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Enzyme ligand screening: an on-flow approach via LC-MS/MS


The identification of substances with biological activity in natural extracts or from synthetic collections is a crucial and complex step and promotes a growing interest in the use of fast and efficient techniques of separation and screening. Such a paradigm stimulates the development of new screening methods that facilitate and reduce the time spent in identifying these substances.Widely diffused, the use of off-line enzymatic assays in solution involves several limitations, among them the low compatibility of the enzymes with organic solvents. The low stability coupled with the high cost of purification makes the assays online using enzymes in solution a viable step for high efficiency screening. In contrast, conducting biological assays online using immobilized enzymes is a valuable alternative. The immobilized enzymes have greater stability in the presence of organic solvents and variations in temperature, besides allowing the reuse and the use of small amounts of enzyme.By employing chromatographic techniques, the isolation of active compounds from a natural extract can rarely be accomplished in a single chromatographic step. The 2D separations by liquid chromatography are already well established and can be classified into comprehensive, pseudo-comprehensive and "heart-cutting". In the comprehensive mode (LCxLC) all effluent from the first dimension, or a representative part thereof, is introduced into the second dimension. In heart-cutting (LC-LC) only selected fractions are transferred to the second column. 2D methods are, therefore, a valuable strategy to obtain greater separation efficiency with reduced analysis time. Classical methods of fractionation of bioguided natural extracts involve high consumption of time, whereas off-line screenings do not result in information about individual compounds and often present false positive or false negative results. Alternatively, the coupling of the techniques of 2D chromatographic separation with the biological assays, called high throughput screening is promising. In this way, it is possible to detect the biological activity directly in the LC eluent and to chemically characterize the biologically active compound (s) online, resulting in a reduction in the time and labor expended in collecting the fractions for subsequent activity detection. The use of selective affinity chromatography using IMERs has been efficiently exploited through frontal, zonal (linear and non-linear) chromatography. In addition, zonal chromatography can be employed with 2D systems hyphenated to different detectors.With the proposed development of new screening models in the identification of active substances in natural and / or synthetic collections using online methods and immobilized enzymes, important targets and immobilization protocols were selected, with emphasis on cyclooxygenase enzymes (COX-1 and COX -2) to be immobilized individually, the kalikreins (KLK1- KLK-6 and KLK-8) immobilized individually but used in parallel online systems (home-made methodology), besides the enzymes Acetylcholinesterase (AChE) and beta-secretase-1 (BACE1 ) that will be co-immobilized for application in studies of dual target inhibitors. (AU)