Spike mutation resilient scFv76 antibody counteracts SARS-CoV-2 lung damage upon aerosol delivery.

The uneven worldwide vaccination coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emergence of variants escaping immunity call for broadly effective and easily deployable therapeutic agents. We have previously described the human single-chain scFv76 antibody, which recognizes SARS-CoV-2 Alpha, Beta, Gamma and Delta variants. We now show that scFv76 also neutralizes the infectivity and fusogenic activity of the Omicron BA.1 and BA.2 variants. Cryoelectron microscopy (cryo-EM) analysis reveals that scFv76 binds to a well-conserved SARS-CoV-2 spike epitope, providing the structural basis for its broad-spectrum activity. We demonstrate that nebulized scFv76 has therapeutic efficacy in a severe hACE2 transgenic mouse model of coronavirus disease 2019 (COVID-19) pneumonia, as shown by body weight and pulmonary viral load data. Counteraction of infection correlates with inhibition of lung inflammation, as observed by histopathology and expression of inflammatory cytokines and chemokines. Biomarkers of pulmonary endothelial damage were also significantly reduced in scFv76-treated mice. The results support use of nebulized scFv76 for COVID-19 induced by any SARS-CoV-2 variants that have emerged so far.

Impairment of SARS-CoV-2 spike glycoprotein maturation and fusion activity by nitazoxanide: an effect independent of spike variants emergence.

SARS-CoV-2, the causative agent of COVID-19, has caused an unprecedented global health crisis. The SARS-CoV-2 spike, a surface-anchored trimeric class-I fusion glycoprotein essential for viral entry, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of SARS-CoV-2 spike variants that facilitate virus spread and may affect vaccine efficacy highlights the need to identify novel antiviral strategies for COVID-19 therapy. Here, we demonstrate that nitazoxanide, an antiprotozoal agent with recognized broad-spectrum antiviral activity, interferes with SARS-CoV-2 spike maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Engineering multiple SARS-CoV-2 variant-pseudoviruses and utilizing quantitative cell-cell fusion assays, we show that nitazoxanide-induced spike modifications hinder progeny virion infectivity as well as spike-driven pulmonary cell-cell fusion, a critical feature of COVID-19 pathology. Nitazoxanide, being equally effective against the ancestral SARS-CoV-2 Wuhan-spike and different emerging variants, including the Delta variant of concern, may represent a useful tool in the fight against COVID-19 infections.

The proteostasis guardian HSF1 directs the transcription of its paralog and interactor HSF2 during proteasome dysfunction.

Protein homeostasis is essential for life in eukaryotes. Organisms respond to proteotoxic stress by activating heat shock transcription factors (HSFs), which play important roles in cytoprotection, longevity and development. Of six human HSFs, HSF1 acts as a proteostasis guardian regulating stress-induced transcriptional responses, whereas HSF2 has a critical role in development, in particular of brain and reproductive organs. Unlike HSF1, that is a stable protein constitutively expressed, HSF2 is a labile protein and its expression varies in different tissues; however, the mechanisms regulating HSF2 expression remain poorly understood. Herein we demonstrate that the proteasome inhibitor anticancer drug bortezomib (Velcade), at clinically relevant concentrations, triggers de novo HSF2 mRNA transcription in different types of cancers via HSF1 activation. Similar results were obtained with next-generation proteasome inhibitors ixazomib and carfilzomib, indicating that induction of HSF2 expression is a general response to proteasome dysfunction. HSF2-promoter analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies unexpectedly revealed that HSF1 is recruited to a heat shock element located at 1.397 bp upstream from the transcription start site in the HSF2-promoter. More importantly, we found that HSF1 is critical for HSF2 gene transcription during proteasome dysfunction, representing an interesting example of transcription factor involved in controlling the expression of members of the same family. Moreover, bortezomib-induced HSF2 was found to localize in the nucleus, interact with HSF1, and participate in bortezomib-mediated control of cancer cell migration. The results shed light on HSF2-expression regulation, revealing a novel level of HSF1/HSF2 interplay that may lead to advances in pharmacological modulation of these fundamental transcription factors.

Remdesivir: From Ebola to COVID-19.

