RESUMO
The continuous interest in discovering new bioactive molecules derived from natural products (NP) has stimulated the development of improved screening assays to help overcome challenges in NP-based drug discovery. Here, we describe a unique platform for the online screening of acetylcholinesterase inhibitors without the need for pre-treating the sample. In the current study, we have demonstrated the ability to combine reversed-phase separation with a capillary immobilized enzyme reactor (cIMER) in two-dimensional liquid chromatography system coupled with mass spectrometry detection. We systematically investigated the effects of method parameters that are of practical significance and are known to affect the enzyme assay and interfere in the analysis such as: bioreactor dimensions, loop sizes, amount of immobilized enzyme, second dimension flow rates, reaction time, substrate concentration, presence of organic modifier, limit of detection and signal suppression. The performance of this new platform was evaluated using a mixture containing three known AChE inhibitors (tacrine, galanthamine and donepezil) and an ethanolic extract obtained from the dry bulbs of Hippeastrum calyptratum (Amaryllidaceae) was investigated to provide a proof of concept of the applicability of the platform for the analysis of complex mixtures such as those derived from NPs.
RESUMO
Functionalized micro- and nano-sized magnetic beads (MBs) have been widely used as versatile supports for proteins, enzymes, and drugs. Immobilized protein on MB surfaces has been successfully applied for ligand fishing assays allowing for direct identification of active ligands from complex mixtures, such as natural products and synthetic libraries. MBs with different properties such as different core compositions, sizes, coatings, and surface modifications are available commercially. Studies have been conducted to understand the role of these properties for ligand fishing assays. Here we evaluated, for the first time, the effect of MB size on the ligand fishing assay for acetylcholinesterase from Electrophorus electricus (AChE). For this purpose, four commercially available amine-terminated magnetic particles with diameters ranging from 4.5 nm to 106 µm were evaluated to fish out galantamine, a well-known AChE inhibitor, from an aqueous solution. All MBs were efficient at using glutaraldehyde to covalently immobilize AChE. The particles with diameters of about 1 µm (small microparticles) presented a higher protein mass capacity per milligram of particle than did those with diameters of about 4.5 nm (nanoparticles) and those with diameters of about 106 µm (large microparticles). The influence of these supports on the produced AChE-MBs with regards to hydrolysis turnover and ligand fishing was evaluated and is fully discussed.