Dual-action gallium-flavonoid compounds for combating Pseudomonas aeruginosa infection.

The opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) causes infections that are difficult to treat, which is due to the bacterial natural resistance to antibiotics. The bacterium is also able to form a biofilm that protects the bacterium from clearance by the human immune system and leads to chronic infection. Herein, we synthesized and characterized a novel gallium compound that interferes with both the iron metabolism and quorum sensing system of P. aeruginosa to achieve a significant bactericidal activity. The compound could substantially reduce the secretion of bacterial virulence factors as well as eliminate biofilm formation. Integrative omics analysis indicates that this compound can significantly disturb the gene transcription and metabolism of P. aeruginosa. The effectiveness of the gallium compound was further validated in mammalian cell and murine skin infection models. Our study offers a new strategy to design new gallium-based antimicrobials to combat P. aeruginosa infection.

Discovery of a Novel Specific Inhibitor Targeting Influenza A Virus Nucleoprotein with Pleiotropic Inhibitory Effects on Various Steps of the Viral Life Cycle.

Influenza A viruses (IAVs) continue to pose an imminent threat to humans due to annual influenza epidemic outbreaks and episodic pandemics with high mortality rates. In this context, the suboptimal vaccine coverage and efficacy, coupled with recurrent events of viral resistance against a very limited antiviral portfolio, emphasize an urgent need for new additional prophylactic and therapeutic options, including new antiviral targets and drugs with new mechanisms of action to prevent and treat influenza virus infection. Here, we characterized a novel influenza A virus nucleoprotein (NP) inhibitor, FA-6005, that inhibited a broad spectrum of human pandemic and seasonal influenza A and B viruses in vitro and protects mice against lethal influenza A virus challenge. The small molecule FA-6005 targeted a conserved NP I41 domain and acted as a potentially broad, multimechanistic anti-influenza virus therapeutic since FA-6005 suppressed influenza virus replication and perturbed intracellular trafficking of viral ribonucleoproteins (vRNPs) from early to late stages. Cocrystal structures of the NP/FA-6005 complex reconciled well with concurrent mutational studies. This study provides the first line of direct evidence suggesting that the newly identified NP I41 pocket is an attractive target for drug development that inhibits multiple functions of NP. Our results also highlight FA-6005 as a promising candidate for further development as an antiviral drug for the treatment of IAV infection and provide chemical-level details for inhibitor optimization.IMPORTANCE Current influenza antivirals have limitations with regard to their effectiveness and the potential emergence of resistance. Therefore, there is an urgent need for broad-spectrum inhibitors to address the considerable challenges posed by the rapid evolution of influenza viruses that limit the effectiveness of vaccines and lead to the emergence of antiviral drug resistance. Here, we identified a novel influenza A virus NP antagonist, FA-6005, with broad-spectrum efficacy against influenza viruses, and our study presents a comprehensive study of the mode of action of FA-6005 with the crystal structure of the compound in complex with NP. The influenza virus inhibitor holds promise as an urgently sought-after therapeutic option offering a mechanism of action complementary to existing antiviral drugs for the treatment of influenza virus infection and should further aid in the development of universal therapeutics.

A curcumin-sophorolipid nanocomplex inhibits Candida albicans filamentation and biofilm development.

Candida albicans is an opportunistic fungal pathogen that is highly resistant to contemporary antifungals, due to their biofilm lifestyle. The ability of C. albicans to invade human tissues is due to its filamentation. Therefore, inhibition of biofilms and filamentation of the yeast are high value targets to develop the next-generation antifungals. Curcumin (CU) is a natural polyphenol with excellent pharmacological attributes, but limitations such as poor solubility, acid, and enzyme tolerance have impeded its practical utility. Sophorolipids (SL) are biologically-derived surfactants that serve as efficient carriers of hydrophobic molecules such as curcumin into biofilms. Here, we synthesised a curcumin-sophorolipid nanocomplex (CUSL), and comprehensively evaluated its effects on C. albicans biofilms and filamentation. Our results demonstrated that sub-inhibitory concentration of CUSL (9.37 µg/mL) significantly inhibited fungal adhesion to substrates, and subsequent biofilm development, maturation, and filamentation. This effect was associated with significant downregulation of a select group of biofilm, adhesins, and hyphal regulatory genes. In conclusion, the curcumin-sophorolipid nanocomplex is a potent inhibitor of the two major virulence attributes of C. albicans, biofilm formation and filamentation, thus highlighting its promise as a putative anti-fungal agent with biofilm penetrative potential.

