Methods and Current Trends in Determination of Neuraminidase Activity and Evaluation of Neuraminidase Inhibitors

Hľasová, Zuzana; Košík, Ivan; Ondrejovič, Miroslav; Miertuš, Stanislav; Katrlı́k, Jaroslav

doi:10.1080/10408347.2018.1531692

Cited by 17 publications

(7 citation statements)

References 117 publications

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“…In addition, we have also established a kinetic model of neuraminidase hydrolysis. By acting neuraminidase on CHO cells, Neu5Gc and Neu5Ac molecules on the cell surface are hydrolyzed to make the cell surface carry different contents of Neu5Gc and Neu5Ac molecules [ 32 ]. The electrochemical detection results show that with the extension of neuraminidase hydrolysis time ( Figure 4(b2) ), the Neu5Gc and Neu5Ac molecules carried on the cell surface will gradually decrease, and the peak currents of PbS QDoligo and CdS QDoligo will gradually decrease ( Figure 4(c2) ), which further explains the sensitivity and accuracy of the electrochemical method.…”

Section: Resultsmentioning

confidence: 99%

Electrochemical Evaluation of Tumor Development via Cellular Interface Supported CRISPR/Cas Trans-Cleavage

et al. 2022

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Evaluating tumor development is of great importance for clinic treatment and therapy. It has been known that the amounts of sialic acids on tumor cell membrane surface are closely associated with the degree of cancerization of the cell. So, in this work, cellular interface supported CRISPR/Cas trans-cleavage has been explored for electrochemical simultaneous detection of two types of sialic acids, i.e., N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Specifically, PbS quantum dot-labeled DNA modified by Neu5Gc antibody is prepared to specifically recognize Neu5Gc on the cell surface, followed by the binding of Neu5Ac through our fabricated CdS quantum dot-labeled DNA modified by Sambucus nigra agglutinin. Subsequently, the activated Cas12a indiscriminately cleaves DNA, resulting in the release of PbS and CdS quantum dots, both of which can be simultaneously detected by anodic stripping voltammetry. Consequently, Neu5Gc and Neu5Ac on cell surface can be quantitatively analyzed with the lowest detection limits of 1.12 cells/mL and 1.25 cells/mL, respectively. Therefore, a ratiometric electrochemical method can be constructed for kinetic study of the expression and hydrolysis of Neu5Gc and Neu5Ac on cell surface, which can be further used as a tool to identify bladder cancer cells at different development stages. Our method to evaluate tumor development is simple and easy to be operated, so it can be potentially applied for the detection of tumor occurrence and development in the future.

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Section: Resultsmentioning

confidence: 99%

Electrochemical Evaluation of Tumor Development via Cellular Interface Supported CRISPR/Cas Trans-Cleavage

et al. 2022

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“…With conventional assays, the neuraminidase activity is quantified with a labeled sialic acid substrate (e.g., MU-NANA), which is optically detectable after the substrate is cleaved . Sialylation in mammals is linked to galactose and N -acetyl-glucosamine residues .…”

Section: Resultsmentioning

confidence: 99%

“…Enzyme activity assays typically use a synthetic substrate labeled with a chromophore that is optically detected upon cleavage. Although several different turn-on assays exist for neuraminidase, 9 4-methylumbelliferyl-α- d - N -acetylneuraminic acid (MUNANA) is the most common reagent used to monitor the cleavage of sialic acid. With this approach, the separation of the MUNANA substrate from the sialic acid product is not required.…”

Section: Introductionmentioning

confidence: 99%

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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“…Therefore, there is an effort to develop antiviral drugs that effectively suppress the infection. Research has mainly been focused on neuraminidase (NA) inhibition [ 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. NA is a sialidase [ 18 ] and helps to release new virions from infected cells.…”

Section: Introductionmentioning

confidence: 99%

Optimization of an Inclusion Body-Based Production of the Influenza Virus Neuraminidase in Escherichia coli

et al. 2022

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Neuraminidase (NA), as an important protein of influenza virus, represents a promising target for the development of new antiviral agents for the treatment and prevention of influenza A and B. Bacterial host strain Escherichia coli BL21 (DE3)pLysS containing the NA gene of the H1N1 influenza virus produced this overexpressed enzyme in the insoluble fraction of cells in the form of inclusion bodies. The aim of this work was to investigate the effect of independent variables (propagation time, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration and expression time) on NA accumulation in inclusion bodies and to optimize these conditions by response surface methodology (RSM). The maximum yield of NA (112.97 ± 2.82 U/g) was achieved under optimal conditions, namely, a propagation time of 7.72 h, IPTG concentration of 1.82 mM and gene expression time of 7.35 h. This study demonstrated that bacterially expressed NA was enzymatically active.

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Methods and Current Trends in Determination of Neuraminidase Activity and Evaluation of Neuraminidase Inhibitors

Cited by 17 publications

References 117 publications

Electrochemical Evaluation of Tumor Development via Cellular Interface Supported CRISPR/Cas Trans-Cleavage

Electrochemical Evaluation of Tumor Development via Cellular Interface Supported CRISPR/Cas Trans-Cleavage

Microscale Quantification of the Inhibition of Neuraminidase Using Capillary Nanogel Electrophoresis

Optimization of an Inclusion Body-Based Production of the Influenza Virus Neuraminidase in Escherichia coli

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