Thiazolides, a New Class of Anti-influenza Molecules Targeting Viral Hemagglutinin at the Post-translational LevelThe 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. 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. Influenza, a highly contagious acute respiratory illness affecting all age groups, is responsible for an average of 36,000 deaths and over 226,000 hospitalizations per year in the United States alone (1Fiore A.E. Shay D.K. Broder K. Iskander J.K. Uyeki T.M. Mootrey G. Bresee J.S. Cox N.S. MMWR Recomm. Rep. 2008; 57: 1-60PubMed Google Scholar). The etiological agent of the disease, the influenza viruses or orthomyxoviruses, are enveloped, negative-stranded RNA viruses classified in three types (A, B, and C), of which the A type is clinically the most important. The genome of influenza A viruses consists of eight single-stranded RNA segments that encode 11 proteins, including the main surface glycoproteins, hemagglutinin (HA), 3The abbreviations used are: HAhemagglutininNTZnitazoxanideTIZtizoxanideTMtunicamycinEndo-Hendo-β-N-acetylglucosaminidase HPNGase-Fpeptide N-glycosidase FMDCKMadin-Darby canine kidneyp.i.post-infectionMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideGFPgreen fluorescent proteinLDLRlow density lipoprotein receptorPBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluoridePICprotease inhibitor mixtureeIFeukaryotic initiation factorHAUhemagglutinating units. and neuraminidase (NA), of which 16 HA (H1–H16) and nine NA (NA1–NA9) subtypes have been identified so far (2Wright P.F. Neumann G. Kawaoka Y. Field's Virology. 5th Ed. Lippincott, Williams and Wilkins, Philadelphia2007: 1691-1740Google Scholar). Influenza virus infection involves a series of steps: the virus attaches to host sialylated glycoproteins via the viral hemagglutinin and enters the cell by endocytosis, followed by pH-dependent fusion and release of viral genomic ribonucleoprotein complexes in the cytoplasm. Ribonucleoproteins then translocate to the nucleus where transcription and replication of viral RNA occurs. During the replication cycle some viral proteins translocate to the nucleus for progeny ribonucleoprotein formation, whereas the viral HA, NA, and M2 proteins reach the plasma membrane via the secretory pathway, an event that is essential for viral particle formation and budding from host cells (3Palese P. Shaw M. Field's Virology. 5th Ed. Lippincott, Williams and Wilkins, Philadelphia2007: 1647-1689Google Scholar). In humans, influenza A virus replicates throughout the respiratory tract, where the viral antigen is found predominantly in the epithelial cells. The typical course of influenza is self-limiting and lasts for about a week; however, clinical responses range from mild disease to fatal viral pneumonia (4Cox N.J. Subbarao K. Lancet. 1999; 354: 1277-1282Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 5Gu J. Xie Z. Gao Z. Liu J. Korteweg C. Ye J. Lau L.T. Lu J. Gao Z. Zhang B. McNutt M.A. Lu M. Anderson V.M. Gong E. Yu A.C. Lipkin W.I. Lancet. 2007; 370: 1137-1145Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Although the mechanisms underlying the expression of symptoms and the development of secondary complications that may result in respiratory failure are still not well understood, excessive inflammation caused by overabundant production of proinflammatory cytokines and lung inflammatory infiltrates is considered an important factor in disease pathogenesis (6Hayden F.G. Fritz R. Lobo M.C. Alvord W. Strober W. Straus S.E. J. Clin. Invest. 1998; 101: 643-649Crossref PubMed Scopus (605) Google Scholar, 7Cheung C.Y. Poon L.L. Lau A.S. Luk W. Lau Y.L. Shortridge K.F. Gordon S. Guan Y. Peiris J.S. Lancet. 2002; 360: 1831-1837Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, 8Bernasconi D. Amici C. La Frazia S. Ianaro A. Santoro M.G. J. Biol. Chem. 2005; 280: 24127-24134Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). hemagglutinin nitazoxanide tizoxanide tunicamycin endo-β-N-acetylglucosaminidase H peptide N-glycosidase F Madin-Darby canine kidney post-infection 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide green fluorescent protein low density lipoprotein receptor phosphate-buffered saline phenylmethylsulfonyl fluoride protease inhibitor mixture eukaryotic initiation factor hemagglutinating units. HA and NA glycoproteins, which are the main targets of the protective immune response, vary continuously as a result of antigenic drift and antigenic shift. Major changes from antigenic shift are caused by the different HA and NA subtypes circulating in birds and other animals that create a reservoir of influenza A genes available for genetic reassortment with the circulating human viruses (9Ghedin E. Sengamalay N.A. Shumway M. Zaborsky J. Feldblyum T. Subbu V. Spiro D.J. Sitz J. Koo H. Bolotov P. Dernovoy D. Tatusova T. Bao Y. St George K. Taylor J. Lipman D.J. Fraser C.M. Taubenberger J.K. Salzberg S.L. Nature. 