Monoamine Transporter Photoaffinity Ligands Based On Methylphenidate and Citalopram: Rational Design, Chemical Synthesis, and Biochemical Application

Monoamine transporters (MATs) are a family of proteins that include the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). Specifically, dysregulation of MAT function is associated with a host of disease states including drug abuse, major depressive disorder, and anxiety. Additionally, several drugs acting as MAT inhibitors are clinically available to treat multiple disorders. However, details regarding the transport inhibition mechanism created by these drugs, as well as their discrete ligand-binding pockets within their target MAT proteins, remains poorly understood. This knowledge gap in turn hinders rational development of novel therapeutics for numerous MAT-associated disorders. The objective of this research dissertation was to develop irreversible chemical probes based on methylphenidate (MP) and citalopram (CIT), two therapeutically significant MAT inhibitors, in order to map their binding sites and poses within their major MAT target protein. The central hypothesis was that MP and CIT could be rationally derivatized, without significant loss in pharmacological activity, to contain a tag moiety and a photoreactive group capable of forming a covalent bond to their target MAT protein, thus allowing application of a "Binding Ensemble Profiling with (f)Photoaffinity Labeling (BEProFL)" experimental approach. Specifically, BEProFL rationally couples photoaffinity labeling, chemical proteomics, and computational molecular modeling in order to map the binding sites and poses of ligands within their target proteins. This central hypothesis was tested by pursuing three specific aims: 1) identification of non-tropane photoprobes based on MP suitable for DAT structure-function studies, 2) identification of photoprobes based on CIT and ( S )-CIT suitable for SERT structure-function studies, and 3) development of a tandem photoaffinity labeling-bioorthogonal conjugation protocol for SERT structure-function studies. In the first aim, MP was structurally modified to contain an aryl azide photoreactive group and a 125 I radioisotope tag. The compounds were then subjected to DAT pharmacological evaluation in order to identify suitable candidates for DAT structure-function studies. In the second aim, CIT and (S )-CIT were structurally modified to contain an aryl azide or benzophenone photoreactive group and 125 I, a terminal alkyne, or an aliphatic azide as a tag. Likewise, these compounds were subjected to SERT pharmacological evaluation in order to identify suitable candidates for SERT structure-function studies. Finally, under the third aim, a tandem photoaffinity labeling-bioorthogonal conjugation protocol was developed to label purified hSERT expressed in HEK-293 cells using a ( S )-CIT-based benzophenone-alkyne clickable photoprobe. Probe-labeled hSERT samples from this protocol are currently being analyzed by high resolution mass spectrometry in order to map the ( S )-CIT-binding site(s) within the hSERT.

Name thesis:degree_name

PhD

Level thesis:degree_level

Immediate Access

Discipline thesis:degree_discipline

Medicinal Chemistry

Year dc:date.available

2015

Author dc:creator

• Yarravarapu, Nageswari

Contributors dc:contributor

• David Lapinsky
• Michael Cascio
• Patrick Flaherty
• Aleem Gangjee
• Marc Harrold

• Pure sciences
• Health and environmental sciences
• Citalopram
• Dopamine transporter
• Methylphenidate
• Monoamine transporter
• Photoaffinity labeling
• Serotonin transporter

Language dc:language

English

Repository record dc:identifier

https://dsc.duq.edu/etd/1388

OAI identifier oai:identifier

oai:dsc.duq.edu:etd-2404