New investigations of cyclosporin A in CDCl, have been performed to obtain additional and more accurate distance restraints than utilized in our previous studies of cyclosporin A. Build-up rates at 600 MHz using 6 different mixing times at low temperatures (252.5 K) were determined and transformed into distances using the two-spin approximation. With the new distance restraints in the MD simulations using the GROMOS package, we can unambiguously conclude the presence of a ,6II'-turn. The new structure resembles the X-ray structure more than the structure previously determined, especially regarding the orientation of the MeBmt side chain. In the new structure and in the solid state, the side chain is folded over the backbone (although there are substantial differences in the xI torsion), in contrast to the old structure, where the side chain is extended away from the backbone.Some years ago, we studied the conformation of cyclosporin A in CDCl, by NMR spectroscopy [I-41. In [lo] and another calculation algorithm which scans the total conformational space better than conventional MD methods [1 11 were applied to this molecule.
This study describes the screening of dynamic combinatorial libraries based on nipecotic acid as core structure with substituents attached to the 5- instead of the common 1-position for the search of novel inhibitors of the GABA transporter GAT1. The generated pseudostatic hydrazone libraries included a total of nearly 900 compounds and were screened for their binding affinities toward GAT1 in competitive mass spectrometry (MS) based Binding Assays. Characterization of the hydrazones with the highest affinities (with cis-configured rac-16gf bearing a 5-(1-naphthyl)furan-2-yl residue and a four atom spacer being the most potent) in binding and uptake experiments revealed an allosteric interaction at GAT1, which was not reported for any other nipecotic acid derivative up to now. Therefore, the herein introduced 5-substituted nipecotic acid derivatives could serve as valuable tools for investigations of allosterically modulated GABA transport mediated by GAT1 and furthermore as starting point for a new class of GAT1 inhibitors.
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.
A three-dimensional model of the human Na+/Cl--dependent y-aminobutyric acid (GABA) transporter hGAT-1 was developed by homology modeling and refined by subsequent molecular modeling using the crystal structure of a bacterial homologue leucine transporter from Aquifex aeolicus (LeuTAa) as the template. Protein structure quality checks show that the resulting structure is particular suited for the analysis of the substrate binding pocket and virtual screening experiments. Interactions of GABA and the substrate binding pocket were investigated using docking studies. The difference of 6 out of 13 substrate interacting side chains between hGAT-1 and LeuTAa lead to the different substrate preference which can be explained using our three-dimensional model of hGAT-1. In particular the replacement of serine 256 and isoleucine 359 in LeuTAa with glycine and threonine in hGAT-1 seems to facilitate the selection of GABA as the main substrate by changing the hydrogen bonding pattern in the active site to the amino group of the substrate. For a set of 12 compounds flexible docking experiments were performed using LigandFit in combination with the Jain scoring function. With few exceptions the obtained rank order of potency was in line with experimental data. Thus, the method can be assumed to give at least a rough estimate of the potency of potential of GABA uptake inhibitors Response to Reviewers: Reviewer #2: Obviously, no PME was used for Gromacs minimization, which is not state-of-the-art.Answer to reviewer: We are very thankful for this suggestion and have repeated the procedure of minimizing the initial protein structure using PME. In addition, we have heated the system using a simulated annealing approach in order to let the protein structure adopt better to GABA as the ligand. The differences in the resulting structure compared to the original structure are very small but, of course, in the paper we report the structure modelled with PME and after simujlated annealing.Reviewer #2:I doubt that LigandFit is the only one of the docking codes mentioned which is able to cope with the sodium-carboxylate-interaction mentioned in the text. the pose of the bound leucine in the leucine transporter LeuTAa showed that LigandFit was the best software to reproduce the orientation of the leucine in the active site and especially the interaction of the Leucine carboxyl group with the sodium atom Na1." In order to select the best-suited docking software for our needs, we have done preliminary docking calculations using the X-Ray structure of LeuTaa. These calculations showed clearly that LigandFit was best suited to reproduce especially the important interaction of the carboxylate to the sodium atom (Na1). Even with modifications in parameter files suggested by support people for FlexX and GOLD, LigandFit performed better in this very special case! And, differrent to the Pallo publication we don't need to introduce the carboxy-sodium interaction as restraint for our calculations! Reviewer #2: The much more extens...
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