Publications by authors named "Sim-Kun Ng"

6 Publications

  • Page 1 of 1

Rhamnose Binding Protein as an Anti-Bacterial Agent-Targeting Biofilm of .

Mar Drugs 2019 Jun 14;17(6). Epub 2019 Jun 14.

Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu 30013, Taiwan.

More than 80% of infectious bacteria form biofilm, which is a bacterial cell community surrounded by secreted polysaccharides, proteins and glycolipids. Such bacterial superstructure increases resistance to antimicrobials and host defenses. Thus, to control these biofilm-forming pathogenic bacteria requires antimicrobial agents with novel mechanisms or properties. , a Gram-negative opportunistic nosocomial pathogen, is a model strain to study biofilm development and correlation between biofilm formation and infection. In this study, a recombinant hemolymph plasma lectin (rHPL) cloned from Taiwanese was expressed in an system. This rHPL was shown to have the following properties: (1) Binding to PA14 biofilm through a unique molecular interaction with rhamnose-containing moieties on bacteria, leading to reduction of extracellular di-rhamnolipid (a biofilm regulator); (2) decreasing downstream quorum sensing factors, and inhibiting biofilm formation; (3) dispersing the mature biofilm of PA14 to improve the efficacies of antibiotics; (4) reducing PA14 cytotoxicity to human lung epithelial cells in vitro and (5) inhibiting PA14 infection of zebrafish embryos in vivo. Taken together, rHPL serves as an anti-biofilm agent with a novel mechanism of recognizing rhamnose moieties in lipopolysaccharides, di-rhamnolipid and structural polysaccharides (Psl) in biofilms. Thus rHPL links glycan-recognition to novel anti-biofilm strategies against pathogenic bacteria.
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http://dx.doi.org/10.3390/md17060355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628293PMC
June 2019

Identification of anti-HBV activities in Andr. using GRP78 as a drug target on Herbochip.

Chin Med 2017 24;12:11. Epub 2017 Apr 24.

Institute of Biotechnology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan.

Background: Herbochip technology is a high throughput drug screening platform in a reverse screening manner, in which potential chemical leads in herbal extracts are immobilized and drug target proteins can be used as probes for screening process [BMC Complementary and Alternative Medicine (2015) 15:146]. While herbal medicines represent an ideal reservoir for drug screenings, here a molecular chaperone GRP78 is demonstrated to serve as a potential target for antiviral drug discovery.

Methods: We cloned and expressed a truncated but fully functional form of human GRP78 (hGRP78) and used it as a probe for anti-HBV drug screening on herbochips. In vitro cytotoxicity and in vitro anti-HBV activity of the herbal extracts were evaluated by MTT and ELISA assays, respectively. Finally, anti-HBV activity was confirmed by in vivo assay using DHBV DNA levels in DHBV-infected ducklings as a model.

Results: Primary screenings using GRP78 on 40 herbochips revealed 11 positives. Four of the positives, namely , , and were subjected to subsequent assays. None of the above extracts was cytotoxic to AML12 cells, but extract (PCE) was found to be cytotoxic to HepG2 2.2.15 cells. Both PCE and extract (PSE) suppressed secretion of HBsAg and HBeAg in HepG2 2.2.15 cells. The anti-HBV activity of PSE was further confirmed in vivo.

Conclusion: We have demonstrated that GRP78 is a valid probe for anti-HBV drug screening on herbochips. We have also shown that PSE, while being non-cytotoxic, possesses in vitro and in vivo anti-HBV activities. Taken together, our data suggest that PSE may be a potential anti-HBV agent for therapeutic use.
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http://dx.doi.org/10.1186/s13020-017-0132-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402670PMC
April 2017

Inhibitory Effect of Multivalent Rhamnobiosides on Recombinant Horseshoe Crab Plasma Lectin Interactions with Pseudomonas aeruginosa PAO1.

Chem Asian J 2016 Dec 3;11(23):3398-3413. Epub 2016 Nov 3.

Department of Pharmaceutical Chemistry, University of Debrecen, H-4032, Debrecen, Egyetem tér 1, Hungary.

To evaluate the molecular interaction of recombinant horseshoe crab plasma lectin (rHPL) with Pseudomonas aeruginosa PAO1, multivalent rhamnobioside derivatives were designed. Eight rhamnoclusters with three or four α(1-3)-rhamnobiosides attached to different central cores, such as methyl gallate, pentaerythritol, and N-Boc Tris, through either an ethylene glycol or a tetraethylene glycol linker, were assembled in two consecutive azide-alkyne cycloaddition click reactions. The synthetic method embraced the preparation of two α(1-3)-rhamnobiosides with different linker arms and their conjugation, in stoichiometric or substoichiometric amounts, to propargyl ether-functionalized tri- or tetravalent scaffolds. A divalent derivative and two self-assembling rhamnobiosides were also prepared. The different architectures and valences of the rhamnoclusters provided an opportunity to evaluate the impact of topology and valency on the binding properties toward rHPL. Inhibitory ELISA data showed that all covalently linked rhamnoclusters could inhibit P. aeruginosa PAO1 recognition activity of rHPL with high efficacy. Trivalent rhamnobiosides showed a stronger inhibitory effect on P. aeruginosa PAO1 binding, and the more flexible clusters on a pentaerythritol or a Tris core were superior to the less flexible methyl gallate-based clusters. Interestingly, the length of the linker arms had a very low impact on the binding ability of the rhamnoclusters. Herein, the two trivalent derivatives on an N-Boc protected Tris central core were the best inhibitors. The self-assembling amphiphilic rhamnobioside derivatives were found to display no multivalent effect.
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http://dx.doi.org/10.1002/asia.201601162DOI Listing
December 2016

