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