Two Bioassays for Cyanobacterial Neuro-active 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”

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.

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

0

5

10

15

20

25

30

35

Am

ount

of

Com

poun

d (m

g)

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 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

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

Part 2. Ability of Cyanobacterial Metabolites to Induce Neuritogenesis

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

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-Br-cAMP, known outgrowth promoters

Neurite Outgrowth Controls

0

5

10

15

20

25

24hrs

48hrs

72hrs

% of cells showing outgrowths

RPMI w/30uL DMSO

5 uMLactacystin

Treated

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 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%

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%

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%

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%

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%

Cont’d

Cymopol 10 ug/mL: toxic 3 ug/mL: 1.5%

Active Compounds

Allolaurinterol 10ug/mL: 3.3% 3ug/mL: 6.2%

CH2

CH3 HCH3 OH

CH3

Br

Cont’d

Methyl 12S-HETE 10ug/mL: 2.3% 3ug/mL: 5.4%

CO2CH3

OH

Cont’d

Sarcolactone A 10ug/mL: 3.7% 3ug/mL: 5.6%

O

O

O

Cont’d

Sarcolactone B 10ug/mL: 3.0% 3ug/mL: 4.2%

O

O

O

Cont’d

Ecklonialactone B 10ug/mL: 4.7% 3ug/mL: 4.2%

O

O

O

H

Cont’d

Constanolactone A 10ug/mL: 2.1% 3ug/mL: 4.7%

OH

OH

H

OH

Cont’d

Lyngbya chlorohydrin (Higa) 10ug/mL: 1.0% 3ug/mL: 8.1%

O

OH

Cl

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