High-throughput screening to identify potential inhibitors of the Zα domain of the adenosine deaminase 1 (ADAR1)

Adenosine deaminases acting on RNA 1 (ADAR1) are enzymes involved in editing adenosine to inosine in the dsRNAs of cells associated with cancer development. The p150 isoform of ADAR1 is the only isoform containing the Zα domain that binds to both Z-DNA and Z-RNA. The Zα domain is suggested to modulate the immune response and could be a suitable target for antiviral treatment and cancer immunotherapy. In this study, we aimed to identify potential inhibitors for ADAR1 protein that bind the Zα domain using molecular docking and simulation tools. Virtual docking and molecular dynamics simulation approaches were used to screen the potential activity of 2115 FDA-approved compounds on the Zα domain of ADAR1 and filtered for to obtain the top-scoring hits. The top three compounds with the best XP Gscore—namely alendronate (−7.045), etidronate (−6.923), and zoledronate (−6.77)—were subjected to 50 ns simulations to characterize complex stability and identify the fundamental interactions that contribute to inhibition of the ADAR1 Zα domain. The three compounds were shown to interact with Lys169, Lys170, Asn173, and Tyr177 of the Zα domain-like helical backbone of Z-RNA. The study provides a comprehensive and novel insights of repurposes drugs for the inhibition of ADAR1 function.     

A fish herpesvirus highlights functional diversities among Zα domains related to phase separation induction and A-to-Z conversion

Zalpha (Zα) domains bind to left-handed Z-DNA and Z-RNA. The Zα domain protein family includes cellular (ADAR1, ZBP1 and PKZ) and viral (vaccinia virus E3 and cyprinid herpesvirus 3 (CyHV-3) ORF112) proteins. We studied CyHV-3 ORF112, which contains an intrinsically disordered region and a Zα domain. Genome editing of CyHV-3 indicated that the expression of only the Zα domain of ORF112 was sufficient for normal viral replication in cell culture and virulence in carp. In contrast, its deletion was lethal for the virus. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion by a broad spectrum of cellular, viral, and artificial Zα domains showed that only those expressing Z-binding activity, the capacity to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, this study reports the ability of some Zα domains to induce LLPS and supports the biological relevance of dsRNA A-to-Z conversion mediated by Zα domains. This study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of proteins containing Zα domains.     

The Structure of the Cyprinid herpesvirus 3 ORF112-Zα·Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L,a Poxvirus Inhibitor of Interferon Response

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zα domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zα domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zα in complex with an 18-bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zα domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.     

The roles of phospholipase C activation and alternative ADAR1 and ADAR2 pre-mRNA splicing in modulating serotonin 2C-receptor editing in vivo

The serotonin 2C receptor (5-HT2CR), a Gq-protein-coupled neurotransmitter receptor, exists in multiple isoforms that result from RNA editing of five exonic adenosines that are converted to inosines. In the adult brain, editing of 5-HT2C pre-mRNA exhibits remarkable plasticity in response to environmental and neurochemical stimuli. Here, we investigated two potential mechanisms underlying these plastic changes in adult 5-HT2CR editing phenotypes in vivo: activation of phospholipase C (PLC) and alternative splicing of pre-mRNA encoding the editing enzymes ADAR1 and ADAR2. Studies on two inbred strains of mice (C57Bl/6 and Balb/c) revealed that sustained stimulation of PLC—a downstream effector of activated Gαq protein—increased editing of forebrain neocortical 5-HT2C pre-mRNA at two sites known to be targeted by ADAR2. Moreover, changes in relative expression of the alternatively spliced “a” and “b” mRNA isoforms of ADAR1 and ADAR2 also correlate with changes in 5-HT2CR editing. The site-specific changes in 5-HT2CR editing detected in mice with different “a” over “b” ADAR mRNA isoform ratios only partially overlap with those evoked by sustained PLC activation and are best explained by the increased editing efficiency of ADAR1. Thus, activation of PLC and alternative splicing of ADAR pre-mRNA have both overlapping and specific roles in modulating 5-HT2CR editing phenotypes.     

