Second-generation nitazoxanide derivatives: thiazolides are effective inhibitors of the influenza A virus.

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 IC50s 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.

Synergistic effect of nitazoxanide with neuraminidase inhibitors against influenza A viruses in vitro.

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 (IC50s) ranging from 0.9 to 3.2 µM, 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 A/Puerto Rico/8/1934 (H1N1), A/WSN/1933 (H1N1), or avian A/chicken/Italy/9097/1997 (H5N9) IAVs; dose-effect analysis and synergism/antagonism quantification were performed using isobologram analysis according to the Chou-Talalay method. Combination index (CI) analysis indicated that NTZ and oseltamivir combination treatment was synergistic against A/Puerto Rico/8/1934 (H1N1) and A/WSN/1933 (H1N1) IAVs, with CI values ranging between 0.39 and 0.63, independently of the MOI used. Similar results were obtained when NTZ was administered in combination with zanamivir (CI=0.3 to 0.48). NTZ-oseltamivir combination treatment was synergistic also against the avian A/chicken/Italy/9097/1997 (H5N9) IAV (CI=0.18 to 0.31). Taken together, the results suggest that regimens that combine neuraminidase inhibitors and nitazoxanide exert synergistic anti-influenza effects.

Thiazolides, a new class of antiviral agents effective against rotavirus infection, target viral morphogenesis, inhibiting viroplasm formation.

Rotaviruses, nonenveloped viruses presenting a distinctive triple-layered particle architecture enclosing a segmented double-stranded 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 (EC50s) ranging from 0.3 to 2 µg/ml and 50% cytotoxic concentrations (CC50s) higher than 50 µg/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.

The non-steroidal anti-inflammatory drug indomethacin activates the eIF2α kinase PKR, causing a translational block in human colorectal cancer cells.

The NSAID (non-steroidal anti-inflammatory drug) indomethacin, a cyclo-oxygenase-1 and -2 inhibitor with anti-inflammatory and analgesic properties, is known to possess anticancer activity against CRC (colorectal cancer) and other malignancies in humans; however, the mechanism underlying the anticancer action remains elusive. In the present study we show that indomethacin selectively activates the dsRNA (double-stranded RNA)-dependent protein kinase PKR in a cyclo-oxygenase-independent manner, causing rapid phosphorylation of eIF2α (the α-subunit of eukaryotic translation initiation factor 2) and inhibiting protein synthesis in colorectal carcinoma and other types of cancer cells. The PKR-mediated translational block was followed by inhibition of CRC cell proliferation and apoptosis induction. Indomethacin did not affect the activity of the eIF2α kinases PERK (PKR-like endoplasmic reticulum-resident kinase), GCN2 (general control non-derepressible-2) and HRI (haem-regulated inhibitor kinase), and induced eIF2α phosphorylation in PERK-knockout and GCN2-knockout cells, but not in PKR-knockout cells or in human PKR-silenced CRC cells, identifying PKR as a selective target for indomethacin-induced translational inhibition. The fact that indomethacin induced PKR activity in vitro, an effect reversed by the PKR inhibitor 2-aminopurine, suggests a direct effect of the drug in kinase activation. The results of the present study identify PKR as a novel target of indomethacin, suggesting new scenarios on the molecular mechanisms underlying the pleiotropic activity of this traditional NSAID.

NEMO-binding domain peptide inhibits proliferation of human melanoma cells.

Melanoma is the most aggressive form of skin cancer, it originates from melanocytes and its incidence has increased in the last decade. Recent advances in the understanding of the underlying biology of the progression of melanoma have identified key signalling pathways that are important in promoting melanoma tumourigenesis, thus providing dynamic targets for therapy. One such important target identified in melanoma tumour progression is the Nuclear Factor-kappaB (NF-kappaB) pathway. In vitro studies have shown that NF-kappaB binding is constitutively elevated in human melanoma cultures compared to normal melanocytes. It has been found that a short cell-permeable peptide spanning the IKK-beta NBD, named NBD peptide, disrupted the association of NEMO with IKKs in vitro and blocked TNFalpha-induced NF-kappaB activation in vivo. In the present study we investigated the effect of the NBD peptide on NF-kappaB activity and survival of A375 human melanoma cells. We found that NBD peptide is able to inhibit the proliferation of A375 cells, which present constitutively elevated NF-kappaB levels. Inhibition of cell proliferation by NBD peptide was associated with direct inhibition of constitutive NF-kappaB DNA-binding activity and induction of apoptosis by activation of caspase-3 as confirmed by the cleavage and consequently inactivation of poly (ADP ribose) polymerase (PARP-1) known as the best marker of this process.

NF-kappaB: a stress-regulated switch for cell survival.

