2. Bringing Back Nature to Drug Discovery
Natural Molecules in an Antibacterial Program
Mark O’Neil-Johnson

4. Parallel Purification Technologies
High-throughput Natural Products Chemistry
Sequoia Sciences, Inc.
State-of-the-art
NMR Probe Technology
Taxonomically
Diverse Plant
Collection
Parallel Purification Technologies

5. Normalized sample weights
Sequoia Sciences, Inc.
Plants to Compound Library 1
Sequoia’s Strengths 3
Analysis performed on each sample
8-channel MUX LC/MS
with 8 ELSD’s
~ 110 samples / plant
1 to 5 compounds per sample
Samples of known quantities
Drug-like compounds
Average MW < 400 daltons
Log P from 0 to 5
State-of-the-art
NMR Probe Technology
Taxonomically
Diverse Plant
Collection
Parallel Purification Technologies
TIC Chromatogram
Mass Spectrum
447.2
506.2
244.9
336.0
200
300
400
m/z 2
Five step purification process to remove
non-drug-like compounds 4
Normalized sample weights
Approximately 90% of plant extracts are composed of “non-drug-like” compounds
Utilize ELSD data to deliver normalized
weights of each sample
ELSD Chromatogram
Screening concentrations between 1 to 20 µM zz
Sequoia’s process removes these “non-drug-like” compounds
Generates approximately 110 preparative HPLC fractions (“sample”) per plant

6. Collaborator rescreens
Sequoia Sciences, Inc.
Biological Activity to Chemical Structure 1
Collaborator identifies an
Active Sample 4
Structure elucidation of an Active Compound
Bruker Avance
test
600 MHz Magnet
Flow Probe
Flow Probe 5 5 m m L L
Flow cell
Flow cell
1.5
1.5 m m L L
Active volume
Active volume
Coil
Coil
Sequoia acquires NMR
spectra on 5 to 100 ug
of an Active Compound 2
Sequoia isolates compounds
from an Active Sample 3
Sequoia delivers compounds to
Collaborator
Sequoia elucidates
the chemical structure
of an Active Compound
Collaborator rescreens
purified compounds

7. What can I do with only micrograms of a sample?

8. Move to Miniaturize 1 mL 100 mL 10 mL 1 L 100 mL 10 mL 1 mL
High-throughput Natural Products Chemistry
Move to Miniaturize
1 mL
100 mL
10 mL
1 L
100 mL
10 mL
1 mL

10. The Ultimate NMR Probe for Samples of Limited Mass
CapNMR Probe
The Ultimate NMR Probe for
Samples of Limited Mass

11. CapNMRTM Probe 2 mg Quinidine in MeOH,d4 5mm TXI probe @ 900 MHz
High-throughput Natural Products Chemistry
CapNMRTM Probe
2 mg Quinidine in MeOH,d4
5mm TXI 900 MHz
2 mg in 550 mL MeOH,d4
CapNMR 600 MHz
2 mg in 8 mL MeOH,d4; Infuse 5 mL

12. CapNMRTM Probe High-throughput Natural Products Chemistry 5 mm Rf Coil
5 mm std. NMR tube
5 mm Rf Coil
270 mlit.
(~17 mm)
1.5 mlit.
(~1 mm)
5 mlit.

13. High-Throughput Natural Products Chemistry
Sequoia Sciences, Inc.
Sample Submission Hardware, Solvent and Waste Recovery
96-well plate
Waste recovery
vial
10 ml gas tight syringe
mini-tube

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

15. Sequoia Sciences, Inc. HPLC / ELSD of Hit 620 of Fraction 8, tube 34
High-throughput Natural Products Chemistry
Sequoia Sciences, Inc.
HPLC / ELSD of Hit 620 of Fraction 8, tube 34

16. Sequoia Sciences, Inc. HPLC / ELSD of Hit 620 of Fraction 8, tube 34
High-throughput Natural Products Chemistry
Sequoia Sciences, Inc.
HPLC / ELSD of Hit 620 of Fraction 8, tube 34

17. Sequoia Sciences, Inc. Active Fractions From Hit 620
High-throughput Natural Products Chemistry
Sequoia Sciences, Inc.
Active Fractions From Hit 620
Active Fractions for Rescreen and NMR

18. SF_4356
SF_4351
SF_4335
SF_4331
SF_4329

19. High-throughput Natural Products Chemistry
Sequoia Sciences, Inc.

20. Plant Family: Orchidaceae
Species: Phragmipedium calurum

21. High-throughput Natural Products Chemistry
Sequoia Sciences, Inc.

22. Plant Family: Asteraceae
Species: Layia platyglossa
Combine tubes F10, G10 and H10
To NMR
SepFraction 15509
6 mg total (2 HPLC runs)

