Different Binding Modes of Small and Large Binders of GAT1

Well-known inhibitors of the γ-aminobutyric acid (GABA) transporter GAT1 share a common scaffold of a small cyclic amino acid linked by an alkyl chain to a moiety with two aromatic rings. Tiagabine, the only FDA-approved GAT1 inhibitor, is a typical example. Some small amino acids such as (R)-nipecotic acid are medium-to-strong binders of GAT1, but similar compounds, such as proline, are very weak binders. When substituted with 4,4-diphenylbut-3-en-1-yl (DPB) or 4,4-bis(3-methylthiophen-2-yl)but-3-en-1-yl (BTB) groups, the resulting compounds have similar pKi and pIC50 values, even though the pure amino acids have very different values. To investigate if small amino acids and their substituted counterparts share a similar binding mode, we synthesized butyl-, DPB-, and BTB-substituted derivatives of small amino acids. Supported by the results of docking studies, we propose different binding modes not only for unsubstituted und substituted, but also for strong- and weak-binding amino acids. These data lead to the conclusion that following a fragment-based approach, not pure but N-butyl-substituted amino acids should be used as starting points, giving a better estimate of the activity when a BTB or DPB substituent is added.