Browsing by Author "Hsu, John Tsu-An"

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Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71
(2020-05-01)
Huang, Peng-Nien
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Wang, Hsiang-Ching
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Hung, Hui-Chen
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Tseng, Sung-Nien
;
Chang, Teng-Yuan
In the past few decades, enterovirus A71 (EVA71) has caused devastating outbreaks in the Asia-Pacific region, resulting in serious sequelae in infected young children. No preventive or therapeutic interventions are currently available for curing EVA71 infection, highlighting a great unmet medical need for this disease. Here, we showed that one novel single-domain antibody (sdAb), F1, isolated from an immunized llama, could alleviate EVA71 infection both in vitro and in vivo. We also confirmed that the sdAb clone F1 recognizes EVA71 through a novel conformational epitope comprising the highly conserved region of VP3 capsid protein by using competitive-binding and overlapping-peptide enzyme-linked immunosorbent assays (ELISAs). Because of the virion's icosahedral structure, we reasoned that adjacent epitopes must be clustered within molecular ranges that may be simultaneously bound by an engineered antibody with multiple valency. Therefore, two singledomain binding modules (F1) were fused to generate an sdAb-in-tandem design so that the capture of viral antigens could be further increased by valency effects. We showed that the tetravalent construct F1xF1-hFc, containing two sdAb-intandem on a fragment crystallizable (Fc) scaffold, exhibits more potent neutralization activity against EVA71 than does the bivalent sdAb F1-hFc by at least 5.8-fold. We also demonstrated that, using a human scavenger receptor class B member 2 (hSCARB2) transgenic mouse model, a half dose of the F1xF1-hFc provided better protection against EVA71 infection than did the F1-hFc. Thus, our study furnishes important insights into multivalent sdAb engineering against viral infection and provides a novel strategic deployment approach for preparedness of emerging infectious diseases such as EVA71.
Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors
(2016-01-01)
Wu, Shou-Cheng
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Chen, Jen
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Wang, Hsiang-Ching
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Chou, Min-Yuan
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Chang, Teng-Yuan
The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T-2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed -94.8 +/- 3.8 and -84.1 +/- 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu-and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu-and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR.
Development of a Whole-Cell Screening System for Evaluation of the Human CYP1A2-Mediated Metabolism
(2011-12-01)
Chu, Chih-Chieh
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Pan, Kao-Lu
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Yao, Hsien-Tsung
;
Hsu, John Tsu-An
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生物科技學系
Cytochrome P450 1A2 (CYP1A2) is an important member of cytochrome P450 involved in drug metabolism. In this study, a cell line, Huh7-1A2-I-E, with high expression level of CYP1A2 is established based on Huh7 cells. To achieve this, we constructed a recombinant lentiviral vector, pLenti-1A2-I-E, containing a single promoter encoding CYP1A2 followed by an internal ribosome entry site (IRES) to permit the translation of enhanced green fluorescence protein (EGFP). Such a design has greatly facilitated the selection of stable cell lines because the translations of CYP1A2 and EGFP proteins would be based on a single bi-cistronic mRNA. The Huh7-1A2-I-E cells were evaluated as a cell-based model for identification of CYP1A2 inhibitors and for studies of cytotoxicity resulted from CYP-mediated drug metabolism. Treatment of Huh7-1A2-I-E cells and the Huh7-E control cells with aflatoxin B1 showed that cells with CYP1A2 expression are much more sensitive to aflatoxin B1 and the cellular toxicity of aflatoxin B1 in Huh7-1A2-I-E cells could be prevented by furafylline, a CYP1A2 inhibitor. A collection of approximately 200 drugs were screened using this system and results indicate that for most drugs the metabolism by CYP1A2 is unlikely to have made a major contribution to the in vitro cytotoxicity except for thimerosal and evoxine. Several previously unidentified CYP1A2 inhibitors such as evoxine and berberine were also identified in this study. Biotechnol. Bioeng. 2011; 108: 2932-2940. (C) 2011 Wiley Periodicals, Inc.
Discovery of M Protease Inhibitors Encoded by SARS-CoV-2
(2020-09-01)
Hung, Hui-Chen
;
Ke, Yi-Yu
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Huang, Sheng Yu
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Huang, Peng-Nien
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Kung, Yu-An
The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (M-pro, also called 3C-like protease [3CL(pro)]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting M-pro in the picornavirus-like supercluster, is a potent inhibitor for the M-pro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 +/- 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 +/- 0.03 mu M. Only a small portion of SARS-CoV-2 M-pro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 M-pro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.
Zika Virus NS3 Protease Pharmacophore Anchor Model and Drug Discovery
(2020-06-02)
Pathak, Nikhil
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Kuo, Yi-Ping
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Chang, Teng-Yuan
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Huang, Chin-Ting
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Hung, Hui-Chen
Zika virus (ZIKV) of the flaviviridae family, is the cause of emerging infections characterized by fever, Guillain-Barre syndrome (GBS) in adults and microcephaly in newborns. There exists an urgent unmet clinical need for anti-ZIKV drugs for the treatment of infected individuals. In the current work, we aimed at the promising virus drug target, ZIKV NS3 protease and constructed a Pharmacophore Anchor (PA) model for the active site. The PA model reveals a total of 12 anchors (E, H, V) mapped across the active site subpockets. We further identified five of these anchors to be critical core anchors (CEH1, CH3, CH7, CV1, CV3) conserved across flaviviral proteases. The ZIKV protease PA model was then applied in anchor-enhanced virtual screening yielding 14 potential antiviral candidates, which were tested by in vitro assays. We discovered FDA drugs Asunaprevir and Simeprevir to have potent anti-ZIKV activities with EC50 values 4.7 mu M and 0.4 mu M, inhibiting the viral protease with IC50 values 6.0 mu M and 2.6 mu M respectively. Additionally, the PA model anchors aided in the exploration of inhibitor binding mechanisms. In conclusion, our PA model serves as a promising guide map for ZIKV protease targeted drug discovery and the identified 'previr' FDA drugs are promising for anti-ZIKV treatments.