Interaction of the Zalpha domain of human ADAR1 with a negatively supercoiled plasmid visualized by atomic force microscopy

Interest to the left-handed DNA conformation has been recently boosted by the findings that a number of proteins contain the Zα domain, which has been shown to specifically recognize Z-DNA. The biological function of Zα is presently unknown, but it has been suggested that it may specifically direct protein regions of Z-DNA induced by negative supercoiling in actively transcribing genes. Many studies, including a crystal structure in complex with Z-DNA, have focused on the human ADAR1 Zα domain in isolation. We have hypothesized that the recognition of a Z-DNA sequence by the Zα_ADAR1 domain is context specific, occurring under energetic conditions, which favor Z-DNA formation. To test this hypothesis, we have applied atomic force microscopy to image Zα_ADAR1 complexed with supercoiled plasmid DNAs. We have demonstrated that the Zα_ADAR1 binds specifically to Z-DNA and preferentially to d(CG)_n inserts, which require less energy for Z-DNA induction compared to other sequences. A notable finding is that site-specific Zα binding to d(GC)₁₃ or d(GC)₂C(GC)₁₀ inserts is observed when DNA supercoiling is insufficient to induce Z-DNA formation. These results indicate that Zα_ADAR1 binding facilities the B-to-Z transition and provides additional support to the model that Z-DNA binding proteins may regulate biological processes through structure-specific recognition.     

ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

Z-DNA binding protein 1 (ZBP1) belongs to a family of proteins that contain the Zα domain, which binds specifically to left-handed Z-DNA and Z-RNA. Like all vertebrate proteins in the Zα family, it contains two Zα-like domains and is highly inducible by immunostimulation. Using circular dichroism spectroscopy and electrophoretic mobility shift assays we show that both Zα domains can bind Z-DNA independently and that substrate binding is greatly enhanced when both domains are linked. Full length ZBP1 and a prominent splice variant lacking the first Zα domain (ΔZα) showed strikingly different subcellular localizations. While the full length protein showed a finely punctate cytoplasmatic distribution, ZBP1ΔZα accumulated in large cytoplasmic granules. Mutation of residues important for Z-DNA binding in the first Zα domain resulted in a distribution comparable to that of ZBP1ΔZα. The ZBP1ΔZα granules are distinct from stress granules (SGs) and processing bodies but dynamically interacted with these. Polysome stabilization led to the disassembly of ZBP1ΔZα granules, indicating that mRNA are integral components. Heat shock and arsenite exposure had opposing effects on ZBP1 isoforms: while ZBP1ΔZα granules disassembled, full length ZBP1 accumulated in SGs. Our data link ZBP1 to mRNA sorting and metabolism and indicate distinct roles for ZBP1 isoforms.     

Proteins that contain a functional Z-DNA-binding domain localize to cytoplasmic stress granules

Long double-stranded RNA may undergo hyper-editing by adenosine deaminases that act on RNA (ADARs), where up to 50% of adenosine residues may be converted to inosine. However, although numerous RNAs may undergo hyper-editing, the role for inosine-containing hyper-edited double-stranded RNA in cells is poorly understood. Nevertheless, editing plays a critical role in mammalian cells, as highlighted by the analysis of ADAR-null mutants. In particular, the long form of ADAR1 (ADAR1^p150) is essential for viability. Moreover, a number of studies have implicated ADAR1^p150 in various stress pathways. We have previously shown that ADAR1^p150 localized to cytoplasmic stress granules in HeLa cells following either oxidative or interferon-induced stress. Here, we show that the Z-DNA-binding domain (Zα^ADAR1) exclusively found in ADAR1^p150 is required for its localization to stress granules. Moreover, we show that fusion of Zα^ADAR1 to either green fluorescent protein (GFP) or polypyrimidine binding protein 4 (PTB4) also results in their localization to stress granules. We additionally show that the Zα domain from other Z-DNA-binding proteins (ZBP1, E3L) is likewise sufficient for localization to stress granules. Finally, we show that Z-RNA or Z-DNA binding is important for stress granule localization. We have thus identified a novel role for Z-DNA-binding domains in mammalian cells.     

