Channel Interactions and Robust Inference for Ratiometric β-lactamase Assay Data: a Tox21 Library Analysis

Ratiometric β-lactamase (BLA) reporters are widely used to study transcriptional responses in a high-throughput screening (HTS) format. Typically, a ratio readout (background/target fluorescence) is used for toxicity assessment and structure-activity modeling efforts from BLA HTS data. This ratio readout may be confounded by channel-specific artifacts. To maximize the utility of BLA HTS data, we analyzed the relationship between individual channels and ratio readouts after fitting 10,000 chemical titration series screened in seven BLA stress-response assays from the Tox21 initiative. Similar to previous observations, we found that activity classifications based on BLA ratio readout alone are confounded by interference patterns for up to 85% (50 % on average) of active chemicals. Most Tox21 analyses adjust for this issue by evaluating target and ratio readout direction. In addition, we found that the potency and efficacy estimates derived from the ratio readouts may not represent the target channel effects and thus complicates chemical activity comparison. From these analyses we recommend a simpler approach using a direct evaluation of the target and background channels as well as the respective noise levels when using BLA data for toxicity assessment. This approach eliminates the channel interference issues and allows for straightforward chemical assessment and comparisons.