A Novel Bromine-Containing Paroxetine Analogue Provides Mechanistic Clues for Binding Ambiguity at the Central Primary Binding Site of the Serotonin Transporter

Abstract

The serotonin transporter (SERT) is the primary target for the selective serotonin reuptake inhibitors (SSRIs). However, the structural basis for the extraordinarily high binding affinity of the widely prescribed SSRI, paroxetine, to human SERT (hSERT) has not yet been fully elucidated. Our previous findings unveiled a plausible ambiguity in paroxetine's binding orientations that may constitute an integral component of this SSRI's high affinity for hSERT. Herein, we investigate factors contributing to paroxetine's high affinity by modifying both the ligand and the protein. We generated a series of bromine (Br)-containing derivatives and found that the one in which the 4-F of paroxetine had been replaced with the chemically similar but more electron-rich Br atom (13) had the highest affinity. By comparatively characterizing the binding of paroxetine and 13 to both wild type (WT) and a construct harboring a paroxetine-sensitive mutation in the binding cavity, we identified a mechanistic determinant responsible for the pose ambiguity of paroxetine, which can guide future drug design.

Chemical and structural investigation of the paroxetine-human serotonin transporter complex

“…1b,c) also reduced paroxetine inhibition and binding, likely also disrupting these interactions. Although the effects were less severe when compared to paroxetine, Br-paroxetine binding and inhibition was also reduced for Ala169Asp 20 . Thus, these mutations highlight the importance of subsite B interactions in paroxetine binding but they cannot be used to demonstrate the inhibitor pose because, in the ABC or ACB poses, either the dioxol or fluorine of paroxetine could act as a hydrogen-bond acceptor in subsite B.…”

“…The binding of paroxetine to SERT has been extensively debated 8,9,[18][19][20] . The first x-ray structure of the ts3-SERT variant demonstrated that the binding pose is such that the piperidine, benzodioxol, and fluorophenyl groups occupy subsites A, B, and C respectively, in the ABC pose 9 (Fig.…”

“…These cross-linking experiments demonstrated that paroxetine binds in a fashion that is similar to the crystal structure 8,9 , where the fluorophenyl group is in proximity to Val501 39 . However, computational docking experiments using wild type SERT showed that the position of benzodioxol and fluorophenyl groups of paroxetine is 'flipped', with paroxetine occupying an ACB pose [18][19][20] (Fig. 1c).…”

“…Despite these efforts, however, the binding pose of paroxetine remains a subject of debate 8,9,[18][19][20] .…”

Chemical and structural investigation of the paroxetine-human serotonin transporter complex

“…In contrast, common methods for (-)-paroxetine synthesis can require the aromatic substituent to be introduced before stereoselective steps or ring construction, reducing flexibility of the process 20,[28][29][30][31][32][33][34] . Nevertheless, during the preparation of this work, the synthesis of Br-paroxetine was reported using an asymmetric conjugate addition and its binding to SERT has been extensively studied 20,30 .…”