Using flux of surrogate ions to assess ion channel activity has dramatically improved the throughput of cell-based assays for ion channels. This approach is particularly useful when considering its general applicability to an entire class of ion channels, for instance, using Tl+ or Rb+ fluxes to assess potassium channel function. This method for detecting ion channel activity can be coupled with various approaches for triggering or modulating channel activity so that assays for channels with different modes of channel gating can be established in high-throughput formats. An ongoing concern when implementing high-throughput ion channel assays using surrogate ion flux to track channel activity is the ability of an assay to reliably monitor ion channel fluxes in a more physiological context. Electrophysiological measurements of ion channel activity are often performed under restrictive or nonphysiological conditions, but are still the most accessible current methods used to directly measure ion channel activity under conditions that may generate relevant pharmacological data.
The data in this report reveal 32% overlap between hits identified by a Tl+
-based surrogate ion assay and those identified by automated patch-clamp recordings using criteria that defined inhibitory hits as signal values lower than −3 SD of the buffer control values on each plate. Although it is recognized that the SD values for different assays were different, the degree of hit overlap between the assays did not improve as the criteria for hit selection was increased to larger than −5 SD. Indeed, this modest level of overlap is surprising. A few factors may contribute to this lower than expected level. First, it is well known that the surrogate ions have considerably different permeabilities from that of physiologic ions. This has been proposed to contribute to a right shift of IC50
values for known compounds inhibiting hERG potassium channels.40
In this case, using a higher concentration of testing compounds may yield a better overlap of hits identified by two different assays. Analysis of the Tl+
assay hits identified at 10
μM testing compound concentration ( and Supplementary Fig. S3
) revealed that, as expected, more hits were identified at each selection criteria compared with 1
μM testing, and fewer Tl+
-negative/IonWorks-positive compounds were identified. However, this approach did not increase the overlap between the Tl+
assay hits and the IonWorks hits because of large increases in the numbers of Tl+
Second, based on studies using known inhibitors of hERG potassium channels, it is clear that different compounds display varying degrees of right shift of potency, as large as 100-fold, in surrogate ion-based flux assays.40
Therefore, the structural features of testing compounds may differentially affect the inhibitory potency in each assay. This factor may be especially more pronounced for hERG than for other channels because hERG is inhibited by a variety of structural classes. Indeed, the Tl-based assay may be further optimized to improve its correlation with electrophysiological recording. Under our assay conditions, IC50
values of dofetilide and terfenadine, two structurally distinct hERG inhibitors, have different degrees of affinity shift when tested in two assays. Based on the Tl+
-based assay, their IC50
values are 35.3
1.3 and 1,256.6
nM. These values are similar to those reported in literature, for example, 26 and 1,278
nM by Bridal et al.29
Both ours and the earlier reports have shown different degrees of right-shift of dose–response curve depending on the given compound tested. It is conceivable that further optimization of the Tl+
-based flux assay could yield a better correlation with electrophysiology, hence arguing for the need of channel-specific assay optimization.
Third, many ion channels display distinct gating characteristics in response to different physiological conditions. For hERG channels, the rapid and precise voltage protocol applied by the automated patch-clamp used to isolate tail currents has considerably higher resolution that is unattainable by a change of extracellular ion concentration introduced via liquid handling, which is commonly used for triggering channel opening in flux assays. As a result, the flux assay, although target specific, may not replicate gating-specific pharmacological effects measured in patch-clamp studies. Of course, the degree of correlation between different assay formats may depend on the target and the properties of active compounds sought by the primary screen.
The present study examines the correlations between compounds inhibiting hERG channels in a fluorescent Tl+
influx assay and an electrophysiological assay performed on an IonWorks 384-well automated patch-clamp instrument. A structurally diverse set of 1,999 compounds assembled from the NIH MLSMR library was used to examine these assays in an HTS format. The validation set contains both diverse compounds and bioactives. With the −3 SD criteria, the hit rate for non-“known bioactives” in the Tl+
assay is 23.3% at 1
μM and 54.9% at 10
μM. The hit rate for “known-bioactives” in the Tl+
assay is 23.4% at 1
μM and 56.9% at 10
μM. In the electrophysiology assay, the hit rate for naïve compounds is 20.4% and 17.2% for known bioactives. Although the validation set does not perfectly represent the MLSMR collection, its diversity compounds and known bioactives show similar performance in the two assays. One may speculate that the naive diversity compounds in MLSMR have uncharacterized bioactivity against hERG. Additionally, because there are considerable data obtained with the validation set available in PubChem, our results may be further analyzed in the context of other assays and targets.
The purpose of these experiments is not to determine the preferred or most reliable predictor of hERG liabilities for cardiac safety screening. That question has been addressed in a number of publications using IC50
values calculated from small sets of compounds typically with defined hERG-blocking properties (e.g.
). The data presented here provide more relevant argument for the importance of electrophysiological assays using statistical data from a much larger collection of naïve compounds. The present experiments indicate that the hERG Tl+
flux assay used in the present format can be used to classify hERG-inhibitory properties of large sets of compounds. A significant fraction (1/4) of the combined hit set was negative in the flux assay and positive in the IonWorks assay when tested at 1
μM in both assays. This fraction decreased to 1/10 when comparing the flux assay results obtained at 10
μM with the IonWorks data at 1
μM. Therefore, a workflow utilizing a Tl+
flux high-density assay at a higher testing concentration followed by electrophysiological recording to validate the active compounds may remain an effective approach for rapid and efficient characterization of hERG inhibitory properties of large sets of compounds. Alternative approaches with lower numbers of false negatives and false positives may be preferable for cardiac safety profiling of limited sets of compounds.