
  • Re-screen compounds showing activity
  • Re-screen toxic compounds at lower concentrations
  • Screen crude extracts and fractions from the
    Gerwick cyanobacterial library

33
Acknowledgements
  • Howard Hughes Medical Institute
  • Dr. Doug Goeger
  • Dr. Bill Gerwick
  • Mirjam Girt
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