mentors: kerry mcphail, phd, phil proteau, phd, and

Mentors: Kerry McPhail, PhD, Phil Proteau, PhD,

and Christopher C. Thornburg, PhD

Undergraduate Honors and Bioresource Research

Thesis Seminar Presentation

May 28, 2013

Roadmap

2

Background and Relevance of Natural Products

Sample Collection by McPhail Lab

Goal of Project

Methods

Culturing

Chromatography

o Compound Hypothesis

Compound Characterization

Results

Known Compounds

New Compounds

Hydrophobic Fraction

Discussion

Relevance of Results

Limitations

Future Work

Acknowledgements

Questions

Roadmap

3

Background and Relevance of Natural Products

Sample Collection by McPhail Lab

Goal of Project

Methods

Culturing

Chromatography

o Compound Hypothesis

Compound Characterization

Results

Known Compounds

New Compounds

Hydrophobic Fraction

Discussion

Relevance of Results

Limitations

Future Work

Acknowledgements

Questions

What are Natural Products?

4

Secondary Metabolites

What produces secondary

metabolites?

Why produce secondary metabolites?

Why are Natural Products Important?

5

www.lipitor.com

www.elucidationimages.com

www.kdna.ucla.edu

www.emory.edu

www.sciencedirect.co

m

Agricultural

Applications

Pharmaceutical Applications

Why Search for More Natural Products?

6

Pharmaceutical Importance: (Newman,

Cragg, 2012)

7

Antibiotic Resistance (Silver, Bostian, 1993)

Anticancer Resistance (Simmons, et al, 2005)

Constant need for new compounds (Strobel,

Daisy, 2003)

Exotic Environments (Baker, et al, 2007)

Why Search for More Natural Products?

Cyanobacteria as Sources of

Natural Products

8

Ancient Phylum

Ubiquitous

Classified alongside Myxobacteria and

Streptomyces as sources of pharmaceuticals (Tan,

2010)

Roadmap

9

Background and Relevance of Natural Products

Sample Collection by McPhail Lab

Goal of Project

Methods

Culturing

Chromatography

o Compound Hypothesis

Compound Characterization

Results

Known Compounds

New Compounds

Hydrophobic Fraction

Discussion

Relevance of Results

Limitations

Future Work

Acknowledgements

Questions

Red Sea Exploratory Expedition

10

OSU Collections

May 2007

12 large collections

28 small live collections

Mangroves

High salinity

Oligotrophic

Fine sediment deposition

Roadmap

11

Background and Relevance of Natural Products

Sample Collection by McPhail Lab

Goal of Project

Methods

Culturing

Chromatography

o Compound Hypothesis

Compound Characterization

Results

Known Compounds

New Compounds

Hydrophobic Fraction

Discussion

Relevance of Results

Limitations

Future Work

Acknowledgements

Questions

Purification Process Brown contaminant

Low light

Overtook brown diatom

Harvest 6 months

Yield: 0.85 g

Culturing Process-Moorea sp.

