Computational study of SERT using new X-ray crystal structures and initial experimental verification of potential GABAB receptor compounds

Abstract

Depression is currently one of the leading causes worldwide of suicide and disability, the most common treatment is antidepressants. Most antidepressants work by increasing the monoamine levels in the central nervous system by inhibition of the reuptake of monoamines into the presynaptic neuron, and thereby ensure accumulation of the monoamines in the synaptic cleft. The main target for most antidepressants is the serotonin transporter, which is responsible for transporting serotonin, a monoamine, from the synaptic cleft back into the presynaptic neuron. The antidepressant (S)-citalopram is an antidepressant targeting the serotonin transporter that is well tolerated among patient populations. It is also the inhibitor that was rendered with best resolution in the recent crystal structures of the serotonin transporter.

A computational study of the serotonin transporter with a focus on key amino acids for its function in both the central and allosteric sites using (S)-citalopram and its substrate serotonin as ligands in four MD simulations was performed. This shed light on some of the internal molecular mechanisms of the serotonin transporter, especially the interactions at the binding sites. Each simulation identified key amino acids between each respective ligand and binding site. For the central site simulation there was identified one key amino acid, TYR95, that both ligands had as their primary interaction and point of contact. There was also seen a disparity between (S)-citalopram and serotonin in terms of interaction types, with their preferences being hydrophobic and H-bonds respectively. For the allosteric site simulations, the interaction type trends were the same as for the central site. In the allosteric simulations the amino acids PHE335 and GLU494 were identified as the strongest interacting partners for (S)-citalopram and serotonin respectively. The interactions between the ligands and the allosteric site were also not as strong overall as the interactions of the central site. A docking study was also performed with verified inhibitors of the serotonin transporter that sought to investigate binding site interactions between the docked inhibitors and the protein. An overview of amino acid interactions was created, which allowed for the identification of amino acids that interacted with the vast majority of ligands docked into the respective binding sites. The data from the docking was also used to attempt to elucidate a connection between in silico and experimental results.

In order for the study to involve experimental work an initial functional cAMP assay screening was incorporated into the project from an ongoing study in the research group. The assay tested 10 compounds that had been identified through a virtual screening approach as being potential GABAB receptor antagonists. None of the 10 compounds showed antagonistic effects on the GABAB receptor, but there were indications that three of them may be agonists. Further assays are required to confirm the three compounds status as agonists. The experimental work provided experience working in a laboratory environment and carrying out every step of a functional cAMP assay.