Browsing by Author "Chang, Teng-Yuan"

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A High-throughput Cell-based Screening for L858R/T790M Mutant Epidermal Growth Factor Receptor Inhibitors
(2012-01-01)
Lin, Wen-Hsing
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Song, Jen-Shin
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Lien, Tzu-Wen
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Chang, Chun-Yu
;
Wu, Szu-Huei
A high-throughput 32D(L858R/T790M) cell-based assay to identify inhibitors of the L858R/T790M mutant epidermal growth factor receptor (EGFR) pathway was established. After screening, ten hits from among 60,000 compounds in our in-house compound library were initially identified. In the secondary assays, one hit, 1-[2-(decyloxy)-2-oxoethyl]-3-methyl-2-[(4-methylphenoxy) methyl]-1H-benzimidazol-3-ium, was confirmed to directly inhibit the kinase activity of recombinant L858R/T790M EGFR and the phosphorylation of EGFR-L858R/T790M in gefitinib-resistant H1975 cells. Thus, this high-throughput assay system may be useful for identifying novel inhibitors which suppress mutant EGFR-T790M signalling and for overcoming T790M-mediated acquired resistance for future anticancer drug discovery.
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.
Design and Synthesis of Tetrahydropyridothieno[2,3-d]pyrimidine Scaffold Based Epidermal Growth Factor Receptor (EGFR) Kinase Inhibitors: The Role of Side Chain Chirality and Michael Acceptor Group for Maximal Potency
(2010-10-28)
Wu, Chia-Hsien
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Coumar, Mohane Selvaraj
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Chu, Chang-Ying
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Lin, Wen-Hsing
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Chen, Yi-Rong
HTS hit 7 was modified through hybrid design strategy to introduce a chiral side chain followed by introduction of Michael acceptor group to obtain potent EGFR kinase inhibitors 11 and 19. Both 11 and 19 showed over 3 orders of magnitude enhanced HCC827 antiproliferative activity compared to HTS hit 7 and also inhibited gefitinib-resistant double mutant (DM, T790M/L858R) EGFR kinase at nanomolar concentration. Moreover, treatment with 19 shrinked tumor in nude mice xenograft model.
Design and Synthesis of Tetrahydropyridothieno[2,3-d]pyrimidine Scaffold Based Epidermal Growth Factor Receptor (EGFR) Kinase Inhibitors: The Role of Side Chain Chirality and Michael Acceptor Group for Maximal Potency
(2010-10-28)
Wu, Chia-Hsien
;
Coumar, Mohane Selvaraj
;
Chu, Chang-Ying
;
Lin, Wen-Hsing
;
Chen, Yi-Rong
HTS hit 7 was modified through hybrid design strategy to introduce a chiral side chain followed by introduction of Michael acceptor group to obtain potent EGFR kinase inhibitors 11 and 19. Both 11 and 19 showed over 3 orders of magnitude enhanced HCC827 antiproliferative activity compared to HTS hit 7 and also inhibited gefitinib-resistant double mutant (DM, T790M/L858R) EGFR kinase at nanomolar concentration. Moreover, treatment with 19 shrinked tumor in nude mice xenograft model.
Diosgenin, a Plant-Derived Sapogenin, Exhibits Antiviral Activity in Vitro against Hepatitis C Virus
(2011-04-01)
Wang, Ya-Jean
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Pan, Kao-Lu
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Hsieh, Tsung-Chih
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Chang, Teng-Yuan
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Lin, Wen-Hsing
Diosgenirr (3 beta-hydroxy-5-spirostene, 1); a plant-derived sapogenin, is used as a dietary: supplement However, the biological effects of 1 related to viral replication remain unexplored. In this study, the effects of 1 on hepatitis C virus (HCV) replication were evaluated. Based on a reporter-based HCV subgenomic replicon system, 1 was found to inhibit HCV replication at low micromolar concentrations. The EC(50) (concentration at which 50% of HCV replication is inhibited) of 1 was 3.8 mu M No cellular toxicity was observed at this concentration.,Diosgenin, (1) also significantly reduced the levels of viral,RNA and viral proteins as evaluated by quantitative real-time reverse :transcriptase PCR and Western blot analysis, respectively. In addition, in an HCV antiviral system more closely aligned to all steps involved in the HCV infection and life cycle, 1 totally abolished HCV replication at 20 mu M. Moreover, 1 reduced the phosphorylation of signal transducer and activator of transcription 3. A combination of 1:and interferon-alpha exerted an additive effect on the resultant anti-HCV activity.
Discovery of M Protease Inhibitors Encoded by SARS-CoV-2
(2020-09-01)
Hung, Hui-Chen
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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.
Identification, SAR Studies, and X-ray Co-crystallographic Analysis of a Novel Furanopyrimidine Aurora Kinase A Inhibitor
(2010-02-01)
Coumar, Mohane Selvaraj
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Tsai, Ming-Tsung
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Chu, Chang-Ying
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Uang, Biing-Jiun
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Lin, Wen-Hsing
Here in we reveal a simple method for the identification of novel Aurora kinase A inhibitors through substructure searching of an in-house compounds for testing. A hydrazone fragment conferring Aurora kinase activity and heterocyclic rings most frequently reported in kinase inhibitors were used as substructure queries to filter the in house compound library collection prior to testing. Five new series of Aurora kinase inhibitors were identified through this strategy with IC(50) values ranging from similar to 300 nm to similar to 15 mu m, by testing only 133 compounds from a database of similar to 125 000 compounds. Structure activity relationship studies and X-ray co-crystallographic analysis of the most potent compound, a furanopyrimidine derivative with an IC(50) value of 309 nm toward Aurora kinase A, were carried out. The knowledge gained through these studies could help in the future design of potent Aurora kinase inhibitors.
The Combination Effects of LiCl and the Active Leflunomide Metabolite, A771726, on Viral-Induced Interleukin 6 Production and EV-A71 Replication
(2014-11-20)
Hung, Hui-Chen
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Shih, Shin-Ru
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Chang, Teng-Yuan
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Fang, Ming-Yu
;
Hsu, John T. -A.
Enterovirus 71 (EV-A71) is a neurotropic virus that can cause severe complications involving the central nervous system. No effective antiviral therapeutics are available for treating EV-A71 infection and drug discovery efforts are rarely focused to target this disease. Thus, the main goal of this study was to discover existing drugs with novel indications that may effectively inhibit EV-A71 replication and the inflammatory cytokines elevation. In this study, we showed that LiCl, a GSK3 beta inhibitor, effectively suppressed EV-A71 replication, apoptosis and inflammatory cytokines production (Interleukin 6, Interleukin-1 beta) in infected cells. Furthermore, LiCl and an immunomodular agent were shown to strongly synergize with each other in suppressing EV-A71 replication. The results highlighted potential new treatment regimens in suppressing sequelae caused by EV-A71 replication.
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.