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Integrated platforms for screening enzyme ligands: from development to application

Abstract

The increasing interest in approaches that use fast and efficient separation and screening techniques to identify active molecules from natural extracts and combinatorial collections has been stimulated the development of new methods and integrated screening platforms that facilitate and reduce time-consuming to identify these molecules. With the proposal to develop new ligand screening models in this project, we propose creating an integrated platform by developing online and offline methods. By using bioaffinity chromatography, we can evaluate the modulation of enzymatic activity and the ligand affinity in high-efficiency screening and ligand fishing approaches. In online models, the coupling of chromatographic separation techniques with biological assays, or high-resolution screening (HRS), involves the inclusion of an efficient enzymatic assay at the end of this separation and, thus, detecting the biological activity directly into the LC eluent, in addition to chemically characterizing the biologically active compound(s) online, reducing time and work. In at-line approaches, one can also include nanofractionation steps followed by off-line assays. In an off-line model of affinity studies, enzymes immobilized on magnetic particles (MBs) and/or sepharose are directly immersed in the sample. Molecules with affinity are retained and are "fished" for future structural identification and their biological activity against the target. These two approaches (affinity and activity) will be integrated, allowing advances in the search for bioactive substances that can be extrapolated to any biological target of interest. Among the targets of interest are the cholinesterases Acetylcholinesterase (AChE) and Butyrylcholinesterase, and the aspartic acid protease beta-secretase-1 (BACE1). Also, the monoamine oxidase (MAO-A and MAO-B) and serine proteases called kallikrein (KLK), with a specific interest in human KLK1, KLK-3, KLK-5, KLK-6 and KLK-8 were selected. (AU)

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