A tailored positively-charged hydrophobic surface reduces the risk of implant associated infections.

Implant-associated infections is one of the most challenging post-operative complications in bone-related implantations. To tackle this clinical issue, we developed a low-cost and durable surface coating for medical grade titanium implants that uses positively charged silane molecules. The in vitro antimicrobial tests revealed that the titanium surface coated with (3-aminopropyl) triethoxysilane, which has the appropriate length of hydrophobic alkyl chain and positive charged amino group, suppressed more than 90% of the initial bacterial adhesion of S. aureus, P. aeruginosa, and E. coli after 30 min of incubation. In terms of growth inhibitory rate, the treated surface was able to reduce 75.7% ± 11.9% of bacterial growth after a 24-hour culturing, thereby exhibiting superior anti-biofilm formation in the late stage. When implanted into the rat model infected by S. aureus, the treated surface eliminated the implant-associated infection through the mechanism of inhibition of bacterial adhesion on the implant surface. Additionally, the treated surface was highly compatible with mammalian cells. In general, our design demonstrated its potential for human clinical trials as a low-cost and effective antibacterial strategy to minimize post-operative implant-related bacterial infection.

Talaromyces marneffei Mp1 Protein, a Novel Virulence Factor, Carries Two Arachidonic Acid-Binding Domains To Suppress Inflammatory Responses in Hosts.

Talaromyces marneffei infection causes talaromycosis (previously known as penicilliosis), a very important opportunistic systematic mycosis in immunocompromised patients. Different virulence mechanisms in T. marneffei have been proposed and investigated. In the sera of patients with talaromycosis, Mp1 protein (Mp1p), a secretory galactomannoprotein antigen with two tandem ligand-binding domains (Mp1p-LBD1 and Mp1p-LBD2), was found to be abundant. Mp1p-LBD2 was reported to possess a hydrophobic cavity to bind copurified palmitic acid (PLM). It was hypothesized that capturing of lipids from human hosts by expressing a large quantity of Mp1p is a virulence mechanism of T. marneffei It was shown that expression of Mp1p enhanced the intracellular survival of T. marneffei by suppressing proinflammatory responses. Mechanistic study of Mp1p-LBD2 suggested that arachidonic acid (AA), a precursor of paracrine signaling molecules for regulation of inflammatory responses, is the major physiological target of Mp1p-LBD2. In this study, we use crystallographic and biochemical techniques to further demonstrate that Mp1p-LBD1, the previously unsolved first lipid binding domain of Mp1p, is also a strong AA-binding domain in Mp1p. These studies on Mp1p-LBD1 support the idea that the highly expressed Mp1p is an effective AA-capturing protein. Each Mp1p can bind up to 4 AA molecules. The crystal structure of Mp1p-LBD1-LBD2 has also been solved, showing that both LBDs are likely to function independently with a flexible linker between them. T. marneffei and potentially other pathogens highly expressing and secreting proteins similar to Mp1p can severely disturb host signaling cascades during proinflammatory responses by reducing the availabilities of important paracrine signaling molecules.

The ribosomal maturation factor P from Mycobacterium smegmatis facilitates the ribosomal biogenesis by binding to the small ribosomal protein S12.

The ribosomal maturation factor P (RimP) is a highly conserved protein in bacteria and has been shown to be important in ribosomal assembly in Escherichia coli Because of its central importance in bacterial metabolism, RimP represents a good potential target for drug design to combat human pathogens such as Mycobacterium tuberculosis However, to date, the only RimP structure available is the NMR structure of the ortholog in another bacterial pathogen, Streptococcus pneumoniae Here, we report a 2.2 Å resolution crystal structure of MSMEG_2624, the RimP ortholog in the close M. tuberculosis relative Mycobacterium smegmatis, and using in vitro binding assays, we show that MSMEG_2624 interacts with the small ribosomal protein S12, also known as RpsL. Further analyses revealed that the conserved residues in the linker region between the N- and C-terminal domains of MSMEG_2624 are essential for binding to RpsL. However, neither of the two domains alone was sufficient to form strong interactions with RpsL. More importantly, the linker region was essential for in vivo ribosomal biogenesis. Our study provides critical mechanistic insights into the role of RimP in ribosome biogenesis. We anticipate that the MSMEG_2624 crystal structure has the potential to be used for drug design to manage M. tuberculosis infections.