2005; 437: 1162-1166Crossref PubMed Scopus (370) Google Scholar). The lack of protective immunity in the human population against new HA and/or NA proteins can result in rapid global spread of the virus. In recent history, the emergence of high pathogenicity avian influenza viruses in domestic poultry and the increasing number of cases of direct transmission of avian influenza viruses to humans represent a major risk, confirmed by the ongoing outbreak of high pathogenicity avian influenza H5N1 viruses in the bird population, which has caused a nearly 50% case fatality rate among the people infected (10Webby R.J. Webster R.G. Science. 2003; 302: 1519-1522Crossref PubMed Scopus (535) Google Scholar, 11Horimoto T. Kawaoka Y. Nat. Rev. Microbiol. 2005; 3: 591-600Crossref PubMed Scopus (591) Google Scholar). In addition, the highly contagious A H1N1 swine flu that recently emerged in Mexico has rapidly spread worldwide, representing a new threat to global human health. Novel antiviral drugs effective against different strains of influenza viruses are therefore greatly needed. Herein we document the anti-influenza activity of nitazoxanide (NTZ), a thiazolide anti-infective licensed in the United States (Alinia®; Romark Laboratories, Tampa, FL) for treating enteritis caused by Cryptosporidium parvum and Giardia lamblia in children and adults (12Fox L.M. Saravolatz L.D. Clin. Infect. Dis. 2005; 40: 1173-1180Crossref PubMed Scopus (346) Google Scholar, 13Rossignol J.F. Kabil S.M. El-Gohary Y. Younis A.M. Clin. Gastroenterol. Hepatol. 2006; 4: 320-324Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 14Rossignol J.F. Ayoub A. Ayers M.S. J. Infect. Dis. 2001; 184: 381-384Crossref PubMed Scopus (146) Google Scholar), its active circulating metabolite tizoxanide (2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide) (TIZ) (see Fig. 1A), and second generation thiazolides. Madin-Darby canine kidney (MDCK), human A549 alveolar type II-like epithelial, Jurkat T-lymphoblastoid, and U397 monocytic leukemia cells were grown at 37 °C in a 5% CO2 atmosphere in RPMI 1640 (Invitrogen), supplemented with 10% fetal calf serum, 2 mm glutamine, and antibiotics. NTZ, TIZ, second generation thiazolides (provided by Romark Laboratories), and swainsonine (Sigma-Aldrich) were dissolved in dimethyl sulfoxide; tunicamycin (TM) and 1-deoxymannojirimicin (Sigma-Aldrich) were dissolved in aqueous solution. Compounds were added immediately after a 1-h adsorption period and kept in the culture medium for the entire time of the experiment, unless differently specified. Controls received equal amounts of vehicle, which did not affect cell viability or virus replication. Cell viability was by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to (Sigma-Aldrich) as Frazia S. Amici C. Santoro M.G. 2006; Google Scholar). of infected or cells was a and were on a cells in were with green fluorescent protein human low density lipoprotein receptor by E. to the different influenza A the H1N1 and and and the low pathogenicity avian as well as influenza clinical were for The and influenza viruses were a from The avian was after an of into S. A. D.J. M. S. Webster R.G. J. 2001; PubMed Scopus Google Scholar). Influenza A viruses were grown in the of at 37 the was and at for to and virus were by hemagglutinin and to D. Amici C. La Frazia S. Ianaro A. Santoro M.G. J. Biol. Chem. 2005; 280: 24127-24134Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, A. A. Amici C. Santoro M.G. PubMed Scopus Google Scholar). cell were infected with influenza virus for at 37 °C at a of infection of unless differently specified. the adsorption period the viral was and cell were three with phosphate-buffered saline The cells were at 37 °C in RPMI 1640 culture medium fetal calf virus the infected cells were in the medium was or post-infection by hemagglutinin D. Amici C. La Frazia S. Ianaro A. Santoro M.G. J. Biol. Chem. 2005; 280: 24127-24134Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). virus cells grown on were with of viral in the of for at 37 and culture was as A. A. Amici C. Santoro M.G. PubMed Scopus Google Scholar). virus were on cells by the of fluorescent cells after infection and with anti-influenza a from E. were as of cells cell by A. were infected with human type 2 for at °C at the adsorption the viral was and the cell were three with medium fetal calf and at °C in medium fetal calf was at after three of by infected cells were and for at the infected or influenza