Anti-influenza A virus activity of uridine derivatives of 2-deoxy sugars Ewelina Krol a,⇑ , Ilona Wandzik b , Beata Gromadzka a , Dawid Nidzworski a , Malgorzata Rychlowska c , Marta Matlacz a , Jolanta Tyborowska a , Boguslaw Szewczyk a a Department of Recombinant Vaccines, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland b Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Faculty of Chemistry, Silesian University of Technology, Krzywoustego 8, 44-101 Gliwice, Poland c Department of Virus Molecular Biology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822 Gdansk, Poland article info Article history: Received 20 April 2013 Revised 6 July 2013 Accepted 23 July 2013 Available online 1 August 2013 Keywords: Influenza A virus Glycoproteins Tunicamycin derivatives Glycosylation inhibition abstract Influenza viruses are important pathogens that cause respiratory infections in humans and animals. Apart from vaccinations, antiviral drugs play a significant role in controlling spread of the disease. Influenza A virus contains two membrane glycoproteins on the external part of viral envelope: hemagglutinin (HA) and neuraminidase (NA), which are crucial for productive infection in target cells. In the present work, two derivatives of tunicamycin – uridine derivatives of 2-deoxy sugars (designated IW3 and IW7), which target the glycan processing steps during maturation of viral glycoproteins, were assayed for their ability to inhibit influenza A virus infection in vitro. Using the cytopathic effect (CPE) inhibition assay and viral plaque reduction assay we showed, that both IW3 and IW7 inhibitors exerted significant inhibitory effect on influenza A virus infection in MDCK cells without significant toxicity for the cells. Moreover, tested compounds selectively suppressed viral protein expression in a dose-dependent manner, suggesting that the mechanism of their antiviral activity may be similar to this shown previously for other viruses. We have also excluded the possibility that both inhibitors act at the replication step of virus life cycle. Using real-time PCR assay it was shown that IW3 and IW7 did not change the level of viral RNA in infected MDCK cells after a single round of infection. Therefore, inhibition of influenza A virus infection by uridine derivatives of 2-deoxy sugars, acting as glycosylation inhibitors, is a promising alternative approach for the development of new anti-influenza A therapy. Ó 2013 Elsevier B.V. All rights reserved. 1. Introduction Each year influenza viruses cause severe respiratory illness in humans and animals with high morbidity and mortality rates worldwide (Miller et al., 2009). Vaccines and antiviral drugs are major options to control new infections. Although it is obvious that vaccination is the best way of prevention, the composition of vac- cines needs to be updated seasonally. Moreover, variable effective- ness of vaccination is the result of antigenic shift and antigenic drift of the viral genome (Nichol and Treanor, 2006). Due to the fact, that vaccines are not effective against rapidly emerging mu- tant viruses, antivirals are still needed to prevent transmission of novel influenza strains. Currently, there are two major classes of anti-influenza drugs approved by the FDA for clinical use: M2 ion-channel inhibitors (amantadine and rimantadine) (Hayden, 1997; Wang et al., 1993) and neuraminidase (NA) inhibitors (oseltamivir and zanamivir) (Monto et al., 1999; Nicholson et al., 2000). M2 ion-channel inhib- itors prevent viral uncoating and are effective only against type A viruses (Luscher-Mattli, 2000). Moreover, the widespread resis- tance of viral strains to both amantadine and rimantadine limits the use of this class of drugs for influenza treatment (Bright et al., 2005, 2006; Deyde et al., 2007). Neuraminidase inhibitors specifically bind to the conserved active site of the enzyme, there- by preventing the release of newly synthesized influenza A and B viruses from infected cells (Garten et al., 2009). Resistance to neur- aminidase inhibitors has been shown to be associated with muta- tions in NA gene occurring rapidly during treatment (Ferraris and Lina, 2008; Thorlund et al., 2011). The increase of oseltamivir- resistant seasonal influenza A (H1N1) viruses has been observed in USA, growing from less than 0.5% in 2006–2007 to 99% in 2008–2009 (Hurt et al., 2011; Renaud et al., 2011). Oseltamivir- resistant H1N1 viruses were also isolated in Europe and other 0166-3542/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.antiviral.2013.07.014 Abbreviations: CC 50 , concentration of the compound required to reduce cell viability by 50%; CPE, cytopathic effect; CSFV, classical swine fever virus; Ct, cycle threshold; ER, endoplasmic reticulum; HA, hemagglutinin; IC 50 , concentration of the compound required to reduce virus plaque formation by 50%; MDCK, Madin– Darby canine kidney cells; MOI, multiplicity of infection; NA, neuraminidase; Sf9, Spodoptera frugiperda insect cell line; S.D., standard deviations; SI, selectivity index; TPCK, L-1-tosylamide-2-phenylethyl chloromethyl ketone. ⇑ Corresponding author. Tel.: +48 58 523 63 36; fax: +48 58 305 73 12. E-mail addresses: ewelina@biotech.ug.gda.pl (E. Krol), ilona.wandzik@polsl.pl (I. Wandzik), g.beata@biotech.ug.gda.pl (B. Gromadzka), dawid.nidzworski@ biotech.ug.gda.pl (D. Nidzworski), ggordon@biotech.ug.gda.pl (M. Rychlowska), mmatlacz@biotech.ug.gda.pl (M. Matlacz), jtybor@biotech.ug.gda.pl (J. Tyborowska), szewczyk@biotech.ug.gda.pl (B. Szewczyk). Antiviral Research 100 (2013) 90–97 Contents lists available at ScienceDirect Antiviral Research journal homepage: www.elsevier.com/locate/antiviral