Nuclear factor-kappaB (NF-kappaB), a stress-regulated transcription factor belonging to the Rel family, has a pivotal role in the control of the inflammatory and the innate immune responses. Its activation rapidly induces the transcription of a variety of genes encoding cell adhesion molecules, inflammatory and chemotactic cytokines, cytokine receptors, and enzymes that produce inflammatory mediators. More recently, NF-kappaB activation has been connected with multiple aspects of oncogenesis, including the control of cell proliferation, migration, cell cycle progression, and apoptosis. Interestingly, NF-kappaB is constitutively activated in several types of cancer cells, including hematological and epithelial malignancies. In addition, activation of NF-kappaB in cancer cells by chemotherapy or radiation therapy has been associated with the acquisition of resistance to apoptosis, which has emerged as a significant impediment to effective cancer treatment. Selective cyclopentenone inhibitors of the IkappaB kinase, the key enzyme controlling NF-kappaB activation, were recently shown to be potent inducers of apoptosis in chemoresistant lymphoid malignancies. Increasing evidence, summarized in this review, indicates that the development of selective NF-kappaB inhibitors may represent a promising therapeutic tool to sensitize tumor cells to apoptosis and increase the efficacy of conventional anticancer drugs in a wide spectrum of malignancies.

15-Deoxy-delta 12,14-prostaglandin J2 induces apoptosis in human malignant B cells: an effect associated with inhibition of NF-kappa B activity and down-regulation of antiapoptotic proteins.

Cyclopentenone prostaglandins are potent inhibitors of nuclear factor-kappa B (NF-kappa B), a transcription factor with a critical role in promoting inflammation and connected with multiple aspects of oncogenesis and cancer cell survival. In the present report, we investigated the role of NF-kappa B in the antineoplastic activity of the cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in multiple myeloma (MM) and Burkitt lymphoma (BL) cells expressing constitutively active NF-kappa B. 15d-PGJ(2) was found to suppress constitutive NF-kappa B activity and potently induce apoptosis in both types of B-cell malignancies. 15d-PGJ(2)-induced apoptosis occurs through multiple caspase activation pathways involving caspase-8 and caspase-9, and is prevented by pretreatment with the pan-caspase inhibitor ZVAD (z-Val-Ala-Asp). NF-kappa B inhibition is accompanied by rapid down-regulation of NF-kappa B-dependent antiapoptotic gene products, including cellular inhibitor-of-apoptosis protein 1 (cIAP-1), cIAP-2, X-chromosome-linked inhibitor-of-apoptosis protein (XIAP), and FLICE-inhibitory protein (cFLIP). These effects were mimicked by the proteasome inhibitor MG-132, but not by the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist troglitazone, suggesting that 15d-PGJ(2)-induced apoptosis is independent of PPAR-gamma. Knockdown of the NF-kappa B p65-subunit by lentiviral-mediated shRNA interference also resulted in apoptosis induction in malignant B cells with constitutively active NF-kappa B. The results indicate that inhibition of NF-kappa B plays a major role in the proapoptotic activity of 15d-PGJ(2) in aggressive B-cell malignancies characterized by aberrant regulation of NF-kappa B.

Inhibition of herpesvirus-induced HIV-1 replication by cyclopentenone prostaglandins: role of IkappaB kinase (IKK).

OBJECTIVES: Herpes simplex virus (HSV) infections have been associated with reactivation of HIV-1 replication and increases of HIV-1-load in plasma of co-infected individuals. The present authors have previously reported that in epithelial cells HSV-1 induces the IkappaB-kinase (IKK) causing persistent activation of NF-kappaB, a critical regulator of HIV-1 replication. The present study was performed to investigate whether HSV-1-infection could induce IKK-mediated NF-kappaB activation and enhance HIV-1 expression in human T cells, and to analyze the effect of the IKK-inhibitor prostaglandin A1 (PGA1) and other prostanoids on the NF-kappaB-mediated HSV-HIV interaction. DESIGN AND METHODS: Induction of IKK and NF-kappaB activity was determined in lymphoblastoid Jurkat cells and HIV-1 chronically-infected H9 and ACH-2 cells by kinase assay and electrophoretic mobility shift assay, respectively. The effect of HSV-1 and different prostanoids on HIV-1 expression and replication was determined in Jurkat cells transfected with HIV-1-LTR-driven reporter genes, and in H9 and ACH-2 cells by p24-antigen level evaluation. The role of NF-kappaB in HSV-1-induced HIV-1 expression was investigated by using the IkappaBalpha dominant-negative IkappaBalpha-AA in co-transfection experiments. RESULTS: In human T lymphoblastoid cells HSV-1 potently induces IKK activity, causing a persistent induction of NF-kappaB. HSV-1-induced IKK and NF-kappaB function results in transactivation of HIV-1-LTR-regulated genes and induction of HIV-1 replication in chronically-infected T cells. The cyclopentenone PGA1 inhibits HSV-1-induced IKK and NF-kappaB activities, blocking HIV-1-LTR-driven expression and preventing HSV-1-induced HIV-1 replication in co-infected cells. CONCLUSIONS: The results indicate that IKK is a key factor in triggering HSV-1-induced HIV-1 transcription in chronically-infected cells and identify cyclopentenone prostanoids as potent inhibitors of HSV-1-induced HIV-1 reactivation.