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

Clickable photoaffinity ligands for the human serotonin transporter based on the selective serotonin reuptake inhibitor (S)-citalopram

To date, the development of photoaffinity ligands targeting the human serotonin transporter (hSERT), a key protein involved in disease states such as depression and anxiety, have been radioisotope-based (i.e., 3H or 125I). This letter instead highlights three derivatives of the selective serotonin reuptake inhibitor (SSRI) (S)-citalopram that were rationally designed and synthesized to contain a photoreactive benzophenone or an aryl azide for protein target capture via photoaffinity labeling and a terminal alkyne or an aliphatic azide for click chemistry-based proteomics. Specifically, clickable benzophenone-based (S)-citalopram photoprobe 6 (hSERT Ki = 0.16 nM) displayed 11-fold higher binding affinity at hSERT when compared to (S)-citalopram (hSERT Ki = 1.77 nM), and was subsequently shown to successfully undergo tandem photoaffinity labeling-biorthogonal conjugation using purified hSERT. Given clickable photoprobes can be used for various applications depending on which reporter is attached by click chemistry subsequent to photoaffinity labeling, photoprobe 6 is expected to find value in structure-function studies and other research applications involving hSERT (e.g., imaging)

Development of a triazole class of highly potent Porcn inhibitors

Учеб. пособие для студентов вузов: в 2-х ч.Доступ к полному тексту открыт из сети СФУ, вне сети доступ возможен для читателей Научной библиотеки СФУ или за плату.Во второй части представлены решения задач с использованием теоретических основ и методик расчетов на прочность и жесткость элементов строительных конструкций при сложном сопротивлении бруса, рассмотрен энергетический метод определения перемещений упругих систем. Подробно представлены расчеты статически неопределимых систем, основы расчета на прочность стержневых систем с учетом развития пластических деформаций, устойчивость сжатых стержней, динамическое действие нагрузок, прочность материалов при переменных напряжениях.http://catalog.sfu-kras.ru/ftext?539.3%2F%D0%9C+294-24099

Azido-iodo-N-benzyl derivatives of threo-methylphenidate (Ritalin, Concerta): Rational design, synthesis, pharmacological evaluation, and dopamine transporter photoaffinity labeling

In contrast to tropane-based compounds such as benztropine and cocaine, non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored. Towards addressing this knowledge gap, ligands were synthesized in which the piperidine nitrogen of 3- and 4-iodomethylphenidate was substituted with a benzyl group bearing a photoreactive azide. Analog (±)-3a demonstrated modest DAT affinity and a radioiodinated version was shown to bind covalently to rat striatal DAT and hDAT expressed in cultured cells. Co-incubation of (±)-3a with nonradioactive d-(+)-methylphenidate or (-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (β-CFT, WIN-35,428, a cocaine analog) blocked DAT labeling. Compound (±)-3a represents the first successful example of a DAT photoaffinity ligand based on the methylphenidate scaffold. Such ligands are expected to assist in mapping non-tropane ligand-binding pockets within plasma membrane monoamine transporters. © 2010 Elsevier Ltd. All rights reserved

Evolution of a Compact Photoprobe for the Dopamine Transporter Based on (±)-<i>threo</i>-Methylphenidate

The development of photoaffinity ligands for determining covalent points of attachment to the dopamine transporter (DAT) has predominantly focused on tropane-based compounds bearing variable-length linkers between the photoreactive group and the inhibitor pharmacophore. To expand the array of photoprobes useful for mapping inhibitor-binding pockets within the DAT, a compact nontropane ligand was synthesized featuring a photoreactive azide and iodine tag directly attached to the aromatic ring of (±)-<i>threo</i>-methylphenidate. (±)-<i>threo</i>-4-Azido-3-iodomethylphenidate [(±)-<b>6</b>; <i>K</i>_<i>i</i> = 4.0 ± 0.8 nM] displayed high affinity for hDAT. Moreover, a radioiodinated analogue of (±)-<b>6</b> demonstrated covalent ligation to the DAT in cultured cells and rat striatal membranes, thus suggesting the potential utility of this photoprobe in DAT structure–function studies

Novel Azido-Iodo Photoaffinity Ligands for the Human Serotonin Transporter Based on the Selective Serotonin Reuptake Inhibitor (<i>S</i>)‑Citalopram

Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (<i>S</i>)-citalopram (<b>1</b>). The classic 4-azido-3-iodo-phenyl group was appended to either the C-1 or C-5 position of the parent molecule, with variable-length linkers, to generate ligands <b>15</b>, <b>22</b>, and <b>26</b>. These ligands retained high to moderate affinity binding (<i>K</i>_i = 24–227 nM) for hSERT, as assessed by [³H]­5-HT transport inhibition. When tested against Ser438Thr hSERT, all three PALs showed dramatic rightward shifts in inhibitory potency, with <i>K</i>_i values ranging from 3.8 to 9.9 μM, consistent with the role of Ser438 as a key residue for high-affinity binding of many SSRIs, including (<i>S</i>)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (<i>S</i>)-citalopram inhibition of labeling by [¹²⁵I]<b>15</b> compared to that by [¹²⁵I]<b>22</b> and [¹²⁵I]<b>26</b> suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug–protein binding interactions for (<i>S</i>)-citalopram at hSERT

Novel Azido-Iodo Photoaffinity Ligands for the Human Serotonin Transporter Based on the Selective Serotonin Reuptake Inhibitor (<i>S</i>)‑Citalopram

Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (<i>S</i>)-citalopram (<b>1</b>). The classic 4-azido-3-iodo-phenyl group was appended to either the C-1 or C-5 position of the parent molecule, with variable-length linkers, to generate ligands <b>15</b>, <b>22</b>, and <b>26</b>. These ligands retained high to moderate affinity binding (<i>K</i>_i = 24–227 nM) for hSERT, as assessed by [³H]­5-HT transport inhibition. When tested against Ser438Thr hSERT, all three PALs showed dramatic rightward shifts in inhibitory potency, with <i>K</i>_i values ranging from 3.8 to 9.9 μM, consistent with the role of Ser438 as a key residue for high-affinity binding of many SSRIs, including (<i>S</i>)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (<i>S</i>)-citalopram inhibition of labeling by [¹²⁵I]<b>15</b> compared to that by [¹²⁵I]<b>22</b> and [¹²⁵I]<b>26</b> suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug–protein binding interactions for (<i>S</i>)-citalopram at hSERT