In contrast to the unmodified sulfated oligosaccharides of muparfostat, compounds possessing dodecyl (3), 12-(4-naphthalen-1-yl-[1,2,3]triazol-1-yl)dodecyl (5) or cholestanyl (14 and PG545) as the aglycone component demonstrated complete or near-complete inhibition of RSV infectivity (Table 1). Moreover, these four glycosides exhibited more favorable IC50 values than muparfostat, and showed virucidal activity, a functional feature absent in muparfostat oligosaccharides (Table 2).
Since PG545 exhibited the most pronounced virucidal activity, this glycoside was selected for detailed evaluation of anti-RSV potency. Note that although both PG545 and 14 are buy Tariquidar composed of a lipophilic cholestanyl group conjugated to a sulfated tetrasaccharide, PG545 contains maltotetraose while 14 possesses a mannose α(1 → 3)/(1 → 2)-linked tetrasaccharide, as found in muparfostat, as the oligosaccharide Selleckchem OSI744 component. The dose response effects of PG545 on the viability of HEp-2 cells and on infection of these cells by RSV are shown in Fig. 1A. The anti-RSV activity of the cholestanol-sulfated
oligosaccharide conjugate (PG545) was ∼5 times greater than that of unmodified sulfated oligosaccharide of muparfostat. PG545 completely blocked RSV infectivity at concentrations of ⩾20 μg/ml while unmodified sulfated oligosaccharides of muparfostat did not demonstrate complete inhibition even at 500 μg/ml. At a concentration range of 0.16–500 μg/ml muparfostat demonstrated no cytotoxicity while PG545 reduced viability of HEp-2 cells with CC50 value of 230 μg/ml. Given the presence in PG545 of cholestanol, a sterol that could interact with many different lipophiles such as serum apolipoproteins, we tested the cytotoxicity and anti-RSV activity of PG545 using serum-free media. Under these conditions, the anti-RSV activity of PG545 was ∼16 times greater than that of muparfostat. Note that the absence
of serum in the culture medium enhanced both the anti-RSV activity and cytotoxicity of PG545 by ∼5-fold (Fig. 1B) as opposed to data obtained in the presence of serum (Fig. 1A). We also tested the effect of PG545 on infectivity of IAV or VSV. The former virus uses sialic acid for initial interaction with cells. While the cellular receptor for VSV is not known (Coil and Miller, 2004) this virus is highly sensitive to GAG mimetics MycoClean Mycoplasma Removal Kit (Baba et al., 1988). PG545 and muparfostat efficiently inhibited infectivity of VSV while showing no effect on IAV infectivity (Fig. 1C). To identify which step of the infectious cell cycle of RSV is affected by PG545, the compound was added to HEp-2 cells at different time points relative to the virus inoculation. The presence of compound during the 2 h period of virus attachment to and entry into the cells resulted in near complete blockade of RSV infectivity (Fig. 2) indicating that one of the initial steps of RSV infection of cells is the major target of PG545 activity.