Nanofractionation LC-MS/MS and at-line Bioassays for identification of bioactive components in Natural products extracts

Abstract

Natural product extracts can be considered an invaluable source of biological active secondary metabolites. The screening of natural products for bioactive compounds has been clearly demonstrated to be a successful approach for the discovery of new drug leads. However, screening biological activities compounds from complex matrices as natural products is laborious, time consuming and costly. This paradigm stimulates the development of fast and efficient techniques as high-throughput and efficient analytical approaches enabling identification and separation of biological activity compounds. A predominant approach used is dereplication with hyphenated techniques for the chemical profiling of crude plant extracts. Natural extracts are complex mixtures and the concentration of the different bioactive compounds in these extracts differs largely, for example between the secondary metabolites, and as such include an additional challenge to screening of these extracts. For this, an optimal combination of separation and activity bioassays is necessary. Classical separation methods are time consuming and costly, while screening offline does not result in information on individual compounds and often gives false positive or false negative results. By employing chromatographic techniques, isolation of active principles derived from a natural extract can exceptionally be achieved in a single chromatographic step. The use of two-dimensional (2D) chromatographic methods is therefore a valuable strategy to obtain grater separation efficiently with reduced analysis time. However, problems arise as the high-resolution chromatographic separation employed is succeeded by a usually low-resolution fractionation process and consequently low- resolution bioactivity profiling. A promising alternative is coupling high-resolution fractionating strategies after separation in which many more microfractions can be collected as shorter time intervals for bioaffinity analysis, thus maintaining the high resolution obtained during the separation step. These fractions are collected into 384 or 1536-well microplates in which biochemical assay reagents are simultaneously or subsequently mixed in. Thus, is possible to detect biological activity directly from the microfractions and determine the chemical characterization of bioactive compounds using mass spectrometry (MS) by using a nanofractionation LC-MS/MS system. For conducting biological assays the use of immobilized enzymes is an attractive option as a high concentration of enzyme can be used in an economic manner since the enhanced stability of immobilized enzyme can allow the enzyme to be reused. This also allows to significantly shorten the incubation time. In this context, the aim of this project is to develop a rapid and efficient methodology for separation, fractionation and identification of bioactive constituents of plant, microbial extracts and venoms. Cycloxygenases (COX-1 and COX-2) are the selected targets. These enzymes are the target of widely used non-steroidal anti-inflammatory drugs (NSAIDs). The enzymes will be individually immobilized by two different approaches: into a fused silica capillary, producing flow type bioreactors, and onto glass bottomed well plates and/or glass micro beads which will provide direct compatibility to nanofractionation (to be tested for production of well plate based immobilized bioreactors in 384 well plate format).Bioaffinity screening assays using activity inhibition will be adopted to identify potential anti-inflammatory leads. After the nanofractionation step for anti-inflammatory assays, additionally also different bioassay formats will be tested including antibiotic activity by a luminescent e-coli assay in 384 well plate format (currently an experimental protocol is successfully running at the VU University) and/or an anti-tuberculosis assay with fluorescence readout (developed and ready to use for natural extract profiling).