Identification of a novel selective serotonin reuptake inhibitor by coupling monoamine transporter-based virtual screening and rational molecular hybridization

Abstract

Ligand virtual screening (VS) using the vestibular binding pocket of a 3-D monoamine transporter (MAT) computational model followed by in vitro pharmacology led to the identification of a human serotonin transporter (hSERT) inhibitor with modest affinity (hSERT K(i) = 284 nM). Structural comparison of this VS-elucidated compound, denoted MI-17, to known SERT ligands led to the rational design and synthesis of DJLDU-3-79, a molecular hybrid of MI-17 and dual SERT/5-HT(1A) receptor antagonist SSA-426. Relative to MI-17, DJLDU-3-79 displayed 7-fold improvement in hSERT binding affinity and a 3-fold increase in [(3)H]-serotonin uptake inhibition potency at hSERT/HEK cells. This hybrid compound displayed a hSERT:hDAT selectivity ratio of 50:1, and a hSERT:hNET (human norepinephrine transporter) ratio of >200:1. In mice, DJLDU-3-79 decreased immobility in the tail suspension test comparable to the SSRI fluvoxamine, suggesting that DJLDU-3-79 may possess antidepressant properties. This proof of concept study highlights MAT virtual screening as a powerful tool for identifying novel inhibitor chemotypes and chemical fragments for rational inhibitor design.

Figure 1

Pharmacological one-point (10 μM) binding assay of virtual screening hits at (A) hDAT-N2A using [³H]-WIN 35,428 and mazindol (10 μM) for nonspecific binding, (B) hSERT-HEK293 using [¹²⁵I]-RTI-55 and paroxetine (10 μM) for nonspecific binding, and (C) hNET-HEK293 using [¹²⁵I]-RTI-55 and desipramine (10 μM) for nonspecific binding. Data represent n = 3 independent experiments. **P < 0.002 vs total binding in that assay; ***P < 0.0001 vs total binding in that assay.

Figure 2

Structural comparison of virtual screening hit MI-17 ((±)-1) with dual SERT/5HT_1A receptor antagonist SSA-426 (2), leading to the rational design of DJLDU-3-79 ((±)-3) via molecular hybridization.

Figure 3

(a) Flexible alignment showing the similar 3-D arrangement for low-energy conformations of (±)-1 (cyan, R-enantiomer pictured) and 2 (orange). (b) High-scoring docking poses of (±)-1 (cyan, R-enantiomer pictured) and 2 (orange) in the vestibular S2 binding site of hSERT (beige cylinders) are superposed; the S1 substrate pocket is defined by a serotonin molecule (magenta). (c) Residues predicted for the hSERT vestibular S2 pocket. Residues within 4.5 Å of (+)-1 (cyan) and 2 (orange) are pictured. The color code indicates residues identical among the MATs and LeuT (green), residues shared among only the MATs (yellow), and nonidentical residues (gray). TM 11 was removed in this panel to facilitate viewing of the relevant side chains. (d) Comparison of predicted S2 pocket residues among hSERT, hDAT, hNET, and LeuT. Colors correspond to panel (c). The local sequence identities for hSERT are 23% between hSERT and LeuT, 38% between hSERT and hDAT, and 42% between hSERT and hNET. Figures were generated using PyMOL.

Scheme 1. Synthesis of MI-17/SSA-426 Hybrid Compound DJLDU-3-79 ((±)-3)

Figure 4

Behavioral effects of MI-17 ((±)-1) and DJLDU-3-79 ((±)-3) in the tail suspension test. (a) Acute administration (30 min) of the selective reuptake inhibitors of serotonin, fluvoxamine (FLVX; 10 mg/kg, i.p.), and norepinephrine, desipramine (DSP; 3 mg/kg, i.p.), decreased immobility times compared to water vehicle-treated (Veh) control animals (n = 7–9). (b) DJLDU-3-79 dose-dependently decreased immobility times compared to DMSO (100%; 0.002 mL/g) vehicle-treated (Veh) animals, indicating antidepressant-like effects (n = 8–14). (c) In contrast, MI-17 dose-dependently increased immobility times compared to DMSO (2%; 0.01 mL/g) vehicle-treated (Veh) animals (n = 8–10). Data are presented as the mean ± SEM and were analyzed by one-way ANOVA with Tukey’s posthoc test with **P < 0.01, ***P < 0.001 vs the appropriate vehicle control.

Figure 5

Locomotor effects of DJLDU-3-79 ((±)-3) and MI-17 ((±)-1). Locomotor activity was measured in animals 30 min following administration of (a) DJLDU-3-79 or (b) MI-17 and the appropriate DMSO vehicle: 100% at 0.002 mL/g or 2% at 0.01 mL/g, respectively. Behavioral responses were measured as the distance traveled (centimeters) and ambulatory time and resting time (seconds). Data are presented as the mean ± SEM (n = 6–10), and were analyzed by one-way ANOVA with Tukey’s posthoc test with **P < 0.01 vs vehicle-treated (Veh) control.