Discovery of Neuro-regenerative Natural Products from Marine Algae and Cyanobacteria

1. Discovery of Neuro-regenerative Natural Products from Marine Algae and Cyanobacteria Amanda Cordes, Dr. Doug Goeger, Dr. William Gerwick

2. Background • At present, there are an estimated 243,000 people living with spinal cord injuries in the United States • 53% of new injuries occur within the 16-30 age group

3. Goals • Identify a small molecule from algae/cyanobacteria which exhibits activity in stimulating nerve regeneration

4. Many algae compounds are similar to retinoic acid and eicosanoids • A marine sponge compound, Lembehyne A, promotes neurite outgrowth • Known outgrowth promoters stop cell cycle progression, as do many of the lab’s characterized compounds

5. 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

6. Neuro 2a Cells with Neurites

7. Experimental Protocol • Neuro 2a cells are cultured in serum supplemented medium • 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

8. Neurite Outgrowth Controls Treated Control

9. Choosing a Concentration Concentration (μg/ml) 1454 Fraction Values are % lethality

10. 52 pure compounds • 27 crude fractions • One worth mentioning…..

11. …Symploca www.sitesatlas.com

12. Symploca 1454 Fractions • 1454 A • 30 μg/mL: 0.53% • 10 μg/mL: 0.27% • 1454 B • 30 μg/mL: .32% • 10 μg/mL: 1.2% • 1454 C • 30 μg/mL: Toxic • 10 μg/mL: .35%

13. Cont’d • 1454 D • 1 μg/mL: 0.55% • 0.3 μg/mL: 1.5% • 1454 E • 10 μg/mL: 1.9% • 3 μg/mL: .88% • 1454 F • 3 μg/mL: 4.0% • 1 μg/mL: .48%

14. Cont’d • 1454 G • 1 μg/mL: 3.1% • 0.3 μg/mL: 4.0% • 1454 H • 0.3 μg/mL: 5.2% • 0.1 μg/mL: 0.9% • 1454 I • 0.3 μg/mL: 3.2% • 0.1 μg/mL: 1.5%

15. Sub fractions • H-1 • 0.003 μg/mL: 4.5% • 0.001 μg/mL: 0.8% • H-2 • 0.1 μg/mL: 1.1% • 0.03 μg/mL: 1.0%

16. H-1 Peak Baseline 1 Baseline 2

17. Assay Results • H-1 Peak • 0.03 μg/mL: 1.0% • 0.01 μg/mL: 1.6% • Baseline 1 • 0.003 μg/mL: 1.8% • 0.001 μg/mL: 0.5% • Baseline 2 • 0.003 μg/mL: 2.0% • 0.001 μg/mL: 0.6%

18. Possible Synergisms? 0.03 μg/mL 0.003 μg/mL 0.03 μg/mL 0.003 μg/mL

19. …..perhaps…. • H-1 Peak and Baseline 1 • 2.3% • H-1 Peak and Baseline 2 • 4.3% • 6.5% • 4.3%

20. …..results, conclusions, and future plans… • Further investigation of Symploca compounds • Continued screening of crude fractions and newly purified compounds

21. Acknowledgements • Rebecca Medina • Gerwick Lab • Kevin Ahern & HHMI

22. References • Blanco et al., Cell Biol. Int. 2001, 25, 909 • Fenteany et al., J. Biol. Chem.1998, 273, 8545 • Aoki et al., Biochem. Biophys. Res. Com. 2001, 289, 558