Molecular Mechanisms of Ligand Recognition by the Human Serotonin Transporter: A Molecular Modeling Approach

The human serotonin transporter (hSERT) is a member of the neurotransmitter: sodium symporter family of membrane proteins and regulates serotonin (5-HT) neurotransmission by reuptake of 5-HT from the synapse into the presynaptic neuron. Dysfunction of this process leads to altered 5-HT activity at post-synaptic 5-HT receptors and is associated with neuropsychiatric disorders. Blockade of hSERT by selective serotonin reuptake inhibitors (SSRIs) is therefore an important therapeutic strategy in the treatment of depression and anxiety. However, there are a number of inhibitors of hSERT that have a high potential for abuse. Among these are cocaine and the amphetamines. Understanding the mechanism of action of these compounds requires information about their binding sites on the monoamine transporters and the structural conformation of the inhibitor-bound transporter complex. Although no atomic-resolution structures from X-ray diffraction studies yet exist for hSERT, several structures of a bacterial homologue of the monoamine transporters, the leucine transporter from Aquifex aeolicus (LeuT) have made it possible to generate homology models of hSERT that account for many aspects of its molecular pharmacology. These homology models were used to predict ligand binding orientations to hSERT in computational docking studies. I have focused my efforts on exploring the selectivity and affinity of a series of cocaine analogs for hSERT. I performed docking calculations on multiple conformations of hSERT to account for the structural flexibility of the protein upon ligand binding, since ligand binding is associated with changes in protein conformation. Conformations were generated by using multiple LeuT conformational templates for building hSERT homology models as well as with computational approaches such as normal mode analysis and snapshots from molecular dynamics simulations. In the case of hSERT, no X-ray structures are available but multiple conformations of the LeuT structure, which my hSERT homology models are based on, are available and these conformations are functionally significant in that the tryptophan-bound LeuT structure (PDB 3F3A) represents binding of an inhibitor at the substrate binding site, yielding an open-to-out conformation. The leucine-bound structure (PDB 2A65) represents a substrate-bound conformation, with the substrate molecule occluded from solvent in the central binding site. Since the correct hSERT conformation bound to cocaine may be a conformation distinct from these crystal structures, I used the homology modeling program Modeller to generate conformations of hSERT with both the occluded LeuT structure and the open-to-out LeuT structure as input templates, using a previously published alignment. By generating an ensemble of structures with a continuum of main-chain heavy atom root mean square deviations (RMSDs) between the open-to-out and occluded structures, docked compounds were be able to explore binding modes during docking runs in a wider range of protein conformations. These conformational refinements in my homology models of hSERT bound to the 3-phenyltropane analogs allowed accurate predictions of binding free energies that reflect experimentally-observed trends in the biological activity of these compounds. My approach was to examine docking solutions for high-scoring poses whose calculated binding free energies match the general rank order of potency from experimental pharmacological analysis of the 3-phenyltropanes at hSERT. High-scoring 3-phenyltropane-hSERT complexes were subjected to short molecular dynamics simulations from which the binding free energy was calculated using the linear interaction energy (LIE) approach. The use of LIE as a post-processing tool for the analysis of docking solutions allowed for more accurate scoring of the binding poses, resulting in a proposed binding mode for the 3-phenyltropane analogs in hSERT that was, in May, 2015, validated by X-ray structures of cocaine and the 3-phenyltropane analog RTI-55 bound to a the dopamine transporter from Drosophila melanogaster (dDAT)