Triple combination of FDA-approved drugs including flufenamic acid, clarithromycin and zanamivir improves survival of severe influenza in mice.

Seasonal influenza virus remains a common cause of mortality despite the use of neuraminidase inhibitors. This study evaluated the efficacy of a triple combination of zanamivir, clarithromycin and flufenamic acid (FFA) in the treatment of influenza virus A(H1N1) infection. An in vitro cell protection assay and a multiple-cycle growth assay showed that the antiviral activity of zanamivir was enhanced when combined with clarithromycin or FFA. A mouse challenge model was used here for the evaluation of the in vivo efficacy of the triple combination treatment. We found that mice receiving the triple combination of FFA, zanamivir, and clarithromycin had a significantly better survival rate than those receiving the double combination of zanamivir and clarithromycin (88% versus 44%, P = 0.0083) or zanamivir monotherapy (88% versus 26%, P = 0.0002). Mice in the FFA-zanamivir-clarithromycin triple combination group also exhibited significantly less body weight loss than those in the zanamivir-clarithromycin double combination group. There was no significant difference in the lung viral titers among the different groups from day 2 to day 6 postinfection. However, the levels of IL-1ß, TNF-α and RANTES in the FFA-zanamivir-clarithromycin triple combination group were significantly lower than those in the zanamivir-clarithromycin double combination group, zanamivir monotherapy group, or solvent group on day 2 postinfection. Our findings showed that the FFA-zanamivir-clarithromycin triple combination improved the inflammatory markers and survival of severe influenza A(H1N1) infection in mice.

Mp1p homologues as virulence factors in Aspergillus fumigatus.

Recently, we showed that Mp1p is an important virulence factor of Talaromyces marneffei, a dimorphic fungus phylogenetically closely related to Aspergillus fumigatus. In this study, we investigated the virulence properties of the four Mp1p homologues (Afmp1p, Afmp2p, Afmp3p, and Afmp4p) in A. fumigatus using a mouse model. All mice died 7 days after challenge with wild-type A. fumigatus QC5096, AFMP1 knockdown mutant, AFMP2 knockdown mutant and AFMP3 knockdown mutant and 28 days after challenge with AFMP4 knockdown mutant (P<.0001). Only 11% of mice died 30 days after challenge with AFMP1-4 knockdown mutant (P<.0001). For mice challenge with AFMP1-4 knockdown mutant, lower abundance of fungal elements was observed in brains, kidneys, and spleens compared to mice challenge with QC5096 at day 4 post-infection. Fungal counts in brains of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.01 and P<.05). Fungal counts in kidneys of mice challenge with QC5096 or AFMP4 knockdown mutant were significantly higher than those challenge with AFMP1-4 knockdown mutant (P<.001 and P<.001) and those of mice challenge with QC5096 were significantly higher than those challenge with AFMP4 knockdown mutant (P<.05). There is no difference among the survival rates of wild-type A. fumigatus, AFMP4 knockdown mutant and AFMP1-4 knockdown mutant, suggesting that Mp1p homologues in A. fumigatus do not mediate its virulence via improving its survival in macrophage as in the case in T. marneffei. Afmp1p, Afmp2p, Afmp3p, and Afmp4p in combination are important virulence factors of A. fumigatus.

A tricyclic pyrrolobenzodiazepine produced by Klebsiella oxytoca is associated with cytotoxicity in antibiotic-associated hemorrhagic colitis.

