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

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Presentation on theme: "Two Bioassays for Cyanobacterial Neuro- active Metabolites Amanda Cordes, Dr. Doug Goeger, Dr. William Gerwick."— Presentation transcript:

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 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.

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)

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 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

11 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

12 Part 2. Ability of Cyanobacterial Metabolites to Induce Neuritogenesis

13 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 First – Defining Some Terms

14 Neuro 2a Cells with Neurites

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 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%

21 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%

22 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%

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 Cont’d Cymopol  10 ug/mL:toxic  3 ug/mL:1.5%

25 Active Compounds Allolaurinterol  10ug/mL:3.3%  3ug/mL:6.2 %

26 Cont’d Methyl 12S-HETE  10ug/mL:2.3%  3ug/mL:5.4%

27 Cont’d Sarcolactone A  10ug/mL:3.7%  3ug/mL:5.6 %

28 Cont’d Sarcolactone B  10ug/mL:3.0%  3ug/mL:4.2%

29 Cont’d Ecklonialactone B  10ug/mL:4.7%  3ug/mL:4.2%

30 Cont’d Constanolactone A  10ug/mL:2.1%  3ug/mL:4.7%

31 Cont’d 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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