12 Large-scale cultures of Moorea sp.

Materials and Methods

13

Brine Shrimp

Assay

Neuroblastoma

Assay

Extraction and

Separation

Further Separation to

Pure Compounds Structure

Elucidation

Chromatography Overview

14

NP-VLC

A

C

20% E/H

E

60% E/H

G

100% E

H

25% MeOH/E

I

100% M

F

80% E/H

B

10% EtOAc/H

D

40% E/H

Hydrophobic

Hydrophilic

100% Hex

Chemical Extraction

3

70% MeOH/H

1

RP-SPE

4

2

100% DCM 100% M 90% M/H

0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2

mA

u

0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

8 0 0

9 0 0

1 0 0 0

1 1 0 0

1 2 0 0

1

2

4 5a

5b

Minutes

3

RP-HPLC

• NP-VLC:Normal Phase

Vacuum Liquid

Chromatography

• RP-SPE: Reversed Phase

Solid Phase Extraction

• RP-HPLC: Reversed

Phase High Performance

Liquid Chromatography

4

Materials and Methods

15

100% Hex

Brine Shrimp Assay (BSA) 1mg/ml: 4 % 0% 100% 100% 89% 100% 100% 85% 0%

Active Fractions

NP-VLC

A 37.2 mg

C 108.1 mg

20% E/H

E 12.7 mg

60% E/H

G 165.0 mg

100% E

H 190.7 mg

25% MeOH/E

I 88.6 mg

100% M

F 131.4 mg

80% E/H

B 62.6 mg

10% EtAc/H

D 144.2 mg

40% E/H

Moorea species

5

Nabq Mangroves near Sharm el Sheikh, Egypt

BSA 0.1mg/ml: 5 % 0% 96% 97% 7% 85% 94% 7% 0%

50 liters collected over

12 months

0.85 g Organic Extract

Mass Spectrometry

and RP-SPE

Hydrophobic

Hydrophilic

16

Moorea sp. Hypothesized Compounds

8 = Unknown: m/z 658

9 = Unknown: m/z 854

7 known compounds & 2 new compounds

1 2 3

4

5 6 7

17

100% Hex

Brine Shrimp Assay (BSA) 1mg/ml: 4 % 0% 100% 100% 89% 100% 100% 85% 0%

Active Fractions

NP-VLC

A 37.2 mg

C 108.1 mg

20% E/H

E 12.7 mg

60% E/H

G 165.0 mg

100% E

H 190.7 mg

25% MeOH/E

I 88.6 mg

100% M

F 131.4 mg

80% E/H

B 62.6 mg

10% EtOAc/H

D 144.2 mg

40% E/H

Moorea species

5

Nabq Mangroves near Sharm el Sheikh, Egypt

BSA 0.1mg/ml: 5 % 0% 96% 97% 7% 85% 94% 7% 0%

50 liters collected over

12 months

0.85 g Organic Extract

Mass Spectrometry

and Reversed Phase-

Solid Phase

Extraction (RP-SPE)

Hydrophobic

Hydrophilic

18

Moorea sp. HPLC Fraction F

3 2.7 mg

70% MeOH/H

F 131.4

mg

1 8.2 mg

RP-SPE

4 2.1 mg

2 91.6 mg

100% DCM 100% M 90% M/H

Minutes

5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

230 nm

330 nm 254 nm

216 nm

C

A D B Minutes

0 1 2 3 4 5 6 7 8 9 10 11 12 13

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

HPLC-80%MeCN-SynergiFusion Column

HPLC-90% MeOH-SynergiFusion Column

1 2

4

3

Mass Spec m/z 840-Apratoxin A

19

Moorea sp. F2C3 Structure Elucidation

11

1H NMR Spectra

for Apratoxin A in

CDCl3 (500 MHz)-

Published

1H NMR Spectra

for Enq004 F2C3 in

CDCl3 (300 MHz)-

Experimental

20

Moorea sp. F2C3 Structure Elucidation

12

13C NMR Spectra for

Enq004 F2C3 in CDCl3

(75 MHz)-Experimental

13C NMR Spectra for

Apratoxin A in CDCl3 (75

MHz)-Published

21

Roadmap

22

Background and Relevance of Natural Products

Sample Collection by McPhail Lab

Goal of Project

Methods

Culturing

Chromatography

o Compound Hypothesis

Compound Characterization

Results

Known Compounds

New Compounds

Hydrophobic Fraction

Discussion

Relevance of Results

Limitations

Future Work

Acknowledgements

Questions

Apratoxin A-Known Compound

13

Compound Characteristics Toxic against oral carcinoma (KB)

and human colon adenocarcinoma

(LoVo) cells

Induce G1 phase cycle arrest

No effect on microfilament network

No effect on microtubule

polymerization or de-

polymerization

Lack of selectivity limits potential

as antitumor agent, but derivatives

are more effective (Luesch, et al,

2001)

23

Lyngbyabellin B-Known Compound

13

Compound Characteristics Toxic against oral carcinoma

(KB) and human colon

adenocarcinoma (LoVo) cells

Antifungal properties

Protease inhibitory activity

(Luesch, et al, 2000)

24

Apratoxin A Sulfoxide-New Compound

13

Compound Characteristics

Similar to Apratoxin A except

slower to display toxicity

effects

Toxic against Neuro-2A

blastoma cells and NCI-H460

lung cancer cells

25

Apratoxin H-New Compound

13

Compound Characteristics

Highly toxic against NCI-H460

lung cancer cells and Neuro-2A

neuroblastoma cells

26

Moorea sp. Hypothesized Compounds

8 = Unknown: m/z 658

9 = Unknown: m/z 854

7 known compounds & 2 new compounds

1 2 3

4

5 6 7

27

Roadmap

28

Background and Relevance of Natural Products

Sample Collection by McPhail Lab

Goal of Project

Methods

Culturing

Chromatography

o Compound Hypothesis

Compound Characterization

Results

Known Compounds

New Compounds

Hydrophobic Fraction

Discussion

Relevance of Results

Limitations

Future Work

Acknowledgements

Questions

100% Hex

Brine Shrimp Assay (BSA) 1mg/ml: 4 % 0% 100% 100% 89% 100% 100% 85% 0%

Active Fractions

NP-VLC

A 37.2 mg

C 108.1 mg

20% E/H

E 12.7 mg

60% E/H

G 165.0 mg

100% E

H 190.7 mg

25% MeOH/E

I 88.6 mg

100% M

F 131.4 mg

80% E/H

B 62.6 mg

10% EtOAc/H

D 144.2 mg

40% E/H

Moorea species

5

Nabq Mangroves near Sharm el Sheikh, Egypt

BSA 0.1mg/ml: 5 % 0% 96% 97% 7% 85% 94% 7% 0%

50 liters collected over

12 months

0.85 g Organic Extract

Mass Spectrometry

and RP-SPE

Hydrophobic

Hydrophilic

29

RP-HPLC

4

6.8 mg

2

70.5 mg

0 %

3

33.9 mg

67 %

1

1.3 mg

0.1mg/ml: 0 % *

(70% MeCN, 3.0 mL min-1,

Synergi-Fusion-RP 10 x 250 mm)

