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Two Bioassays for Cyanobacterial Neuro-active Metabolites
Amanda Cordes, Dr. Doug Goeger, Dr. William Gerwick
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The Gerwick Group
Purpose: Study of marine algae to discover novel compounds and develop biomedicinal agents
Focus: Marine Cyanobacteria = “blue green algae”
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Antillatoxin Kalkitoxin Curacin A
Anticancer agents Anesthetics Agrichemicals
Discoveries: Applications:
∙ Nogle LM, Okino T, Gerwick WH. "Antillatoxin B, a neurotoxic lipopeptide from the marine cyanobacterium Lyngbya majuscula." Journal of Natural Products 2001, 64:983-985.
∙ 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.
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Part 1. Detection and Characterization of Cyanobacterial Neurotoxins using Zebrafish Behavior
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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
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Zebrafish(Danio rerio)
http://edtech.tph.wku.edu/~jbilotta/neuro.htm
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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
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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
0
5
10
15
20
25
30
35
Am
ount
of
Com
poun
d (m
g)
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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
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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
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Conclusions on Zebrafish Model
Fish to fish variability is high
Large quantities of toxin required to induce response
Zebrafish are not a viable model for detection and characterization of cyanobacterial neurotoxins
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Part 2. Ability of Cyanobacterial Metabolites to Induce Neuritogenesis
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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
First – Defining Some Terms
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Neuro 2a Cells with Neurites
http://users.jagunet.com/~meledy/cell2.jpg
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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.
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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
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Neurite Outgrowth Controls
0
5
10
15
20
25
24hrs
48hrs
72hrs
% of cells showing outgrowths
RPMI w/30uL DMSO
5 uMLactacystin
Treated
Control
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Screening for Pure Cyanobacterial Natural Products that Induce Neurite Outgrowth
Based on % of cells with outgrowths after 24 hours
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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%
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Cont’d
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%
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Cont’d
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%
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Cont’d
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%
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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%
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Cont’d
Cymopol 10 ug/mL: toxic 3 ug/mL: 1.5%
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Active Compounds
Allolaurinterol 10ug/mL: 3.3% 3ug/mL: 6.2%
CH2
CH3 HCH3 OH
CH3
Br
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Cont’d
Methyl 12S-HETE 10ug/mL: 2.3% 3ug/mL: 5.4%
CO2CH3
OH
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Cont’d
Sarcolactone A 10ug/mL: 3.7% 3ug/mL: 5.6%
O
O
O
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Cont’d
Sarcolactone B 10ug/mL: 3.0% 3ug/mL: 4.2%
O
O
O
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Cont’d
Ecklonialactone B 10ug/mL: 4.7% 3ug/mL: 4.2%
O
O
O
H
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Cont’d
Constanolactone A 10ug/mL: 2.1% 3ug/mL: 4.7%
OH
OH
H
OH
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Cont’d
Lyngbya chlorohydrin (Higa) 10ug/mL: 1.0% 3ug/mL: 8.1%
O
OH
Cl
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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
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Acknowledgements
Howard Hughes Medical Institute Dr. Doug Goeger Dr. Bill Gerwick Mirjam Girt