DOI
https://doi.org/10.25772/6BYP-6P66
Defense Date
2019
Document Type
Thesis
Degree Name
Master of Science
Department
Physiology and Biophysics
First Advisor
Dr. Jose-Miguel Eltit
Second Advisor
Dr. Charles Anderson
Third Advisor
Dr. Sidney Negus
Abstract
Ligands of the human monoamine transporters encompass a wide range of both illicit and therapeutic drugs that act upon neural circuitry related to reward, motivation, and the processing of salient stimuli. The present study utilizes two methods for analyzing transporter substrates and inhibitors in order to characterize activity and assess potency. The first measures transient changes in intracellular calcium as a surrogate for transporter activity by harnessing the electrical coupling of monoamine transporters and L-type calcium channels. This is used to analyze novel chimera of the strong hDAT inhibitors methylphenidate and ��-PPP in order to assess the contribution of specific moieties to potency. The observed reduction in potency suggests that methylphenidate may bind to the transporter in a manner distinct from ��-PPP, as chimera would otherwise be expected to show similar activity to parent compounds. These results highlight the importance of ��-carbon substituents and the relatively small contribution of beta-carbon groups to inhibitor potency at hDAT, while the lack of activity at hSERT suggests potency is not strongly influenced by beta-carbon or N-alkyl substituents. In order to further characterize drug-transporter interaction, a method was developed to analyze the kinetics of binding and unbinding using both known and novel hNET ligands, including a series of N-alkyl derivatives of 4-methylamphetamine. The study emphasizes the importance of both association and dissociation kinetics to affinity and sets up a methodological framework with two ways for determining Kd, with notable advantages over current models. The results indicate that lengthening the N-alkyl chain of 4-methylamphetamine leads to a decrease in potency and a shift in activity from substrate to blocker, with the results of N-propyl 4-methylamphetamine in particular indicating the potential existence of multiple low-affinity binding sites, each with distinct on and off kinetics. The implications of these results help elucidate the mechanism of action of transporter ligands and set up a framework for future studies that can more specifically classify the interaction between transporters and inhibitors or releasing agents.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-9-2019
Included in
Amino Acids, Peptides, and Proteins Commons, Biological and Chemical Physics Commons, Medical Biophysics Commons, Medical Physiology Commons, Other Chemicals and Drugs Commons, Physiological Processes Commons