Green Tea and One of Its Constituents,Epigallocatechine-3-gallate,Are Potent Inhibitors of Human 11β-hydroxysteroid Dehydrogenase Type 1

The microsomal enzyme 11β-hydroxysteroid deydrogenase type 1 (11β-HSD1) catalyzes the interconversion of glucocorticoid receptor-inert cortisone to receptor- active cortisol, thereby acting as an intracellular switch for regulating the access of glucocorticoid hormones to the glucocorticoid receptor. There is strong evidence for an important aetiological role of 11β-HSD1 in various metabolic disorders including insulin resistance, diabetes type 2, hypertension, dyslipidemia and obesity. Hence, modulation of 11β-HSD1 activity with selective inhibitors is being pursued as a new therapeutic approach for the treatment of the metabolic syndrome. Since tea has been associated with health benefits for thousands of years, we sought to elucidate the active principle in tea with regard to diabetes type 2 prevention. Several teas and tea specific polyphenolic compounds were tested for their possible inhibition of cortisone reduction with human liver microsomes and purified human 11β-HSD1. Indeed we found that tea extracts inhibited 11β-HSD1 mediated cortisone reduction, where green tea exhibited the highest inhibitory potency with an IC50 value of 3.749 mg dried tea leaves per ml. Consequently, major polyphenolic compounds from green tea, in particular catechins were tested with the same systems. (−)-Epigallocatechin gallate (EGCG) revealed the highest inhibition of 11β-HSD1 activity (reduction: IC50 = 57.99 µM; oxidation: IC50 = 131.2 µM). Detailed kinetic studies indicate a direct competition mode of EGCG, with substrate and/or cofactor binding. Inhibition constants of EGCG on cortisone reduction were Ki = 22.68 µM for microsomes and Ki = 18.74 µM for purified 11β-HSD1. In silicio docking studies support the view that EGCG binds directly to the active site of 11β-HSD1 by forming a hydrogen bond with Lys187 of the catalytic triade. Our study is the first to provide evidence that the health benefits of green tea and its polyphenolic compounds may be attributed to an inhibition of the cortisol producing enzyme 11β-HSD1.     

Dual conformational recognition by Z-DNA binding protein is important for the B–Z transition process

Left-handed Z-DNA is radically different from the most common right-handed B-DNA and can be stabilized by interactions with the Zα domain, which is found in a group of proteins, such as human ADAR1 and viral E3L proteins. It is well-known that most Zα domains bind to Z-DNA in a conformation-specific manner and induce rapid B–Z transition in physiological conditions. Although many structural and biochemical studies have identified the detailed interactions between the Zα domain and Z-DNA, little is known about the molecular basis of the B–Z transition process. In this study, we successfully converted the B–Z transition-defective Zα domain, vvZα_E3L, into a B–Z converter by improving B-DNA binding ability, suggesting that B-DNA binding is involved in the B–Z transition. In addition, we engineered the canonical B-DNA binding protein GH5 into a Zα-like protein having both Z-DNA binding and B–Z transition activities by introducing Z-DNA interacting residues. Crystal structures of these mutants of vvZα_E3L and GH5 complexed with Z-DNA confirmed the significance of conserved Z-DNA binding interactions. Altogether, our results provide molecular insight into how Zα domains obtain unusual conformational specificity and induce the B–Z transition.     

A left-handed RNA double helix bound by the Z alpha domain of the RNA-editing enzyme ADAR1

The A form RNA double helix can be transformed to a left-handed helix, called Z-RNA. Currently, little is known about the detailed structural features of Z-RNA or its involvement in cellular processes. The discovery that certain interferon-response proteins have domains that can stabilize Z-RNA as well as Z-DNA opens the way for the study of Z-RNA. Here, we present the 2.25 A crystal structure of the Zalpha domain of the RNA-editing enzyme ADAR1 (double-stranded RNA adenosine deaminase) complexed to a dUr(CG)(3) duplex RNA. The Z-RNA helix is associated with a unique solvent pattern that distinguishes it from the otherwise similar conformation of Z-DNA. Based on the structure, we propose a model suggesting how differences in solvation lead to two types of Z-RNA structures. The interaction of Zalpha with Z-RNA demonstrates how the interferon-induced isoform of ADAR1 could be targeted toward selected dsRNAs containing purine-pyrimidine repeats, possibly of viral origin.     

Molecular modeling of Ruellia tuberosa L compounds as a-amylase inhibitor: an in silico comparation between human and rat enzyme model

Inhibition of α-amylase is an important strategy to control post-prandial hyperglycemia. The present study on Ruellia tuberosa, known as traditional anti-diabetic agent, is being provided in silico study to identify compounds inhibiting α-amylase in rat and human. Compounds were explored from PubChem database. Molecular docking was studied using the autodock4. The interactions were further visualized and analyzed using the Accelrys Discovery Studio version 3.5. Binding energy of compounds to α-amylase was varying between -1.92 to -6.66 kcal/mol in rat pancreatic alpha amylase and -3.06 to -8.42kcal/mol in human pancreatic alpha amylase, and inhibition konstanta (ki) was varying between 13.12- 39460µM in rat and 0.67-5600µM in human. The docking results verify that betulin is the most potential inhibitor of all towards rat model alpha amylase and human alpha amylase. Further analysis reveals that betulin could be a potential inhibitor with non-competitive pattern like betulinic acid. In comparison, betulin has smaller Ki (0.67µM) than acarbose (2.6 µM), which suggesting that betulin is more potential as inhibitor than acarbose, but this assumption must be verified in vitro.     

