인플루엔자 바이러스 검출을 위한 종이 기반 neuraminidase 효소 활성 평가 센서 개발

황철환 ( Cheol-hwan Hwang ),정성근 ( Seong-geun Jeong ),박한규 ( Han-kyu Park ),이창수 ( Chang-soo Lee ),김윤곤 ( Yun-gon Kim ) 한국화학공학회 2016 Korean Chemical Engineering Research(HWAHAK KONGHA Vol.54 No.3

In this study, we described a paper-based neuraminidase assay sensor (PNAS) which can be applied to detect the infection by influenza viruses. The PNAS was designed and manufactured to quantitatively identify the levels of neuraminidase in the sample, which is based on colorimetric analysis using the X-Neu5Ac substrate. The limit of detection of the PNAS was determined as 0.004 U/mL of neuraminidase. According to the amount of neuraminidase in human serum, the PNAS could monitor the enzyme activity with a good linearity (R2 > 0.99). In addition, the initial performance of the PNAS has been maintained up to 70 days in the 4 ℃. Finally, we demonstrated whether the Michaelis- Menten kinetics is applied to the PNAS, which can show the reliability of the enzyme reactions. The kinetic studies indicated that the PNAS provides the good condition for enzyme reactions (Km=8.327×10.3 M), but they were performed on paper chip nonetheless. The paper-based neuraminidase assay sensor may be useful in a wide range of rapid and safe detection of influenza virus.

Neuraminidase Treatment Enhances Allogeneic Stimulation of Unprimed CD8+ T Cells

Kim,Kilhyoun The Korea Science and Technology Center 1997 BMB Reports Vol.30 No.6

Many cell types are known to stimulate CD8+ T cells in allogeneic recognition such as mixed lymphocyte reaction (MLR). Whereas dendritic cells are most potent among them. T cells are usually considered very poor in stimulating CD8+ T cells although there are some tumor cells that are weakly stimulatory. T cells, as a stimulator, cultured in the presence of concanavalin A that were otherwise nonstimulatory to CD8+ T cells appeared to stimulate CD8+ T cells strongly when they were pretreated with neuraminidase. The enhancement of MLR by neuraminidase could be achieved by treating either the stimulators or responders with neuraminidase. Removal of negatively-charged sialic acid moieties from the cell surface, which reduced electrostatic repulsion between responders and stimulators to give better cell-cell contact might be responsible for the enhanced MLR. In addition, neuraminidase treatment also appeared to deliver activation signal to responding T cells since it could activate CD8+ T cells in synergy with phorbol myristate acetate. The maximal responses were observed when both responders and stimulators were treated with neuraminidase.

Neuraminidase Inhibitors from the Fruiting Body of Glaziella splendens

( Ji-yul Kim ),( E-eum Woo ),( Lee Su Ha ),( Dae-won Ki ),( In-kyoung Lee ),( Bong-sik Yun ) 한국균학회 2019 Mycobiology Vol.47 No.2

Neuraminidase (NA) cleaves the glycosidic bond linkages of sialic acids to release the mature virions from infected cells and has been an attractive therapeutic target for anti-influenza agents. In our ongoing investigation of NA inhibitors in mushroom extracts, we found that the extract the fruiting body of Glaziella splendens potently inhibited neuraminidase. The fruiting bodies of G. splendens were extracted and partitioned successively with hexane, ethyl acetate, and butanol. The ethyl acetate soluble-layer was subjected to silica gel and Sephadex LH-20 column chromatographies, and MPLC to obtain five compounds (1-5). Their structures were determined by spectroscopic methods. NA inhibitory activity of these compounds was evaluated using NAs from recombinant rvH1N1, H3N2, and H5N1 influenza A viruses. One compound (1) was elucidated as a new azaphilone derivative, and four compounds (2-5) were identified as entonaemin A, comazaphilone D, rubiginosin A, and entonaemin B, respectively. Compounds 3 and 4 showed considerable inhibitory activity against three types of neuraminidases with the IC₅₀ values of 30.9, 41.8, and 35.7 μM for 3 and 46.5, 50.4, and 29.9 μM for 4, respectively. This study reveals that the fruiting bodies of G. splendens possess azaphilone derivatives with the NA inhibitory activity. This is the first report on the isolation of neuraminidase inhibitors from the fruiting bodies of G. splendens.