0 %

ESI-MS m/z 419, 838

[M+Na]+

RP-HPLC

(98% EtOH, 0.6 mL min-1,

Chirobiotic TAG 4.6 x 250 mm)

0 %

D

C

46 %

B

0.1mg/ml: 0 %

E

100 %

Moorea sp. Fraction C3

6

ESI-MS m/z 431

[M+Na]+

Cytotoxicity

Cell Line

Testing

30

Fraction C-Cytotoxicity Assay-Viability

7

0

20

40

60

80

100

120

140

C2

C2A

C2

B

C2

B3

C2

C2B

C2

D2

C2

E

C2

E2

C2

F2

C2

F3

C2

G

C3

C

C3

D

C3

E

% o

f co

ntr

ol r

esp

on

se (

Ne

uro

-2

A C

ells

)

Fraction

Moorea sp. Fraction C Neuro2A Blastoma Assay-Viable Cells

p<0.01

31

Hypothesized Masses and Applications

8

Mass Spectrometry

Determination Structural Components

No published compounds

with same masses

o MarinLit

ESI-Mass Spectrometry-Direct Inject C2C2B

o m/z 403, [M+Na]+

C2E2

o m/z 431 [M+Na]+

C2F2

o m/z 471, [M+Na]+

0

20

40

60

80

100

120

140

C2

C2A

C2

B

C2

B3

C2

C2B

C2

D2

C2

E

C2

E2

C2

F2

C2

F3

C2

G

C3

C

C3

D

C3

E

% o

f co

ntr

ol r

esp

on

se

(N

eu

ro-2

A c

ells

)

Fraction

Moorea sp. Fraction C Neuro2A Blastoma Assay-Viable Cells

32

Results Summary

33

2 Known Compounds Characterized

Apratoxin A

Lyngbyabellin B

2 New Compounds Characterized

Apratoxin A Sulfoxide

Apratoxin H

New Hydrophobic Fractions

0

50

100

150

C2

C2A

C2

B

C2

B3

C2

C2B

C2

D2

C2

E

C2

E2

C2

F2

C2

F3

C2

G

C3

C

C3

D

C3

E

%o

f co

ntr

ol r

esp

on

se

(Ne

uro

-2A

ce

lls)

Fraction

Moorea sp. Fraction C Neuro2A Blastoma Assay-Viable Cells

Roadmap

34

Background and Relevance of Natural Products

Sample Collection by McPhail Lab

Goal of Project

Methods

Culturing

Chromatography

o Compound Hypothesis

Compound Characterization

Results

Known Compounds

New Compounds

Hydrophobic Fraction

Discussion

Relevance of Results

Limitations

Future Work

Acknowledgements

Questions

9

Abundance of Apratoxin Production

Gene Cluster

Regulated by Environment

More Knowns

Thornburg classified two more

apratoxins and heptachlorin

Conclusions-Known Compounds

35

(Chen, Liu, Luesch, 2011)

9

New apratoxins

Medicinal Applications

New Chemical Motifs

More Unknowns?

More separation

Conclusions-Unknown Compounds

36

9

Predicted new

hydrophobic

compounds

Applications

Gerwick Group

Curacin A

FDA Clinical

Trials

www.ucsd.edu

Conclusions-Hydrophobic Fraction

37

9

Prolific secondary metabolite producer

Source of potent and selective analogues

for synthetic scaffolds

Regulated by Environment

Culturable

Produces secondary metabolites in

absence of environmental stresses

More with stress?

Conclusions-Moorea sp. Overall

38

Roadmap

39

Background and Relevance of Natural Products

Sample Collection by McPhail Lab

Goal of Project

Methods

Culturing

Chromatography

o Compound Hypothesis

Compound Characterization

Results

Known Compounds

New Compounds

Hydrophobic Fraction

Discussion

Relevance of Results

Limitations

Future Work

Acknowledgements

Questions

15

Limitations of the Project

40

Relying on slow growing organism

Sensitive to culture media

Low resolution LC-MS can’t definitively

identify compounds

Not suitable for untargeted compound

screening

Roadmap

41

Background and Relevance of Natural Products

Sample Collection by McPhail Lab

Goal of Project

Methods

Culturing

Chromatography

o Compound Hypothesis

Compound Characterization

Results

Known Compounds

New Compounds

Hydrophobic Fraction

Discussion

Relevance of Results

Limitations

Future Work

Acknowledgements

Questions

15

Spectroscopic analysis of

pure compounds from

HPLC

High interest in obtaining

enough of the unknown m/z

658 for characterization

Obtain larger quantities of

pure unknowns for

biological testing on

human cell lines

Future Directions of the Project

42

10

Funding Howard Hughes Medical Institute, facilitated by Dr. Kevin Ahern

Oregon State University Honors College

Oregon State College of Pharmacy

Sigma Xi

E.R. Jackman Internship Grant Research Lab Dr. Kerry McPhail

Dr. Phil Proteau

Christopher Thornburg

Justyna Sikorska

Oliver Vining

Mariko Nonogaki

Acknowledgements

43

Support Wanda Crannell

Perakis Lab

15

Questions?

44