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

Bupropion Binds to Two Sites in the <i>Torpedo</i> Nicotinic Acetylcholine Receptor Transmembrane Domain: A Photoaffinity Labeling Study with the Bupropion Analogue [¹²⁵I]-SADU-3-72

Bupropion, a clinically used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analogue, (±)-2-(<i>N</i>-<i>tert</i>-butylamino)-3′-[¹²⁵I]-iodo-4′-azidopropiophenone (SADU-3-72). Based on inhibition of [¹²⁵I]­SADU-3-72 binding, SADU-3-72 binds with high affinity (IC₅₀ = 0.8 μM) to the <i>Torpedo</i> nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with 3-fold higher affinity in the desensitized (IC₅₀ = 1.2 μM) than in the resting state. Photolabeling of <i>Torpedo</i> nAChRs with [¹²⁵I]­SADU-3-72 followed by limited <i>in-gel</i> digestion of nAChR subunits with endoproteinase Glu-C established the presence of [¹²⁵I]­SADU-3-72 photoincorporation within nAChR subunit fragments containing M1–M2–M3 helices (αV8-20K, βV8-22/23K, and γV8-24K) or M1–M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K, and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu²⁶⁵, βLeu²⁵⁷) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr²¹³ in αM1. These results establish the presence of two distinct bupropion binding sites within the <i>Torpedo</i> nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket

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