Neuraminidase Treatment Enhances Allogeneic Stimulation of Unprimed CD8^+ T Cells

Kim, Kilhyoun 梨花女子大學校 藥學硏究所 1998 藥學硏究論文集 Vol.- No.7

Many cell types are know to stimulate CD8^+ T cells in allogeneic recognition such as mixed lymphocyte reaction (MLR). Whereas dendritic cells are most potent among them, T cells are usually considered very poor in stimulating CD8^+ T cells although there are some tumor cells that are weakly stimulatory. T cells, as a stimulator, cultured in the presence of concanavalin A that were otherwise nonstimulatory to CD8^+ T cells appeared to stimulate CD8^+ T cells strongly when they were pretreated with neuraminidase. The enhancement of MLR by neuraminidase could be achieved by treating either the stimulators or responders with neuraminidase. Removal of negatively-charged sialic acid moieties from the cell surface, which reduced electrostatic repulsion between responders and stimulators to give better cell-cell contact might be responsible for the enhanced MLR In addition, neuraminidase treatment also appeared to deliver activation signal to responding T cells since it could activate CD8^+ T cells in synergy with phorbol myristate acetate. The maximal responses were observed when both responders and stimulators were treated with neuraminidase.

Neuraminidase Treatment Enhances Allogeneic Stimulation of Unprimed $CD8^+$ T Cells

Kim, Kil-Hyoun Korean Society for Biochemistry and Molecular Biol 1997 Journal of biochemistry and molecular biology Vol.30 No.6

Many cell types are known to stimulate $CD8^+$ T cells in allogeneic recognition such as mixed lymphocyte reaction (MLR). Whereas dendritic cells are most potent among them. T cells are usually considered very poor in stimulating $CD8^+$ T cells although there are some tumor cells that are weakly stimulatory. T cells, as a stimulator, cultured in the presence of concanavalin A that were otherwise nonstimulatory to $CD8^+$ T cells appeared to stimulate $CD8^+$ T cells strongly when they were pretreated with neuraminidase. The enhancement of MLR by neuraminidase could be achieved by treating either the stimulators or responders with neuraminidase. Removal of negatively-charged sialic acid moieties from the cell surface, which reduced electrostatic repulsion between responders and stimulators to give better cell-cell contact might be responsible for the enhanced MLR. In addition, neuraminidase treatment also appeared to deliver activation signal to responding T cells since it could activate $CD8^+$ T cells in synergy with phorbol myristate acetate. The maximal responses were observed when both responders and stimulators were treated with neuraminidase.

Neuraminidase Inhibitors from Reynoutria elliptica

Chu-HyunLee,Sang-ImKim,이경복,Yung-ChoonYoo,Si-YoungRyu,송경식 대한약학회 2003 Archives of Pharmacal Research Vol.26 No.5

In the course of screening neuraminidase inhibitors from herbal medicines, Reynoutria elliptica exhibited high inhibitory activity. Four active compounds were isolated from the ethyl acetate soluble fraction by consecutive purification using sillica gel, Sephadex LH-20 chromatography, and recrystallization. The chemical structures of these compounds were identified as 1,3,8-trihydroxy- 6-methylanthraquinone (emodin) 1,8-dihydroxy-3-methoxy-6-methylanthraquinone (emodin 3-methyl ether; physcion), 1,3,8-trihydroxy-6-hydoxymethylanthraquinone (w-hydroxyemodin), and 3,5,4'-trihydroxystilbene ( trans-resvertrol) by spectral data including MS, 1H-, and 13C-NMR. The IC50 values of emodin, emodin 3-methyl ether, w-hydroxyemodin, and trans-resvertrol were 2.81, 74.07, 10.49, and 8.77 mM, respectively. They did not inhibit other glycosidase such as glucosidase, mannosidase, and galactosidase, indicating that they were relatively specific inhibitors of neuraminidase.

Neuraminidase Inhibitors from Reynoutria elliptica

Lee, Chu-Hyun,Kim, Sang-In,Lee, Kyung-Bok,Yoo, Yung-Choon,Ryu, Si-Young,Song, Kyung-Sik The Pharmaceutical Society of Korea 2003 Archives of Pharmacal Research Vol.26 No.5

In the course of screening neuraminidase inhibitors from herbal medicines, Reynoutria elliptica exhibited high inhibitory activity. Four active compounds were isolated from the ethyl acetate soluble fraction by consecutive purification using sillica gel, Sephadex LH-20 chromatography, and recrystallization. The chemical structures of these compounds were identified as 1,3,8-trihydroxy-6-methylanthraquinone (emodin) 1,8-dihydroxy-3-methoxy-6-methylanthraquinone (emodin 3-methyl ether; physcion), 1,3,8-trihydroxy-6-hydoxymethylanthraquinone ($\omega$-hydroxyemodin), and 3,5,4 -trihydroxystilbene (trans-resvertrol) by spectral data including MS, $^1 H-, and ^{13}C-NMR. The IC_{50}$ values of emodin, emodin 3-methyl ether, $\omega$-hydroxyemodin, and trans-resvertrol were 2.81, 74.07, 10.49, and 8.77 $\mu$M, respectively. They did not inhibit other glycosidase such as glucosidase, mannosidase, and galactosidase, indicating that they were relatively specific inhibitors of neuraminidase.

