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

Abstract

Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (S)-citalopram (1). 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 15, 22, and 26. These ligands retained high to moderate affinity binding (Ki = 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 Ki 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 (S)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (S)-citalopram inhibition of labeling by [125I]15 compared to that by [125I]22 and [125I]26 suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug–protein binding interactions for (S)-citalopram at hSERT.

Photochemical Reactions in the Synthesis of Protein–Drug Conjugates

“…used photochemistry to radiolabel plasmid DNA with 111 In 4‐( p ‐azidosalicylamido)butylamine diethylenetriaminepentaacetic acid (DTPA‐ASBA) . In addition, other groups have prepared photoactivatable ligands radiolabelled with 3 H, 14 C, and 125 I for studying binding‐site interactions on various plasma proteins …”

“…[53][54][55][56] Nishikawa et al used photochemistry to radiolabel plasmid DNA with 111 In 4-(p-azidosalicylamido)butylamine diethylenetriaminepentaacetic acid (DTPA-ASBA). [57] In addition, otherg roups have prepared photoactivatable ligandsr adiolabelled with 3 H, [58] 14 C, [59] and 125 I [60] for studying binding-site interactions on variousp lasma proteins. [61] Our laboratory recently reported the synthesis, radiolabelling, and bimolecular couplingo fp hotoactivatable chelates bearingt he photoactive aryl azide (ArN 3 )g roup to antibodies for use in immuno-PET and RIT ( Figure 2).…”

Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

“…However, these studies do not provide direct experimental information about inhibitor binding, and it has been difficult to assign specific inhibitor-binding functionalities unambiguously based on these mutational studies due to potential indirect and long-range allosteric effects on protein structure. Photoaffinity analogues of tropane-based inhibitors, such as cocaine, and SSRIs, including escitalopram, have been developed to define drug–protein interactions at the molecular level in DAT and SERT, respectively 47 48 49 50 . However, although photoaffinity ligands can provide a defined anchor point for ligand orientation within a binding pocket, addition of the relatively large photoreactive functional group to a drug likely affects its binding mode.…”

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

“…Traditionally, photoprobes have contained radiolabeled atoms (e.g., 3 H, 125 I), serving as a detection method. Several radioactive hSERT photoprobes have been reported based on serotonin, 24 tropanes, 25–26 the tricyclic antidepressant (TCA) imipramine, 27–28 and the selective serotonin reuptake inhibitors (SSRIs) paroxetine 29 and ( S )-citalopram; 30 however, none of these photoprobes have elucidated the structural basis for non-covalent interactions with hSERT. Using radioactive isotopes as reporter tags for detection purposes after photoaffinity labeling has several drawbacks, including practical concerns of special handling, the lack of an affinity handle for enrichment of probe-labeled biological macromolecules, and potentially short half-lives due to chemical degradation.…”

“…To begin our work, we postulated that the C-1 and C-5 positions of ( S )-citalopram could tolerate incorporation of an “all-in-one moiety” 23, 31 (i.e., a structural motif that contains a photoreactive functional group for protein capture via photoaffinity labeling and a click chemistry functional group as a latent affinity handle) without significant loss in hSERT binding affinity. This hypothesis, used in the design of radioactive azido-iodo ( S )-citalopram PALs 2 – 4 30 (Figure 1), was based on previously described structureactivity relationships for citalopram analogs 37–39 that suggested these two positions could accommodate the significant steric bulk associated with known “all-in-one moieties” without an appreciable decrease in hSERT binding affinity. To test this, novel clickable ( S )-citalopram PALs 5 – 7 (Figure 1) were chemically synthesized and pharmacologically evaluated for binding affinity to hSERT as described below.…”