Cytotoxin-producing Klebsiella oxytoca is the causative agent of antibiotic-associated hemorrhagic colitis (AAHC). Recently, the cytotoxin associated with AAHC was identified as tilivalline, a known pentacyclic pyrrolobenzodiazepine (PBD) metabolite produced by K. oxytoca Although this assertion of tilivalline's role in AAHC is supported by evidence from animal experiments, some key aspects of this finding appear to be incompatible with toxicity mechanisms of known PBD toxins. We therefore hypothesized that K. oxytoca may produce some other uncharacterized cytotoxins. To address this question, we investigated whether tilivalline alone is indeed necessary and sufficient to induce cytotoxicity or whether K. oxytoca also produces other cytotoxins. LC-MS- and NMR-based metabolomic analyses revealed the presence of an abundant tricyclic PBD, provisionally designated kleboxymycin, in the supernatant of toxigenic K. oxytoca strains. Moreover, by generating multiple mutants with gene deletions affecting tilivalline biosynthesis, we show that a tryptophanase-deficient, tilivalline-negative K. oxytoca mutant induced cytotoxicity in vitro similar to tilivalline-positive K. oxytoca strains. Furthermore, synthetic kleboxymycin exhibited greater than 9-fold higher cytotoxicity than tilivalline in TC50 cell culture assays. We also found that the biosynthetic pathways for kleboxymycin and tilivalline appear to overlap, as tilivalline is an indole derivative of kleboxymycin. In summary, our results indicate that tilivalline is not essential for inducing cytotoxicity observed in K. oxytoca-associated AAHC and that kleboxymycin is a tilivalline-related bacterial metabolite with even higher cytotoxicity.

Talaromyces marneffei Mp1p Is a Virulence Factor that Binds and Sequesters a Key Proinflammatory Lipid to Dampen Host Innate Immune Response.

Talaromyces (Penicillium) marneffei is one of the leading causes of systemic mycosis in immunosuppressed or AIDS patients in Southeast Asia. How this intracellular pathogen evades the host immune defense remains unclear. We provide evidence that T. marneffei depletes levels of a key proinflammatory lipid mediator arachidonic acid (AA) to evade the host innate immune defense. Mechanistically, an abundant secretory mannoprotein Mp1p, shown previously to be a virulence factor, does so by binding AA with high affinity via a long hydrophobic central cavity found in the LBD2 domain. This sequesters a critical proinflammatory signaling lipid, and we see evidence that AA, AA's downstream metabolites, and the cytokines interleukin-6 and tumor necrosis factor α are downregulated in T. marneffei-infected J774 macrophages. Given that Mp1p-LBD2 homologs are identified in other fungal pathogens, we expect that this novel class of fatty-acid-binding proteins sequestering key proinflammatory lipid mediators represents a general virulence mechanism of pathogenic fungi.

Identification of a novel small-molecule compound targeting the influenza A virus polymerase PB1-PB2 interface.

The PB1 C-terminal domain and PB2 N-terminal domain interaction of the influenza A polymerase, which modulates the assembly of PB1 and PB2 subunits, may serve as a valuable target for the development of novel anti-influenza therapeutics. In this study, we performed a systematic screening of a chemical library, followed by the antiviral evaluation of primary hits and their analogues. Eventually, a novel small-molecule compound PP7 that abrogated the PB1-PB2 association and impaired viral polymerase activity was identified. PP7 exhibited antiviral activities against influenza virus subtypes A (H1N1)pdm09, A(H7N9) and A(H9N2) in cell cultures and partially protected mice against lethal challenge of mouse-adapted influenza A (H1N1)pdm09 virus. Surprisingly, a panel of other subtypes of influenza virus, including A(H5N1) and A(H7N7), showed various degrees of resistance to the compound. Biochemical studies revealed a similar pattern of resistance on the impairment of polymerase activity. Molecular docking analyses suggested a PP7-binding site that appeared to be completely conserved among the subtypes of the virus mentioned above. Thus, we propose that alternative/additional binding site (s) may exist for the regulation of PB1-PB2 subunits assembly of influenza A virus.

Identification of catabolite control protein A from Staphylococcus aureus as a target of silver ions.

