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A Comprehensive 2D-LC/MS Online Platform for Screening of Acetylcholinesterase Inhibitors

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Seidl, Claudia ; de Lima, Juliana Maria ; Leme, Gabriel Mazzi ; Pires, Ananda Ferreira ; Stoll, Dwight R. ; Cardoso, Carmen Lucia
Total Authors: 6
Document type: Journal article
Source: FRONTIERS IN MOLECULAR BIOSCIENCES; v. 9, p. 14-pg., 2022-03-16.

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. (AU)

FAPESP's process: 14/50249-8 - Green chemistry: sustainable synthetic methods employing benign solvents, safer reagents, and bio-renewable feedstock
Grantee:Arlene Gonçalves Corrêa
Support Opportunities: Research Grants - Research Centers in Engineering Program
FAPESP's process: 14/11640-3 - LC-MS / 2D methods for screening ligands for evaluating enzyme inhibitory activity and identification of active compounds in crude extracts
Grantee:Cláudia Seidl
Support Opportunities: Scholarships in Brazil - Post-Doctoral
FAPESP's process: 14/06907-0 - Immobilization nucleoside diphosphate kinase (NDK) enzyme Leishmania major: the study of conditions for the application in the screening of ligands
Grantee:Juliana Maria de Lima
Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
FAPESP's process: 19/05363-0 - Enzyme ligand screening: an on-flow approach via LC-MS/MS
Grantee:Carmen Lúcia Cardoso
Support Opportunities: Regular Research Grants
FAPESP's process: 16/02941-5 - Development and optimization of two-dimensional liquid chromatography methodologies coupled with bioassays for discovery of enzyme inhibitors in natural product extracts
Grantee:Cláudia Seidl
Support Opportunities: Scholarships abroad - Research Internship - Post-doctor