23. Sequoia Sciences, Inc. High-throughput Natural Products Chemistry
64 Scans
512 Scans
Data base peak search

24. Bacterial Biofilms
Nasty Stuff!

25. Bacteria…..Some Nasty Stuff!
High-throughput Natural Products Chemistry
Bacteria…..Some Nasty Stuff!
Test Summary: Pseudomonas Disinfection and Regrowth in Pipes
R. Anderson (Center for Disease Control) filled plastic pipes with water
contaminated with two strains of Pseudomonas.
Incubate for 8 weeks
Empty and douse pipes with germ-killing biocides, include chlorine, for
7 days
Refill with sterile water and periodically sample “clean water”
Both strains of Pseudomonas survived and reestablished colonies!

26. High-throughput Natural Products Chemistry
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

27. Type of Biofilms Industrial Biofilms Medical Biofilms
High-throughput Natural Products Chemistry
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

28. High-throughput Natural Products Chemistry
Biofilms
Resistant to antibiotics at concentrations from to 1500 times higher than conventional use
Involved in two-thirds of human bacterial infections
No specific biofilm inhibitor is commercially available

29. Screening for Biofilms
High-throughput Natural Products Chemistry
Screening for Biofilms
96 Well plate format for initial screen
(initial screen at 10 mg/ml)
Asiatic Acid
Corosolic Acid
Ursolic Acid

30. High-throughput Natural Products Chemistry
Screening Challenge
Minimum Inhibitory Concentrations (MIC) against planktonic bacteria not relevant to biofilms
New biofilm susceptibility methods need to be developed: biofilm inhibition conc. (BIC) or minimum biofilm eradication conc. (MBEC)
Variability in BIC and MBEC values suggest characteristics of biofilms is related to model used to grow them in
96 Well plate format
Rotating Disk Reactor (RDR)

31. Screening Challenge Bacterial strain: Pseudomonas aeruginosa (PA01)
High-throughput Natural Products Chemistry
Screening Challenge
Bacterial strain: Pseudomonas aeruginosa (PA01)
Ciprofloxacin active against planktonic bacteria at 1 mg/ml
Ciprofloxacin active against a bacterial biofilm at 50 mg/ml
Develop a valid physiological screening assay

32. Rotating Disk Reactor (RDR)
High-throughput Natural Products Chemistry
Rotating Disk Reactor (RDR)
Figure 1
Rotating Disk Reactor showing test coupon
holder with growth coupons inserted
Figure 2
Test coupons: disk on left is polycarbonate,
Disk on right is stainless steel

33. Airway Infections in CF Patients
High-throughput Natural Products Chemistry
Airway Infections in CF Patients
Pseudomonas aeruginosa airway infections: leading cause of death in Cystic Fibrosis patients
Chronic infection that is resistant to antibiotic therapies
Standard treatment: tobramycin and azithromycin
RDR, a physiological valid screening method?
Test Sequoia compounds in RDR with tobramycin and azithromycin or ciprofloxacin combination

35. Rotating Disk Reactor (RDR)
High-throughput Natural Products Chemistry
Rotating Disk Reactor (RDR)
255+toby water toby control

36. Rotating Disk Reactor (RDR)
High-throughput Natural Products Chemistry
Rotating Disk Reactor (RDR)

37. Rotating Disk Reactor (RDR)
High-throughput Natural Products Chemistry
Rotating Disk Reactor (RDR)
C = ciprofloxacin
T = tobramycin

38. Screening for Biofilms
High-throughput Natural Products Chemistry
Screening for Biofilms
Summary
Asiatic Acid and Corosolic Acid in a physiological relevant biofilm screen
are not true biofilm inhibitors but biofilm modulators
Synergistic effect on P. aeruginosa biofilm by Sequoia compounds indicate
that they do affect the tolerance of antibiotic to PA
Sequoia compounds not true biofilm inhibitors, suggesting a different MOA
by modification of biofilm
P. aeruginosa grown in presence of 10 mg/ml Asiatic Acid makes biofilm sensitive to
10 mg/ml ciprofloxacin
Sequoia Compounds are excellent biofilm potentiator candidates

39. Sequoia Sciences, Inc. Special Thanks to: Jin-Feng Hu Eliane Garo
High-throughput Natural Products Chemistry
Sequoia Sciences, Inc.
Special Thanks to:
Jin-Feng Hu
Eliane Garo
Gary Eldridge
James Norcross
Tim Peck
Dean Olson
Bill Costerton
Garth James