Redox-regulated Intracellular Pathways as New Potential Targets for Anti-influenza Drugs Summer School on Influenza 2012 16-21 July, Siena Anna Teresa Palamara Summary • Redox balance of eukariotic cells • Effects of influenza virus infection on intracellular redox state and redox regulated pathways •Effects of redox modulating compounds on influenza virus replication ROS/RNS: sources and cellular response Endogenous sources Mitochondria Peroxisomes Lipooxygenases NADPH oxidase Cytochrome P450 Nitric Oxide synthase Regulation of cell functions Antioxidant defences CAT, SOD, GSH-Px, Trx, GSH Vitamins (A,C,E) Homeostasis Exogenous sources UV Radiation Chemiotherapy Inflammatory cytokine Environmental toxins Alteration in cellular functions General cell damages Cell proliferation and differentiation, Immune responses inflammatory responses Alteration In redox regulated metabolic pathways Cell death, Ageing Cancer, Neurodegeneration Virus/host cell interactions play a key role in the pathogenesis of viral-induced diseases Fas L Fa s Redox imbalance NOXs ? FADD NO ROS GSH Caspases P Apoptosis NFkB P P Kinases K K NF-kB AP1 INFLAMMATION FATE OF INFECTED CELLS (death/survival) IRFs/STATs VIRAL REPLICATION IRF, STAT IMMUNE RESPONSE CHRONIC-DEGENERATIVE EFFECTS Redox state and viral infections HIV Garaci E. et al. (1997). J.Leuk. Biol. Stehbens W. (2004). Exp. Mol. Pathol. Wallace DR., (2006). J. Biomed. Biotechnol. Savarino A., et al., (2009) Retrovirology Huang W. et al., (2011) FASEB J. Palamara A.T. et al. (1992). B.B.R.C INFLUENZA/ Flory E., et al. (2000). J. Biol. Chem.. PARAINFLUENZA Nencioni L., et al. (2003). FASEB J. Sgarbanti R., et al., (2011) Antiox.& Redox Sign. Vlahos R., et al., (2011) PLoS Pathogen HSV-1 HEPATITIS C RHINOVIRU S Palamara A.T. et al. (1995). Antiviral Res. Stehbens W. (2004). Exp. Mol. Pathol. Tardif K.D. (2005). Trends Microbiol. Steingart RA., et al., (2006). Mol. Cell. Endocrinol.. Seronello, S., et al., (2007). Free Radic Biol Med. De Mochel N., et al. (2010) Hepatology Kaul P., et al. (2000). J. Infect. Dis. Papi A., et al. (2002). FASEB J. Kaul P., et al. (2002). Free Radic Res. RED OX Different viruses induce an intracellular PRO-OXIDANT STATE via decrease of GSH Parainfluenza 1 Herpes Simplex 1 HIV 18 40 1,5 RED 30 12 1 20 10 * 0 * 1 3 hours 6 24 * * 0 * 6 0,5 * 1 3 hours 6 24 OX 0 0 5 10 days 15 *p<0.001 *p<0.001 *p<0.001 Palamara et al. BBRC, 1992. Palamara et al. Antiv. Res., 1995. Garaci et al. J.Leuk. Biol., 1997 Mechanisms involved in parainfluenza1 Sendai virus induced GSH decrease 20 minutes during viral challenge 24 hours after viral challenge Ciriolo et al., J Biol Chem (1997) 300 250 200 * * ** ** *** *** 150 100 50 0 0 * ** *** 24 48 MDCK NCI-H292 MDCKBcl-2 SH-SY5Y U937 72 Time (hrs) *P< 0.05; **P< 0.01; ***P< 0.0001 vs. MDCK cells IS IT POSSIBLE TO BLOCK VIRAL REPLICATION AND ITS EFFECTS BY INTERFERING WITH INTRACELLULAR REDOX STATE? Inhibits Sendai and HSV-1 virus… GSH-C4 Palamara A.T. et al., Antiv. Chem. Chemoth.,2004 Fraternale A., et al. Curr Med Chem, 2006 Redox steps of folding are mediated by oxidoreductases in the ER N-glycosylation Disulfide bonds formation Ellgaard and Helenius, Nature Rev. 2004 GSH-C4 affects the expression of viral hemagglutinin HA= hemagglutinin NP= nucleoprotein; M= matrix protein Sgarbanti R. et al., Antioxidants &Redox Signaling 2011 GSH-C4 inhibits influenza virus replication in MDCK cells *,**P< 0.05 A/ULSTER/H7N1 Sgarbanti R. et al., Antioxidants &Redox Signaling 2011 GSH-C4 affects the redox state of PDI without affecting cellular glycoprotein secretion in uninfected cells Intracellular Secreted Sgarbanti R. et al., Antioxidants &Redox