cells were with of in cell for the after of in the cells were with for after of in the of the cells were in medium mm and mm for different in the or of The were by the cells on cell in mm mm mm mm mm phenylmethylsulfonyl fluoride and a protease inhibitor mixture the of were by 10% and for as A. A. Amici C. Santoro M.G. PubMed Scopus Google Scholar). were and in a and were of proteins into virus or infected cells with TIZ, or after virus adsorption were at with in the of the drugs. the cell culture were to at for to and then to at for 2 The viral particles were in and viral proteins were by 10% and by after to were as the cells were with high A. P. G. T. Y. M. Santoro M.G. Nature. PubMed Scopus Google Scholar), 2 mm mm mm mm mm and or with mm and cell were by and to and were with and anti-influenza or and followed by with or of proteins was by the A. S. M. P. Santoro M.G. 2006; PubMed Scopus Google Scholar). or infected cells with or control after virus adsorption were at or with after of in in in the of and mm cell were by at for were with in mm and protein (Sigma-Aldrich) at °C for 16 the were three with and in E. S. Amici C. D. A. Santoro M.G. 2007; PubMed Scopus Google at °C for were to endoglycosidase H and/or for 10% and after to were in and the were as infected or influenza cells were with of or for at and then for 10% and as endoglycosidase cells were infected with influenza of and in the or of the cells were with after of in the of the medium was and cells were on in mm mm mm in the of and mm and at for the of were for or peptide N-glycosidase F digestion, the with or were in of and mm in mm and for at of mm and the were into equal and was with of for 16 at 37 N-glycosidase was with of to the were with the of The were at °C for 10% of of HA was by of dimethyl mm to cell from infected and cells. at the were by the of at a of and the were to Cell Biol. PubMed Scopus Google Scholar). The were by with or and A549 cells grown on were with in phosphate-buffered saline for at at 16 or infected cells were cells were with for at 37 °C for plasma membrane or were with for at and then with and or Cell for at 37 followed by with or and The were with or The were and with a the the or the and the of a of three with results are of plasma membrane of human cells in were with human after with or for the 16 blocking protein with (Sigma-Aldrich) for the plasma were plasma membrane the cells were a with The were with a or cell were with TIZ, or after virus the cells were three with and with of human cells in for at °C to inhibit neuraminidase of by three with cells on cell surface were by The were with a with a were in mm for 2 at and by at G. J. P. 2007; PubMed Scopus Google Scholar). was for The are as the of of were considered The of thiazolide was in human and canine cells after infection with different strains of influenza A the H1N1 and and and the low pathogenicity avian cells infected with or influenza viruses were with different of NTZ, TIZ, or immediately after the virus adsorption and virus was at caused a of virus replication with of and for and was active against all influenza A strains with an of and and was effective in the replication of influenza A and influenza viruses Fig. A and were at the effective antiviral for cells In to the canine cells used for influenza virus was effective in influenza A virus replication at in different types of human including monocytic and alveolar type II-like A549 cells The anti-influenza activity of was of the of and a of H1N1 virus replication was of and virus Fig. and In to and TIZ, the antiviral activity of different second generation thiazolides against influenza A virus is in the novel thiazolides the was found to effective and TIZ, with and activity of thiazolides thiazolide virus adsorption host cells from viral cells were with for or the the drug was and cell were three infection with virus. in Fig. tizoxanide of cells to viral infection on influenza virus replication. of the viral not or of cells the adsorption period did not inhibit virus replication that the drug is not affecting virus its or into cells. and was the most effective in virus replication at was effective still to inhibit virus whereas the drug was at A of the drug after virus adsorption was effective in virus replication for at after infection the anti-influenza activity of thiazolides was caused by protein infected or cells with after virus adsorption were with at different and the proteins were by and or in Fig. did not inhibit host protein in the of the in addition, did not affect of in or cells. The main influenza virus proteins were found to in amounts in cells at major changes in influenza virus protein were in with the of the of