Human coronaviruses (HCoV) were discovered in the 1960s and were originally thought to cause only mild upper respiratory tract diseases in immunocompetent hosts. This view changed since the beginning of this century, with the 2002 SARS (severe acute respiratory syndrome) epidemic and the 2012 MERS (Middle East respiratory syndrome) outbreak, two zoonotic infections that resulted in mortality rates of approximately 10% and 35%, respectively. Despite the importance of these pathogens, no approved antiviral drugs for the treatment of human coronavirus infections became available. However, remdesivir, a nucleotide analogue prodrug originally developed for the treatment of Ebola virus, was found to inhibit the replication of a wide range of human and animal coronaviruses in vitro and in preclinical studies. It is therefore not surprising that when the highly pathogenic SARS-CoV-2 coronavirus emerged in late 2019 in China, causing global health concern due to the virus strong human-to-human transmission ability, remdesivir was one of the first clinical candidates that received attention. After in vitro studies had shown its antiviral activity against SARS-CoV-2, and a first patient was successfully treated with the drug in the USA, a number of trials on remdesivir were initiated. Several had encouraging results, particularly the ACTT-1 double blind, randomized, and placebo controlled trial that has shown shortening of the time to recovery in hospitalized patients treated with remdesivir. The results of other trials were instead negative. Here, we provide an overview of remdesivir discovery, molecular mechanism of action, and initial and current clinical studies on its efficacy.

The biogenesis of SARS-CoV-2 spike glycoprotein: multiple targets for host-directed antiviral therapy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 (coronavirus disease-19), represents a far more serious threat to public health than SARS and MERS coronaviruses, due to its ability to spread more efficiently than its predecessors. Currently, there is no worldwide-approved effective treatment for COVID-19, urging the scientific community to intense efforts to accelerate the discovery and development of prophylactic and therapeutic solutions against SARS-CoV-2 infection. In particular, effective antiviral drugs are urgently needed. With few exceptions, therapeutic approaches to combat viral infections have traditionally focused on targeting unique viral components or enzymes; however, it has now become evident that this strategy often fails due to the rapid emergence of drug-resistant viruses. Targeting host factors that are essential for the virus life cycle, but are dispensable for the host, has recently received increasing attention. The spike glycoprotein, a component of the viral envelope that decorates the virion surface as a distinctive crown ("corona") and is essential for SARS-CoV-2 entry into host cells, represents a key target for developing therapeutics capable of blocking virus invasion. This review highlights aspects of the SARS-CoV-2 spike biogenesis that may be amenable to host-directed antiviral targeting.

Inhibition of viral protein translation by indomethacin in vesicular stomatitis virus infection: role of eIF2α kinase PKR.

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.

The proteasome inhibitor bortezomib is a potent inducer of zinc finger AN1-type domain 2a gene expression: role of heat shock factor 1 (HSF1)-heat shock factor 2 (HSF2) heterocomplexes.

The zinc finger AN1-type domain 2a gene, also known as arsenite-inducible RNA-associated protein (AIRAP), was recently identified as a novel human canonical heat shock gene strictly controlled by heat shock factor (HSF) 1. Little is known about AIRAP gene regulation in human cells. Here we report that bortezomib, a proteasome inhibitor with anticancer and antiangiogenic properties used in the clinic for treatment of multiple myeloma, is a potent inducer of AIRAP expression in human cells. Using endothelial cells as a model, we unraveled the molecular mechanism regulating AIRAP expression during proteasome inhibition. Bortezomib induces AIRAP expression at the transcriptional level early after treatment, concomitantly with polyubiquitinated protein accumulation and HSF activation. AIRAP protein is detected at high levels for at least 48 h after bortezomib exposure, together with the accumulation of HSF2, a factor implicated in differentiation and development regulation. Different from heat-mediated induction, in bortezomib-treated cells, HSF1 and HSF2 interact directly, forming HSF1-HSF2 heterotrimeric complexes recruited to a specific heat shock element in the AIRAP promoter. Interestingly, whereas HSF1 has been confirmed to be critical for AIRAP gene transcription, HSF2 was found to negatively regulate AIRAP expression after bortezomib treatment, further emphasizing an important modulatory role of this transcription factor under stress conditions. AIRAP function is still not defined. However, the fact that AIRAP is expressed abundantly in primary human cells at bortezomib concentrations comparable with plasma levels in treated patients suggests that AIRAP may participate in the regulatory network controlling proteotoxic stress during bortezomib treatment.