Identification of anti-inflammatory fractions of Geranium wilfordii using tumor necrosis factor-alpha as a drug target on Herbochip® - an array-based high throughput screening platform.

BMC Complement Altern Med 2015 May 12;15:146. Epub 2015 May 12.

Yunnan Baiyao-Herbcopoeia Laboratory Inc, 51 Xi-Ba Road, Kunming, Yunnan, China.

Background: Geranium wilfordii is one of the major species used as Herba Geranii (lao-guan-cao) in China, it is commonly used solely or in polyherbal formulations for treatment of joint pain resulted from rheumatoid arthritis (RA) and gout. This herb is used to validate a target-based drug screening platform called Herbochip® and evaluate anti-inflammatory effects of Geranium wilfordii ethanolic extract (GWE) using tumor necrosis factor-alpha (TNF-α) as a drug target together with subsequent in vitro and in vivo assays.

Methods: A microarray-based drug screening platform was constructed by arraying HPLC fractions of herbal extracts onto a surface-activated polystyrene slide (Herbochip®). Using TNF-α as a molecular probe, fractions of 82 selected herbal extracts, including GWE, were then screened to identify plant extracts containing TNF-α-binding agents. Cytotoxicity of GWE and modulatory effects of GWE on TNF-α expression were evaluated by cell-based assays using TNF-α sensitive murine fibrosarcoma L929 cells as an in vitro model.

Results: The in vivo anti-inflammatory effects of GWE were further assessed by animal models including carrageenan-induced hind paw edema in rats and xylene-induced ear edema in mice, in comparison with aspirin. The hybridization data obtained by Herbochip® analysis showed unambiguous signals which confirmed TNF-α binding activity in 46 herbal extracts including GWE. In L929 cells GWE showed significant inhibitory effect on TNF-α expression with negligible cytotoxicity. GWE also significantly inhibited formation of carrageenan-induced hind paw edema and xylene-induced ear edema in animal models, indicating that it indeed possessed anti-inflammatory activity.

Conclusion: We have thus validated effectiveness of the Herbochip® drug screening platform using TNF-α as a molecular target. Subsequent experiments on GWE lead us to conclude that the anti-RA activity of GWE can be attributed to inhibitory effect of GWE on the key inflammatory factor, TNF-α. Our results contribute towards validation of the traditional use of GWE in the treatment of RA and other inflammatory joint disorders.
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http://dx.doi.org/10.1186/s12906-015-0665-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443519PMC
May 2015

A recombinant horseshoe crab plasma lectin recognizes specific pathogen-associated molecular patterns of bacteria through rhamnose.

PLoS One 2014 26;9(12):e115296. Epub 2014 Dec 26.

Institute of Molecular and Cellular Biology & Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.

Horseshoe crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of horseshoe crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant horseshoe crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115296PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277298PMC
August 2015

Functional characterization of ECP-heparin interaction: a novel molecular model.

PLoS One 2013 11;8(12):e82585. Epub 2013 Dec 11.

Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China ; Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.

Human eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are two ribonuclease A (RNaseA) family members secreted by activated eosinophils. They share conserved catalytic triad and similar three dimensional structures. ECP and EDN are heparin binding proteins with diverse biological functions. We predicted a novel molecular model for ECP binding of heparin hexasaccharide (Hep6), [GlcNS(6S)-IdoA(2S)]3, and residues Gln(40), His(64) and Arg(105) were indicated as major contributions for the interaction. Interestingly, Gln(40) and His(64) on ECP formed a clamp-like structure to stabilize Hep6 in our model, which was not observed in the corresponding residues on EDN. To validate our prediction, mutant ECPs including ECP Q40A, H64A, R105A, and double mutant ECP Q40A/H64A were generated, and their binding affinity for heparins were measured by isothermal titration calorimetry (ITC). Weaker binding of ECP Q40A/H64A of all heparin variants suggested that Gln(40)-His(64) clamp contributed to ECP-heparin interaction significantly. Our in silico and in vitro data together demonstrate that ECP uses not only major heparin binding region but also use other surrounding residues to interact with heparin. Such correlation in sequence, structure, and function is a unique feature of only higher primate ECP, but not EDN.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082585PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859622PMC
October 2014