分类： 生物学 >> 生物物理学 提交时间： 2016-05-11
Shang, Luqing
Zhang, Shumei
Yang, Xi
Sun, Jixue
Li, Linfeng
Cui, Zhengjie
Guo, Yu
Yin, Zheng
Shang, Luqing
Zhang, Shumei
Yang, Xi
Sun, Jixue
Li, Linfeng
Cui, Zhengjie
Guo, Yu
Yin, Zheng
Shang, Luqing
Zhang, Shumei
Yang, Xi
Li, Linfeng
摘要： Enterovirus 71 (EV71), a primary pathogen of hand, foot, and mouth disease (HFMD), affects primarily infants and children. Currently, there are no effective drugs against HFMD. EV71 3C protease performs multiple tasks in the viral replication, which makes it an ideal antiviral target. We synthesized a small set of fluorogenic model peptides derived from cleavage sites of EV71 polyprotein and examined their efficiencies of cleavage by EV71 3C protease. The novel peptide P08 [(2-(N-methylamino)benzoyl) (NMA)-IEALFQGPPK(DNP)FR] was determined to be the most efficiently cleaved by EV71 3C protease, with a kinetic constant k(cat)/K-m of 11.8 +/- 0.82mM(-1) min(-1). Compared with literature reports, P08 gave significant improvement in the signal/background ratio, which makes it an attractive substrate for assay development. A Molecular dynamics simulation study elaborated the interactions between substrate P08 and EV71 3C protease. Arg39, which is located at the bottom of the S2 pocket of EV71 3C protease, may participate in the proteolysis process of substrates. With an aim to evaluate EV71 3C protease inhibitors, a reliable and robust biochemical assay with a Z' factor of 0.87 +/- 0.05 was developed. A novel compound (compound 3) (50% inhibitory concentration [IC50] = 1.89 +/- 0.25 mu M) was discovered using this assay, which effectively suppressed the proliferation of EV 71 (strain Fuyang) in rhabdomyosarcoma (RD) cells with a highly selective index (50% effective concentration [EC50] = 4.54 +/- 0.51 mu M; 50% cytotoxic concentration [CC50] > 100 mu M). This fast and efficient assay for lead discovery and optimization provides an ideal platform for anti-EV71 drug development targeting 3C protease.
分类： 生物学 >> 生物物理学 提交时间： 2016-05-11
Zhai, Yangyang
Zhao, Xiangshuai
Cui, Zhengjie
Wang, Man
Li, Linfeng
Yang, Xi
Zeng, Debin
Liu, Ying
Shang, Luqing
Yin, Zheng
Zhai, Yangyang
Zhao, Xiangshuai
Cui, Zhengjie
Wang, Man
Li, Linfeng
Yang, Xi
Zeng, Debin
Liu, Ying
Shang, Luqing
Yin, Zheng
摘要： Cyanohydrin derivatives as enterovirus 71 (EV71) 3C protease (3C(pro)) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. Compared with the reported inhibitors, cyanohydrins (1S,2S,2'S,5S)-16 and (1R,2S,2'S,5S)-16 exhibited significantly improved activity and attractive selectivity profiles against other proteases, which were a result of the specific interactions between the cyanohydrin moiety and the catalytic site of 3C(pro). Cyanohydrin as an anchoring group with high selectivity and excellent inhibitory activity represents a useful choice for cysteine protease inhibitors.
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