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.

The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein.

Coronaviridae is recognized as one of the most rapidly evolving virus family as a consequence of the high genomic nucleotide substitution rates and recombination. The family comprises a large number of enveloped, positive-sense single-stranded RNA viruses, causing an array of diseases of varying severity in animals and humans. To date, seven human coronaviruses (HCoV) have been identified, namely HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, which are globally circulating in the human population (seasonal HCoV, sHCoV), and the highly pathogenic SARS-CoV, MERS-CoV and SARS-CoV-2. Seasonal HCoV are estimated to contribute to 15-30% of common cold cases in humans; although diseases are generally self-limiting, sHCoV can sometimes cause severe lower respiratory infections and life-threatening diseases in a subset of patients. No specific treatment is presently available for sHCoV infections. Herein we show that the anti-infective drug nitazoxanide has a potent antiviral activity against three human endemic coronaviruses, the Alpha-coronaviruses HCoV-229E and HCoV-NL63, and the Beta-coronavirus HCoV-OC43 in cell culture with IC50 ranging between 0.05 and 0.15 µg/mL and high selectivity indexes. We found that nitazoxanide does not affect HCoV adsorption, entry or uncoating, but acts at postentry level and interferes with the spike glycoprotein maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Altogether the results indicate that nitazoxanide, due to its broad-spectrum anti-coronavirus activity, may represent a readily available useful tool in the treatment of seasonal coronavirus infections.

Thiazolide Prodrug Esters and Derived Peptides: Synthesis and Activity.

Amino acid ester prodrugs of the thiazolides, introduced to improve the pharmacokinetic parameters of the parent drugs, proved to be stable as their salts but were unstable at pH > 5. Although some of the instability was due to simple hydrolysis, we have found that the main end products of the degradation were peptides formed by rearrangement. These peptides were stable solids: they maintained significant antiviral activity, and in general, they showed improved pharmacokinetics (better solubility and reduced clearance) compared to the parent thiazolides. We describe the preparation and evaluation of these peptides.

Long Covid: where we stand and challenges ahead.

Post-acute sequelae of SARS-CoV-2 (PASC), also known as Post-Covid Syndrome, and colloquially as Long Covid, has been defined as a constellation of signs and symptoms which persist for weeks or months after the initial SARS-CoV-2 infection. PASC affects a wide range of diverse organs and systems, with manifestations involving lungs, brain, the cardiovascular system and other organs such as kidney and the neuromuscular system. The pathogenesis of PASC is complex and multifactorial. Evidence suggests that seeding and persistence of SARS-CoV-2 in different organs, reactivation, and response to unrelated viruses such as EBV, autoimmunity, and uncontrolled inflammation are major drivers of PASC. The relative importance of pathogenetic pathways may differ in different tissue and organ contexts. Evidence suggests that vaccination, in addition to protecting against disease, reduces PASC after breakthrough infection although its actual impact remains to be defined. PASC represents a formidable challenge for health care systems and dissecting pathogenetic mechanisms may pave the way to targeted preventive and therapeutic approaches.

AIRAP, a new human heat shock gene regulated by heat shock factor 1.

Heat shock factor-1 (HSF1) is the central regulator of heat-induced transcriptional responses leading to rapid expression of molecular chaperones that protect mammalian cells against proteotoxic stress. The main targets for HSF1 are specific promoter elements (HSE) located upstream of heat shock genes encoding a variety of heat shock proteins, including HSP70, HSP90, HSP27, and other proteins of the network. Herein we report that the zinc finger AN1-type domain-2a gene, also known as AIRAP, behaves as a canonical heat shock gene, whose expression is temperature-dependent and strictly controlled by HSF1. Transcription is triggered at temperatures above 40 degrees C in different types of human cancer and primary cells, including peripheral blood monocytes. As shown by ChIP analysis, HSF1 is recruited to the AIRAP promoter rapidly after heat treatment, with a kinetics that parallels HSP70 promoter HSF1-recruitment. In transfection experiments HSF1-silencing abolished heat-induced AIRAP promoter-driven transcription, which could be rescued by exogenous Flag-HSF1 expression. The HSF1 binding HSE sequence in the AIRAP promoter critical for heat-induced transcription was identified. Because its expression is induced at febrile temperatures in human cells, AIRAP may represent a new potential component of the protective response during fever in humans.

