Modeling and Mutational Analysis of the Binding Mode for the Multimodal Antidepressant Drug Vortioxetine to the Human 5-HT_3A Receptor

Abstract

5-Hydroxytryptamine 3 (5-HT 3) receptors are ligand-gated ion channels that mediate neurotransmission by serotonin in the central nervous system. Pharmacological inhibition of 5-HT 3 receptor activity has therapeutic potential in several psychiatric diseases, including depression and anxiety. The recently approved multimodal antidepressant vortioxetine has potent inhibitory activity at 5-HT 3 receptors. Vortioxetine has an inhibitory mechanism that differs from classic 5-HT 3 receptor competitive antagonists despite being believed to bind in the same binding site. Specifically, vortioxetine shows partial agonist activity followed by persistent and insurmountable inhibition. We have investigated the binding mode of vortioxetine at the human 5-HT 3A receptor through computational and in vitro experiments to provide insight into the molecular mechanisms behind the unique pharmacological profile of the drug. We find that vortioxetine binds in a manner different from currently known 5-HT 3A orthosteric ligands. Specifically, while the binding pattern of vortioxetine mimics some aspects of both the setron class of competitive antagonists and 5-hydroxytryptamine (5-HT) with regards to interactions with residues of the aromatic box motif in the orthosteric binding site, vortioxetine also forms interactions with residues not previously described to be important for the binding of either setrons or 5-HT such as Val202 on Loop F. Our results expand the framework for understanding how orthosteric ligands drive 5-HT 3 receptor function, which is of importance for the potential future development of novel classes of 5-HT 3 receptor antagonists.

Conformational Changes in the 5-HT3A Receptor Extracellular Domain Measured by Voltage-Clamp Fluorometry

“…6). In general, the obtained EC 50 values for ΔF were within the 10-fold range of previously reported IC 50 or K d values for all of the antagonists (Hope et al, 1996;Lankiewicz et al, 1998;Ladefoged et al, 2018); except for the Q146C mutant that displayed decreased EC 50 values for ΔF compared with Y89C and M223C mutants, which suggests that Cys mutation and/or TAMRA conjugation decreases ligand affinity. In general, we did not attempt to quantify the rate by which the fluorescence signal decayed back toward baseline during the ligand postapplication washout phase due to the design of our VCF recording chamber, which did not allow for solution exchange rates likely required to isolate the rate of ensemble receptor transitions upon removal of extracellular ligand.…”

“…cDNA encoding h5-HT 3A was kindly provided by Beate Niesler (Department of Human Molecular Genetics, University of Heidelberg, Heidelberg, Germany). For expression in Xenopus laevis oocytes, the h5-HT 3A coding region was subcloned into the pXOON expression vector as described previously (Ladefoged et al, 2018). Cysteine mutations were introduced using the QuickChange site-directed mutagenesis kit from Stratagene (La Jolla, CA).…”

“…Molecular Modeling. A previously generated homology model of h5-HT 3A (Ladefoged et al, 2018) was used as a starting point for the creation of models of h5-HT 3A receptor Cys mutants with TAMRA conjugated. Specifically, for each reporter position (Tyr89, Met223, and Gln146), the wild-type residue was mutated to a Cys using the mutate tool within Maestro 10.1 (Schrödinger Suite, 2015 release).…”

“…Identification of h5-HT 3A residues as voltage-clamp fluorometry reporter positions. (A) Surface contoured cartoon representation of a homology model structure of the pentameric h5-HT 3A receptor (left) from Ladefoged et al (2018). The principal and complementary subunits are highlighted in cyan and green, respectively, with indications of the ECD and TMD.…”

CSTI-300 (SMP-100); a Novel 5-HT3Receptor Partial Agonist with Potential to Treat Patients with Irritable Bowel Syndrome or Carcinoid Syndrome

“…Of relevance to the present studies, vortioxetine, a multimodal, well-tolerated, and efficacious antidepressant (Cipriani et al, 2018) displays 5-HT 3 receptor partial agonist actions (Bang-Andersen et al, 2011;Ladefoged et al, 2018), and a case report describes the symptomatic relief of a patient with IBSd by this drug (Aydin and Ünal-Aydin, 2017). This further strengthens the argument that CSTI-300 would make a good therapeutic for IBS-d.…”