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

Abstract

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.

On-flow enzymatic inhibitor screening: The emerging success of liquid chromatography-based assays

“…More recently, an innovative platform for ligand screening in natural products that do not require pre-treatment steps of the sample was described by Seidl et al (2022). An analytical column inserted in the first dimension (Figure 4C) provided the online microfractionation of the extract components, while the AChE-IMER in the second dimension yielded the inhibition profile of each chromatogram zone.…”

“…Different solid supports can be employed for enzyme immobilization in the development of LC-based on-flow screening assays. Silica-based supports (de Moraes et al, 2012Vanzolini et al, 2013;da Silva et al, 2013;Hu et al, 2015;Silva et al, 2015;Vilela et al, 2015Vilela et al, , 2018Calil et al, 2016;Magalhães et al, 2016;Sarria et al, 2016;Ferreira Lopes Vilela and Cardoso, 2017;Cornelio et al, 2018;Lima et al, 2019;Medina et al, 2019;Qian et al, 2019;Seidl et al, 2019Seidl et al, , 2022Ferey et al, 2019;Chapla et al, 2020;Hou et al, 2020;de Castro et al, 2022) present a versatile and largely explored motif due presence of many hydroxyl groups on its surface. For example, those groups are readily reactive with 3aminopropyltriethoxysilane (APTES), furnishing an amine- rich surface that can further react with glutaraldehyde (GA), yielding a terminal reactive aldehyde surface susceptible reactions with nucleophilic groups present on the enzyme surface (Figure 2B).…”

“…For example, those groups are readily reactive with 3aminopropyltriethoxysilane (APTES), furnishing an amine- rich surface that can further react with glutaraldehyde (GA), yielding a terminal reactive aldehyde surface susceptible reactions with nucleophilic groups present on the enzyme surface (Figure 2B). Seidl et al (2022) successfully explored this approach by immobilizing AChE in a fused silica capillary for inhibitors screening in bulbs of Hippeastrum calyptratum. Moreover, the readily available fused silica capillary might justify the majority of the works (>80%) employing silica-based supports for on-flow assays (Table 1).…”