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1. chinaXiv:201605.01737 [pdf]

Baicalin Inhibits the Lethality of Shiga-Like Toxin 2 in Mice

Dong, Jing; Zhang, Yong; Zhang, Yu; Deng, Xuming; Dong, Jing; Chen, Yutao; Wang, Quan; Li, Xuemei; Niu, Xiaodi; Yang, Cheng
Subjects: Biology >> Biophysics

Shiga-like toxins (Stxs), produced by pathogenic Escherichia coli, are a major virulence factor involved in severe diseases in human and animals. These toxins are ribosome-inactivating proteins, and treatment for diseases caused by them is not available. Therefore, there is an urgent need for agents capable of effectively targeting this lethal toxin. In this study, we identified baicalin, a flavonoid compound used in Chinese traditional medicine, as a compound against Shiga-like toxin 2 (Stx2). We found that baicalin significantly improves renal function and reduces Stx2-induced lethality in mice. Further experiments revealed that baicalin induces the formation of oligomers by the toxin by direct binding. We also identified the residues important for such interactions and analyzed their roles in binding baicalin by biophysical and biochemical analyses. Our results establish baicalin as a candidate compound for the development of therapeutics against diseases caused by Stxs.

submitted time 2016-05-15 Hits3402Downloads1413 Comment 0

2. chinaXiv:201605.01512 [pdf]

Baicalin Inhibits the Lethality of Ricin in Mice by Inducing Protein Oligomerization

Dong, Jing; Zhang, Yong; Zhang, Yu; Li, Rui; Deng, Xuming; Dong, Jing; Chen, Yutao; Wang, Quan; Li, Xuemei; Niu, Xiaodi; Yang, Cheng
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Toxic ribosome-inactivating proteins abolish cell viability by inhibiting protein synthesis. Ricin, a member of these lethal proteins, is a potential bioterrorism agent. Despite the grave challenge posed by these toxins to public health, post-exposure treatment for intoxication caused by these agents currently is unavailable. In this study, we report the identification of baicalin extracted from Chinese herbal medicine as a compound capable of inhibiting the activity of ricin. More importantly, post-exposure treatment with baicalin significantly increased the survival of mice poisoned by ricin. We determined the mechanism of action of baicalin by solving the crystal structure of its complex with the A chain of ricin (RTA) at 2.2 angstrom resolution, which revealed that baicalin interacts with two RTA molecules at a novel binding site by hydrogen bond networks and electrostatic force interactions, suggesting its role as molecular glue of the RTA. Further biochemical and biophysical analyses validated the amino acids directly involved in binding the inhibitor, which is consistent with the hypothesis that baicalin exerts its inhibitory effects by inducing RTA to form oligomers in solution, a mechanism that is distinctly different from previously reported inhibitors. This work offers promising leads for the development of therapeutics against ricin and probably other ribosome-inactivating proteins.

submitted time 2016-05-12 Hits2407Downloads1299 Comment 0

3. chinaXiv:201605.01322 [pdf]

Studies on Inhibition of Proliferation of Enterovirus-71 by Compound YZ-LY-0

Yang, Qingzhan; Shaw, Neil; Rao, Zihe; Lou, Zhiyong; Jie, Qing; Shaw, Neil; Rao, Zihe; Yin, Zheng; Yin, Zheng; Yin, Zheng; Li, Lei; Rao, Zihe; Lou, Zhiyong; Li, Lei; Rao, Zihe; Lou, Zhiyong; Li, Lei; Rao, Zihe; Lou, Zhiyong
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

In recent years, hand-foot-and-mouth disease (HFMD), which is caused by Enteroviruses, has emerged as a serious illness. It affects mainly children under the age of five and results in high fatality rates. Enterovirus 71 (EV71) is the main causative agent of HFMD in China and currently there are no effective anti-viral drugs available to treat HFMD. In the present study, we screened compounds for inhibition of proliferation of EV71. Compound YZ-LY-0 stalled the life cycle of EV71. The inhibitor exhibited EC50 value of 0.29 mu m against SK-EV006 strain of EV71. Notably, YZ-LY-0 had low cytotoxicity (CC50 > 100 mu M) and a high selectivity index (over 300) in Vero and RD cells. YZ-LY-0 in combination with an EV71 RdRp inhibitor or an entry inhibitor showed an antagonistic effect at very low concentrations. However, at higher concentrations the inhibitors exhibited a synergistic effect in inhibiting viral replication. Preliminary results on investigation of the mechanism of inhibition indicate that YZ-LY-0 does not block the entry of the virus in the host cell, but instead inhibits an early stage of EV71 replication. Our studies provide a potential clinical therapeutic option against EV71 infections and suggest that a combined application of YZ-LY-0 with other inhibitors could be more effective in the treatment of HFMD.

submitted time 2016-05-11 Hits1807Downloads1054 Comment 0

4. chinaXiv:201605.01281 [pdf]

