BILD 1351 SE is a selective peptidomimetic subunit association inhibitor of the herpes simplex virus (HSV) ribonucleotide reductase (RR) with potent antiviral activity both in cell culture assays and animal models of HSV disease. The ability of BILD 1351 SE to inhibit the replication of HSV-1 when used in combination with acyclovir (ACV) for the treatment of HSV infections was investigated in baby hamster kidney cells using a 96-well enzyme-linked immunosorbent assay. The effective concentrations to achieve 50% inhibition (EC50) of virus replication by BILD 1351 SE in serum-starved and non serum-starved cells were 2 +/- 0.9 and 4.1 + 1.6 microM, respectively. The EC50 of ACV under both assay conditions was equal to 2.7 +/- 0.9 microM when tested alone. However, upon addition of BILD 1351 SE, the antiviral activity of ACV was potentiated in a synergistic manner as determined by the isobole method. At a concentration of BILD 1351 SE that produced 30% inhibition of HSV-1 replication, the EC50 of ACV decreased by about 15-fold in confluent cells and 17-fold in serum-starved cells. Similar conclusions were reached when evaluating drug interactions by the median dose-effect. Assuming mutually non-exclusive conditions at a drug ratio of ACV/BILD 1351 SE of 1/2, synergy was demonstrated in confluent cells with a drug enhancement index at EC50 of 14 and a combination index of 0.25. None of the drug combinations tested showed increased cytotoxicity in comparison with each drug alone. These results are consistent with the expected mode of action of a selective HSV RR inhibitor and support the strategy of combining these inhibitors with ACV for improved therapy of HSV infections.