Impaired Antiviral Responses to Extracellular Double-Stranded RNA and Cytosolic DNA, but Not to Interferon-α Stimulation, in TRIM56-Deficient Cells

The physiologic function of tripartite motif protein 56 (TRIM56), a ubiquitously expressed E3 ligase classified within the large TRIM protein family, remains elusive. Gene knockdown studies have suggested TRIM56 as a positive regulator of the type I interferon (IFN-I) antiviral response elicited via the Toll-like receptor 3 (TLR3) and cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathways, which detect and respond to danger signals—extracellular double-stranded (ds) RNA and cytosolic dsDNA, respectively. However, to what extent these pathways depend on TRIM56 in human cells is unclear. In addition, it is debatable whether TRIM56 plays a part in controlling the expression of IFN-stimulated genes (ISGs) resulting from IFN-I based antiviral treatment. In this study, we created HeLa-derived TRIM56 null cell lines by gene editing and used these cell models to comprehensively examine the impact of endogenous TRIM56 on innate antiviral responses. Our results showed that TRIM56 knockout severely undermined the upregulation of ISGs by extracellular dsRNA and that loss of TRIM56 weakened the response to cytosolic dsDNA. ISG induction and ISGylation following IFN-α stimulation, however, were not compromised by TRIM56 deletion. Using a vesicular stomatitis virus-based antiviral bioactivity assay, we demonstrated that IFN-α could efficiently establish an antiviral state in TRIM56 null cells, providing direct evidence that TRIM56 is not required for the general antiviral action of IFN-I. Altogether, these data ascertain the contributions of TRIM56 to TLR3- and cGAS–STING-dependent antiviral pathways in HeLa cells and add to our understanding of the roles this protein plays in innate immunity.

Discovery of small-molecule modulators of melanogenesis by docking-based virtual screening

Background: Vitiligo is a relatively common depigmenting skin disorder. UV light stimulation is often used to obtain repigmentation. Wnt signaling regulates melanocyte differentiation, and expression of TYR is upregulated in narrow-band UVB-treated epidermis. Manipulation of these two pathways by drugs could serve as one of the therapeutic approaches for durable repigmentation. Methods and results: CD9 was identified as a novel TYR activator by virtual screening and bioactivity assay. CD9 activated the Wnt signaling pathway through triggering translocation of β-catenin from cytoplasm to nucleus. Conclusion: The pathogenesis of vitiligo is complicated and varies with each individual, so combination therapy may be much more suitable for treatment of vitiligo. CD9 could synergize with other anti-inflammatory compounds or autoimmune suppressors to shorten repigmentation time and improve efficacy.

Design and Synthesis of a Highly Simplified Pateamine Analogue

<p>Pateamine (1) is a natural product from the marine sponge Mycale hentscheli that exhibits potent anticancer properties, and has potential as an antiviral agent, and in preventing the muscle wasting disorder cachexia. This biological activity of pateamine is due to its ability to inhibit the eukaryotic initiation factor eIF4A, which leads to the formation of stress granules, the inhibition of protein synthesis, and ultimately cell death. Unfortunately, pateamine is obtained in very small amounts from Mycale hentscheli; thus, it is necessary to synthesise pateamine and novel structural analogues in the laboratory. Previously a separate binding and scaffolding domain of pateamine was proposed, which led to the synthesis of a simplified des-methyl des- amino analogue that reduced the number of synthetic steps compared to pateamine while retaining its biological activity. This was followed by the synthesis of a simplified triazole- containing analogue 9 6 ; unfortunately, this exhibited substantially reduced bioactivity compared to pateamine, and it is therefore necessary to determine if the reduction in bioactivity was due to the replacement of the thiazole ring with a triazole ring, or due to the removal of key methyl groups of pateamine. Thus, the thiazole-containing analogue of 96 is deemed to be an important synthetic target. In this Master’s project a highly simplified side chain-free analogue 130 was synthesised, which laid the groundwork for future synthesis of a thiazole-containing analogue of 96. The synthesis of 130 was achieved through a convergent synthesis with one commercially available and two prepared fragments. Particular attention was paid to the development of an efficient thiazole formation methodology, as well as optimising fragment synthesis and coupling reactions. Determination of the binding of analogue 130 with eIF4A using a competitive bioactivity assay in the presence of pateamine was then undertaken, which showed that either 130 does not bind to eIF4A or that it binds non-covalently and is then displaced by pateamine.</p>

