The emergence of highly contagious influenza A virus strains, such as the new H1N1 swine influenza, represents a serious threat to global human health. Efforts to control emerging influenza strains focus on surveillance and early diagnosis, as well as development of effective vaccines and novel antiviral drugs. Herein we document the anti-influenza activity of the anti-infective drug nitazoxanide and its active circulating-metabolite tizoxanide and describe a class of second generation thiazolides effective against influenza A virus. Thiazolides inhibit the replication of H1N1 and different other strains of influenza A virus by a novel mechanism: they act at post-translational level by selectively blocking the maturation of the viral hemagglutinin at a stage preceding resistance to endoglycosidase H digestion, thus impairing hemagglutinin intracellular trafficking and insertion into the host plasma membrane, a key step for correct assembly and exit of the virus from the host cell. Targeting the maturation of the viral glycoprotein offers the opportunity to disrupt the production of infectious viral particles attacking the pathogen at a level different from the currently available anti-influenza drugs. The results indicate that thiazolides may represent a new class of antiviral drugs effective against influenza A infection.

Paramyxoviridae, a large family of enveloped viruses harboring a nonsegmented negative-sense RNA genome, include important human pathogens as measles, mumps, respiratory syncytial virus (RSV), parainfluenza viruses, and henipaviruses, which cause some of the deadliest emerging zoonoses. There is no effective antiviral chemotherapy for most of these pathogens. Paramyxoviruses evolved a sophisticated membrane-fusion machine consisting of receptor-binding proteins and the fusion F-protein, critical for virus infectivity. Herein we identify the antiprotozoal/antimicrobial nitazoxanide as a potential anti-paramyxovirus drug targeting the F-protein. We show that nitazoxanide and its circulating-metabolite tizoxanide act at post-entry level by provoking Sendai virus and RSV F-protein aggregate formation, halting F-trafficking to the host plasma membrane. F-protein folding depends on ER-resident glycoprotein-specific thiol-oxidoreductase ERp57 for correct disulfide-bond architecture. We found that tizoxanide behaves as an ERp57 non-competitive inhibitor; the putative drug binding-site was located at the ERp57-b/b′ non-catalytic domains interface. ERp57-silencing mimicked thiazolide-induced F-protein alterations, suggesting an important role of this foldase in thiazolides anti-paramyxovirus activity. Nitazoxanide is used in the clinic as a safe and effective antiprotozoal/antimicrobial drug; its antiviral activity was shown in patients infected with hepatitis-C virus, rotavirus and influenza viruses. Our results now suggest that nitazoxanide may be effective also against paramyxovirus infection.

dRotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented doublestranded RNA genome, exhibit a unique morphogenetic pathway requiring the formation of cytoplasmic inclusion bodies called viroplasms in a process involving the nonstructural viral proteins NSP5 and NSP2. In these structures the concerted packaging and replication of the 11 positive-polarity single-stranded RNAs take place to generate the viral double-stranded RNA (dsRNA) genomic segments. Rotavirus infection is a leading cause of gastroenteritis-associated severe morbidity and mortality in young children, but no effective antiviral therapy exists. Herein we investigate the antirotaviral activity of the thiazolide anti-infective nitazoxanide and reveal a novel mechanism by which thiazolides act against rotaviruses. Nitazoxanide and its active circulating metabolite, tizoxanide, inhibit simian A/SA11-G3P[2] and human Wa-G1P[8] rotavirus replication in different types of cells with 50% effective concentrations (EC 50 s) ranging from 0.3 to 2 g/ml and 50% cytotoxic concentrations (CC 50 s) higher than 50 g/ml. Thiazolides do not affect virus infectivity, binding, or entry into target cells and do not cause a general inhibition of viral protein expression, whereas they reduce the size and alter the architecture of viroplasms, decreasing rotavirus dsRNA formation. As revealed by protein/protein interaction analysis, confocal immunofluorescence microscopy, and viroplasm-like structure formation analysis, thiazolides act by hindering the interaction between the nonstructural proteins NSP5 and NSP2. Altogether the results indicate that thiazolides inhibit rotavirus replication by interfering with viral morphogenesis and may represent a novel class of antiviral drugs effective against rotavirus gastroenteritis. R otaviruses are complex nonenveloped viruses belonging to the Reoviridae family. The rotavirion has a distinctive triple-layered particle (TLP) architecture that surrounds a genome composed of 11 segments of double-stranded RNA (dsRNA) encoding six structural viral proteins (VPs) and six nonstructural proteins (NSPs) (1, 2). The capsid structure comprises an inner-core shell of VP2 dimers and an intermediate shell formed by trimers of the major structural protein VP6, which interacts with both the VP2 core protein and the outer shell constituted by the VP4 protein (the rotavirus spikes, which express P-serotype epitopes), and VP7 glycoprotein trimers, which express G-serotype epitopes (2). The P and G serotypes represent independently segregating neutralization epitopes imparting immunity to infection. VP7, which is the second most abundant protein in the virion, is cotranslationally glycosylated as it is inserted into the endoplasmic reticulum (ER) membrane via a cleavable signal sequence found at the N terminus of the protein (1, 2). Rotaviruses exhibit a unique morphogenetic pathway. Double-layered particles (DLPs) are assembled in the cytoplasm at special areas termed viroplasms and the...

