Computational studies of molecular interactions in the human serotonin and dopamine transporters

Die humanen Serotonin- und Dopamintransporter (hSERT und hDAT) transportieren ihre endogenen Substrate, die Neurotransmitter Serotonin und Dopamin, schnell und selektiv in presynaptische Neuronen. Beide Transporter gehören zur Solute Carrier 6 Familie, weisen eine hohe Sequenzidentität auf, zeigen die gleiche Proteinfaltung und arbeiten mit dem gleichen Transportmechanismus. Viele Substanzklassen interagieren mit diesen Transportern, manche werden therapeutisch genutzt und andere illegal konsumiert. Diese Arbeit befasst sich mit den molekularen Interaktionsprofilen von hSERT und hDAT mittels verschiedener computergestützter Methoden. Die Ergebnisse und Herausforderungen in diesem Gebiet werden in drei peer-reviewed Studien präsentiert. Zu Beginn wurde das Spektrum der interagierenden Verbindungen in öffentlichen Datenbanken anhand ihrer strukturellen Grundgerüste und biologischen Messdaten untersucht. An einem einheitlichen Datensatz wurden die Selektivitätstrends in hSERT und hDAT durch Struktur Wirkungsbeziehungen, Protein-Ligand-Docking und Moleküldynamiksimulationen untersucht. Anschließend wurden die molekularen Selektivitätsdeterminanten in beiden Transportern an konkreten Beispielen der gleichen Substanzklasse mit biochemischen, biophysikalischen und elektrophysiologischen Methoden analysiert. Die Computeranalyse beinhaltete die Charakterisierung der Substituenten mit molekularen Deskriptoren, Induced-Fit-Docking und Solvatisierungsanalysen in den Bindungstaschen der Liganden. Abschließend wurde ein serotoningebundenes Homologiemodell von hSERT in einer außen okkludierten Konformation generiert, die eine tragende Rolle im Transportzyklus spielt und für die Untersuchung der Substratinteraktionen besonders geeignet ist. Mit moleküldynamischen Simulationen dieser und einer außen offenen Transporterkonformation wurden wichtige molekulare Merkmale miteinander verglichen, dabei wurden die Interaktionen entscheidender Aminosäuren im extra- und intrazellulären Bereich, in der zentralen Bindungstasche und die Transportersolvatisierung studiert. Die Arbeit leistet einen hilfreichen Beitrag zum Verständnis molekularer Interaktionen in Bezug auf Transporterselektivität und Transportmechanismus, allerdings bleiben noch viele Fragen für zukünftige Studien offen.The human serotonin and dopamine transporters (hSERT and hDAT) facilitate the fast and selective reuptake of their endogenous substrates, the neurotransmitters serotonin and dopamine, respectively. Both proteins belong to the solute carrier 6 family and share high sequence identity, the same protein fold and transport mechanism. Numerous compound classes have been identified to interact with these transporters, which are used in therapeutic settings or abused as illicit drugs. This thesis focuses on the molecular interaction profiles of hSERT and hDAT by us-ing different computational methods. The results and challenges in this field are presented in form of three peer-reviewed studies. At first, the chemical compound space was explored by retrieving common scaffold structures and biological measurements from linked open data sources. A consistent data set was chosen from the results to investigate the selectivity trends in hSERT and hDAT with studies of structure-activity relationships, protein-ligand docking, and molecular dynamics simulations. Subsequently, the molecular determinants for selectivity in both transporters were studied in detail with exemplary representatives from the same compound class by biochemical, computational, and electrophysiological approaches. The computational part comprised characterization of the substituents with molecular descriptors, induced-fit docking, and a solvent analysis in the ligand binding pocket. Finally, a serotonin-bound homology model of the human serotonin transporter was built in an outward-occluded conformation, a key intermediate in the physiological transport cycle, where the substrate interactions can be optimally studied. In microsecond-long molecular dynamics simulations of the outward-occluded and outward-open transporter conformations, important molecular features of these states were compared by monitoring the extra- and intracellular gating residues, permeation pathway solvation, and the protein-ligand interactions in the central binding site. Taken together, this work provides a useful contribution to enhance our understanding of molecular interactions regarding transporter selectivity and transport mechanism, but many questions remain elusive for upcoming future studies

A structural model of the human serotonin transporter in an outward-occluded state.

The human serotonin transporter hSERT facilitates the reuptake of its endogenous substrate serotonin from the synaptic cleft into presynaptic neurons after signaling. Reuptake regulates the availability of this neurotransmitter and therefore hSERT plays an important role in balancing human mood conditions. In 2016, the first 3D structures of this membrane transporter were reported in an inhibitor-bound, outward-open conformation. These structures revealed valuable information about interactions of hSERT with antidepressant drugs. Nevertheless, the question remains how serotonin facilitates the specific conformational changes that open and close pathways from the synapse and to the cytoplasm as required for transport. Here, we present a serotonin-bound homology model of hSERT in an outward-occluded state, a key intermediate in the physiological cycle, in which the interactions with the substrate are likely to be optimal. Our approach uses two template structures and includes careful refinement and comprehensive computational validation. According to microsecond-long molecular dynamics simulations, this model exhibits interactions between the gating residues in the extracellular pathway, and these interactions differ from those in an outward-open conformation of hSERT bound to serotonin. Moreover, we predict several features of this state by monitoring the intracellular gating residues, the extent of hydration, and, most importantly, protein-ligand interactions in the central binding site. The results illustrate common and distinct characteristics of these two transporter states and provide a starting point for future investigations of the transport mechanism in hSERT