Exploring the interaction of myricetin with human alpha-2-macroglobulin: biophysical and in-silico analysis

Myricetin (MYR) is a bioactive secondary metabolite found in plants that is recognized for its nutraceutical value and is an essential constituent of various foods and beverages. It is reported to exhibit a plethora of activities, including antioxidant, antimicrobial, antidiabetic, anticancer, and anti-inflammatory. Alpha-2-macroglobulin (α2M) is a major plasma anti-proteinase that can inhibit proteinases of both human and non-human origin, regardless of their specificity and catalytic mechanism. Here, we explored the interaction of MYR-α2M using various biochemical and biophysical techniques. It was found that the interaction of MYR brings subtle change in its anti-proteolytic potential and thereby alters its structure and function, as can be seen from absorbance and fluorescence spectroscopy. UV spectroscopy of α2M in presence of MYR indicated the occurrence of hyperchromism, suggesting complex formation. Fluorescence spectroscopy reveals that MYR reduces the fluorescence intensity of native α2M with a shift in the wavelength maxima. At 318.15 K, MYR binds to α2M with a binding constant of 2.4 × 10³ M⁻¹, which indicates significant binding. The ΔG value was found to be − 7.56 kcal mol⁻¹ at 298.15 K, suggesting the interaction to be spontaneous and thermodynamically favorable. The secondary structure of α2M does not involve any major change as was confirmed by CD analysis. The molecular docking indicates that Asp-146, Ser-172, Glu-174, and Tyr-180 were the key residues involved in α2M-MYR complex formation. This study contributes to our understanding of the function and mechanism of protein and flavonoid binding by providing a molecular basis of the interaction between MYR and α2M.     

Inhibitory evaluation of Curculigo latifolia on α-glucosidase,DPP (IV) and in vitro studies in antidiabetic with molecular docking relevance to type 2 diabetes mellitus

The inhibition of α-glucosidase and DPP enzymes capable of effectively reducing blood glucose level in the management of type 2 diabetes. The purpose of the present study is to evaluate the inhibitory potential of α-glucosidase and DPP (IV) activity including with the 2-NBDG uptake assay and insulin secretion activities through in vitro studies. The selected of active compounds obtained from the screening of compounds by LC-MS were docked with the targeted enzyme that involved in the mechanism of T2DM. From the results, root extracts displayed a better promising outcome in α-glucosidase (IC₅₀ 2.72 ± 0.32) as compared with the fruit extracts (IC₅₀ 3.87 ± 0.32). Besides, root extracts also displayed a better activity in the inhibition of DPP (IV), enhance insulin secretion and glucose uptake activity. Molecular docking results revealing that phlorizin binds strongly with α-glucosidase, DPP (IV) and Insulin receptor (IR) enzymes with achieving the lowest binding energy value. The present work suggests several of the compounds have the potential that contribute towards inhibiting α-glucosidase and DPP (IV) and thus effective in lowering post-prandial hyperglycaemia.     

Characterization of DNA-binding activity of Z alpha domains from poxviruses and the importance of the beta-wing regions in converting B-DNA to Z-DNA

The E3L gene is essential for pathogenesis in vaccinia virus. The E3L gene product consists of an N-terminal Zα domain and a C-terminal double-stranded RNA (dsRNA) binding domain; the left-handed Z-DNA-binding activity of the Zα domain of E3L is required for viral pathogenicity in mice. E3L is highly conserved among poxviruses, including the smallpox virus, and it is likely that the orthologous Zα domains play similar roles. To better understand the biological function of E3L proteins, we have investigated the Z-DNA-binding behavior of five representative Zα domains from poxviruses. Using surface plasmon resonance (SPR), we have demonstrated that these viral Zα domains bind Z-DNA tightly. Ability of Zα_E3L converting B-DNA to Z-DNA was measured by circular dichroism (CD). The extents to which these Zαs can stabilize Z-DNA vary considerably. Mutational studies demonstrate that residues in the loop of the β-wing play an important role in this stabilization. Notably the Zα domain of vaccinia E3L acquires ability to convert B-DNA to Z-DNA by mutating amino acid residues in this region. Differences in the host cells of the various poxviruses may require different abilities to stabilize Z-DNA; this may be reflected in the observed differences in behavior in these Zα proteins.     