Staphylococcus aureus is one of the most common pathogenic bacteria that causes human infectious diseases. The emergence of antibiotic-resistant strains of S. aureus promotes the development of new anti-bacterial strategies. Silver ions (Ag+) have attracted profound attention due to their broad-spectrum antimicrobial activities. Although the antibacterial properties of silver have been well known for many centuries, its mechanism of action remains unclear and its protein targets are rarely reported. Herein, we identify the catabolite control protein A (CcpA) of S. aureus as a putative target for Ag+. CcpA binds 2 molar equivalents of Ag+via its two cysteine residues (Cys216 and Cys242). Importantly, Ag+ binding induces CcpA oligomerization and abolishes its DNA binding capability, which further attenuates S. aureus growth and suppresses α-hemolysin toxicity. This study extends our understanding of the bactericidal effects of silver.

Mp1p Is a Virulence Factor in Talaromyces (Penicillium) marneffei.

BACKGROUND: Talaromyces marneffei is an opportunistic dimorphic fungus prevalent in Southeast Asia. We previously demonstrated that Mp1p is an immunogenic surface and secretory mannoprotein of T. marneffei. Since Mp1p is a surface protein that can generate protective immunity, we hypothesized that Mp1p and/or its homologs are virulence factors. METHODOLOGY/PRINCIPAL FINDINGS: We examined the pathogenic roles of Mp1p and its homologs in a mouse model. All mice died 21 and 30 days after challenge with wild-type T. marneffei PM1 and MP1 complemented mutant respectively. None of the mice died 60 days after challenge with MP1 knockout mutant (P<0.0001). Seventy percent of mice died 60 days after challenge with MP1 knockdown mutant (P<0.0001). All mice died after challenge with MPLP1 to MPLP13 knockdown mutants, suggesting that only Mp1p plays a significant role in virulence. The mean fungal loads of PM1 and MP1 complemented mutant in the liver, lung, kidney and spleen were significantly higher than those of the MP1 knockout mutant. Similarly, the mean load of PM1 in the liver, lung and spleen were significantly higher than that of the MP1 knockdown mutant. Histopathological studies showed an abundance of yeast in the kidney, spleen, liver and lung with more marked hepatic and splenic necrosis in mice challenged with PM1 compared to MP1 knockout and MP1 knockdown mutants. Likewise, a higher abundance of yeast was observed in the liver and spleen of mice challenged with MP1 complemented mutant compared to MP1 knockout mutant. PM1 and MP1 complemented mutant survived significantly better than MP1 knockout mutant in macrophages at 48 hours (P<0.01) post-infection. The mean fungal counts of Pichia pastoris GS115-MP1 in the liver (P<0.001) and spleen (P<0.05) of mice were significantly higher than those of GS115 at 24 hours post-challenge. CONCLUSIONS/SIGNIFICANCE: Mp1p is a key virulence factor of T. marneffei. Mp1p mediates virulence by improving the survival of T. marneffei in macrophages.

Structural Characterization of H1N1 Nucleoprotein-Nucleozin Binding Sites.

Influenza viruses are among the most common pathogens that threaten the health of humans and animals worldwide. Various anti-viral therapeutic agents are currently used for treatment and prophylaxis of influenza virus, but the targets of these drugs are easily mutated and result in resistance. Therefore, medications that have broad spectrum coverage are urgently needed to combat with the disease. Since nucleoprotein is regarded as a druggable target due to its conserved sequence and important functions during influenza virus life cycle, numerous studies are focused on this protein in attempts to develop broad-spectrum anti-influenza therapeutics. Recently, a novel small molecule compound, nucleozin, was found to induce large aggregates of nucleoprotein, which in turn caused cessation of virus replication. However, the aggregation-inducing mechanism of nucleozin has not been unveiled. Here we report the crystal structure of nucleoprotein-nucleozin complex at 3 Å resolution, which shows the binding sites of nucleozin at nucleoprotein for the first time. The complex structure reveals how nucleoprotein and nucleozin interact with each other and hence result in nucleoprotein aggregates. The structural information is envisaged to help accelerate the development of anti-influenza therapeutic agents.

A novel small-molecule compound disrupts influenza A virus PB2 cap-binding and inhibits viral replication.

