Combining In Vitro and In Silico New Approach Methods to Investigate Type 3 Iodothyronine Deiodinase Chemical...

Area of Science:

• Ecotoxicology and risk assessment
• Bioinformatics and computational biology
• Molecular toxicology

Background:

• New Approach Methodologies (NAMs) aim to replace animal testing in chemical safety evaluations.
• The type 3 iodothyronine deiodinase (DIO3) enzyme is a target for understanding thyroid hormone disruption.
• Assessing cross-species chemical susceptibility requires understanding protein target variations.

Purpose of the Study:

• To demonstrate the application of NAMs using the SeqAPASS tool for evaluating species-specific differences in the DIO3 enzyme.
• To investigate the impact of amino acid variations on chemical inhibition of DIO3.
• To guide the development of in vitro assays for ecotoxicological risk assessment.

Main Methods:

• Utilized the SeqAPASS bioinformatic tool to assess amino acid conservation in DIO3 across species.
• Identified critical amino acids for thyroid hormone binding and cofactor interaction.
• Employed site-directed mutagenesis to create DIO3 variants and tested their in vitro inhibition by chemicals.
• Constructed and mutated a molecular model of human DIO3 using MOE software.

Main Results:

• SeqAPASS identified varying degrees of amino acid matches for critical DIO3 residues across species.
• In vitro assays revealed significant differences in chemical inhibition sensitivity among DIO3 variants.
• Molecular modeling identified potential catalytic, cofactor, and alternative binding sites.

Conclusions:

• SeqAPASS is a valuable NAM for predicting species-specific protein target susceptibility.
• In vitro testing of engineered protein variants can reveal differences in chemical interactions.
• Consideration of amino acid characteristics and protein complexity is crucial for NAM development in ecotoxicology.