Synthesis and bioactivities evaluation of oleanolic acid oxime ester derivatives as α-glucosidase and α-amylase inhibitors

Different oleanolic acid (OA) oxime ester derivatives (3a-3t) were designed and synthesised to develop inhibitors against α-glucosidase and α-amylase. All the synthesised OA derivatives were evaluated against α-glucosidase and α-amylase in vitro. Among them, compound 3a showed the highest α-glucosidase inhibition with an IC₅₀ of 0.35 µM, which was ∼1900 times stronger than that of acarbose, meanwhile compound 3f exhibited the highest α-amylase inhibitory with an IC₅₀ of 3.80 µM that was ∼26 times higher than that of acarbose. The inhibition kinetic studies showed that the inhibitory mechanism of compounds 3a and 3f were reversible and mixed types towards α-glucosidase and α-amylase, respectively. Molecular docking studies analysed the interaction between compound and two enzymes, respectively. Furthermore, cytotoxicity evaluation assay demonstrated a high level of safety profile of compounds 3a and 3f against 3T3-L1 and HepG2 cells.

Highlights

1. Oleanolic acid oxime ester derivatives (3a–3t) were synthesised and screened against α-glucosidase and α-amylase.
2. Compound 3a showed the highest α-glucosidase inhibitory with IC50 of 0.35 µM.
3. Compound 3f presented the highest α-amylase inhibitory with IC50 of 3.80 µM.
4. Kinetic studies and in silico studies analysed the binding between compounds and α-glucosidase or α-amylase.

     

Stepwise binding of inhibitors to human cytochrome P450 17A1 and rapid kinetics of inhibition of androgen biosynthesis

Cytochrome P450 (P450) 17A1 catalyzes the 17α-hydroxylation of progesterone and pregnenolone as well as the subsequent lyase cleavage of both products to generate androgens. However, the selective inhibition of the lyase reactions, particularly with 17α-hydroxy pregnenolone, remains a challenge for the treatment of prostate cancer. Here, we considered the mechanisms of inhibition of drugs that have been developed to inhibit P450 17A1, including ketoconazole, seviteronel, orteronel, and abiraterone, the only approved inhibitor used for prostate cancer therapy, as well as clotrimazole, known to inhibit P450 17A1. All five compounds bound to P450 17A1 in a multistep process, as observed spectrally, over a period of 10 to 30 s. However, no lags were observed for the onset of inhibition in rapid-quench experiments with any of these five compounds. Furthermore, the addition of substrate to inhibitor–P450 17A1 complexes led to an immediate formation of product, without a lag that could be attributed to conformational changes. Although abiraterone has been previously described as showing slow-onset inhibition (t_1/2 = 30 min), we observed rapid and strong inhibition. These results are in contrast to inhibitors of P450 3A4, an enzyme with a larger active site in which complete inhibition is not observed with ketoconazole and clotrimazole until the changes are completed. Overall, our results indicate that both P450 17A1 reactions—17α-hydroxylation and lyase activity—are inhibited by the initial binding of any of these inhibitors, even though subsequent conformational changes occur.     

Absence of Macrophage Inflammatory Protein-1α Prevents the Development of Blinding Herpes Stromal Keratitis

