Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase ( Lic NTPDase-2-ICER-LC/UV)

Abstract

Nucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme belonging to the apyrase family that participates in the hydrolysis of the nucleosides di- and triphosphate to the corresponding nucleoside monophosphate. This enzyme underlies the virulence of parasites such as Leishmania. Recently, an NTPDase from Leishmania infantum (LicNTPDase-2) was cloned and expressed and has been considered as a new drug target for the treatment of leishmaniasis. With the intent of developing label-free online screening methodologies, LicNTPDase-2 was covalently immobilized onto a fused silica capillary tube in the present study to create an immobilized capillary enzyme reactor (ICER) based on LicNTPDase-2 (LicNTPDase-2-ICER). To perform the activity assays, a multidimensional chromatographic method was developed employing the LicNTPDase-2-ICER in the first dimension, and an analytical Ascentis C8 column was used in the second dimension to provide analytical separation of the substrates and products. The validated LicNTPDase-2-ICER method provided the following kinetic parameters of the immobilized enzyme: KM of 2.2 and 1.8mmolL(-1) for the ADP and ATP substrates, respectively. Suramin (1mmolL(-1)) was also shown to inhibit 32.9% of the enzymatic activity. The developed method is applicable to kinetic studies and enables the recognition of the ligands. Furthermore, a comparison of the values of LicNTPDase-2-ICER with those obtained with an LC method using free enzyme in solution showed that LicNTPDase-2-ICER-LC/UV was an accurate and reproducible method that enabled automated measurements for the rapid screening of ligands.

100 Years of Suramin

“…For caspases, cysteine proteases involved in apoptosis, suramin was described as acting as either inhibitor or activator (120,121). Suramin further inhibits the Na ϩ ,K ϩ -ATPase and other ATPases (122)(123)(124), certain classes of GABA receptors (125,126), and several G protein-coupled receptors (127), including P2 purinoceptors and follicle-stimulating hormone receptor (128,129). Suramin also showed inhibitory effects against components of the coagulation cascade (71,130) and the complement system (131-133) and against deubiquitinating enzymes (PubChem BioAssay no.…”

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

“…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.…”

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

“…This approach overcomes the need for constant use of enzyme solution allowing the enzyme to be reused, while also enhancing enzyme stability in the presence of organic modifiers and pH variations ( Liu et al, 2018 ; De Simone et al, 2019 ). Our group has explored different applications of immobilized capillary enzyme reactor (cIMER) as a powerful tool for online ligand screening when coupled with UV ( da Silva et al, 2013 ; Vilela et al, 2014 ; Lima et al, 2016 ; Magalhães et al, 2016 ) and MS detection ( Vanzolini et al, 2013 ; Ferreira Lopes Vilela and Cardoso, 2017 ; Seidl et al, 2019 ). More recently, Yuan et al ( Yuan et al, 2020 ) developed a method that coupled LC separation with MS detection and an immobilized enzyme reactor (HPLC-IMER-MS) for screening of AChE inhibitors in NP crude extracts.…”