Tiagabine association.

<p>(a) Graphs following the trajectory of the association of tiagabine (using distance restraints). The individual figures from top to bottom show: the distance of tiagabine relative to the selected point in the bottom of the S1 binding site towards which tiagabine was pulled (I); the biasing potential energy profile (II); the sum of the non-bonded interaction energy profiles between tiagabine and the protein, water, sodium- and chloride ions (III); the electrostatic energy contribution to the non-bonded interaction energies between tiagabine and the protein, water, sodium- and chloride ions (IV); the van der Waals energy contribution to the non-bonded interaction energies between tiagabine and the protein, water, sodium- and chloride ions (V). Tiagabine is moving unhindered with the target movements through the extracellular vestibule until it binds to the S2 site after circa 34 ns. From this point the biasing potential energy is repeatedly accumulating though only by small amounts until tiagabine reaches the S1 site from where it cannot move any further into the protein. Compared to the biasing potential energy accumulated during translocation of GABA to the cytoplasm (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039360#pone-0039360-g004" target="_blank">Figure 4</a>) the potential energy barriers for tiagabine reaching the S1 site are considerable lower than translocation of a substrate. (b) 2D ligand interaction diagram sketching the binding mode obtained from the association of tiagabine towards the S1 site in GAT-1. Residues are colored according to their properties: charged (pink or purple), polar (blue) and hydrophobic (green). Hydrogen bonds are shown as lines and water molecules as red open circles.</p

Dissociation and re-association of GABA.

<p>SMD profiles for GABA dissociation using displacement restraints (a), and GABA re-association using distance restraints (b). For both (a) and (b) the individual figures from top to bottom show the displacement of GABA relative to the center of mass (COM) of the chemical system (I); the biasing potential energy profile (II); non-bonded interaction energy profiles between GABA and the protein, water, sodium ions, and chloride ions (III); non-bonded interaction energy profiles between GABA and residues interacted with during simulation (IV-VII). Figures on the left-hand side share the same time-axis as does figures on the right-hand side. Note, for the displacement restrained simulation depicted in (A) the displacement vector is specified by its components, hence the biasing potential energy is resolved into its x-, y-, and z-components, while the biasing potential energy is given for the vector in space in the distance restrained simulation in (B). Generally, changes in the interaction pattern between GABA and the protein are reflected in e.g. the biasing potential energy profile and vice versa. Most notably is the departure from the primary binding site, S1. During the initial 6 ns of the dissociation simulation (left-hand side) GABA is firmly keeping the initial binding mode (i.e. interactions with particularly Y60, G65, Y140, S396 and Na1), while the biasing potential energy (II) is accumulating. After circa 6 ns the direct ionic or hydrogen bonding interactions to the protein and sodium ion (Na1) are solvated by water molecules (III) thereby facilitating GABA to depart from the binding site, which is visualized by a sudden change in the COM movements depicted in (I) and in the drop in the biasing potential energy in (II). At the S2 site the interactions between GABA and the protein are reinforced due to particularly the interactions to R69 and D451.</p

GAT-1 upon substrate translocation.

<p>(a) Cartoon representation of GAT-1 after 33 ns simulation with GABA (yellow spheres) located at the cytoplasmic gate. The orange surface shows the shape of the revealed channel leading from the S1 site to the cytoplasm. (b) Cartoon showing the backbone of GAT-1 in the initial occluded state conformation, with GABA (yellow spheres) located in the S1 site. The green and red arrows indicate the direction and size of the principal backbone movements observed during the translocation simulation (from the simulation applying displacement restraints) (This Figure was prepared with Pymol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039360#pone.0039360-Schrodinger1" target="_blank">[59]</a>. In both figures the four TM helices forming the S1 site are highlighted, i.e. TM1 (blue), TM3 (purple), TM6 (pink), and TM8 (red).</p

GAT-1 and GAT-1 ligand structures.

<p>(a) Three-dimensional representation of the homology model of the human GABA transporter GAT-1. Structural features mentioned in the text are marked. S1, the substrate binding site; S2, the secondary/interim binding site; TM1 (TM3, TM6 and TM8), transmembrane region 1 (3, 6 and 8); and EL4, extracellular loop 4. (b) Structures of the endogenous ligand gamma-aminobutyric acid, GABA, <i>R</i>-nipecotic acid, and the anti-convulsive drug tiagabine.</p

GABA binding upon re-association.

<p>(a) Comparison of the initial binding mode of GABA (from which the dissociation simulation was started) and the binding mode obtained upon re-association to the S1 binding site. The initial binding mode is color-coded with grey carbon atoms, and the binding mode obtained by SMD is color-coded with cyan carbon atoms. Red spheres represent water molecules involved in GABA binding upon re-association (see text), the blue spheres represent the two structural sodium ions, and the green sphere represents the structural chloride ion. Nitrogen and oxygen atoms are color-coded blue and red, respectively. (b) 2D ligand interaction diagram sketching the binding mode obtained from the association simulation (prepared via 2D ligand interaction available through Maestro <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039360#pone.0039360-Schrdinger1" target="_blank">[55]</a>. Red circles represents water molecules involved in GABA binding, a dashed line represents a hydrogen bond to a protein side chain while a solid line represents an interaction with a protein backbone atom. Protein residues are color-coded as: green – hydrophobic, blue – polar, gray – glycine, pink – metal.</p

List of ORs predicted to interact with molecules carrying the “anis” note.

<p>List of ORs predicted to interact with molecules carrying the “anis” note.</p

Workflow of the study.

<p>Strategy to improve knowledge of olfactory perception and biological roles of odorant molecules. First an OR-OR association network identifies novel odorant-OR interactions for odorant candidates. Second, pathways linked to proteins are integrated in the OR-OR network allowing deciphering odor-disease connections. The last step involves scoring and ranking of odorant candidates for biological targets within the pharmacological space.</p

Concentration-response curves of odorants for human ORs.

<p>Odorants predicted as agonists ( =  predicted compounds) and odorants previously shown to be agonists by Saito et al. 2009 (positive controls) activated four human ORs: (a) OR2W1, (b) OR51E1 (c) OR5P3. Data points and EC₅₀ values are means ± s.e.m. from at least three experiments.</p

Concentration-response curves of odorants for the human cannabinoid receptor CB1.

<p>Predicted compounds, tributyl acetyl citrate, 2-nonanone and 2-phenylethyl hexanoate acted as inverse agonists. GloSensor assays were carried out in the absence (•) or in the presence (○) of pertussis toxin-treated cells. Data points and EC₅₀ values are means ± s.e.m. from three experiments.</p

Pharmacologically relevant PPARγ ligands.

<p>Pharmacologically relevant PPARγ ligands.</p