The development of new antiviral molecules derived from acyclovir increases the selection pressure risk of resistant strains (Danve-Szatanek et al., 2004) that have been observed in vivo since the first large therapeutic trials ( McLaren et al., 1985). Therefore, the search for new antiviral agents, especially those with different mechanisms of action, is a crucial goal ( Butler, 2008). Alisertib Cardiac glycosides belong to a group of naturally derived compounds that bind to and inhibit Na+K+ATPase (Lingrel et al., 1997). Members of this group have been traditionally used for the treatment of heart
failure and atrial arrhythmia, such as digoxin, digitoxin and ouabain (Rahimtoola and Tak, 1996). Recently, other important applications have been suggested for these compounds related to their potential anticancer (Prassas and Diamandis, 2008) and antiviral activity (Dodson et al., 2007,
Hartley et al., 2006, Hoffmann et al., 2008 and Su et al., 2008). In this report, we screened 65 cardenolide derivatives obtained from plants, by synthesis or by fungi biotransformation, for anti HSV-1 and HSV-2 activity. Among them, glucoevatromonoside (Fig. 1), isolated from a Brazilian cultivar of Digitalis lanata ( Braga et al., 1996) was chosen for its lower IC50 against Wnt tumor HSV to further elucidate its mechanism of action. The 65 tested cardenolide derivatives were obtained from plants (Braga et al., 1996 and Braga et al., 1997), by synthesis (Extrasynthèse, Genay, France; Merck, Darmstadt, Germany; Boehringer, Mannheim, Germany; Carl Roth, Karlsruhe, Germany), Dipeptidyl peptidase or by fungi biotransformation (Pádua
et al., 2005 and Pádua et al., 2007). Acyclovir, digoxin, dextran sulfate and furosemide were obtained from Sigma (St. Louis, MO, USA). All compounds were dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany), not exceeding the minimum non cytotoxic concentration of 1% DMSO and were further diluted in culture medium prior its use. Vero (ATCC: CCL 81) and GMK-AH1 (Department of Clinical Virology, University of Göteborg, Sweden) cells were grown in Eagle’s minimum essential medium (MEM; Cultilab, Campinas, Brazil) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA), 100 U/mL penicillin G, 100 μg/mL streptomycin and 25 μg/mL amphotericin B (Cultilab) and maintained at 37 °C in a humidified 5% CO2. HSV-1 [KOS and 29R (acyclovir-resistant) strains] (Faculty of Pharmacy, University of Rennes, France), and HSV-2 [333 strain (Department of Clinical Virology, Göteborg University, Sweden)] were propagated in Vero and GMK AH1 cells, respectively. Viral stocks were stored at −80°C and titrated based on plaque forming units (PFU) count by plaque assay as previously described (Burleson et al., 1992). Firstly, cytotoxicity was determined by MTT assay (Mosmann, 1983).