In Vitro Combination of Anti-Cytomegalovirus Compounds Acting through Different Targets: Role of the Slope Parameter and Insights into Mechanisms of Action

Conventional therapy for human cytomegalovirus (CMV) relies on inhibition of the viral DNA polymerase. Ganciclovir (GCV) is the first-line therapy, but when GCV-resistant strains emerge, alternative therapies are extremely limited and are associated with significant toxicities. Combination of anti-CMV agents that act on different targets or stages of virus replication has not been well studied, mostly because of the limited number of anti-CMV agents. We report our investigation of combinations of agents that inhibit CMV by targeting the viral DNA polymerase, cellular kinases, or other cell/virus mechanisms yet to be discovered. The selected compounds differed by the slopes of their dose-response curve: compounds with a slope of 1 (GCV) representing one target or noncooperativity and compounds with high slopes indicating positive cooperativity. Analysis of anti-CMV drug combinations using the Bliss model (which accounts for the slope parameter) distinguished between combinations with synergistic, antagonistic, and additive activities. The combination of GCV and foscarnet was slightly synergistic; strong synergism was found when GCV was used with artemisinin-derived monomers or dimers or the MEK inhibitor U0126. The combination of GCV and cardiac glycosides (digoxin, digitoxin, and ouabain) was additive. The monomeric artemisinin artesunate was synergistic when combined with U0126 or the multikinase inhibitor sunitinib. However, the combination of artemisinin-derived dimers (molecular weights, 606 and 838) and U0126 or sunitinib was antagonistic. These results demonstrate that members of a specific drug class show similar patterns of combination with GCV and that the slope parameter plays an important role in the evaluation of drug combinations. Lastly, antagonism between different classes of CMV inhibitors may assist in target identification and improve the understanding of CMV inhibition by novel compounds.