Biochemical Characterization of Recombinant Enterovirus 71 3C Protease with Fluorogenic Model Peptide Substrates and Development of a Biochemical Assay

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
Subjects: Biology >> Biophysics

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.

submitted time 2016-05-11 Hits1698Downloads957 Comment 0

5. chinaXiv:201605.01279 [pdf]

Peptidyl Aldehyde NK-1.8k Suppresses Enterovirus 71 and Enterovirus 68 Infection by Targeting Protease 3C

Wang, Yaxin; Sun, Yuna; Rao, Zihe; Yang, Ben; Zhai, Yangyang; Yin, Zheng; Rao, Zihe; Zhai, Yangyang; Yin, Zheng; Rao, Zihe; Zhai, Yangyang; Yin, Zheng; Rao, Zihe; Wang, Yaxin; Rao, Zihe; Rao, Zihe
Subjects: Biology >> Biophysics

Enterovirus (EV) is one of the major causative agents of hand, foot, and mouth disease in the Pacific-Asia region. In particular, EV71 causes severe central nervous system infections, and the fatality rates from EV71 infection are high. Moreover, an outbreak of respiratory illnesses caused by an emerging EV, EV68, recently occurred among over 1,000 young children in the United States and was also associated with neurological infections. Although enterovirus has emerged as a considerable global public health threat, no antiviral drug for clinical use is available. In the present work, we screened our compound library for agents targeting viral protease and identified a peptidyl aldehyde, NK-1.8k, that inhibits the proliferation of different EV71 strains and one EV68 strain and that had a 50% effective concentration of 90 nM. Low cytotoxicity (50% cytotoxic concentration, >200 mu M) indicated a high selective index of over 2,000. We further characterized a single amino acid substitution inside protease 3C (3C(pro)), N69S, which conferred EV71 resistance to NK-1.8k, possibly by increasing the flexibility of the substrate binding pocket of 3C(pro). The combination of NK-1.8k and an EV71 RNA-dependent RNA polymerase inhibitor or entry inhibitor exhibited a strong synergistic anti-EV71 effect. Our findings suggest that NK-1.8k could potentially be developed for anti-EV therapy.

submitted time 2016-05-11 Hits1868Downloads1162 Comment 0

6. chinaXiv:201605.01253 [pdf]

Cyanohydrin as an Anchoring Group for Potent and Selective Inhibitors of Enterovirus 71 3C Protease

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
Subjects: Biology >> Biophysics

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.

submitted time 2016-05-11 Hits1705Downloads1016 Comment 0

7. chinaXiv:201605.00730 [pdf]

Crystal Structure of the Core Region of Hantavirus Nucleocapsid Protein Reveals the Mechanism for Ribonucleoprotein Complex Formation

Guo, Yu; Ma, Chao; Wang, Xu; Wang, Xin; Liu, Pi; Lin, Jianping; Guo, Yu; Ma, Chao; Wang, Xu; Wang, Xin; Liu, Pi; Lin, Jianping; Lou, Zhiyong; Lou, Zhiyong; Sun, Yuna; Lou, Zhiyong; Shen, Shu; Deng, Fei; Wang, Hualin; Wang, Wenming
Subjects: Biology >> Biophysics

Hantaviruses, which belong to the genus Hantavirus in the family Bunyaviridae, infect mammals, including humans, causing either hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in humans with high mortality. Hantavirus encodes a nucleocapsid protein (NP) to encapsidate the genome and form a ribonucleoprotein complex (RNP) together with viral polymerase. Here, we report the crystal structure of the core domains of NP (NPcore) encoded by Sin Nombre virus (SNV) and Andes virus (ANDV), which are two representative members that cause HCPS in the New World. The constructs of SNV and ANDV NPcore exclude the N- and C-terminal portions of full polypeptide to obtain stable proteins for crystallographic study. The structure features an N lobe and a C lobe to clamp RNA-binding crevice and exhibits two protruding extensions in both lobes. The positively charged residues located in the RNA-binding crevice play a key role in RNA binding and virus replication. We further demonstrated that the C-terminal helix and the linker region connecting the N-terminal coiled-coil domain and NPcore are essential for hantavirus NP oligomerization through contacts made with two adjacent protomers. Moreover, electron microscopy (EM) visualization of native RNPs extracted from the virions revealed that a monomer-sized NP-RNA complex is the building block of viral RNP. This work provides insight into the formation of hantavirus RNP and provides an understanding of the evolutionary connections that exist among bunyaviruses. IMPORTANCE Hantaviruses are distributed across a wide and increasing range of host reservoirs throughout the world. In particular, hantaviruses can be transmitted via aerosols of rodent excreta to humans or from human to human and cause HFRS and HCPS, with mortalities of 15% and 50%, respectively. Hantavirus is therefore listed as a category C pathogen. Hantavirus encodes an NP that plays essential roles both in RNP formation and in multiple biological functions. NP is also the exclusiv

submitted time 2016-05-05 Hits1713Downloads948 Comment 0

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