mark o’neil-johnson advanced analytical technologies as applied to natural products for drug...

Mark O’Neil-Johnson

Advanced Analytical Technologies as Applied to Natural Products for

Drug Discovery:

Segment Deux

Parallel Purification Technologies

State-of-the-art NMR Probe Technology

Taxonomically Diverse Plant

Collection

High-throughput Natural Products Chemistry

Sequoia Sciences, Inc.

Plants to Compound LibrarySequoia Sciences, Inc.

Analysis performed on each sample3

Normalized sample weights

4

zz

Utilize ELSD data to deliver normalized weights of each sample

ELSD Chromatogram

Screening concentrations between 1 to 20 µM

Sequoia’s Platform1

Parallel Purification Technologies

State-of-the-art NMR Probe Technology

Taxonomically Diverse Plant

Collection

2Five step purification process to removenon-drug-like compounds

• Approximately 90% of plant extracts are composed of “non-drug-like” compounds

Highly Lipophilic Compounds > Log P of 5

Highly Hydrophilic Compounds < Log P of 0

High Molecular Weight Compounds > 1000 Daltons

Miscellaneous Compounds

i.e. Carbohydrates, Tannins

• Sequoia’s process removes these “non-drug-like” compounds• Generates approximately 110 preparative HPLC fractions

(“sample”) per plant

8-channel MUX LC/MS with 8 ELSD’s

Mass SpectrumTIC Chromatogram

200 300 400 500 m/z

506.2447.2

244.9336.0

~ 110 samples / plant

1. 1 to 5 compounds per sample2. Samples of known quantities3. Drug-like compounds

a.Average MW < 400 daltons

b.Log P from 0 to 5

Flow Probe

Coil

5 LFlow cell1.5 L

Active volume

Bruker Avance

Flow Probe

Coil

5 LFlow cell1.5 L

Active volume

O

OOCH3

HO

OH3C

O

H

O

O

Biology rescreens purified compounds

Biological Activity to Chemical StructureSequoia Sciences, Inc.

Biology identifies anActive Sample

•Sequoia acquires NMR

spectra on 5 to 100 ug

of an Active Compound

Structure elucidation of an Active Compound

Sequoia sends pure compounds to Biology

Sequoia isolates compounds from an Active Sample

•Sequoia elucidates the chemical structure of an Active Compound

600 MHz Magnet

1

2

3

4

test

Ground Plant

8765

Extract

A

E F GC

Extremelylipophilicfraction is discarded

Preparative HPLC4 x 40 20 mL chromatographic fractions

D

A - 50:50 ethyl acetate : ethanolB - 100 methanolC - 75:25 hexane : ethyl acetateD - 25:75 hexane : ethyl acetateE - 100 ethyl acetateF - 70:30 ethyl acetate : methanolG - 50:50 ethyl acetate : methanolH – 50:50 methanol:water 1% acetic acid

C18 SPE to remove

lipophilics

LC/MS analysis, and removal of tannins with polyamide SPE

Flash Chromatography

Extract

B

H

9


Sequoia Sciences, Inc.HPLC / ELSD of Hit 620 of Fraction 8, tube 34



Active Fractions From Hit 620

Active Fractions for Rescreen and NMR

SF_4329

SF_4331

SF_4335

SF_4351

SF_4356

CapNMRTMProbe

4 g.

• Amount isolated: 4 mg. • Dilute with 7mL MeOD• Infuse 5 mL• Center in flow cell with 7 mL



C7

D7E7

F7

Combine tubes C7, D7, E7 and F7

SepFraction 15827 9 g total (2 HPLC Collections)



SF_15818_001001r

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0Chemical Shift (ppm)

0

0.005

0.010

0.015

0.020

No

rma

lize

d In

ten

sity

METHANOL-d4

METHANOL-d4 Impurity

Impurity

Impurity

Impurity

Impurity

Impurity

Impurity

Impurity

1.2

7

1.2

91

.31

1.3

41

.38

1.4

71

.54

1.7

41

.76

1.9

7

1.9

8

2.0

0

2.0

0

2.4

0

2.4

1

2.4

3

3.2

3

3.3

5

5.7

5

Compound 553

O

O

HO



Compound 553

From Hit 1212IC50: 0.38 M

O

O

HO

Plant Families Underrepresented in a Drug Discovery Program

Courtesy of Michelle Goering

http://www.mobot.org/default.asp

Botanical/Chemical diversity of plant collection?

