Maribavir

Kinase experiment:

Enzyme kinetic analysis is performed on the purified wild type and mutant UL97 protein species using increasing concentrations of ATP (2 μM to 20 μM). The amount of incorporated radiolabelled phosphate is plotted against the concentration of ATP in a Lineweaver Burke plot to determine the Km for ATP for each UL97 species. The effect of Maribavir upon the rate of radiolabelled phosphate incorporation by wild type or mutant UL97 is determined by protein kinase assays at a fixed concentration of Maribavir (0.5 μM) as above, or with increasing concentrations of Maribavir (0.01 μM to 5.0 μM) to determine the IC50 of Maribavir for each UL97 species. In order to determine the nature of the inhibition mediated by Maribavir, plots of 1/v vs 1/ATP with increasing concentrations of Maribavir are constructed. Competitive inhibition is evident if the family of lines cconverged on the y-axis at 1/Vmax. The change in slope caused by the addition of Maribavir is used to calculate the Ki[1].

Cell experiment:

For these studies MRC-5 cells are seeded in 24-well plates at ~5×104 cells/well and grown for 3 days in MEM 8-1-1 to confluence (~1.1×105 cells/well). The cells are infected with AD169 in MEM 2-1-1 at an MOI ranging from 1 to 3 and incubated at 37°C for 90 min to allow viral adsorption. The unadsorbed virus is removed and replaced with 1 mL of MEM 2-1-1. To test the effect of compounds on viral DNA synthesis or maturation, Maribavir, BDCRB, or GCV is added to the medium at the concentrations indicated for each experiment[2].

References:

[1]. Shannon-Lowe CD, et al. The effects of Maribavir on the autophosphorylation of ganciclovir resistant mutants of the cytomegalovirus UL97 protein. Herpesviridae. 2010 Dec 7;1(1):4.
[2]. Biron KK, et al. Potent and selective inhibition of human cytomegalovirus replication by 1263W94, a benzimidazole L-riboside with a unique mode of action. Antimicrob Agents Chemother. 2002 Aug;46(8):2365-72.