Two Bioassays for Cyanobacterial Neuroactive Metabolites Amanda Cordes, Dr. Doug Goeger, Dr. William Gerwick.
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Transcript Two Bioassays for Cyanobacterial Neuroactive Metabolites Amanda Cordes, Dr. Doug Goeger, Dr. William Gerwick.
Two Bioassays for
Cyanobacterial Neuroactive Metabolites
Amanda Cordes, Dr. Doug
Goeger, Dr. William Gerwick
The Gerwick Group
Purpose: Study of marine algae to
discover novel compounds and
develop biomedicinal agents
Focus: Marine Cyanobacteria = “blue
green algae”
Discoveries:
Antillatoxin
Kalkitoxin
Curacin A
Applications:
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, 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 voltagegated 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.
Part 1. Detection and
Characterization of
Cyanobacterial Neurotoxins using
Zebrafish Behavior
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
Zebrafish
(Danio rerio)
http://edtech.tph.wku.edu/~jbilotta/neuro.htm
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
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
35
Amount of Compound (mg)
30
25
20
15
10
5
0
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
Results of Blind Tests
One 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
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
Part 2. Ability of Cyanobacterial
Metabolites to Induce
Neuritogenesis
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
Neuro 2a Cells with Neurites
http://users.jagunet.com/~meledy/cell2.jpg
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.
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-BrcAMP, known outgrowth promoters
% of cells showing outgrowths
Neurite Outgrowth Controls
25
Treated
20
RPMI w/
30uL DMSO
5 uM
Lactacystin
15
10
5
0
24
hrs
48
hrs
72
hrs
Control
Screening for Pure
Cyanobacterial Natural Products
that Induce Neurite Outgrowth
Based on % of cells with outgrowths
after 24 hours
Inactive Compounds
Octadec-5-yne-7Z,9Z,12Z-trienoic Acid
0.65%
0.86%
Malhamensilipin A
10 ug/mL:
3 ug/mL:
10 ug/mL:
3 ug/mL:
2.2%
2.%
Avrainvilleol
10 ug/mL:
3 ug/mL:
2.2%
4.2%
Cont’d
Gloiosiphone A Dimethyl Ether
2.6%
3.8%
Pacifenol
10 ug/mL:
3 ug/mL:
10 ug/mL:
3 ug/mL:
0.68%
3.3%
Dilophic Acid
10 ug/mL:
3 ug/mL:
1.2%
2.8%
Cont’d
Cymathere Lactone
1.5%
2.4%
Malyngolide
10 ug/mL:
3 ug/mL:
10 ug/mL:
3 ug/mL:
0.59%
2.8%
Spiro-bis-pinnaketal
10 ug/mL:
3 ug/mL:
1.6%
2.4%
Cont’d
Palisadin A
2.4%
2.9%
Carmabin A
10 ug/mL:
3 ug/mL:
10 ug/mL:
3 ug/mL:
0%
2.8%
Martensia Indole
10 ug/mL:
3 ug/mL:
0%
1.2%
Toxic Compounds
Hormothamnione
toxic
1.1%
Malyngamide F Acetate
10 ug/mL:
3 ug/mL:
10 ug/mL:
3 ug/mL:
toxic
toxic
Ptilodene Methyl Ester
10 ug/mL:
3 ug/mL:
toxic
1.8%
Cont’d
Cymopol
10 ug/mL: toxic
3 ug/mL: 1.5%
Active Compounds
Allolaurinterol
10ug/mL:
3ug/mL:
CH3
CH2
3.3%
6.2%
H
CH3
OH
CH3
Br
Cont’d
Methyl 12S-HETE
10ug/mL:
2.3%
3ug/mL:
5.4%
OH
CO2CH3
Cont’d
Sarcolactone A
10ug/mL:
3ug/mL:
3.7%
5.6%
O
O
O
Cont’d
Sarcolactone B
10ug/mL:
3ug/mL:
3.0%
4.2%
O
O
O
Cont’d
Ecklonialactone B
10ug/mL:
3ug/mL:
4.7%
4.2%
H
O
O
O
Cont’d
Constanolactone A
10ug/mL:
3ug/mL:
2.1%
4.7%
OH
H
H
OH
O
Cont’d
Lyngbya chlorohydrin (Higa)
10ug/mL:
3ug/mL:
1.0%
8.1%
O
Cl
OH
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
Acknowledgements
Howard Hughes Medical Institute
Dr. Doug Goeger
Dr. Bill Gerwick
Mirjam Girt