Abstract:
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.