Design and Synthesis of a Highly Simplified Pateamine Analogue

<p>Pateamine (1) is a natural product from the marine sponge Mycale hentscheli that exhibits potent anticancer properties, and has potential as an antiviral agent, and in preventing the muscle wasting disorder cachexia. This biological activity of pateamine is due to its ability to inhibit the eukaryotic initiation factor eIF4A, which leads to the formation of stress granules, the inhibition of protein synthesis, and ultimately cell death. Unfortunately, pateamine is obtained in very small amounts from Mycale hentscheli; thus, it is necessary to synthesise pateamine and novel structural analogues in the laboratory. Previously a separate binding and scaffolding domain of pateamine was proposed, which led to the synthesis of a simplified des-methyl des- amino analogue that reduced the number of synthetic steps compared to pateamine while retaining its biological activity. This was followed by the synthesis of a simplified triazole- containing analogue 9 6 ; unfortunately, this exhibited substantially reduced bioactivity compared to pateamine, and it is therefore necessary to determine if the reduction in bioactivity was due to the replacement of the thiazole ring with a triazole ring, or due to the removal of key methyl groups of pateamine. Thus, the thiazole-containing analogue of 96 is deemed to be an important synthetic target. In this Master’s project a highly simplified side chain-free analogue 130 was synthesised, which laid the groundwork for future synthesis of a thiazole-containing analogue of 96. The synthesis of 130 was achieved through a convergent synthesis with one commercially available and two prepared fragments. Particular attention was paid to the development of an efficient thiazole formation methodology, as well as optimising fragment synthesis and coupling reactions. Determination of the binding of analogue 130 with eIF4A using a competitive bioactivity assay in the presence of pateamine was then undertaken, which showed that either 130 does not bind to eIF4A or that it binds non-covalently and is then displaced by pateamine.</p>

Identification of Broad-Spectrum MMP Inhibitors by Virtual Screening

Matrix metalloproteinases (MMPs) are the family of proteases that are mainly responsible for degrading extracellular matrix (ECM) components. In the skin, the overexpression of MMPs as a result of ultraviolet radiation triggers an imbalance in the ECM turnover in a process called photoaging, which ultimately results in skin wrinkling and premature skin ageing. Therefore, the inhibition of different enzymes of the MMP family at a topical level could have positive implications for photoaging. Considering that the MMP catalytic region is mostly conserved across different enzymes of the MMP family, in this study we aimed to design a virtual screening (VS) workflow to identify broad-spectrum MMP inhibitors that can be used to delay the development of photoaging. Our in silico approach was validated in vitro with 20 VS hits from the Specs library that were not only structurally different from one another but also from known MMP inhibitors. In this bioactivity assay, 18 of the 20 compounds inhibit at least one of the assayed MMPs at 100 μM (with 5 of them showing around 50% inhibition in all the tested MMPs at this concentration). Finally, this VS was used to identify natural products that have the potential to act as broad-spectrum MMP inhibitors and be used as a treatment for photoaging.