OBJECTIVES: The conserved residues 318-483 in the PB2 subunit of influenza A polymerase is an independently folded cap-binding domain (PB2cap) that exhibits a distinct binding mode from other host cap-binding proteins, which suggests that PB2cap might be an ideal drug target. This study aimed to identify a new class of anti-influenza inhibitors that specifically disrupts the interaction between PB2cap and host cap structures. METHODS: An innovative fluorescence polarization assay was established for primary screening, followed by cap-binding inhibitory activity, antiviral efficacy and cytotoxicity evaluations of the selected compounds. The best compound was characterized by multi-cycle virus growth assay, cross-protection test, synergism evaluation, mini-replicon assay, binding affinity analysis, docking simulation and mouse study. RESULTS: Several PB2 cap-binding inhibitors were discovered. The compound 7-(4-hydroxy-2-oxo-2H-chromen-3-yl)-6H,7H,8H-chromeno[3',4':5,6]pyrano[3,2-c]chromene-6,8-dione, designated PB2-39, was identified as a potent inhibitor of replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 in vitro and H1N1, H5N1 and H7N9 in vivo. Combinational treatment with the influenza virus release inhibitor zanamivir and PB2-39 exerted a synergistic anti-influenza effect. Mechanistic experiments supported that PB2-39 suppressed viral polymerase activity. Docking and binding affinity analyses demonstrated that PB2-39 interacted with the PB2 cap-binding pocket, suggesting its role as a cap-binding competitor. CONCLUSIONS: Our study provides new insights for the strategic development of novel cap-binding inhibitors of influenza A viruses.

Mycophenolic acid, an immunomodulator, has potent and broad-spectrum in vitro antiviral activity against pandemic, seasonal and avian influenza viruses affecting humans.

Immunomodulators have been shown to improve the outcome of severe pneumonia. We have previously shown that mycophenolic acid (MPA), an immunomodulator, has antiviral activity against influenza A/WSN/1933(H1N1) using a high-throughput chemical screening assay. This study further investigated the antiviral activity and mechanism of action of MPA against contemporary clinical isolates of influenza A and B viruses. The 50 % cellular cytotoxicity (CC50) of MPA in Madin Darby canine kidney cell line was over 50 µM. MPA prevented influenza virus-induced cell death in the cell-protection assay, with significantly lower IC50 for influenza B virus B/411 than that of influenza A(H1N1)pdm09 virus H1/415 (0.208 vs 1.510 µM, P=0.0001). For H1/415, MPA interfered with the early stage of viral replication before protein synthesis. For B/411, MPA may also act at a later stage since MPA was active against B/411 even when added 12 h post-infection. Virus-yield reduction assay showed that the replication of B/411 was completely inhibited by MPA at concentrations ≥0.78 µM, while there was a dose-dependent reduction of viral titer for H1/415. The antiviral effect of MPA was completely reverted by guanosine supplementation. Plaque reduction assay showed that MPA had antiviral activity against eight different clinical isolates of A(H1N1), A(H3N2), A(H7N9) and influenza B viruses (IC50 <1 µM). In summary, MPA has broad-spectrum antiviral activity against human and avian-origin influenza viruses, in addition to its immunomodulatory activity. Together with a high chemotherapeutic index, the use of MPA as an antiviral agent should be further investigated in vivo.

A novel small-molecule inhibitor of influenza A virus acts by suppressing PA endonuclease activity of the viral polymerase.

The RNA-dependent RNA polymerase of influenza A virus comprises conserved and independently-folded subdomains with defined functionalities. The N-terminal domain of the PA subunit (PA(N)) harbors the endonuclease function so that it can serve as a desired target for drug discovery. To identify a class of anti-influenza inhibitors that impedes PA(N) endonuclease activity, a screening approach that integrated the fluorescence resonance energy transfer based endonuclease inhibitory assay with the DNA gel-based endonuclease inhibitory assay was conducted, followed by the evaluation of antiviral efficacies and potential cytotoxicity of the primary hits in vitro and in vivo. A small-molecule compound ANA-0 was identified as a potent inhibitor against the replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2, in cell cultures. Combinational treatment of zanamivir and ANA-0 exerted synergistic anti-influenza effect in vitro. Intranasal administration of ANA-0 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. In summary, ANA-0 shows potential to be developed to novel anti-influenza agents.