Neuraminidase Inhibitors from Mushroom Microphorus affinis

( Kim Kyung Bum ),( Sang In Kim ),( Kyung Sik Song ) 한국미생물생명공학회 2003 Journal of microbiology and biotechnology Vol.13 No.5

유산균으로 발효한 침향공진단으로부터 분리한 Nodakenetin의 Neuraminidase 활성 억제 효능

서지현 ( Ji Hyun Seo ),박동준 ( Dong Jun Park ),이소영 ( So Young Lee ),조호성 ( Ho Song Cho ),진무현 ( Mu Hyun Jin ) 한국미생물 · 생명공학회 2020 한국미생물·생명공학회지 Vol.48 No.3

대표적인 한방 보약 처방인 원방공진단을 재해석한 침향공진단 (당귀, 녹용, 산수유, 및 침향의 혼합추출물)을 유산균으로 발효하고, 침향공진단 성분 중 발효를 통해 증가하는 성분을 분리, 정제하고 nodakenetin임을 동정하였다. 발효전 침향공진단(침향공진단 농축액 1% 함유 MRS 배지, unfermented Gongjin-dan, GD) 및 발효후 침향공진단(침향공진단 발효액, fermented Gongjin-dan, FGD)에서의 nodakenetin 함량 분석 결과, 각각 6 μg/ml과 70 μg/ml으로 발효를 통해 nodakenetin이 약 10배 이상 증가하였다. 한편, 고서에 전해지는 공진단의 면역력 강화 효능에 근거하여, GD 및 FGD의 인플루엔자 바이러스 증식 억제 효능을 확인하고자 Neuraminidase (NA) 활성 평가법(NA activity assay)을 실시하였다. 실험 결과, GD는 NA 활성을 억제하지 못하였으나, FGD는 농도의존적으로 NA 활성을 억제하였으며 500 μg/ml에서 대조군 대비 약 92%의 억제율을 보였다. 또한, 발효를 통해 증가한 침향공진단의 성분인 nodakenetin과 그 배당체인 nodakenin에 대한 NA 활성 평가 결과, nodakenin은 NA 활성을 거의 억제하지 못하였으나, nodakenetin은 농도의존적으로 NA 활성을 억제하였으며 250 μg/ml에서 대조군 대비 약 68%의 억제율을 보였다. 이상의 결과들을 종합하여, 유산균 발효를 통해 침향공진단 내에 미량 존재하던 nodakenetin이 nodakenin의 가수분해로 인해 증가하였으며, NA 활성 억제 성분인 nodakenetin이 증가함으로 인해 FGD도 높은 NA 활성 억제 효능을 보였다고 판단할 수 있었다. The purpose of this study was to identify the changes in the components of unfermented Gongjin-dan (GD) and fermented Gongjin-dan (FGD) and to confirm whether GD or FGD has an inhibitory effect on viral neuraminidase (NA) activity. A major component of FGD was isolated and identified as nodakenetin, which is the aglycone of nodakenin. After fermentation, the nodakenetin content in FGD was approximately 10- fold higher than that in GD. Then, we examined the viral NA-inhibitory activity of GD, FGD, nodakenin, and nodakenetin. At a concentration of 500 μg/ml, FGD inhibited viral NA activity by 92% compared to the DMSO-treated control, while GD barely inhibited viral NA activity. In addition, 250 μg/ml of nodakenetin inhibited viral NA activity by 68% compared to the control, while nodakenin inhibited viral NA activity by only 4% at the same concentration as nodakenetin. Collectively, these results suggest that FGD has a more remarkable viral NA-inhibitory activity than GD because the content of the anti-viral component nodakenetin was higher in FGD due to the hydrolysis of nodakenin by Lactobacillus plantarum KCTC 3104.

Molecular Docking Study of Naturally-derived Neuraminidase Inhibitors Isolated from Phellinus Baumii

Babu, Sathya The Basic Science Institute Chosun University 2015 조선자연과학논문집 Vol.8 No.3

Influenza A virus (H1N1) causes and spreads infectious diseases and becomes a major health threat in humans. Among the subtypes of influenza virus, neuraminidase (NA) plays an important role in viral life cycle and becomes an attractive therapeutic target. Currently two NA inhibitors namely Zanamivir and Oseltamivir are available for treating infectious diseases. Recently five naturally derived polyphenols extracted from Phellinus baumii was reported as inhibitors against NA. Molecular docking is powerful tool in computer aided drug designing which aids in exploring and elucidating the properties of the molecules from their 3D structure. Hence, in the present study, molecular docking was carried out on reported polyphenols isolated from ethanolic extract of fruiting bodies of Phellinus baumii. The objective of this work was to study the interaction and to propose the binding mode of these compounds within the binding site of H1N1 neuraminidase. The results showed these compounds had better binding energy and H-bond interactions with the important active site residues of the receptor which authenticate these compounds contributes to inhibitory activity of neuraminidase to treat influenza infection.