Cathepsin D immobilized capillary reactors for on-flow screening assays

Cornelio, Vivian E.; Moraes, Marcela Cristina de; Domingues, Vanessa de Cássia; Fernandes, João B.; Silva, Maria Fátima das Graças Fernandes da; Cass, Quézia B.; Vieira, Paulo C.

doi:10.1016/j.jpba.2018.01.001

Cited by 13 publications

(8 citation statements)

References 28 publications

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“…The enzyme immobilization on magnetic particles proved to be a versatile approach in which offline and inline modes could be applied using the same target-support system. Other biomolecules were immobilized by covalent bond through the formation of Schiff bases, like as trypsin (Liu et al, 2019), human Thymidylate Kinase (hTMPK) and the human Nucleoside Diphosphate Kinase (hNDPK) (Ferey et al, 2019), cathepsin D (Cornelio et al, 2018), acetylcholinesterase (AChE) (Vandeput et al, 2015;Seidl et al, 2019), butyrylcholinesterase (BChE) (Seidl et al, 2019), Phospholipase A2 (PLA2) (Wei et al, 2021), and others.…”

Section: Immobilization Methodsmentioning

confidence: 99%

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

et al. 2022

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Enzymes are targets commonly explored in screening assays aiming to discover new leads in the drug development process. Among the diverse assay models to identify new enzymatic inhibitors, on-flow assays based on liquid chromatography (LC) can be highlighted. In these approaches, the ligand-enzyme interaction can be examined by monitoring the catalytic activity or the affinity/retention. Most applications use the biological target immobilized in solid supports resulting in the acquisition of an immobilized enzymatic reactor (IMER). Coupling IMERs to LC or mass spectrometry (MS) systems allows monitoring enzyme activity online and studying binding events between target and ligands. On-flow screening assays present many advantages for the hit-to-lead process, such as the possibility of system automation, reusability, and high stability. This review covers articles from the last decade that combine the use of varied immobilization methods on different solid supports and several equipment setups in on-flow systems, emphasizing the performance and capacity of recognizing and identifying biologically active compounds in various matrices.

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Section: Immobilization Methodsmentioning

confidence: 99%

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

et al. 2022

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“…Most of the capillary-based µ-IMERs utilize commercially available fused silica capillaries to accommodate the enzymes. A large variety of methods exist for the derivatization of these capillaries, which mostly rely on surface-modification [46][47][48][49] or in situ monolith formation [50][51][52][53][54][55][56][57]. The greater flexibility regarding reactor layout and architecture in the case of chips is enabled by (often) in-house created devices made by means of microfabrication.…”

Section: Designs Materials and Fabrication Technologiesmentioning

confidence: 99%

“…Since LC-MS and CE-MS apparatus are commercially available on-line coupled systems, the main difficulty lies in creating an automated, continuous fluidic connection between the µ-IMER and the separation platform. The on-line coupling of digestion and LC separation can be achieved with the use of switching valves [46,47,56,[66][67][68]. In such configurations, the µ-IMER and the analytical column are connected via a valve, where the microreactor can be considered as the first dimension of the setup.…”

Section: Coupling To Downstream Processing Unitsmentioning

confidence: 99%

Microfluidic Immobilized Enzymatic Reactors for Proteomic Analyses—Recent Developments and Trends (2017–2021)

2022

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Given the strong interdisciplinary nature of microfluidic immobilized enzyme reactor (μ-IMER) technology, several branches of science contribute to its successful implementation. A combination of physical, chemical knowledge and engineering skills is often required. The development and application of μ-IMERs in the proteomic community are experiencing increasing importance due to their attractive features of enzyme reusability, shorter digestion times, the ability to handle minute volumes of sample and the prospect of on-line integration into analytical workflows. The aim of this review is to give an account of the current (2017–2021) trends regarding the preparation of microdevices, immobilization strategies, and IMER configurations. The different aspects of microfabrication (designs, fabrication technologies and detectors) and enzyme immobilization (empty and packed channels, and monolithic supports) are surveyed focusing on μ-IMERs developed for proteomic analysis. Based on the advantages and limitations of the published approaches and the different applications, a probable perspective is given.

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“…Zonal elution has also been applied to monitor on-line enzyme activity in ligand screening assays, resulting in reliable and specific assays that allow the biocatalysis product to be directly quantified (da Silva et al, 2012; de Moraes et al, 2013; Calil et al, 2016; Lima et al, 2016; Magalhães et al, 2016; Ferreira Lopes Vilela and Cardoso, 2017; Cornelio et al, 2018; Vilela et al, 2018; Seidl et al, 2019). This approach prevents interferences in inhibitors screening and furnishes reliable data concerning the substrate- and inhibitor-enzyme binding.…”

Section: On-line Approachesmentioning

confidence: 99%

Solid-Supported Proteins in the Liquid Chromatography Domain to Probe Ligand-Target Interactions

2019

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Ligand-target interactions play a central role in drug discovery processes because these interactions are crucial in biological systems. Small molecules-proteins interactions can regulate and modulate protein function and activity through conformational changes. Therefore, bioanalytical tools to screen new ligands have focused mainly on probing ligand-target interactions. These interactions have been evaluated by using solid-supported proteins, which provide advantages like increased protein stability and easier protein extraction from the reaction medium, which enables protein reuse. In some specific approaches, precisely in the ligand fishing assay, the bioanalytical method allows the ligands to be directly isolated from complex mixtures, including combinatorial libraries and natural products extracts without prior purification or fractionation steps. Most of these screening assays are based on liquid chromatography separation, and the binding events can be monitored through on-line or off-line methods. In the on-line approaches, solid supports containing the immobilized biological target are used as chromatographic columns most of the time. Several terms have been used to refer to such approaches, such as weak affinity chromatography, high-performance affinity chromatography, on-flow activity assays, and high-performance liquid affinity chromatography. On the other hand, in the off-line approaches, the binding event occurs outside the liquid chromatography system and may encompass affinity and activity-based assays in which the biological target is immobilized on magnetic particles or monolithic silica, among others. After the incubation step, the supernatant or the eluate from the binding assay is analyzed by liquid chromatography coupled to various detectors. Regardless of the selected bioanalytical approach, the use of solid supported proteins has significantly contributed to the development of automated and reliable screening methods that enable ligands to be isolated and characterized in complex matrixes without purification, thereby reducing costs and avoiding time-laborious steps. This review provides a critical overview of recently developed assays.

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Cathepsin D immobilized capillary reactors for on-flow screening assays

Cited by 13 publications

References 28 publications

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

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

Microfluidic Immobilized Enzymatic Reactors for Proteomic Analyses—Recent Developments and Trends (2017–2021)

Solid-Supported Proteins in the Liquid Chromatography Domain to Probe Ligand-Target Interactions

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