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Bioinformatics for cross-species chemical susceptibility prediction and interpretation of in vitro screening results: Case study using a thyroid deiodinase enzyme
Citation:
Mayasich, S., M. Goldsmith, S. Vliet, D. Blatz, AND C. LaLone. Bioinformatics for cross-species chemical susceptibility prediction and interpretation of in vitro screening results: Case study using a thyroid deiodinase enzyme. SETAC North America 42nd Annual meeting, Portland, OR, November 14 - 18, 2021. https://doi.org/10.23645/epacomptox.17102537
Impact/Purpose:
Presentation to the SETAC North America 42nd Annual meeting November 2021. As part of EPA’s efforts to reduce live-animal testing, new approach methods (NAMs) are being developed including in vitro assays and computational (in silico) methods. The study being presented is an example of how these methods can complement each other, aiding in interpretation and providing an additional measure of confidence in the data that is generated. These methods, including the SeqAPASS (Sequence Alignment to Predict Across Species Susceptibility) tool, are especially useful in cross-species comparisons of chemical sensitivity and can be applied to any of thousands of species, including many endangered or threatened species, for which genomic sequence data exists in any of the publicly available databases. Our molecular modeling enabled us to gain a better understanding of the mechanisms of inhibition of a thyroid deiodinase enzyme by chemicals that were identified through in vitro screening as potent inhibitors. This information can be used for ecological risk assessment and adverse outcome pathway (AOP) development to further sort chemicals into categories as specific inhibitors that should be considered as thyroid disruptors, or non-specific inhibitors that may disrupt many types of proteins or cellular integrity. Further, designing recombinant proteins that can be produced and assayed in the lab and comparing those results to computer predictions can refine techniques for both approaches, as well as yielding meaningful cross-species comparisons.
Description:
New Approach Methodologies (NAMs) are being developed to reduce animal use and to evaluate greater numbers of chemicals more efficiently. In vitro chemical screening assays can be used to evaluate thousands of chemicals rapidly at lower costs than in vivo assays. Such approaches can specifically target the endocrine system. However, due to varying assay conditions and chemical characteristics, interpretation of in vitro results can be challenging, especially when comparing across species. To better understand results from an in-vitro screening study comparing chemical inhibitory activity between recombinant human and amphibian (Xenopus laevis) Type 3 iodothyronine deiodinase (DIO3) enzymes, the putative inhibitors were evaluated using molecular modeling and virtual docking. The DIO3 enzyme inactivates the thyroid hormone (TH) by catalyzing removal of iodine. To function, DIO3 must form a homodimer anchored in the cell membrane. Therefore, a variety of protein modeling strategies were needed. Some chemicals with low in vitro IC50 values matched the native TH-substrate-based pharmacophore and showed affinity for the catalytic site. Other chemicals may be allosteric inhibitors, showing affinity for a location on the DIO3 enzyme outside the catalytic site. However, in a model that included the cell membrane lipid bilayer, many of the chemicals exhibited greater affinity for the lipids than the protein, potentially indicating inhibition through lipid bilayer disruption. In another approach to understand how cross-species variations in protein sequence affect chemical susceptibility, the human DIO3 sequence was modified using in silico site-directed mutagenesis. Single amino acid modifications were made to represent variations in other species at positions critical to enzyme catalytic function. The species-specific amino acid sequence variations were identified using the US EPA Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool. These variations were then introduced in the laboratory through recombinant human DIO3 genes inserted into plasmid vectors to produce modified enzymes in cell culture. A suite of inhibitors selected from the previous in vitro screening were assayed to evaluate the in-silico predictions of chemical susceptibility for each DIO3 variant. These studies establish the basis for a pipeline using bioinformatics and molecular laboratory methods to advance NAMs for cross-species extrapolation. The contents of this abstract neither constitute nor necessarily reflect US EPA policy.
URLs/Downloads:
DOI: Bioinformatics for cross-species chemical susceptibility prediction and interpretation of in vitro screening results: Case study using a thyroid deiodinase enzyme
SETAC2021 DEIODINASE SDM MAYASICH_FINAL SLIDES.PDF (PDF, NA pp, 2417.136 KB, about PDF)