Lectin-based assay for the determination of the inhibition activity of small molecule inhibitors of neuraminidases

Hľasová, Zuzana; Pažitná, Lucia; Ondrejovič, Miroslav; Katrlı́k, Jaroslav

doi:10.1016/j.jbiotec.2020.11.016

Cited by 3 publications

(4 citation statements)

References 32 publications

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“…Given the concentration of the substrate and a K m of 2.0 ± 0.3 mM determined with capillary nanogel electrophoresis, the IC 50 values are converted to K i values of 4.3, 4.5, and 6.0, with an average K i of 5.0 ± 0.9 μM (see Tables S10 and S11 in the Supporting Information). An IC 50 of 13 ± 3 μM measured in this experiment is in agreement with a literature report of an IC 50 value of 13.4 μM . In a separate literature report, a K i value of 8 μM was reported; however, this measurement was made in an acetate buffer at a pH value of 5.5, which was above the optimum pH for this enzyme.…”

Section: Resultssupporting

confidence: 92%

Section: Resultssupporting

confidence: 91%

“…An IC 50 of 13 ± 3 μM measured in this experiment is in agreement with a literature report of an IC 50 value of 13.4 μM. 44 In a separate literature report, a K i value of 8 μM was reported; 45 however, this measurement was made in an acetate buffer at a pH value of 5.5, which was above the optimum pH for this enzyme. The Siastatin B IC 50 experiments are performed with substrate concentrations of 3.3 mM (curves 1 and 2) or 3.0 mM (curve 3) sialyllactose and an enzyme concentration of 350 μU/μL.…”

Section: ■ Materials and Methodssupporting

confidence: 91%

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Microscale Quantification of the Inhibition of Neuraminidase Using Capillary Nanogel Electrophoresis

et al. 2022

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Neuraminidase inhibitors modulate infections that involve sialic acids, making quantitative analyses of this inhibitory effect important for selecting and designing potential therapeutics. An automated nanogel capillary electrophoresis system is developed that integrates a 5 nL enzyme inhibition reaction in line with a 5 min separation-based assay of the enzymatic product to quantify inhibition as the half maximal inhibitory concentration (IC50) and inhibitor constant (K i). A neuraminidase enzyme from Clostridium perfringens is non-covalently immobilized in a thermally tunable nanogel positioned in the thermally controlled region of the capillary by increasing the capillary temperature to 37 °C. Aqueous inhibitor solutions are loaded into the capillary during the nanogel patterning step to surround the enzyme zone. The capillary electrophoresis separation provides a means to distinguish the de-sialylated product, enabling the use of sialyllactose which contains the trisaccharide motif observed on serine/threonine-linked (O-linked) glycans. A universal nanogel patterning scheme is developed that does not require pre-mixing of enzymes with inhibitors when an automated capillary electrophoresis instrument is used, thus reducing the consumption of enzymes and enabling adaption of the method to different inhibitors. The universal approach is successfully applied to two classical neuraminidase inhibitors with different electrophoretic mobilities. The IC50 and K i values obtained for N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (DANA) are 13 ± 3 and 5.0 ± 0.9 μM, respectively, and 28 ± 3 and 11 ± 1 μM, respectively, for Siastatin B. These values agree with literature reports and reflect the weaker inhibition anticipated for Siastatin B in comparison to DANA.

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Section: Resultssupporting

confidence: 92%

Section: Resultssupporting

confidence: 91%

Section: ■ Materials and Methodssupporting

confidence: 91%

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Microscale Quantification of the Inhibition of Neuraminidase Using Capillary Nanogel Electrophoresis

et al. 2022

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“…Several diseases require the use of enzyme inhibitors, indeed, a substantial part of the drugs applied for clinical purposes are enzyme inhibitors (Drews, 2000;Hopkins and Groom, 2002;Robertson, 2007). The clinical treatment of many diseases such as cancer (Dhokne et al, 2021), neurological disorders (Saxena and Dubey, 2019), infections (Zhang et al, 2021), viruses (Hľasová et al, 2021;Kharkwal et al, 2021), and others, usually involves enzymes as the drug target. In fact, in the last few years (2015-2020), FDA has approved several drugs that act as enzyme inhibitors, those classes include kinase inhibitors for cancer therapy, CY3PA4 inhibitors for neurological, metabolic disorders, and infections, and others (Bhutani et al, 2021a).…”

Section: Molecular Basis Of Enzyme Inhibitionmentioning

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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Systematic review on lectin-based electrochemical biosensors for clinically relevant carbohydrates and glycoconjugates

Abrantes-Coutinho

Santos

Moura

et al. 2021

Colloids and Surfaces B: Biointerfaces

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Lectin-based assay for the determination of the inhibition activity of small molecule inhibitors of neuraminidases

Cited by 3 publications

References 32 publications

Microscale Quantification of the Inhibition of Neuraminidase Using Capillary Nanogel Electrophoresis

Microscale Quantification of the Inhibition of Neuraminidase Using Capillary Nanogel Electrophoresis

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

Systematic review on lectin-based electrochemical biosensors for clinically relevant carbohydrates and glycoconjugates

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