Courtesy of Missouri Botanical Garden

Study of NCI collection in Madagascar:Representative families genera in flora versus collected

Number of Genera

Num

ber

of G

ener

a Sa

mpl

ed

Ref: Biologically active natural products: pharmaceuticals, 2000, CRC pressCourtesy of Jim Miller

Conclusion of NCI collection survey

Collection could be improved by greater attention to rare plants with provisions to allow for smaller samples to be collected for certain taxa

Advanced technology that allow the analysis of small samples

Plants grown in greenhouses

What can I do with only micrograms of a sample?


Move to Miniaturize

100 mL1 L 10 mL 1 mL

1 mL 100 L 10 L

CapNMRTM ProbeHigh-throughput Natural Products Chemistry

1.5 lit.(~1 mm)

5 lit.

5 mm std. NMR tube

270 lit.(~17 mm)

5 mm Rf Coil

Sequoia Sciences, Inc.High-Throughput Natural Products Chemistry

Sample Submission Hardware, Solvent and Waste Recovery

mini-tube10 l gas tight syringe

96-well plateWaste recoveryvial

Courtesy of Missouri Botanical Garden

Orchidaceae

Plant Family: OrchidaceaeSpecies: Phragmipedium calurum

Stilbenes isolated from Phragmipedium sp.

OH

R5O

OR4

R1

R2

R3

14

67

82'

4'

6'

1''

3''4''

1 R1=R2=R3=R4=H, R5=CH32 R1=R2=R3=R5=H, R4=CH33 R1=OH, R2=R3=R4=H, R5=CH34 R1=OH, R2=R3=R5=H, R4=CH37 R1=R2=OH, R3=H, R4=R5=CH38 R1=OH, R2=R5=H, R3=OCH3, R4=CH3

OH

O

OH

R

HO

2'

1''

1'''

3''

3'''

4'

5 R=H6 R=OH

O

O

OH

OH

O

R2

R1

10

12

2'

7'8'

10'

A1

A2

B2

B1

9 R1=H, R2=OH10 R1=OH, R2=H

R4O

O

R1

R2

R3

R5

11 R1=R2=R3=R5=R4=H12 R1=OH, R2=R3=R4=R5=H13 R1=OH, R2=R3=R5=H, R4=CH314 R1=R2=OH, R3=R5=H, R4=CH315 R2=OH, R1=R3=R4=R5=H16 R1=OH, R2=H, R3=OCH3, R4=R5=H17 R1=R2=R3=R4=H, R5=OH


Nepenthes


Sequoia Sciences, Inc.Combine tubes C7, D7, E7 and F7

SepFraction 1668115 g per collection

Secoiridoids isolated from Sarracenia alata


Sequoia Sciences, Inc.Secoiridoids isolated from Sarracenia alata

Combine tubes F10, G10 and H10

SepFraction 155096 mg total (2 HPLC runs)

To NMR

Plant Family: AsteraceaeSpecies: Layia platyglossa


ACD Software

512 Scans

64 Scans

Data base peak search

Bacterial Biofilms


What is a Biofilm? Bacteria attach on surface, begin to secrete polysaccharide matrix

Cells grow into a community, complete with nutrient channels

Community thrives, protected by polysaccharide matrix


Type of Biofilms

Industrial Biofilms

Ship bottoms and pipes Cooling towers Air ventilation systems Drinking water filtration and

distribution systems Air filtration units Dental water lines Hemodialysis units Food processing systems

Medical Biofilms

Urinary tract infections Lung infections Sinus infections Ear infections Chronic wounds Dental caries Medical device implants Acne


Biofilms

Resistant to antibiotics at concentrations from 1000 to 1500 times higher than conventional use

Involved in two-thirds of human bacterial infections

No specific biofilm inhibitor is commercially available


Screening Challenge

Bacterial strain: Pseudomonas aeruginosa (PA01)

Ciprofloxacin active against planktonic bacteria at 1 g/ml

Ciprofloxacin active against a bacterial biofilm at 50 g/ml

Develop a valid physiological screening assay


Screening for Biofilms

96 Well plate format for initial screen (initial screen at 10 g/ml)

Asiatic Acid Corosolic Acid Ursolic Acid

O

OH

HO

HO

OH

Asiatic Acid Corosolic Acid

O

OH

HO

HO

Ursolic Acid

O

OH

HO



Very Special Thanks to: Brent Lefebvre and Ryan Sasaki of ACD

Eliane GaroJin-Feng HuGary Eldridge

James NorcrossTim Peck Dean Olson

Peter RavenJames Miller

http://www.mobot.org/default.asp

mark o’neil-johnson advanced analytical technologies as applied to natural products for drug...

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