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Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase
Kanyarat Thueng-in Jeeraphong Thanongsaksrikul Surasak Jittavisutthikul Watee Seesuay Monrat Chulanetra Yuwaporn Sakolvaree Potjanee Srimanote Wanpen Chaicumpa 《MABS-AUSTIN》2014,6(5):1327-1339
A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV''s NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes. 相似文献
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Kedsarin Fong-ngern Kanyarat Sueksakit Visith Thongboonkerd 《Journal of biological inorganic chemistry》2016,21(4):463-474
Adhesion of calcium oxalate monohydrate (COM) crystals on renal tubular epithelial cells is a crucial step in kidney stone formation. Finding potential crystal receptors on the apical membrane of the cells may lead to a novel approach to prevent kidney stone disease. Our previous study identified a large number of crystal-binding proteins on the apical membrane of MDCK cells. However, their functional role as potential crystal receptors had not been validated. The present study aimed to address the potential role of heat shock protein 90 (HSP90) as a COM crystal receptor. The apical membrane was isolated from polarized MDCK cells by the peeling method and recovered proteins were incubated with COM crystals. Western blot analysis confirmed the presence of HSP90 in the apical membrane and the crystal-bound fraction. Immunofluorescence staining without permeabilization and laser-scanning confocal microscopy confirmed the surface HSP90 expression on the apical membrane of the intact cells. Crystal adhesion assay showed that blocking surface HSP90 by specific anti-HSP90 antibody and knockdown of HSP90 by small interfering RNA (siRNA) dramatically reduced crystal binding on the apical surface of MDCK cells (by approximately 1/2 and 2/3, respectively). Additionally, crystal internalization assay revealed the presence of HSP90 on the membrane of endocytic vesicle containing the internalized COM crystal. Moreover, pretreatment of MDCK cells with anti-HSP90 antibody significantly reduced crystal internalization (by approximately 1/3). Taken together, our data indicate that HSP90 serves as a potential receptor for COM crystals on the apical membrane of renal tubular epithelial cells and is involved in endocytosis/internalization of the crystals into the cells. 相似文献
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Adisakwattana S Ngamrojanavanich N Kalampakorn K Tiravanit W Roengsumran S Yibchok-Anun S 《Journal of enzyme inhibition and medicinal chemistry》2004,19(4):313-316
Cyanidin-3-rutinoside, a natural anthocyanin, inhibited alpha-glucosidase from baker's yeast in dose-responsive manner. The IC50 value was 19.7 microM +/- 0.24 microM, compared with the IC50 value of voglibose (IC50 = 23.4 +/- 0.30 microM). Cyanidin-3-rutinoside was found to be a non-competitive inhibitor for yeast alpha-glucosidase with a Ki value in the range of 1.31-1.56 x 10(-5)M. These results indicated that cyanidin-3-rutinoside could be classed as a new alpha-glucosidase inhibitor. 相似文献
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Kanyarat Thueng-in Jeeraphong Thanongsaksrikul Potjanee Srimanote Kunan Bangphoomi Ornnuthchar Poungpair Santi Maneewatch Kiattawee Choowongkomon Wanpen Chaicumpa 《PloS one》2012,7(11)
NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/VHH) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/VHH from a humanized-camel VH/VHH display library. VH/VHH from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3′di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed VHH hallmark and were designated VHH6 and VHH24; other clones were conventional VH, designated VH9 and VH13. All VH/VHH were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, VHH6 and VHH24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/VHH mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication. 相似文献
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Kanyarat Ruangrung Ornpreya Suptawiwat Kittipong Maneechotesuwan Chompunuch Boonarkart Warunya Chakritbudsabong Jirawatna Assawabhumi Parvapan Bhattarakosol Mongkol Uiprasertkul Pilaipan Puthavathana Witthawat Wiriyarat Anan Jongkaewwattana Prasert Auewarakul 《Journal of virology》2016,90(9):4637-4646
