Citation:

Mayasich, S., M. Goldsmith, K. Mattingly, AND C. Lalone. Combining In Vitro and In Silico New Approach Methods to Investigate Type 3 Iodothyronine Deiodinase Chemical Inhibition Across Species. ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY. Society of Environmental Toxicology and Chemistry, Pensacola, FL, 42(5):1032-1048, (2023). https://doi.org/10.1002/etc.5591

Impact/Purpose:

As part of EPA’s efforts to reduce live-animal testing, new approach methods (NAMs) are being developed including in vitro assays and computational methods. The study being presented is an example of how these methods can complement each other. The SeqAPASS (Sequence Alignment to Predict Across Species Susceptibility) tool is useful in cross-species comparisons of critical amino acids involved in protein function at ligand-protein interaction sites. Molecular modeling supported the in vitro finding of potential competitive inhibitors binding at the putative catalytic site identified from the literature, and other chemicals interacting at the proposed cofactor site. Differences in response to chemicals among the variants representing cross-species variations in critical amino acids were partially explained by the SeqAPASS prediction. The study demonstrates the value in further development of computational methods to advance effective and efficient cross-species comparisons for ultimate use in ecological risk assessments and Adverse Outcome Pathway (AOP) development.

Description:

New approach methodologies (NAMs) are being developed to reduce and replace vertebrate animal testing in support of ecotoxicology and risk assessment. The US Environmental Protection Agency's Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) bioinformatic tool was used to evaluate amino acid sequence conservation of the type 3 iodothyronine deiodinase (DIO3) enzyme across species to demonstrate NAM applications for understanding effects of chemical interactions with a specific protein target. Existing literature was used to identify critical amino acids for thyroid hormone binding and interaction with a reducing cofactor. The SeqAPASS tool identifies whether known critical amino acids involved in ligand binding are exact, partial, or not matches across species compared with a template species based on molecular weight and side chain classification. This evaluation guided the design of variant proteins representing critical amino acid substitutions found in various species. Site-directed mutagenesis of the wild-type (WT) human DIO3 gene sequence was used to create six variant proteins expressed in cell culture, which were then tested in vitro for chemical inhibition. Significant differences in in vitro median inhibitory concentration results were observed among variants for potential competitive inhibitors. A molecular model representing the WT human DIO3 was constructed using Molecular Operating Environment (MOE) software and mutated in silico to create the six variants. The MOE Site Finder tool identified the proposed catalytic and cofactor sites and potential alternative binding sites. Virtual docking did not provide affinity scores with sufficient resolution to rank the potency of the chemical inhibitors. Chemical characteristics, function and location of substituted amino acids, and complexities of the protein target are important considerations in developing NAMs to evaluate chemical susceptibility across species.

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