When Does the IC(50) Accurately Assess the Blocking Potency of a Drug?

[Image: see text] Preclinical assessment of drug-induced proarrhythmicity is typically evaluated by the potency of the drug to block the potassium human ether-à-go-go-related gene (hERG) channels, which is currently quantified by the IC(50). However, channel block depends on the experimental conditions. Our aim is to improve the evaluation of the blocking potency of drugs by designing experimental stimulation protocols to measure the IC(50) that will help to decide whether the IC(50) is representative enough. We used the state-of-the-art mathematical models of the cardiac electrophysiological activity to design three stimulation protocols that enhance the differences in the probabilities to occupy a certain conformational state of the channel and, therefore, the potential differences in the blocking effects of a compound. We simulated an extensive set of 144 in silico I(Kr) blockers with different kinetics and affinities to conformational states of the channel and we also experimentally validated our key predictions. Our results show that the IC(50) protocol dependency relied on the tested compounds. Some of them showed no differences or small differences on the IC(50) value, which suggests that the IC(50) could be a good indicator of the blocking potency in these cases. However, others provided highly protocol dependent IC(50) values, which could differ by even 2 orders of magnitude. Moreover, the protocols yielding the maximum IC(50) and minimum IC(50) depended on the drug, which complicates the definition of a “standard” protocol to minimize the influence of the stimulation protocol on the IC(50) measurement in safety pharmacology. As a conclusion, we propose the adoption of our three-protocol IC(50) assay to estimate the potency to block hERG in vitro. If the IC(50) values obtained for a compound are similar, then the IC(50) could be used as an indicator of its blocking potency, otherwise kinetics and state-dependent binding properties should be accounted.