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Weeraya Thongkum Kanyarat Thongheang Chatchai Tayapiwatana 《Current issues in molecular biology》2022,44(7):2842
Concerns over vaccine efficacy after the emergence of the SARS-CoV-2 Delta variant prompted revisiting the vaccine design concepts. Monoclonal antibodies (mAbs) have been developed to identify the neutralizing epitopes on spike protein. It has been confirmed that the key amino acid residues in epitopes that induce the formation of neutralizing antibodies do not have to be on the receptor-binding domain (RBD)- angiotensin-converting enzyme 2 (ACE2) contact surface, and may be conformationally hidden. In addition, this epitope is tolerant to amino acid mutations of the Delta variant. The antibody titers against RBD in health care workers in Thailand receiving two doses of CoronaVac, followed by a booster dose of BNT162b2, were significantly increased. The neutralizing antibodies against the Delta variant suggest that the overall neutralizing antibody level against the Wuhan strain, using the NeutraLISA, was consistent with the levels of anti-RBD antibodies. However, individuals with moderate anti-RBD antibody responses have different levels of a unique antibody population competing with a cross-neutralizing mAb clone, 40591-MM43, determined by in-house competitive ELISA. Since 40591-MM43 mAb indicates cross-neutralizing activity against the Delta variant, this evidence implies that the efficiency of the vaccination regimen should be improved to facilitate cross-protective antibodies against Delta variant infections. The RBD epitope recognized by 40591-MM43 mAb is hidden in the close state. 相似文献
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Teeranai Poti Kanyarat Mahawan Ratchadawan Cheewangkoon Hatthaya Arunothayanan Kazuya Akimitsu Sarunya Nalumpang 《Journal of Phytopathology》2020,168(5):267-278
A loss of fungicide efficacy, particularly for carbendazim, was noted in soybean fields in Thailand and was considered to be due to the development of Colletotrichum truncatum resistance. The carbendazim sensitivity of C. truncatum populations isolated from various soybean fields in Thailand was thus evaluated with in vitro sensitivity assays and molecular characterization of mutations in the sequences of the ß2-tubulin (TUB2) gene that confer carbendazim resistance in the pathogen. Among 52 isolates, 46 isolates were classified as highly resistant (HR) to carbendazim (EC50 > 1,000 µg/ml). All HR isolates grew on PDA amended with carbendazim at 1,000 µg/ml. Six isolates were classified as carbendazim sensitive (S) (EC50 < 1 µg/ml). Mycelial growth on PDA amended with 1 µg/ml carbendazim was inhibited by over 50% compared with growth on PDA alone. When a partial TUB2 gene from the isolates was amplified and analysed using predicted amino acid sequences, an alteration from glutamic acid to alanine at codon 198 (E198A) was found in 45 HR isolates for which the EC50 was higher than 2000 µg/ml. This mutation resulted from a nucleotide substitution from adenine to cytosine (GA G → GC G). The other HR isolate, CtPhS_1, with EC50 of 1,127 µg/ml, had an alteration at codon 200 (F200Y) (TT C → TA C). 相似文献
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The objective of this study was to compare the performance of anaerobic baffled reactor (ABR) treating concentrated rubber latex wastewater under different pH adjustment substances and recycling ratios (R). Two ABRs, one received wastewater pretreated with NaOH and the other with ash, were operated at 35 degrees C under identical HRTs from 10 to 1.25d. Results show that both ABRs had highest COD and sulfate removal efficiencies at HRT 10d (averaged 82.71% and 96.16% of ABR-NaOH, and 80.77% and 96.60% of ABR-Ash, respectively), where majority of the influent COD and sulfate were removed by the first compartment of the ABR at all conditions tested. Increasing R (0, 0.3 and 0.5) raised the hydraulic loading on the system and resulted in a drop of organic removal efficiency and methane yield. Translocation of sulfate reducing bacteria and methanogens in the ABRs caused by increased organic loading and effluent recycle is discussed. The results show great potential of parawood ash as a pH adjustment substance for acidic wastewaters. 相似文献
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