a of identified as the of the hemagglutinin and the of a of selectively HA infected or cells with were at and proteins were with and then for and in Fig. the protein is by as the viral the was infected or cells with or the inhibitor were at for the and proteins were by and cells were at and then in the of mm and mm for the in Fig. post-translational was still at and to from as by a different of the in addition, whereas caused a in as K. PubMed Scopus Google Scholar), did not intracellular in infected cells. from did not the expression of the protein a of the protein response, in cells from the that in cells the and after whereas in the of the to after and in was in the not is cells were with or tunicamycin after virus adsorption at were with or in Fig. whereas with did not and into the the thiazolide to act differently from the of and as by the different of as with the in cells with the is that HA maturation is by the host cell D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and the virus Rev. 2002; PubMed Scopus Google Scholar). the was for virus or was human lung epithelial A549 cells were infected with the influenza A human whereas cells were infected with the avian In in maturation to the for the were and that is to inhibit of the type of host cell and influenza A as in Fig. and nitazoxanide caused in the hemagglutinin of human and avian influenza of HA, other cell surface glycoproteins, is in the the B. A. Full Text PDF PubMed Scopus Google Scholar). The is in the the to the cell and in of the C. A. J. PubMed Scopus Google Scholar). affect the we of proteins and to with an that that have not been R. M. W. J. PubMed Google or with an that all D. F.G. J. 2005; PubMed Scopus Google Scholar). of the protein were to however, whereas from control cells was from cells to with the protease to after A and in Fig. the did not the of to of resistance is a for into the and D. F.G. J. 2005; PubMed Scopus Google Scholar), results indicate that the may trafficking the and the its to the plasma of to the was in by that HA to the host cell plasma membrane, the exit of viral infected and cells were with or tunicamycin after virus and of and plasma membrane viral hemagglutinin were by at 16 confirmed whereas in cells were to control plasma membrane of the viral protein were in cells A in HA plasma membrane after was confirmed by the of plasma HA by of In after of cells with a human low density lipoprotein receptor was found that did not inhibit plasma membrane of a of thiazolides Fig. results were after of cells and cells with a different plasma membrane the human not In infected and cells were with at for the and were from the of infected cells. into viral particles were by and in Fig. viral proteins not in the of cells. The of viral particles was confirmed by virus from by or at nitazoxanide and did not affect the replication of the human type a maturation not viral protein trafficking to the cell membrane Fig. The emergence of highly influenza A virus strains represents a serious threat to global human health. Efforts to control emerging influenza strains are on surveillance and early diagnosis, as well as on the development of effective vaccines and novel antiviral drugs. The in vaccines against emerging influenza viruses with have been recently K. T. Nat. Rev. 2007; PubMed Scopus Google Scholar). for antiviral of drugs are currently available for and of the NA and which the release of viruses from the infected host and and which the viral M2 protein for virus K. T. Nat. Rev. 2007; PubMed Scopus Google Scholar, S. Science. 2006; PubMed Scopus Google Scholar). with M2 the of symptoms of clinical influenza, major were S. Science. 2006; PubMed Scopus Google Scholar). Novel have however, they have on the infection S. Science. 2006; PubMed Scopus Google Scholar). to effective of influenza with drugs is by the high level drug resistance to types of by in the M2 and NA proteins S. Science. 2006; PubMed Scopus Google Scholar). the disease is by and novel antiviral drugs against different influenza virus targets are greatly in of the emergence of new Herein we document that the anti-infective drug nitazoxanide and its active circulating metabolite tizoxanide anti-influenza A activity that is of the viral and/or the describe a of novel thiazolides with anti-influenza the was found to the most active the act via a novel The thiazolides not affect virus or into cells and not a of viral protein whereas they selectively the maturation and intracellular of the viral Influenza A virus hemagglutinin is a glycoprotein that three to nine on the A. Full Text PDF PubMed Scopus Google Scholar, J. Infect. Dis. PubMed Scopus Google Scholar). HA is and in the as a that into The are rapidly to the and reach the plasma membrane, where HA insertion the of assembly and maturation of the viral particles J. Infect. Dis. PubMed Scopus Google Scholar, Webster R.G. A. J. Cell Biol. PubMed Scopus Google Scholar, H. Zhang J. J. PubMed Scopus Google Scholar). to or with insertion into the plasma membrane, is into glycoproteins, and which by a (3Palese P. Shaw M. Field's Virology. 