Development and optimization of two-dimensional liquid chromatography methodologies coupled with bioassays for discovery of enzyme inhibitors in natural product extracts

Abstract

The identification of substances with biological activity in natural extracts or synthetic collections is a crucial step and requires the use of fast and efficient techniques for separation and screening. This paradigm stimulates the development of new screening methods to facilitate and reduce the time taken to identify such substances. 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 a valuable strategy to obtain greater separation efficiency with reduced analysis time. Classical separation methods of natural extracts involve high consumption of time, while screening offline does not result in information on individual compounds and often have false positive or false negative results. A promising alternative is the coupling of chromatographic separation techniques, particularly the multi-dimensional ones, with biological assays (i.e., high throughput screening). Thus, it is possible to detect biological activity directly in the LC effluent and chemically characterize the compounds with biological activity online, using mass spectrometry (MS), resulting in reduced time and labor spent in collecting fractions for subsequent biological activity determination. For conducting online biological assays, the use of immobilized enzymes is a great option. In this context, the aim of this project is to develop a rapid method for online identification of bioactive constituents of plant extracts without the need of prior purification. Plant extracts will first be separated in the first dimension and the separated compounds will be transferred to the bioreactor, where the biological activity of each compound or small group of compounds will be measured. ICERs (Immobilized capillary enzyme reactors) containing the cholinesterase enzyme acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), were developed in our research group and were selected as model enzyme targets for this project. These enzymes are serine hydrolases responsible for the catalyzing the hydrolysis of the neurotransmitter acetylcholine at nerve endings and in synapses. These are targets of interest in studies of the development of drugs for Alzheimer's disease. (AU)