Aquatic Sciences Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Office of Research and Development, Center for Computational Toxicology and Ecology, Great Lakes Toxicology and Ecology Division, US Environmental Protection Agency, Duluth, Minnesota.
Environ Toxicol Chem. 2023 May;42(5):1032-1048. doi: 10.1002/etc.5591. Epub 2023 Apr 10.
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. Environ Toxicol Chem 2023;42:1032-1048. © 2023 University of Wisconsin-Madison. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.
正在开发新的方法学 (NAMs) 来减少和替代脊椎动物测试，以支持生态毒理学和风险评估。美国环境保护署的序列比对预测跨物种易感性 (SeqAPASS) 生物信息学工具被用于评估 3 型甲状腺素脱碘酶 (DIO3) 酶在物种间的氨基酸序列保守性，以展示用于理解化学物质与特定蛋白质靶标相互作用的 NAM 应用。利用现有文献确定了与甲状腺激素结合和与还原辅因子相互作用的关键氨基酸。SeqAPASS 工具根据分子量和侧链分类，确定与模板物种相比，涉及配体结合的已知关键氨基酸是否完全匹配、部分匹配或不匹配。这种评估指导了代表各种物种中发现的关键氨基酸取代的变异蛋白的设计。通过定点突变野生型 (WT) 人 DIO3 基因序列，创建了在细胞培养中表达的六种变体蛋白，然后在体外进行化学抑制测试。对于潜在的竞争性抑制剂，变体之间观察到体外中值抑制浓度结果存在显著差异。使用 Molecular Operating Environment (MOE) 软件构建了代表 WT 人 DIO3 的分子模型，并在计算机中进行了突变，以创建六种变体。MOE Site Finder 工具确定了提议的催化和辅因子位点以及潜在的替代结合位点。虚拟对接没有提供足够分辨率的亲和力分数来对化学抑制剂的效力进行排序。化学特性、取代氨基酸的功能和位置以及蛋白质靶标的复杂性是开发 NAMs 以评估跨物种化学易感性的重要考虑因素。环境毒理学与化学 2023；42:1032-1048。© 2023 威斯康星大学麦迪逊分校。环境毒理学与化学由 Wiley Periodicals LLC 代表 SETAC 出版。本文由美国政府雇员贡献，其工作在美国公共领域。
