Structure-based discovery of antiviral inhibitors targeting the E dimer interface of Japanese encephalitis virus.

Flaviviruses are emerging arthropod-borne viruses posing a great threat to human beings worldwide. The E dimer configuration of the flavivirus was prominent during viral assembly, maturation and entry. Neutralization antibodies targeting E dimer played the important role in controlling the flavivirus infection. Previously, the ideal drug target of small molecular inhibitors of JEV was viral proteases and polymerases. The crystal structure of JEV E protein showed a conserved pocket in it is important at membrane fusion step. Recently, a set of anti-virus drugs has been found by virtual screening. Here, we show that the fusion-loop pocket of JEV E protein was a conservative region and an ideal drug target. ChemDiv-3 from virtual screening as the lead compound was found to show a relatively modest inhibition effect for JEV in vitro and in vivo test and could interfere with the formation of JEV sE dimer. ChemDiv-3 interacts with the amino acid residues ASN 313, PRO 314, ALA 315, and VAL 323 in E protein via hydrogen bonds for occupation of the fusion-loop pocket. The key binding sites LYS 312, ALA 513 and THR 317 forming the fusion-loop pocket are the same and other auxiliary sites are similar among the flavivirus. Taken together, the fusion-loop pocket of the flavivirus could be one promising target for drug discovery.

Outer membrane lipoprotein Lpp is Gram-negative bacterial cell surface receptor for cationic antimicrobial peptides.

Antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms. Here we demonstrate that the outer membrane lipoprotein, Lpp, of Enterobacteriaceae interacts with and promotes susceptibility to the bactericidal activities of AMPs. The oligomeric Lpp was specifically recognized by several cationic α-helical AMPs, including SMAP-29, CAP-18, and LL-37; AMP-mediated bactericidal activities were blocked by anti-Lpp antibody blocking. Blebbing of the outer membrane and increase in membrane permeability occurred in association with the coordinate internalization of Lpp and AMP. Interestingly, the specific binding of AMP to Lpp was resistant to divalent cations and salts, which were able to inhibit the bactericidal activities of some AMPs. Furthermore, using His-tagged Lpp as a ligand, we retrieved several characterized AMPs, including SMAP-29 and hRNase 7, from a peptide library containing crude mammalian cell lysates. Overall, this study explores a new mechanism and target of antimicrobial activity and provides a novel method for screening of antimicrobials for use against drug-resistant bacteria.

Development and validation of a yeast high-throughput screen for inhibitors of Aß42 oligomerization.

Recent reports point to small soluble oligomers, rather than insoluble fibrils, of amyloid ß (Aß), as the primary toxic species in Alzheimer's disease. Previously, we developed a low-throughput assay in yeast that is capable of detecting small Aß(42) oligomer formation. Specifically, Aß(42) fused to the functional release factor domain of yeast translational termination factor, Sup35p, formed sodium dodecyl sulfate (SDS)-stable low-n oligomers in living yeast, which impaired release factor activity. As a result, the assay for oligomer formation uses yeast growth to indicate restored release factor activity and presumably reduced oligomer formation. We now describe our translation of this assay into a high-throughput screen (HTS) for anti-oligomeric compounds. By doing so, we also identified two presumptive anti-oligomeric compounds from a sub-library of 12,800 drug-like small molecules. Subsequent biochemical analysis confirmed their anti-oligomeric activity, suggesting that this form of HTS is an efficient, sensitive and cost-effective approach to identify new inhibitors of Aß(42) oligomerization.

De-repression of heat shock transcription factor-1 in interleukin-6- treated hepatocytes is mediated by downregulation of glycogen synthase kinase 3beta and MAPK/ERK-1.

Expression of inducible heat shock protein (HSP70) requires activation of heat shock transcription factor-1 (HSF-1). Recent evidence suggests that interleukin-6 (IL-6) can modify the response of HSF-1 to heat. We hypothesized that IL-6 would prime the HSP response by causing de-repression of HSF-1 resulting in augmented HSP expression in stressed cells. In this study we show that IL-6 has no direct effect on HSP70 expression at 37 degrees C but does augment HSP70 expression in response to heat. IL-6 treatment decreased active MAPK/pERK and glycogen synthase kinase 3beta (GSK3beta) expression and GSK3beta kinase activity. In IL-6-treated cells, monomeric HSF-1 accumulated in the cytoplasm and nucleus, bound DNA but was transcriptionally inactive. On exposure to heat shock this modified monomer assumed the transcriptionally active phenotype with trimerization and hyperphosphorylation evident. The increased induction of HSP70 in IL-6 and heat-treated cells was inhibited using PI3-kinase inhibitors or Akt inhibition and was HSF-1 dependent. IL-6, via the PI3-kinase/Akt pathway leads to inhibition of the repressive kinases MAPK/pERK and GSK3beta, and this converts inactive HSF-1 to an intermediate DNA-binding form augmenting transcriptional activation in the presence of a second stressor.