Synthesis, antiviral activity, preliminary pharmacokinetics and structural parameters of thiazolide amine salts.

Background: The thiazolides, typified by nitazoxanide, are an important class of anti-infective agents. A significant problem with nitazoxanide and its active circulating metabolite tizoxanide is their poor solubility. Results: We report the preparation and evaluation of a series of amine salts of tizoxanide and the corresponding 5-Cl thiazolide. These salts demonstrated improved aqueous solubility and absorption, as shown by physicochemical and in vivo measurements. They combine antiviral activity against influenza A virus with excellent cell safety indices. We also report the x-ray crystal structural data of the ethanolamine salt. Conclusion: The ethanol salt of thiazolide retains the activity of the parent together with an improved cell safety index, making it a good candidate for further evaluation.

Thiazolides, a new class of anti-influenza molecules targeting viral hemagglutinin at the post-translational level.

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.

Coronaviruses and stress: from cellular to global.

Near the end of 2019, SARS-CoV-2, a novel highly contagious coronavirus phylogenetically related to the SARS virus, entered the human population with lethal consequences. This special issue devoted to the resulting disease COVID-19 was not planned but instead the articles accumulated organically as researchers in the cell stress response field noticed similarities among the pathophysiology of COVID-19 infections and the responses that they studied in contexts unrelated to viral infection. We preface the issue with an introductory article which begins with a brief review of the structure and biology of SARS-CoV-2. As we collected and compared the COVID-19 articles, several shared themes emerged. In the second part of the introduction, each article is summarized briefly and the common themes that link each into a spontaneously arising chain of ideas and hypotheses are emphasized. These themes include growing evidence of molecular mimicry among the viral proteins and the proteins of patients. The realization that much of the consequences of such immune mimicry may play out on the plasma membrane of vascular endothelial cells raised the specter of autoimmune-induced vascular endothelial damage in multiple organs. Proposals of new therapeutic approaches have coalesced around the theme of inducing protection of the vascular endothelium. New chemical treatments that are proposed include stannous chloride, inducers of the gasotransmitter hydrogen sulfide such as sodium thiosulfate and inducers of the cytoprotective stress protein heme oxygenase. Oxygen delivered by ventilators is already in extensive use to provide life support for patients with severe COVID-19. Two articles propose to advance the use of oxygen to the level of a therapeutic treatment early in the detection of the virus in infected patients by delivering oxygen under elevated pressure in hyperbaric chambers. At elevated blood plasma concentrations, hyperbaric oxygen is capable of achieving results far beyond the capability of ventilators as it promotes the activation of transcription factors that control the establishment of inducible cellular defense systems.

The Zinc-Finger AN1-Type Domain 2a Gene Acts as a Regulator of Cell Survival in Human Melanoma: Role of E3-Ligase cIAP2.

The zinc-finger AN1-type domain-2a gene, also known as AIRAP (arsenite-inducible RNA-associated protein), was initially described as an arsenite-inducible gene in Caenorhabditis elegans and mammalian cells. Differently from the AIRAP worm homologue, aip-1, a gene known to play an important role in preserving animal lifespan and buffering arsenic-induced proteotoxicity, mammals have a second, constitutively expressed, AIRAP-like gene (AIRAPL), recently implicated in myeloid transformation. We have identified human AIRAP as a canonical heat-shock gene, whose expression, differently from AIRAPL, is strictly dependent on the proteotoxic-stress regulator heat-shock factor 1 (HSF1). AIRAP function is still not well defined and there is no information on AIRAP in cancer. Herein we show that bortezomib and next-generation proteasome inhibitors ixazomib and carfilzomib markedly induce AIRAP expression in human melanoma at concentrations comparable to plasma-levels in treated patients. AIRAP-downregulation leads to bortezomib sensitization, whereas AIRAP-overexpression protects melanoma cells from the drug, identifying AIRAP as a novel HSF1-regulated marker of chemotherapy resistance. More importantly, this study unexpectedly revealed that, also in the absence of drugs, AIRAP-silencing hinders melanoma clonogenic potential and spheroid growth, promoting caspase activation and apoptotic cell death, an effect independent of AIRAPL and linked to downregulation of the antiapoptotic protein cIAP2. Interestingly, AIRAP was found to interact with cIAP2, regulating its stability in melanoma. Taken together, the results identify AIRAP as a novel HSF1-dependent regulator of prosurvival networks in melanoma cells, opening new therapeutic perspectives in chemoresistant melanoma treatment. IMPLICATIONS: The findings identify ZFAND2A/AIRAP as a novel stress-regulated survival factor implicated in the stabilization of the antiapoptotic protein cIAP2 and as a new potential therapeutic target in melanoma.

