Channel Interactions and Robust Inference for Ratiometric β-Lactamase Assay Data: A Tox21 Library Analysis
Fjodor Melnikov, Jui-Hua Hsieh, Nisha S. Sipes, and Paul T. Anastas
ACS Sustainable Chemistry & Engineering (2018). DOI: https://doi.org/10.1021/acssuschemeng.7b03394 PMID: Not Available
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.
Figure 1. Set up and mechanism of β-Lactamase (BLA) assays.
Broadly, the left panel shows the cell before BLA transcription is upregulated, while the right panel shows the BLA activity when stimulated by chemical exposure.
Specifically, A) Cell culture is grown and exposed to test chemical.
B) Chemical (“the star”) enters the cell and activates target transcription factors (TF).
C) TF activates BLA transcription through target promoter and thus results in BLA production.
D) CCF2/4-AM reagent is added.
E) CCF2/4-AM is absorbed into the cell.
F) CCF2/4-AM is converted to CCF2/4 by the cytoplasmic esterases and is trapped in the cell.
G) Esterase activity is assessed by CCF2/4 green FRET fluorescence at 530 nm.
H) BLA cleaves CCF2/4.
G) BLA activity is measured by fluorescence at 460nm.
In summary, the chemical effect on the target TF is quantitatively measured by 460nm fluorescence (channel 2) that indicates BLA activity and substrate loading is measured by FRET fluorescence at 530 nm (channel 1). The cell color represents the expected fluorescence effects.
- Figure 1 (130 KB)
Figure 2. Titration series and concentration response curves (CRCs).
Titration series and concentration response curves (CRCs) representative of:
A) an RFP chemical (e.g. CAS# 639-58-7; Tox21 ID: 303984);
B) an EUC chemical (e.g. CAS# 207-08-9, Tox21 ID: 200721);
C) an EOC chemical (CAS# 14866-33-2, Tox21 ID: 201028);
D) a PUC chemical (e.g. CAS# 100-56-1, ; Tox21 ID: 300878);
E) a POC chemical (CAS# 602-38-0, Tox21 ID: 202886);
F) a RFN chemical (CAS# 50-65-7, Tox21 ID: 300749);
G-H) CCA chemicals with non-monotonic and monotonic CRCs that are not flagged in any of the categories of potential concern (G: CAS# 1024009-92-7, Tox21 ID: 303340; H: CAS# 103577-45-3, Tox21 ID: 110184_1).
- Figure 2 (166 KB)
Figure 3. The number of channel 1 activator, inactive, and repressor chemicals.
The number of channel 1 activator, inactive, and repressor chemicals for ratio false positive (RFP), efficacy underestimation of concern (EUC), efficacy overestimation of concern (EOC), potency underestimation of concern (PUC), potency overestimation of concern (POC), ratio false negative (RFN), and all other active substances (All Other). The numbers above bars indicate % channel 1 activators, repressors, and inactive curves within each category.
- Figure 3 (100 KB)
Table 1. Tox21 β-Lactamase stress response assays, their targets, positive control chemicals, and background variability.
- Table 1 (286 KB)
Table 2. Activity Classification Statistics for Seven BLA Assays.
- Table 2 (319 KB)
- Supplemental Data (44 MB)