The Mini-B used here was designed to maintain several important structural features of full-length human SP-B

t require considerably more time and effort. In addition, there are several inherent caveats associated with testing NPs in HTS. Crude extracts from various species of plants, fungi, and bacteria, herein after called NP extracts, are complex mixtures of mostly uncharacterized compounds, some of which might have undesired effects. The chemical properties of certain secondary metabolites might hinder the test Microscale Natural Product Discovery in Zebrafish readout and interfering constituents present in the crude extract can either mask the biological activity or cause toxic effects that lead to false positives, e.g. in enzymatic assays. Nevertheless, a considerable advantage of NPs is their chemical diversity. The chemical space occupied by NPs is different from the one occupied by synthetic compounds often with far greater degrees of 3dimensionality and structural complexity. NPs are a promising source of diverse molecular scaffolds for the discovery of novel lead compounds against original targets and recently, combinatorial libraries with NP-like compounds have been used for HTS. Bioassay-guided fractionation has proven successful as a wellestablished platform to isolate and characterize active constituents present in NP extracts, which are then suitable for HTS. However, such an approach requires multiple chromatographic steps and large amounts of biological material. Recent technological improvements in the area of chromatographic separation methods have nevertheless provided new possibilities to accelerate the overall process of bioassay-guided fractionation. In particular, the development of microfractionation approaches based on advanced high performance liquid chromatography techniques is now enabling the systematic separation of complex plant extracts using more widely applicable protocols. The increasing sophistication of such techniques by linking them directly or indirectly by adding an additional step of sample concentration with analytical assays allows the more rapid dereplication of extracts identifying known NPs prior to thorough characterization thereby focusing resources on novel molecules. Although active constituents present in NP extracts can now be identified more quickly as less time is expended on the purification of inactive constituents, still appreciable amount of time is invested if the bioactive compounds need to be isolated for the determination of their structure and in-depth biological testing. This is the bottleneck of bioassay-guided isolation since the de novo structure elucidation of small molecules relies on NMR 17496168 spectroscopy, which has intrinsically low sensitivity. Nevertheless, with the emergence of microflow NMR and cryo and microcryo NMR technologies used routinely in NP drug discovery, the boundaries could be pushed to the low microgram scale of sample needed for the acquisition 23713790 of 1H-13C and 13C spectra. When working with HPLC-based biological profiling, another issue is to order PHA-793887 quantify the potency of a given extract constituent in a given bioassay since the microgram quantities obtained by microfractionation have to be correctly estimated. Weighing of the individual microfractions is not only impractical but also inaccurate at sub-milligram quantities. Furthermore, compound purity is not taken into account. Since NMR gives an absolute signal response, it can not only provide unambiguous compound identification but allows precise quantification even of unknown compounds and estimate ratios in fr

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