Green tea (-)-epigallocatechin-gallate modulates early events in huntingtin misfolding and reduces toxicity in Huntington's disease models.

Huntington's disease (HD) is a progressive neurodegenerative disorder for which only symptomatic treatments of limited effectiveness are available. Preventing early misfolding steps and thereby aggregation of the polyglutamine (polyQ)-containing protein huntingtin (htt) in neurons of patients may represent an attractive therapeutic strategy to postpone the onset and progression of HD. Here, we demonstrate that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) potently inhibits the aggregation of mutant htt exon 1 protein in a dose-dependent manner. Dot-blot assays and atomic force microscopy studies revealed that EGCG modulates misfolding and oligomerization of mutant htt exon 1 protein in vitro, indicating that it interferes with very early events in the aggregation process. Also, EGCG significantly reduced polyQ-mediated htt protein aggregation and cytotoxicity in an yeast model of HD. When EGCG was fed to transgenic HD flies overexpressing a pathogenic htt exon 1 protein, photoreceptor degeneration and motor function improved. These results indicate that modulators of htt exon 1 misfolding and oligomerization like EGCG are likely to reduce polyQ-mediated toxicity in vivo. Our studies may provide the basis for the development of a novel pharmacotherapy for HD and related polyQ disorders.

Functional characterization of Musca glutamate- and GABA-gated chloride channels expressed independently and coexpressed in Xenopus oocytes.

Ligand-gated chloride channels (LGICs) are important targets for insecticides and parasiticides. Genes encoding subunits of two LGICs, a glutamate-gated chloride channel (MdGluCl-alpha) and a gamma-aminobutyric acid (GABA)-gated chloride channel (MdRdl), were cloned from house-flies (Musca domestica L.). These genes were first expressed independently in Xenopus laevis oocytes by cRNA injection in order to investigate the pharmacology of these ligand-gated channels using two-electrode voltage-clamp electrophysiology. It was found that L-glutamate and GABA activated the MdGluCl-alpha homo-oligomers with an EC(50) value of 30 microM and the MdRdl homo-oligomers with an EC(50) value of 101 microM, respectively. Both channels were chloride ion-permeable, and the MdRdl channel was more sensitive to chloride channel blockers, such as gamma-hexachlorocyclohexane (gamma-HCH), fipronil and picrotoxinin, than the MdGluCl-alpha channel. MdGluCl-alpha required only 1-2 days of incubation after cRNA injection to be expressed in oocytes, whereas 4-7 days of incubation was necessary to achieve MdRdl expression. However, when the cRNA of MdGluCl-alpha was injected at a dose of 1% (w/w) 1 day after the injection of the cRNA of MdRdl, a significant increase in the current amplitude of responses to GABA was observed, and the incubation period necessary for MdRdl expression became shorter. These results suggest that MdGluCl-alpha assists in the expression of MdRdl when the two are coexpressed.

Structure of alpha-latrotoxin oligomers reveals that divalent cation-dependent tetramers form membrane pores.

We report here the first three-dimensional structure of alpha-latrotoxin, a black widow spider neurotoxin, which forms membrane pores and stimulates secretion in the presence of divalent cations. We discovered that alpha-latrotoxin exists in two oligomeric forms: it is dimeric in EDTA but forms tetramers in the presence of Ca2+ or Mg2+. The dimer and tetramer structures were determined independently at 18 A and 14 A resolution, respectively, using cryo-electron microscopy and angular reconstitution. The alpha-latrotoxin monomer consists of three domains. The N- and C-terminal domains have been identified using antibodies and atomic fitting. The C4-symmetric tetramers represent the active form of alpha-latrotoxin; they have an axial channel and can insert into lipid bilayers with their hydrophobic base, providing the first model of alpha-latrotoxin pore formation.