The Role of Natural Products in

1
The Role of Natural Products in Source Organisms
Chemical Defense -antifeedant -antimicrobial -a
ntifouling -allelochemicals Chemical
Offense -toxins
Communication -hormones -pheromones -colors S
ymbiotic Relationships -provide a benefit to
the host
2
NCI Natural Products Repository
3
(No Transcript)
4
Crude Organic Extracts Plated in 96 Well Plates
(0.5 mg/well)
5
High throughput natural product chemistry
techniques allow rapid characterization of
extracts and pure compounds (smaller, faster,
better!)
Small-scale solid phase extraction
Robotic sample handling
Automated HPLC
Fractions assayed directly from 96-well plates
Coupled LC-MS
Capillary NMR
6
Examples of HTS Campaigns

• Protein-Protein MDM2 interactions
• Cell-Based ABCG2

7
MDM2

• A member of the E3 ring-finger ubiquitin ligase
  family
• Originally identified as an oncoprotein
• MDM2 activity increased in 33 of human sarcomas
  and 50 of human leukemia
• Ubiquitinates p53 and other targets leading to
  altered cell trafficking and proteosomal
  degradation
• Oncogenic activity attributed to its inhibitory
  effect on the tumor suppressor p53

8
Screen for Inhibitors of MDM2 Mediated
Ubiquitination

• Complex assay system (3 enzymes E1, E2 E3)
• GST-MDM2 (E3) bound to the surface
• E2-Ub treated as a substrate for E3
• Visualization of polyubiquitin chains on MDM2
• using Ruthenium-tagged antibody which generates
  an electrochemiluminescent signal

9
Secondary Evaluations of a Natural Product Lead
Compound
Dose-dependent accumulation of p53
p53
b-Actin
10
ABCG2 is a Multidrug Resistance Pump
- in addition to Pgp and MRP1, ABCG2 plays a
central role in drug resistance - acute
myelogenous leukemia, breast cancer - ABCG2 is
associated with resistance of tumor cells to many
cytotoxic agents - mitoxantrone - topotecan -
flavopiridol - methotrexate
11
ABCG2 Expression Results in Reduced Accumulation
of Mitoxantrone and Topotecan
(photomicrographs courtesy of Susan Bates, NCI)
12
Cell-Based HTS Assay for ABCG2 Inhibitors
13
Cell culture NCI-H460 non-small cell lung
carcinoma cells that overexpress wild-type
ABCG2 (mitoxantrone resistant) Assay end
point fluorescent measurement of cell associated
Ph A (395 nm excitation, 670 nm emission)
Assay was run in 384 well format (50 ?L/well)
Fumitremorgin C (FTC) positive control for the
assay
Pheophorbide A (Ph A) an ABCG2 specific substrate