In another example of the EFI’s early outreach efforts, Computation Core strategies were used to predict ligands of the norepinephrine transporter NET in a collaboration with the Pharmacogenetics of Membrane Transporters (PMT) and Center Structures of Membrane Proteins (CSMP) centers. As for Carlsson et al., the effort here extended the utility of modeling expertise and strategies that EFI members initially developed for EFI projects.
The norepinephrine transporter (NET) transports norepinephrine from the synapse into presynaptic neurons, where norepinephrine regulates signaling pathways associated with cardiovascular effects and behavioral traits via binding to various receptors (e.g., β2-adrenergic receptor). NET is a known target for a variety of prescription drugs, including antidepressants and psychostimulants, and may mediate off-target effects of other prescription drugs. Here, we identify prescription drugs that bind NET, using virtual ligand screening followed by experimental validation of predicted ligands. We began by constructing a comparative structural model of NET based on its alignment to the atomic structure of a prokaryotic NET homolog, the leucine transporter LeuT. The modeled binding site was validated by confirming that known NET ligands can be docked favorably compared to nonbinding molecules. We then computationally screened 6,436 drugs from the Kyoto Encyclopedia of Genes and Genomes (KEGG DRUG) against the NET model. Ten of the 18 high-scoring drugs tested experimentally were found to be NET inhibitors; five of these were chemically novel ligands of NET. These results may rationalize the efficacy of several sympathetic (tuaminoheptane) and antidepressant (tranylcypromine) drugs, as well as side effects of diabetes (phenformin) and Alzheimer's (talsaclidine) drugs. The observations highlight the utility of virtual screening against a comparative model, even when the target shares less than 30% sequence identity with its template structure and no known ligands in the primary binding site.
Figure 1. Validation of modeling and docking. (A) Predicted structure of the NET–norepinephrine complex. Norepinephrine is colored in orange, with oxygen, nitrogen, and hydrogen atoms in red, blue, and white, respectively. Sodium ions are visualized as purple spheres. NET’s transmembrane helices are depicted as white ribbons. Key residues are displayed as sticks; the three hydrogen bonds between norepinephrine and NET (involving residues Ala145, Phe72, and Asp75) are shown as dotted gray lines. (B) Enrichment plots for various structures: the refined NET model (blue), random selection (red), the initial NET model (green), and the LeuT template structure (orange).
Figure 2. Predicted binding modes for NET ligands. Predicted binding modes of the known substrate norepinephrine (A), and four ligands discovered in the docking screen (B–E). Residues making polar interactions with the ligand are illustrated with sticks; carbon atoms are colored in white, nitrogen atoms in blue, and oxygen atoms in red; hydrogen bonds are represented by dotted gray lines. The predicted pose of the known ligand is shown in orange sticks in A, and in green lines in B–E). The compounds are adrenalone (B), tranylcypromine (C), phenformin (D), and tuaminoheptane (E). The proposed key interactions between NET and norepinephrine are highlighted. (F) Protein–ligand hydrogen bonds are represented by dashed lines. Hydrophobic effect is represented by green lines. Interactions involving π electrons are represented by dotted green lines.
Figure 3. Uptake experiments. Inhibition of [3H]norepinephrine uptake by the identified inhibitors in NET stable transfected HEK (HEK-NET) cells. HEK-NET cells were incubated with 200 nM unlabeled NE (50 nM radiolabeled [3H]norepinephrine) and either 10 μM (blue) or 100 μM (red) of inhibitor for 3 min. Drugs that are chemically dissimilar to known NET ligands (Table S2) are annotated in bold font.
Figure 4. Uptake kinetic experiment. Concentration-dependent effect of tranylcypromine on [3H]norepinephrine uptake under the same conditions as in Fig. 3. Data are expressed as the percent [3H]norepinephrine uptake in the absence of inhibitor, and are the mean ± standard error of the mean of three independent experiments.
© 2011 National Academy of Sciences, USA