Targeting the heat shock factor 1 by RNA interference: a potent tool to enhance hyperthermochemotherapy efficacy in cervical cancer.

Carcinoma of the uterine cervix is one of the highest causes of mortality in female cancer patients worldwide, and improved treatment options for this type of malignancy are highly needed. Local hyperthermia has been successfully used in combination with systemic administration of cisplatin-based chemotherapy in phase I/II clinical studies. Heat-induced expression of cytoprotective and antiapoptotic heat shock proteins (HSP) is a known complication of hyperthermia, resulting in thermotolerance and chemoresistance and hindering the efficacy of the combination therapy. Heat shock transcription factor 1 (HSF1) is the master regulator of heat-induced HSP expression. In the present report, we used small interfering RNA (siRNA) to silence HSF1 and to examine the effect of HSF1 loss of function on the response to hyperthermia and cisplatin-based chemotherapy in HeLa cervical carcinoma. We have identified the 322-nucleotide to 340-nucleotide HSF1 sequence as an ideal target for siRNA-mediated HSF1 silencing, have created a pSUPER-HSF1 vector able to potently suppress the HSF1 gene, and have generated for the first time human cancer cell lines with stable loss of HSF1 function. We report that, although it surprisingly does not affect cancer cell sensitivity to cisplatin or elevated temperatures up to 43 degrees C when administered separately, loss of HSF1 function causes a dramatic increase in sensitivity to hyperthermochemotherapy, leading to massive (>95%) apoptosis of cancer cells. These findings indicate that disruption of HSF1-induced cytoprotection during hyperthermochemotherapy may represent a powerful strategy to selectively amplify the damage in cancer cells and identify HSF1 as a promising therapeutic target in cervical carcinoma.

The second-generation thiazolide haloxanide is a potent inhibitor of avian influenza virus replication.

The emergence of new avian influenza virus (AIV) strains able to infect humans represents a serious threat to global human health. In addition to surveillance and vaccine development, antiviral therapy remains crucial for AIV control; however, the increase in drug-resistant AIV strains underscores the need for novel approaches to anti-influenza chemotherapy. We have previously shown that the thiazolide anti-infective nitazoxanide (NTZ) inhibits influenza A/PuertoRico/8/1934(H1N1) virus replication, and this effect was associated with inhibition of viral hemagglutinin (HA) maturation. Herein we investigated the activity of the second-generation thiazolide haloxanide (HLN) against H5N9, H7N1 and H1N1 AIV infection in vitro, and explored the mechanism of the antiviral action. Using the A/chicken/Italy/9097/1997(H5N9) AIV as a model, we show that HLN and its precursor p-haloxanide are more effective than NTZ against AIV, with IC50 ranging from 0.03 to 0.1 µg/ml, and SI ranging from 200 to >700, depending on the multiplicity of infection. Haloxanide did not affect AIV entry into target cells and did not cause a general inhibition of viral protein expression, whereas it acted at post-translational level by inhibiting HA maturation at a stage preceding resistance to endoglycosidase-H digestion. Importantly, this effect was independent of the AIV-HA subtype and the host cell. Immunomicroscopy and receptor-binding studies confirmed that HLN-induced alterations impair AIV-HA trafficking to the host cell plasma membrane, a key step for viral morphogenesis. The results indicate that haloxanide could provide a new tool for treatment of avian influenza virus infections.