Prior studies in our laboratory have suggested that the CC chemokine macrophage inflammatory protein-1α (MIP-1α) may be an important mediator in the blinding ocular inflammation which develops following herpes simplex virus type 1 (HSV-1) infection of the murine cornea. To directly test this hypothesis, MIP-1α-deficient (−/−) mice and their wild-type (+/+) counterparts were infected topically on the scarified cornea with 2.5 × 10⁵ PFU of HSV-1 strain RE and subsequently graded for corneal opacity. Four weeks postinfection (p.i.), the mean corneal opacity score of −/− mice was 1.1 ± 0.3 while that of the +/+ mice was 3.7 ± 0.5. No detectable infiltrating CD4⁺ T cells were seen histologically at 14 or 21 days p.i. in −/− animals, whereas the mean CD4⁺ T-cell count per field (36 fields counted) in +/+ hosts was 26 ± 2 (P < 0.001). In addition, neutrophil counts in the −/− mouse corneas were reduced by >80% in comparison to the wild-type controls. At 2 weeks p.i., no interleukin-2 or gamma interferon could be detected in six of seven −/− mice, whereas both T-cell cytokines were readily demonstrable in +/+ mouse corneas. Also, MIP-2 and monocyte chemoattractant protein-1 protein levels were significantly lower in MIP-1α −/− mouse corneas than in +/+ host corneas, suggesting that MIP-1α directly, or more likely indirectly, influences the expression of other chemokines. Interestingly, despite the paucity of infiltrating cells, HSV-1 clearance from the eyes of −/− mice was not significantly different from that observed in +/+ hosts. We conclude that MIP-1α is not needed to control virus growth in the cornea but is essential for the development of severe stromal keratitis.     

Coumarins effectively inhibit bacterial α-carbonic anhydrases

Coumarins are known to act as prodrug inhibitors of mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) but they were not yet investigated for the inhibition of bacterial α-CAs. Here we demonstrate that such enzymes from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) are inhibited by a panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system. The nature and the position of the substituents in the coumarin ring were the factors which strongly influenced inhibitory efficacy. NgCAα was inhibited with K_Is in the range of 28.6–469.5 µM, whereas VchCAα with K_Is in the range of 39.8–438.7 µM. The two human (h)CA isoforms included for comparison reason in the study, hCA I and II, were less prone to inhibition by these compounds, with K_Is of 137–948.9 µM for hCA I and of 296.5–961.2 µM for hCA II, respectively. These findings are relevant for discovering coumarin bacterial CA inhibitors with selectivity for the bacterial over human isoform, with potential applications as novel antibacterial agents.     

Variola virus E3L Zα domain,but not its Z-DNA binding activity,is required for PKR inhibition

Responding to viral infection, the interferon-induced, double-stranded RNA (dsRNA)–activated protein kinase PKR phosphorylates translation initiation factor eIF2α to inhibit cellular and viral protein synthesis. To overcome this host defense mechanism, many poxviruses express the protein E3L, containing an N-terminal Z-DNA binding (Zα) domain and a C-terminal dsRNA-binding domain (dsRBD). While E3L is thought to inhibit PKR activation by sequestering dsRNA activators and by directly binding the kinase, the role of the Zα domain in PKR inhibition remains unclear. Here, we show that the E3L Zα domain is required to suppress the growth-inhibitory properties associated with expression of human PKR in yeast, to inhibit PKR kinase activity in vitro, and to reverse the inhibitory effects of PKR on reporter gene expression in mammalian cells treated with dsRNA. Whereas previous studies revealed that the Z-DNA binding activity of E3L is critical for viral pathogenesis, we identified point mutations in E3L that functionally uncouple Z-DNA binding and PKR inhibition. Thus, our studies reveal a molecular distinction between the nucleic acid binding and PKR inhibitory functions of the E3L Zα domain, and they support the notion that E3L contributes to viral pathogenesis by targeting PKR and other components of the cellular anti-viral defense pathway.     

Synthesis and biological evaluation of 2-arylbenzofuran derivatives as potential anti-Alzheimer’s disease agents

Alzheimer''s disease (AD) is a type of progressive dementia caused by degeneration of the nervous system. A single target drug usually does not work well. Therefore, multi-target drugs are designed and developed so that one drug can specifically bind to multiple targets to ensure clinical effectiveness and reduce toxicity. We synthesised a series of 2-arylbenzofuran derivatives and evaluated their in vitro activities. 2-Arylbenzofuran compounds have good dual cholinesterase inhibitory activity and β-secretase inhibitory activity. The IC₅₀ value of compound 20 against acetylcholinesterase inhibition (0.086 ± 0.01 µmol·L⁻¹) is similar to donepezil (0.085 ± 0.01 µmol·L⁻¹) and is better than baicalein (0.404 ± 0.04 µmol·L⁻¹). And most of the compounds have good BACE1 inhibitory activity, of which 3 compounds (8, 19 and 20) show better activity than baicalein (0.087 ± 0.03 µmol·L⁻¹). According to experimental results, 2-arylbenzofuran compounds provide an idea for drug design to develop prevention and treatment for AD.