Structural Features Contributing to Methylenedioxypyrovalerone (MDPV) Analog Inhibition of the Human Dopamine Transporter

Author

Zachary R. Spires, Virginia Commonwealth UniversityFollow

DOI

https://doi.org/10.25772/DRQS-M836

Defense Date

2020

Document Type

Thesis

Degree Name

Master of Science

Department

Physiology and Biophysics

First Advisor

José-Miguel Eltit, Ph.D

Second Advisor

Malgorzata Dukat, Ph.D

Third Advisor

I. Scott Ramsey, Ph.D

Abstract

Monoamine transporters (MATs) are transmembrane neurotransmitter sodium symporters consisting of the dopamine, norepinephrine, and serotonin transporters: DAT, NET, and SERT, respectively. Following an action potential and neurotransmitter vesicular release, MAT-mediated reuptake of these neurotransmitters in the presynaptic membrane contributes to the termination of monoaminergic neurotransmission. MATs are the targets of many drugs providing therapeutic benefits or taken recreationally. One of these drugs is methylenedioxypyrovalerone (MDPV), an abused synthetic cathinone that is a high potency uptake inhibitor of the human DAT (hDAT). In contrast, MDPV is a low potency uptake inhibitor of the Drosophila melanogaster dopamine transporter (dDAT). The reasons for the differing potencies of MDPV at hDAT versus dDAT remain unclear. The three-dimensional crystal structure of dDAT has been elucidated complexed with several different ligands, while there is no available crystal structure of hDAT. A previous study identified unique residues within the S1 binding site of hDAT that increase potency to MDPV when inserted in the dDAT backbone. Through site-directed mutagenesis, a triple and a quadruple mutant were constructed to partially recreate the “hDAT” S1 site in the dDAT isoform template. Utilizing a novel assay involving intracellular Ca²⁺ as a reporter of MAT activity, these dDAT mutants caused up to a ~100 fold increase in MDPV potency versus wild type dDAT. Using the same assay, this work tested various analogs of MDPV. We found that all tested analogs demonstrate significant increases in potency at the mutant constructs when compared to wild type dDAT. Specifically, unlike at hDAT, the absence of the methylenedioxy ring appears to decrease recognition at the reconstructed S1 site. Additionally, α-alkyl chain length is important for binding at both hDAT and dDAT mutants, but in contrast to hDAT, increased bulk at this position appears to cause slightly decreased recognition at the dDAT mutants. Overall, however, while there are disparities in fine interactions of these analogs at the reconstructed S1 site, these results indicate that the S1 binding site is the place of binding for these compounds in the chimeric hDAT/dDAT constructs and likely hDAT.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-17-2020