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Different methods for studying the concurrent effects of two linear inhibitors on a single enzyme have been published, including the fractional product of Webb, the Yonetani-Theorell plot or the method of Chou and Talalay. Recently the use of combination plots has also been advocated for this purpose. We have evaluated the applicability of these methods and found that most of them depend on assumptions about the mechanism of action of the inhibitors. If the mechanism of action is not completely understood, or if some assumptions about the mechanism are unfounded, the parameters obtained may be meaningless. Even if these assumptions are correct, the interaction can be advantageously measured using an alternative representation that does not require a knowledge of the inhibition constants and allows experimental data to be retrieved from the plot. In other cases it is the interpretation of the results rather than the validity of the method that is misleading. A common mistake is to take the exclusivity of the effects of two inhibitors as exclusivity of their binding. We show that this assumption is seldom justified. In any case, it is possible to decide whether the combination of two or more inhibitors is more effective than their individual use by means of isobolographic analysis, even when no information about their mechanism of action is available.

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Synergistic inhibition of HIV replication in cell culture has been reported for many combinations of reverse transcriptase inhibitors. However, the biochemical basis underlying this interaction is in most cases unknown. It has been previously shown that combinations of L-697,661 or U-90152s with AZT or ddC synergistically inhibit HIV-1 replication in cell culture. The combination of AZT with ddC is also favorable with respect to the inhibition of viral replication. However, the corresponding combinations showed no synergy in inhibiting enzyme activity when tested on conventional polymerase assays using homo- or heteropolymeric RNA and DNA as template. Data obtained suggest that amplification of the effect of chain terminators, a consequence of the high potential number of termination sites present on the template, override the synergistic effect expected for the combination of two independent nucleotide analogs. When a saturating amount of enzyme over template:primer was used, and a single site on the template was available for each chain terminator, the combination of AZTTP and ddCTP synergistically inhibited enzyme activity, whereas, as expected, the combination of AZTTP and ddTTP behaved as merely additive. Under similar conditions the combination of U-90152s and AZTTP was also synergistic. These results suggest that synergy found in antiviral assays with combinations having nucleosidic inhibitors is not related to the synergistic inhibition of reverse transcriptase and might be due to the presence in the viral population of virus strains with different sensitivity to the inhibitors.