Signaling 2011 GSH-C4 increases plasma GSH levels with no sign of toxicity in mice Sgarbanti R. et al., Antioxidants &Redox Signaling 2011 GSH-C4 increases the survival of PR8-infected mice †GSH‐C4 or PBS was administered intraperitoneally once daily to 6‐week‐old BALB/c mice, after inoculation with 1 PFU of PR8 virus. Survival was observed for 15 days. *P < 0.05, ** P= 0.001 compared with Placebo treated‐control group Sgarbanti R. et al., Antioxidants &Redox Signaling 2011 Day 9 Uninfected Infected GSH‐C4 Days 40 Uninfected Infected GSH‐C4 8360 Days p.i. 6 9 18 40 Uninfected Alveolar area NI* score 59.85 ± 0.8 0.3 60.27 ± 2.0 0.3 58.98 ± 1.4 0.3 59.10 ± 1.0 0.3 *= necroinflammatory score **P< 0.05 (two-way ANOVA) compared with Placebo treated-control group. *** P< 0.05 (t test) compared with control Placebo Alveolar area NI score 47.14 ± 2.6 *** 3 46.91 ± 2.6 *** 4 51.44 ± 2.3 *** 3 50.86 ± 3.4 3.5 GSH-C4 Alveolar area NI score 51.28 ± 1.8 3 54.42 ± 1.9** 4 54.76 ± 2.1 2.5 56.54 ± 2.0 2.5 Sgarbanti R. et al., Antioxidants &Redox Signaling 2011 Summary • Redox balance of eukariotic cells • Effects of influenza virus infection on intracellular redox state and redox regulated pathways • Effect of other redox modulating compounds on influenza virus replication Other redox modulating compounds… ...only partially affect viral replication * *P< 0.05 * Survival X X Placebo RV 20 g/mouse/day Days of infection J Infect Dis. 2005; 191(10):1719‐29 RV anti-influenza effect is due to the inhibition of p38 MAPK mediated VRNP export Resveratrol p38MAPK i ERK i % of 24 h p.i. viral yield (HAU/mL) DAPI NP 100 75 * * 50 25 ** 0 CI 1 5 10 15 20 ** 40 RV (g/ml) *P<0.05 and **P<0.001 vs. Control Infected cells (CI) J Infect Dis. 2005; 191(10):1719‐29 Is RV an antioxidant drug…? …No, when administered to infected cells! Influenza virus induces different waves of oxidative stress in the epithelial cells This oxidative stress seems to be essential for vRNP nuclear export and HA maturation Modulation of host-cell functions essential for viral replication could offer some important advantages: It affects viral replication indipendently from virus type or strain It is more difficult for the virus to adapt to It could block virus-induced inflammatory cascades Cell-based anti-influenza strategies Adsorption siRNA Zhang et al Budding BBRC 2009 GSH THIAZOLIDES LC3 FASEB J 2003 •Fraternale et al AAAA AAAA AAAA Autophagosome INHIBITION OF AUTOPHAGIC PATHWAY •Nencioni L et al Assembly LC3 Endocitosis Golgi J Biol Chem 2009 Translation Matarrese P et al J Cell Physiol 2011 Amphisome Mol Aspects Med 2009 •Rossignol et al NP •Sgarbanti R et al ER ARS 2011 Post-translational modifications Fusion and RNA release CHLOROQUINE mRNA vRNA (-) Di Trani L et al Virol J 2007 Import cRNA (+) RNP export RV p38MAPK i ERK i •Pleschka et al Nat Cell Biol 2001 •Palamara AT et al J Infect Dis 2005 •Nencioni L et al J Biol Chem 2009 REMEMBER… Antioxidant does not mean Antiviral... The “Sapienza” Team Thanks to “Sapienza” University of Rome Dept of Public Health and Infectious Diseases Lucia Nencioni Rossella Sgarbanti Donatella Amatore Paola Checconi Maria Elena Marcocci Livia Civitelli Ignacio Celestino Simona Panella Marta Aleandri Dolores Limongi Simona Anticoli University of Rome “Tor Vergata” Dept of Biology Katia Aquilano Sara Baldelli Maria Ciriolo Giuseppe Rotilio Dept of Exp. Medicine and Biochemical Sciences Enrico Garaci Istituto Superiore di Sanità University of Urbino Institute of Biochemistry “G. Fornaini” Mauro Magnani Alessandra Fraternale Dept of Cell Biology and Neuroscience Giovanna De Chiara Animal care service Paolo Coluccio Mauro Valeri Emanuela D’Amore