Potential Antibacterial Activity of Bioactive β-sitosterol from Root Bark of Rhizophora apiculata from Lampung Coastal

β-sitosterol is an essential bioactive phytosterol naturally present in plant cell membranes. It has a coincident structure with animal cholesterol. This investigation reported isolation, structure analysis, and an antimicrobial assay of β-sitosterol from the root bark of Bakau Minyak (Rhizophora apiculata) from Lampung coastal. The isolation of β-sitosterol was carried out through maceration using methanol, separation by vacuum liquid chromatography (VLC), and purification by column chromatography (CC) using ethyl acetate/n-hexane (2:8) as eluent. The structure of β-sitosterol was determined using spectroscopic analysis (UV-Vis, FT-IR, 13C-NMR, 1H-NMR, DEPT, and GC-MS). The pure β-sitosterol has 107.4 mg of white needle crystalline compound, the compound melting point about 140.7-141.2oC, the molecular mass confirmed by m/z 414, and UV absorption detected at λ 203.9 nm. The β-sitosterol antimicrobial bioactivity assay has shown potential activity to be developed as a lead compound against E. coli.

Wheat root protection from cereal cyst nematode (Heterodera avenae) by fluopyram seed treatment

Cereal cyst nematode (Heterodera avenae), an important plant-parasitic nematode causing yield losses on wheat, has been found in many provinces in China. It is urgent to develop an effective method to protect wheat from H. avenae damage. With a novel mode of action, fluopyram has been registered for controlling root-knot nematodes on cucumber and tomato in China. However, the bioactivity of fluopyram against H. avenae and whether seed treatment is effective in controlling H. avenae on wheat remains unknown. In this study, a bioactivity assay revealed that fluopyram increased the mortality of H. avenae second stage juveniles (J2), with LC50 and LC90 values of 0.92 mg L-1 and 2.92 mg L-1, respectively. Hatching tests showed that H. avenae egg hatching percent decreased 35.2-69.2% with fluopyram at the rate of 1.6-6.4 mg L-1, and the egg hatching period was delayed by 3-9 days compared with the control. In pot and field trials, fluopyram seed treatment significantly reduced H. avenae population density and increased wheat yield by 3.0-13.7%. Therefore, fluopyram seed treatment is an effective approach for the management of H. avenae on wheat in China.

Anti-Syncytium (MC99+1A2) and Anti-Bacterial Activities from Twigs and Stems of Ficus dubia

Aims: To study the anti-HIVs and anti-bacterial activity and investigate the chemical compositions of the crude extracts and isolated compounds from stems and twig of Ficus dubia. Methodology: Twigs and stems of Ficus dubia were collected from Si Sawat District, Kanchanaburi Province, Thailand. Bioactivity assay and phytochemical analysis of ficus dubia were processed under standard method including, anti-HIV1-RT, antibacterial activities, and CC chromatography procedures. Structures of the compound were elucidated by spectroscopic techniques. Results: Bioactivities assay exposed that, the hexane, ethyl acetate, and methanol extracts of Ficus dubia were verified for anti-HIV and anti-bacterial activities. The ethyl acetate and methanol extract showed evidence of anti-HIVs-1RT inhibition at 69.02 % and 69.24 %, respectively. Anti-syncytium (MC99+1A2) among evaluated all three extracts: it afforded the lowest EC50; hexane 9.65 (TI 4.39), ethyl acetate 11.15 (TI 3.84), and methanol 43.61 (TI 3.04). Moreover, an antibacterial study on extract was also performed. The antibacterial study was evaluated using nine strains (Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli 0157: H7, Escherichia coli (ETEC), Escherichia coli (EPEC), Proteus mirabilis, Salmonella typhimurium, Shigella flexneri, and Vibrio cholera) by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) method. The MeOH extract was the most effective antibacterial with a MIC in the range >0.1 mg/mL and MBC in the range >0.2 mg/mL. However, the isolated compounds from this plant had not been studied in all bioassay. Phytochemical examination of stems and twigs from Ficus dubia had directed to the isolation of four known compounds, epifriedelanol, β-sitosterol, stigmasterol, and bergapten. Conclusion: The crude extract of twigs and stems of Ficus fubia were specifically active toward MC99+1A2. With these results, it could be concluded that Ficus dubia may be an accomplished candidate in pill and future medicine.