Defense Date

2021

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmaceutical Sciences

First Advisor

Dr. Douglas H. Sweet

Abstract

Organic anion transporters (OATs) are known to interact with a wide variety of negatively charged drugs and can impact their clinical safety and efficacy profiles. The U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) have recognized and highlighted the importance of evaluating the potential interactions with transporters, specifically hOAT1 and hOAT3, during the development of new drug entities. Little is known about OAT-drug interactions as they are difficult to discern on a molecular level in the absence of any solved crystal structures for OATs. Therefore, in a previous study, in silico homology models of hOAT1 and hOAT3 were generated based on the solved crystal structure for Piriformospora indica phosphate transporter (PiPT). The models were docked with their respective prototypical substrates, amino acid contacts involved in substrate recognition predicted, and single point mutations generated. Following mutagenesis, singly mutated hOAT1 and hOAT3 transporters were subject to accumulation and saturation studies to determine their role in substrate binding and subsequent translocation. The findings from this previous study indicated singly mutated constructs did not result in altered binding affinity (Km). However, the question remained whether these predicted amino acid contacts would significantly alter affinity when present in various double and triple combinations.

In this study, multiple combination hOAT1 (Arg15Lys/Ile19Leu, Ile19Leu/Tyr230Phe, Arg15Lys/Tyr230Phe, and Arg19Lys/Ile19Leu/Tyr230Phe) and hOAT3 mutants (Phe426Tyr/Phe430Ser and Phe426Tyr/Phe430Tyr) were generated and functional accumulation screens were conducted to determine the impact on overall transport activity. Mutants that retained transport activity were then further assessed by kinetic assays to determine any changes in Km. Functional accumulation screens showed none of the hOAT1 multiple mutants retained PAH transport activity. In contrast, the generated hOAT3 double mutants retained ES transport activity. Subsequent kinetic analysis revealed the hOAT3 double mutants exhibited no statistically significant changes in estimated Km values as compared to hOAT3 wild-type.

This study provides further insight as to the importance of these predicted critical amino acid residues in substrate binding interactions. Further characterizing these molecular interactions will allow for improved manipulation of drug substrate pharmacokinetics as well as prediction of drug-drug interactions, both of which can be utilized in drug design and development.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-14-2021

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