Analysis of the combined effect of two linear inhibitors on a single enzyme

Abstract

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.

Non-nucleoside Inhibitors of HIV-1 Reverse Transcriptase Inhibit Phosphorolysis and Resensitize the 3′-Azido-3′-deoxythymidine (AZT)-resistant Polymerase to AZT-5′-triphosphate

“…6B). As expected, both methods of evaluating the synergy between RT inhibitors are fully consistent (24). In fact, the combination of 0.2 M of 4-arylmethylpyridinone with 1 M on the AZT r -RT showed a 90% of inhibition (Fig.…”

“…6). In this plot, parallel lines are found if inhibitors do no interact, whereas intersecting lines are found if both inhibitors act synergistically (24). From this plot it can be concluded that in the presence of ATP the interaction is at least 10-fold greater than in its absence, as judged by the abscissa intersection points of both graphs.…”

“…The interaction index was calculated as explained previously (20,24). Briefly, dose-response curves for each inhibitor alone were obtained within a wide range of effects by fitting experimental data to Equation 1 by unweighted nonlinear regression.…”

Adenoviral expression of XIAP antisense RNA induces apoptosis in glioma cells and suppresses the growth of xenografts in nude mice

“…The combination of Ad-XIAP-as and Apo2L.0 increased the median survival to 130 days (P ¼ 0.0006, compared with Ad-DE1). Synergy is difficult to evaluate in vivo, but the fractional product method 33 may be applied when the single effects are expressed as ratios of gains in survival, which correspond to values in the range of 1.3 for either treatment alone. The predicted increase in survival with co-treatment can then be calculated as 1.69, which is below the doubling of median survival observed with co-treatment ( Figure 9).…”

Inhibitory activity of chlorogenic acid on enzymes involved in the fatty acid synthesis in animals and bacteria

“…If the two inhibitors bind in mutually exclusive fashion, a will be infinite (a ¼ ? ), and in the Yonetani-Theorell method, the most popular way of evaluating the interaction between two enzyme inhibitors, it will be observed that plotting 1/v ij against [I] at fixed [J] would result in parallel straight lines (34). In this case, I and J respectively represent NADP þ and chlorogenic acid.…”