5th Ed. Lippincott, Williams and Wilkins, Philadelphia2007: 1647-1689Google Scholar). different we that HA at a stage preceding resistance to endoglycosidase H digestion, which is a for into the and D. F.G. J. 2005; PubMed Scopus Google Scholar). and of viral particles by infected cells that the trafficking the and the its and insertion into the host cell plasma membrane and blocking the exit of from host cells. the of HA maturation is caused by direct of to the viral glycoprotein or is to a to Thiazolides have been to antiviral activity against different RNA a RNA and a RNA and a the virus J.F. M. A. Santoro M.G. Lancet. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, K. K. S. Ayers M.S. J.F. 2008; PubMed Scopus Google Scholar, J.F. Microbiol. 2008; 3: PubMed Scopus Google Scholar, J.F. A. El-Gohary Y. Full Text Full Text PDF PubMed Scopus Google Scholar). The antiviral activity a a viral The that maturation of viral glycoproteins may in the antiviral activity against and viruses is currently In the case of of the viral glycoprotein has been recently G. Santoro and J. the that maturation and of key viral glycoproteins a of the antiviral activity of new class of drugs. The that thiazolides not affect the replication of human a maturation not viral glycoprotein trafficking to the cell membrane, In the case of influenza the maturation of the viral a key step for correct assembly and exit of the virus from the host 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. is a licensed in the United States for the of infectious and is clinical in the United States and in the of J.F. M. A. Santoro M.G. Lancet. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, J.F. A. El-Gohary Y. Full Text Full Text PDF PubMed Scopus Google Scholar). The drug has been to and effective over a and clinical in the of influenza at time in the have recently activity of available of in treating J.F. M. A. Santoro M.G. Lancet. 2006; Full Text Full Text PDF PubMed Scopus Google and and J.F. Microbiol. 2008; 3: PubMed Scopus Google Scholar, J.F. A. El-Gohary Y. Full Text Full Text PDF PubMed Scopus Google Scholar). anti-influenza activity can in humans, a to the of drugs used for and of are to for and and for the and influenza and Amici for in the and production of virus and for with the with
In vitro evaluation of activities of nitazoxanide and tizoxanide against anaerobes and aerobic organismsL. Dubreuil, Isabelle Houcke, Y Mouton et al.|Antimicrobial Agents and Chemotherapy|1996 The antibacterial activities of nitazoxanide and its main metabolite, tizoxanide, were tested against a broad range of bacteria, including anaerobes. Metronidazole, amoxicillin, amoxicillin-clavulanic acid, piperacillin, cefoxitin, imipenem, and clindamycin were used as positive controls. MICs were determined by reference agar dilution methods. The 241 anaerobes were all inhibited by nitazoxanide, with the MICs at which 90% of isolates are inhibited (MIC90S) being between 0.06 and 4 mg/liter with the exception of those for Propionibacterium species, for which the MIC90 was 16 mg/liter. The MIC90s of nitazoxanide were 0.5 mg/liter for the Bacteroides fragilis group (80 strains), 0.06 mg/liter for Clostridium difficile (21 strains), and 0.5 mg/liter for Clostridium perfringens (16 strains). Metronidazole showed a level of activity comparable to that of nitazoxanide except against Bifidobacterium species, against which it was poorly active, and Propionibacterium species, which were resistant to metronidazole. The other antibiotics showed various levels of activity against anaerobes, with imipenem along with nitazoxanide being the most active agents tested. Tizoxanide was less effective than nitazoxanide except against the B. fragilis group, against which its activity was similar to that of nitazoxanide. Under aerobic conditions, nitazoxanide demonstrated poor activity against members of the family Enterobacteriacae and Pseudomonas, Staphylococcus, and Enterococcus species. The same results were obtained when culture was performed under anaerobic conditions with the notable exception of the results against Staphylococcus aureus. The MICs of nitazoxanide were in the range of 2 to 4 mg/liter for 34 clinical isolates of S. aureus, 12 of which were methicillin resistant, while tizoxanide was not effective.