Integration of Microfractionation, qNMR and Zebrafish Screening for the In Vivo Bioassay-Guided Isolation and Quantitative Bioactivity Analysis of Natural Products

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.

Biological Screening of Novel Derivatives of Valproic Acid for Anticancer and Antiangiogenic Properties

“…As shown in Figure 2 B&D and Table 2, most of these compounds failed to affect the inter-segmental blood vessels formation process. However, three compounds in this series (10a-d) completely blocked the formation of sub-intestinal vein (Bohni et al, 2013) within the concentration range of 40-50µM in 100% of treated embryos ( …”

“…It suppressed 72% of inter-segmental (Se) and 100% of subintestinal vein formation process at 2µM in all treated embryos. The compound 12c inhibited more than 83% of intersegmental (Se) and 100% of subinntestinal vein (Bohni et al, 2013) formation but with at 10µM (table1). The intersegmental blood vessels (Se) formed normally in compound 12d treated embryos; however, subinntestinal vein (Bohni et al, 2013) did not form at all in these embryos (table 1).…”

“…The compound 12c inhibited more than 83% of intersegmental (Se) and 100% of subinntestinal vein (Bohni et al, 2013) formation but with at 10µM (table1). The intersegmental blood vessels (Se) formed normally in compound 12d treated embryos; however, subinntestinal vein (Bohni et al, 2013) did not form at all in these embryos (table 1). The compound 12e failed to show any significant level of antiangiogenic activity in treated zebrafish embryos even using a 10X (50µM) more concentration as compared to 12a.…”

“…We have scored the antiangiogenic activity of the compounds in live transgenic zebrafish embryos by observing the reduction in the outgrowth of blood vessels at two time points i) Inter-segmental blood vessels (ISV) at 48 hpf and then ii) subintestinal vein (Bohni et al, 2013) out growth at 72 hpf. The embryos were anesthetized using 0.003% tricane (Sigma Aldrich cat#E10521) for counting the blood vessels and subsequently photographed.…”

“…Drug screening and development could be cost effective and save lot of time if relevant in vivo models are used. Zebrafish become an important model organism for the study of development, genetics, immunity, cancer and other diseases (Amatruda Patton, 2008;Huang Zon, 2008;Ingham, 2009;Chan Mably, 2011;Littleton Hove, 2013;Terriente Pujades, 2013) and at the same time variety of potent natural and synthetic antiangiogenic molecules have been identified using this animal model (Crawford et al, 2011;Liu et al, 2011a;Lin et al, 2012;Bohni et al, 2013;Yang et al, 2014) The objective of this study was to synthesize hydrazide-hydarzone derivatives of VPA which could have improved antiangiogenic and anticancer potential. Among the synthesized compounds, the valproylglycine hydrazide-hydrazone derivatives (compounds 12 a-c, Table 1) exhibited strong anti-angiogenic and anti cancer activity.…”