Integration of Microfractionation, qNMR and Zebrafish Screening for the In Vivo Bioassay-Guided Isolation and Quantitative Bioactivity Analysis of Natural Products
- Authors
- Bohni, N., Cordero-Maldonado, M.L., Maes, J., Siverio-Mota, D., Marcourt, L., Munck, S., Kamuhabwa, A.R., Moshi, M.J., Esguerra, C.V., de Witte, P.A., Crawford, A.D., and Wolfender, J.L.
- ID
- ZDB-PUB-130703-14
- Date
- 2013
- Source
- PLoS One 8(5): e64006 (Journal)
- Registered Authors
- Cordero-Maldonado, Maria Lorena, Crawford, Alexander, Esguerra, Camila V.
- Keywords
- none
- MeSH Terms
-
- Angiogenesis Inhibitors/chemistry
- Angiogenesis Inhibitors/pharmacology
- Animals
- Animals, Genetically Modified
- Anti-Inflammatory Agents/chemistry
- Anti-Inflammatory Agents/pharmacology
- Biological Assay/methods*
- Biological Products/chemistry
- Biological Products/isolation & purification
- Biological Products/pharmacology*
- Blood Vessels/drug effects
- Blood Vessels/growth & development
- Cell Movement/drug effects
- Chemical Fractionation
- Drug Discovery
- Fabaceae/chemistry
- Inhibitory Concentration 50
- Leukocytes/drug effects
- Leukocytes/immunology
- Mass Spectrometry
- Microfluidic Analytical Techniques*
- Nuclear Magnetic Resonance, Biomolecular*
- Plant Extracts/chemistry
- Plant Extracts/isolation & purification
- Plant Extracts/pharmacology
- Plants, Medicinal/chemistry
- Zebrafish
- PubMed
- 23700445 Full text @ PLoS One
Natural products (NPs) are an attractive source of chemical diversity for small-molecule drug discovery. Several challenges nevertheless persist with respect to NP discovery, including the time and effort required for bioassay-guided isolation of bioactive NPs, and the limited biomedical relevance to date of in vitro bioassays used in this context. With regard to bioassays, zebrafish have recently emerged as an effective model system for chemical biology, allowing in vivo high-content screens that are compatible with microgram amounts of compound. For the deconvolution of the complex extracts into their individual constituents, recent progress has been achieved on several fronts as analytical techniques now enable the rapid microfractionation of extracts, and microflow NMR methods have developed to the point of allowing the identification of microgram amounts of NPs. Here we combine advanced analytical methods with high-content screening in zebrafish to create an integrated platform for microgram-scale, in vivo NP discovery. We use this platform for the bioassay-guided fractionation of an East African medicinal plant, Rhynchosia viscosa, resulting in the identification of both known and novel isoflavone derivatives with anti-angiogenic and anti-inflammatory activity. Quantitative microflow NMR is used both to determine the structure of bioactive compounds and to quantify them for direct dose-response experiments at the microgram scale. The key advantages of this approach are (1) the microgram scale at which both biological and analytical experiments can be performed, (2) the speed and the rationality of the bioassay-guided fractionation – generic for NP extracts of diverse origin – that requires only limited sample-specific optimization and (3) the use of microflow NMR for quantification, enabling the identification and dose-response experiments with only tens of micrograms of each compound. This study demonstrates that a complete in vivo bioassay-guided fractionation can be performed with only 20 mg of NP extract within a few days.