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

Amanda Cordes, Dr. Doug Goeger, Dr. William Gerwick

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

Goals

Identify a small molecule from algae/cyanobacteria which exhibits activity in stimulating nerve regeneration

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

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

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

0

5

10

15

20

25

24hrs

48hrs

72hrs

% o

f ce

lls

sho

win

g o

utg

row

ths

RPMI w/30μL DMSO

5 μMLactacystin

Neurite Outgrowth Controls

Treated

Control

Choosing a Concentration

Concentration (μg/ml)

30 10 3 1 0.3 0.1

F 91 74 19

G 95 90 64

H 97 96 95 66 21 11

I 97 96 96 61 2114

54

Fra

ctio

n

Values are % lethality

52 pure compounds

27 crude fractions

One worth mentioning…..

…Symploca

www.sitesatlas.com

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%

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%

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%

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%

AU

0.00

0.50

1.00

1.50

2.00

2.50

3.00

Minutes10.00 20.00 30.00 40.00 50.00 60.00 70.00

996 at 340.00996 at 216.00996 at 230.00996 at 254.00

Baseline 1

H-1 Peak

Baseline 2

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%

Possible Synergisms?

0.03 μg/mL0.03 μg/mL

0.003 μg/mL

0.003 μg/mL

…..perhaps….

H-1 Peak and Baseline 1 2.3%

H-1 Peak and Baseline 2 4.3% 6.5% 4.3%

…..results, conclusions, and future plans…

Further investigation of Symploca compounds

Continued screening of crude fractions and newly purified compounds

Acknowledgements

Rebecca Medina

Gerwick Lab

Kevin Ahern & HHMI

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