Development of novel implants with self-antibacterial performance through in-situ growth of 1D ZnO nanowire.

To prevent the attachment of bacteria to implant surfaces, the 1D zinc oxide nanowire-coating has been successfully developed on material surfaces by using a custom-made hydrothermal approach. The chemical nature, surface topography and wettability of spike-like 1D ZnO nanowire-coating are comprehensively investigated. The anti-adhesive and antimicrobial properties of 1D nanowire-coating are tested against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli by using in vitro live/dead staining and scanning electron microscopy. We find that the adhesion of bacteria can be reduced via the special spike-like topography and that the release of Zn(2+) ions can help suppress the growth of attached bacteria. Furthermore, the antimicrobial effect is also evaluated under in vivo conditions by using a rat model infected with bioluminescent S. aureus. The amount of live bacteria in the rat implanted with a nanowire-coated sample is less than that of the control at various time points. Hence, it is believed that the nanowire-coated material is promising for application in orthopaedic implantation after the long-term animal studies have been completed.

Identification of a small-molecule inhibitor of influenza virus via disrupting the subunits interaction of the viral polymerase.

Assembly of the heterotrimeric influenza virus polymerase complex from the individual subunits PB1, PA, and PB2 is a prerequisite for viral replication, in which the interaction between the C terminal of PA (PAC) and the N-terminal of PB1 (PB1N) may be a desired target for antiviral development. In this study, we compared the feasibility of high throughput screening by enzyme-linked immunosorbent assay (ELISA) and fluorescence polarization assay. Among the two, ELISA was demonstrated to own broader dynamic range so that it was used for screening inhibitors that blocked PAC and PB1N interaction. Several binding inhibitors of PAC-PB1N were identified and subsequently tested for the antiviral efficacy. Apparently, 3-(2-chlorophenyl)-6-ethyl-7-methyl[1,2,4]triazolo[4,3-a]pyrimidin-5-ol, designated ANA-1, was found to be a strong inhibitor of viral polymerase activity and act as a potent antiviral agent against the infections of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9 and H9N2 subtypes, in cell cultures. Intranasal administration of ANA-1 protected mice from lethal challenge and reduced lung viral loads in H1N1 virus infected BALB/c mice. Docking analyses predicted that ANA-1 bound to an allosteric site of PAC, which might cause conformational changes thereby disrupting the PAC-PB1N interaction. Overall, our study has identified a novel compound with potential to be developed as an anti-influenza drug.

Identification of Novel Small Organic Compounds with Diverse Structures for the Induction of Epstein-Barr Virus (EBV) Lytic Cycle in EBV-Positive Epithelial Malignancies.

Phorbol esters, which are protein kinase C (PKC) activators, and histone deacetylase (HDAC) inhibitors, which cause enhanced acetylation of cellular proteins, are the main classes of chemical inducers of Epstein-Barr virus (EBV) lytic cycle in latently EBV-infected cells acting through the PKC pathway. Chemical inducers which induce EBV lytic cycle through alternative cellular pathways may aid in defining the mechanisms leading to lytic cycle reactivation and improve cells' responsiveness towards lytic induction. We performed a phenotypic screening on a chemical library of 50,240 novel small organic compounds to identify novel class(es) of strong inducer(s) of EBV lytic cycle in gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Five hit compounds were selected after three successive rounds of increasingly stringent screening. All five compounds are structurally diverse from each other and distinct from phorbol esters or HDAC inhibitors. They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers. Two of the five compounds with rapid lytic-inducing action were further studied for their mechanisms of induction of EBV lytic cycle. Unlike HDAC inhibitors, lytic induction by both compounds was not inhibited by rottlerin, a specific inhibitor of PKCδ. Interestingly, both compounds could cooperate with HDAC inhibitors to enhance EBV lytic cycle induction in EBV-positive epithelial cancer cells, paving way for the development of strategies to increase cells' responsiveness towards lytic reactivation. One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways. These structurally diverse novel organic compounds may represent potential new classes of chemicals that can be used to investigate any alternative mechanism(s) leading to EBV lytic cycle reactivation from latency.