The emergence of drug-resistant influenza A virus (IAV) strains represents a serious threat to global human health and underscores the need for novel approaches to anti-influenza chemotherapy. Combination therapy with drugs affecting different IAV targets represents an attractive option for influenza treatment. We have previously shown that the thiazolide anti-infective nitazoxanide (NTZ) inhibits H1N1 IAV replication by selectively blocking viral hemagglutinin maturation. Herein we investigate the anti-influenza activity of NTZ against a wide range of human and avian IAVs (H1N1, H3N2, H5N9, H7N1), including amantadine-resistant and oseltamivir-resistant strains, in vitro. We also investigate whether therapy with NTZ in combination with the neuraminidase inhibitors oseltamivir and zanamivir exerts synergistic, additive, or antagonistic antiviral effects against influenza viruses. NTZ was effective against all IAVs tested, with 50% inhibitory concentrations (IC 50 s) ranging from 0.9 to 3.2 M, and selectivity indexes (SIs) ranging from >50 to >160, depending on the strain and the multiplicity of infection (MOI). Combination therapy studies were performed in cell culture-based assays using

Influenza A viruses continue to represent a severe threat worldwide, causing large epidemics and pandemics responsible for thousands of deaths every year. Excessive inflammation due to overabundant production of proinflammatory cytokines by airway epithelial cells is considered an important factor in disease pathogenesis. Here we report that influenza A virus induced IB kinase (IKK) activity in human airway epithelial A549 cells, resulting in persistent activation of nuclear factor-B (NF-B), a critical regulator of the inflammatory response. Although lung epithelial cells are highly sensitive to stimulation of the IKK/NF-B pathway by influenza virus infection, NF-B was not activated in several non-pulmonary cells permissive to the virus, indicating a cell-specific response. Moreover, NF-B was not essential for virus replication but triggered the expression of proinflammatory cytokines in infected lung cells and was directly responsible for production of high levels of interleukin-8, a chemokine associated with influenzainduced inflammation and airway pathology. We also report that 9-deoxy-⌬ 9 ,⌬ 12 -13,14-dihydro-prostaglandin D 2 , a cyclopentenone prostanoid with therapeutic efficacy against influenza in preclinical studies, was a powerful inhibitor of influenza virus-induced IKK activity and interleukin-8 production by human pulmonary cells. The results identify IKK as an important factor in triggering influenza virus-induced inflammatory reactions in pulmonary epithelium, suggesting novel therapeutic approaches in the treatment of influenza.

Background: AIRAP is a recently identified heat shock protein whose function and regulation are still not well defined. Results: Proteasome inhibition causes abundant AIRAP expression in human primary cells. Conclusion:The anticancer drug bortezomib regulates AIRAP expression through HSF1-HSF2 interplay. Significance: This study reveals how persistent proteasomal inhibition by clinically relevant concentrations of bortezomib affects heat shock response regulation and HSF-mediated AIRAP induction.

Aim:The only small molecule drugs currently available for treatment of influenza A virus (IAV) are M2 ion channel blockers and sialidase inhibitors. The prototype thiazolide, nitazoxanide, has successfully completed Phase III clinical trials against acute uncomplicated influenza.Results: We report the activity of seventeen thiazolide analogs against A/PuertoRico/8/1934(H1N1), a laboratory-adapted strain of the H1N1 subtype of IAV, in a cell culture-based assay. A total of eight analogs showed IC 50 s in the range of 0.14-5.0 μM. Additionally a quantitative structure-property relationship study showed high correlation between experimental and predicted activity based on a molecular descriptor set. Conclusion: A range of thiazolides show useful activity against an H1N1 strain of IAV. Further evaluation of these molecules as potential new small molecule therapies is justified. Graphical abstract:OR 2 A number of thiazolides (R 1 = a 3, 4 or 5-substituent; R 2 = H or CH 3 CO; R 3 = a 4´ -or 5´-substituent) are active with IC 50 = 0.14-5.0 µM against a prototypical H1N1 strain of influenza A virus in MDCK cells; a QSAR regression model was developed, showing good correlation between predicted and measured in vitro activity.Since the prototype nitazoxanide has successfully completed Phase III clinical trials against acute uncomplicated influenza, this molecule series has considerable potential for future development.

Indomethacin, a cyclooxygenase-1 and -2 inhibitor widely used in the clinic for its potent anti-inflammatory/analgesic properties, possesses antiviral activity against several viral pathogens; however, the mechanism of antiviral action remains elusive. We have recently shown that indomethacin activates the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) in human colon cancer cells. Because of the important role of PKR in the cellular defence response against viral infection, herein we investigated the effect of indomethacin on PKR activity during infection with the prototype rhabdovirus vesicular stomatitis virus. Indomethacin was found to activate PKR in an interferon- and dsRNA-independent manner, causing rapid (< 5 min) phosphorylation of eukaryotic initiation factor-2 α-subunit (eIF2α). These events resulted in shutting off viral protein translation and blocking viral replication (IC50  = 2 μM) while protecting host cells from virus-induced damage. Indomethacin did not affect eIF2α kinases PKR-like endoplasmic reticulum-resident protein kinase (PERK) and general control non-derepressible-2 (GCN2) kinase, and was unable to trigger eIF2α phosphorylation in the presence of PKR inhibitor 2-aminopurine. In addition, small-interfering RNA-mediated PKR gene silencing dampened the antiviral effect in indomethacin-treated cells. The results identify PKR as a critical target for the antiviral activity of indomethacin and indicate that eIF2α phosphorylation could be a key element in the broad spectrum antiviral activity of the drug.