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.
Highlights
Natural products (NPs) are an important source of drug-like compounds for the discovery of new therapeutic candidates and over time their chemical diversity has contributed significantly to the development of drugs for a wide range of diseases
The anti-inflammatory effect of the crude extract of R. viscosa was evident at 50 mg/mL – a concentration at which a relative leukocyte migration (RLM) value of 0.39 was achieved (Figure 1D), in comparison with an RLM of 0.24 achieved by 100 mM indomethacin as a positive control (Figure 1E)
The known anti-inflammatory and anti-angiogenic activities of genistein provide an initial validation of our NP discovery approach
Summary
Natural products (NPs) are an important source of drug-like compounds for the discovery of new therapeutic candidates and over time their chemical diversity has contributed significantly to the development of drugs for a wide range of diseases. Most drug discovery programs are based on highthroughput screening (HTS) to rapidly query the bioactivity of large libraries of synthetic compounds. The isolation and characterization of bioactive secondary metabolites present in complex NP extracts involves the application of several complementary methodologies that require considerably more time and effort [4,5]. The chemical properties of certain secondary metabolites might hinder the test readout and interfering constituents present in the crude extract can either mask the biological activity [6] or cause toxic effects that lead to false positives, e.g. in enzymatic assays. NPs are a promising source of diverse molecular scaffolds for the discovery of novel lead compounds against original targets [7] and recently, combinatorial libraries with NP-like compounds have been used for HTS [3]
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