DOI
https://doi.org/10.25772/14WB-VK23
Defense Date
2020
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Douglas Sweet
Abstract
A goal in the drug development process, as indicated by the FDA, is to evaluate a drug’s ADME profile, as potential drug interactions could exist, leading to adverse drug reactions or loss of efficacy. Transport proteins, specifically organic anion transporters (OATs) are involved in the absorption, distribution, and elimination of small, negatively charged compounds. Although there is an exhaustive list of structurally diverse organic anions which interact with OATs, interactions at a molecular level are still shrouded in mystery particularly due to the lack of a solved crystal structure. Therefore, in silico homology models (hOAT1, 2, 3) were generated using a crystalized protein as template. Amino acid contacts predicted to be involved in compound recognition were then altered through mutagenesis, followed by accumulation and kinetic studies to evaluate their role in compound translocation (hOAT1 and hOAT3).
Three-dimensional (3-D) homology models were generated for hOAT1, hOAT2 and hOAT3 utilizing Piriformospora indica high affinity phosphate transporter (PiPT) as template. The prototypical substrates para-aminohippuric acid (PAH) and estrone sulfate (ES) were docked into hOAT1 and hOAT3, respectively. Five amino acid contacts were identified after docking within hOAT1-PAH (Arg15, Ile19, Tyr230, Asn439 and Arg466) and hOAT3-ES (Tyr342, Phe426, Phe430, Leu431, and Arg454). Initial accumulation studies revealed hOAT1 substitutions at positions Arg15Ala, Ile19Ala, Tyr230Ala, Asn439Gln, Asn439Ala, and Arg466Ala abolished PAH transport mediated by hOAT1. Initial accumulation studies revealed hOAT3 substitutions at positions Tyr342Phe, Tyr342Ala, Phe426Ser, Leu431Ala, and Arg454Lys abolished ES transport mediated by hOAT3. Kinetic analysis revealed hOAT3 Phe430Ser substitution had a statistically significant increase in Km as compared to hOAT3 WT. Additionally, numerous structurally divergent compounds were docked within the generated hOAT1, hOAT2, and hOAT3 models, revealing additional amino acid contacts potentially critical to compound recognition and translocation.
Initial in silico studies revealed amino acid contacts potentially critical in hOAT1, hOAT2, and hOAT3 compound recognition. hOAT1 and hOAT3 in vitro studies further validated the generated in silico models, as well as emphasized significance in residues involved in substrate recognition. Development of these homology models could serve as an invaluable tool to support targeted rational drug design in addition to predicting drug-drug interactions.
Rights
© Christopher Edward Jay
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
6-2-2020