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Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris.
Nguyen, Thi Thanh Hanh,Woo, Hye-Jin,Kang, Hee-Kyoung,Nguyen, Van Dao,Kim, Young-Min,Kim, Do-Won,Ahn, Sul-Ah,Xia, Yongmei,Kim, Doman Kluwer Academic Publishers 2012 Biotechnology letters. Vol.34 No.5
<P>The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Recombinant 3CL(pro) was expressed in Pichia pastoris GS115 as a 42??kDa protein that displayed a K ( m ) of 15??±??2??관M with Dabcyl-KTSAVLQSGFRKME-Edans as substrate. Purified 3CL(pro) was used for inhibition and kinetic assays with seven flavonoid compounds. The IC(50) of six flavonoid compounds were 47-381??관M. Quercetin, epigallocatechin gallate and gallocatechin gallate (GCG) displayed good inhibition toward 3CL(pro) with IC(50) values of 73, 73 and 47??관M, respectively. GCG showed a competitive inhibition pattern with K ( i ) value of 25??±??1.7??관M. In molecular docking experiments, GCG displayed a binding energy of -14??kcal??mol(-1) to the active site of 3CL(pro) and the galloyl moiety at 3-OH position was required for 3CL(pro) inhibition activity.</P>
Inhibition Effect of Flavonoid Compounds against Neuraminidase Expressed in Pichia pastoris
Nguyen, Thi Thanh Hanh,Kang, Hee-Kyoung,Kim, Young-Min,Jang, Tae-Su,Kim, Doman 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.1
Neuraminidase (NA) is one of the two glycoproteins on the surface of influenza virus, which cleaves terminal sialic acid residues and facilitates the release of virions from infected cells. The recombinant NA from H5N1 influenza virus strain A/Vietnam/1203/04 was expressed in Pichia pastoris X33 as a 45 kDa protein that displayed a $K_m$ of $9.96{\pm}1.26{\mu}M$ with fluorogenic substrate, 2'-(4-methylumbelliferyl)-${\alpha}$-D-N-acetyl neuraminic acid. Partially purified NA was used for the inhibition and kinetic assays with eight flavonoid compounds and gallic acid. Among them, gallocatechin gallate (GCG) showed the best inhibition against NA with the $IC_{50}$ of $8.98{\pm}0.46{\mu}M$ and showed a competitive inhibition pattern with Ki value of $8.34{\pm}0.25{\mu}M$. In molecular docking experiments, GCG displayed a binding energy of -13.71 kcal/mol to the active site of NA and the galloyl moiety was required for NA inhibition activity.
Nguyen, Thi Thanh Hanh,Jung, Sun-Hwa,Lee, Sun,Ryu, Hwa-Ja,Kang, Hee-Kyoung,Moon, Young-Hwan,Kim, Young-Min,Kimura, Atsuo,Kim, Doman 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.5
Human intestinal maltase (HMA) is an ${\alpha}$-glucosidase responsible for the hydrolysis of ${\alpha}$-1,4-linkages from the non-reducing end of malto-oligosaccharides. HMA has become an important target in the treatment of type-2 diabetes. In this study, epigallocatechin gallate (EGCG) and EGCG glucoside (EGCG-G1) were identified as inhibitors of HMA by an in vitro assay with $IC_{50}$ of $20{\pm}1.0$ and $31.5{\pm}1.0{\mu}M$, respectively. A Lineweaver-Burk plot confirmed that EGCG and EGCG-G1 were competitive inhibitors of maltose substrate against HMA and inhibition kinetic constants ($K_i$) calculated from a Dixon plot were $5.93{\pm}0.26$ and $7.88{\pm}0.57{\mu}M$, respectively. Both EGCG and EGCG-G1 bound to the active site of HMA with numerous hydrophobic and hydrogen bond interactions.