Immobilization of NTPDase-1 fromTrypanosoma cruziand Development of an Online Label-Free Assay

Abstract

The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the TcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave K M of 0.317 ± 0.044 mmol·L−1, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L−1, which is in accordance with the data for the enzyme in solution.

Purinergic Antagonist Suramin Aggravates Myocarditis and Increases Mortality by Enhancing Parasitism, Inflammation, and Reactive Tissue Damage in Trypanosoma cruzi‐Infected Mice

“…From in vitro evidence, it is understandable that ecto -NTPDase has been considered a promising molecular target in the treatment of Chagas disease [ 10 , 37 , 38 ]. However, in vitro studies also showed a contradictory response.…”

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

“…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).…”

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.…”