Subscriber access provided by McMaster University Library Article A binding mode hypothesis of tiagabine confirms liothyronine effect on #-aminobutyric acid transporter 1 (GAT1) Andreas Jurik, Barbara Zdrazil, Marion Holy, Thomas Stockner, Harald H. Sitte, and Gerhard F. Ecker J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5015428 • Publication Date (Web): 13 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Page 1 of 47 Journal of Medicinal Chemistry
1 2 3 4 5 6 7 8 A binding mode hypothesis of tiagabine confirms 9 10 11 12 liothyronine effect on γ aminobutyric acid 13 14 15 16 transporter 1 (GAT1) 17 18 19 20 21 Andreas Jurik 1, Barbara Zdrazil 1, Marion Holy 2, Thomas Stockner 2, Harald H. Sitte 2, Gerhard 22 23 F. Ecker*1 24 25 26 1 27 University of Vienna, Department of Pharmaceutical Chemistry, Division of Drug Design and 28 29 Medicinal Chemistry, Althanstraße 14, 1090 Vienna, Austria. 30 31 32 2 33 Medical University of Vienna, Institute of Pharmacology, Center of Biomolecular Medicine 34 35 and Pharmacology, Währingerstraße 13a, 1090 Vienna, Austria. 36 37 38 KEYWORDS 39 40 41 42 tiagabine, GAT1, homology modeling, ligand docking, pharmacophore modeling, liothyronine. 43 44 45 46 ABSTRACT 47 48 Elevating GABA levels in the synaptic cleft by inhibiting its reuptake carrier GAT1 is an 49 50 established approach for the treatment of CNS disorders like epilepsy. With the increasing 51 52 53 availability of crystal structures of transmembrane transporters, structure based approaches to 54 55 elucidate the molecular basis of ligand transporter interaction also become feasible. 56 57 Experimental data guided docking of derivatives of the GAT1 inhibitor tiagabine into a protein 58 59 60 ACS Paragon Plus Environment 1 Journal of Medicinal Chemistry Page 2 of 47
1 2 3 homology model of GAT1 allowed derivation of a common binding mode for this class of 4 5 6 inhibitors, that is able to account for the distinct structure activity relationship pattern of the 7 8 dataset. Translating essential binding features into a pharmacophore model followed by in silico 9 10 11 screening of the DrugBank identified liothyronine as a drug potentially exerting a similar effect 12 13 on GAT1. Experimental testing further confirmed the GAT1 inhibiting properties of this thyroid 14 15 hormone. 16 17 18 19 INTRODUCTION 20 21 22 Imbalances in the levels of excitatory and inhibitory neurotransmitters such as serotonin, 23 24 dopamine, and GABA, can lead to severe CNS disorders like epilepsy, schizophrenia, anxiety 25 26 27 and depression. Tackling CNS diseases related to the GABAergic system is most commonly 28 29 achieved by using drugs of the benzodiazepine family (e.g. diazepam), which allosterically 30 31 1 32 modulates the pentameric GABA A receptor (GABA A R). However, an alternative way of 33 2 4 34 enhancing GABA action is inhibition of the corresponding neurotransmitter uptake system. In 35 36 case of the GABA transporter (GAT) family, four GABA reuptake transporter subtypes (GAT1 37 38 5 39 3, BGT1) and one vesicular carrier exist in mammalian organisms. The GAT family belongs to 40 41 the neurotransmitter:sodium symporters (NSS) which is organized as oligomers at the plasma 42 43 6 7 membrane while, in contrast to the GABA A R, functions as a monomer. Usually, NSS 44 45 46 transporters use a sodium gradient for uphill transport of neurotransmitters out of the synaptic 47 48 cleft. In certain cases, a reverse transport mode is also known, releasing neurotransmitter in a 49 50 non vesicular way. 8 At present, only one drug targeting this receptor, the anticonvulsant 51 52 53 tiagabine, is on the market. Tiagabine selectively inhibits GAT1, the most abundant GAT 54 55 subtype in the human brain.8 An X ray crystallographic structure is not yet available for any 56 57 58 member of the GAT family, but a number of homology models have been constructed. Further 59 60 ACS Paragon Plus Environment 2 Page 3 of 47 Journal of Medicinal Chemistry
1 2 3 docking studies indicated distinct modes of drug transporter interaction. 9 15 The molecular basis 4 5 6 of tiagabine action, however, remains elusive, as experimental evidence for proposed binding 7 8 modes is still lacking. Furthermore, ligand based exploration of inhibitor scaffolds is limited by 9 10 11 the low tolerance of this transporter for inhibitor modification. Based on a set of tiagabine 12 13 analogs from literature sources, we recently investigated ligand based structure activity 14 15 relationships of the compound class 16 . Briefly, binary QSAR allowed classification of GABA 16 17 18 uptake inhibitors into active and inactive bins by using the degree of rigidity and polarity 19 20 distribution as main descriptors. With the increasing knowledge provided by the X ray structures 21 22 of analogous transport proteins 17 , structure based approaches for elucidating the molecular basis 23 24 25 of drug transporter interaction also become feasible. In the present study, we describe a binding 26 27 hypothesis of tiagabine in GAT1 and its successful validation by in silico screening. 28 29 30 RESULTS AND DISCUSSION 31 32 33 34 COMPARATIVE MODELING 35 36 37 The closest transporter proteins related to hGAT1 for which structures are available are the 38 39 40 bacterial leucine amino acid transporter protein LeuT Aa and the drosophila dopamine transporter, 41 42 dDAT. Despite its lower overall sequence identity, closer substrate relationship and significantly 43 44 higher resolution of 2.00 vs. 2.95 Å favored the use of LeuT as template structure. 18, 19 Several 45 46 47 sequence alignments between hGAT1 and LeuT Aa have been published and all alignments are 48 49 almost identical within the conserved central substrate binding cavity.11, 20, 21 Both template 50 51 candidates were available in an open to out conformation, thus granting access to bulky inhibitor 52 53 54 molecules. Suitable templates for the intracellular N and C terminal domains of hGAT1 are not 55 56 available and thus were not included in the final homology model. Due to the differing 57 58 59 60 ACS Paragon Plus Environment 3 Journal of Medicinal Chemistry Page 4 of 47
1 2 3 stoichiometry of eukaryotic NSS family members for Cl , the LeuT structure (PDB code: 3F3A) 4 5 6 was modified by engineering a chloride binding site using structural information from crystal 7 8 structure of the dDAT and topological information from the literature.22 24 Based on a 9 10 11 combination of low B values and proximity to the binding site or stabilization of adjacent 12 13 domains, several water molecules were selected and kept in the template file. Finally, a known 14 15 disulfide bridge between C164 and C173 of EL 2 was defined.3 Modeller 25 was used to generate 16 17 18 100 models, which were ranked according to their respective DOPE (Discrete Optimized Protein 19 20 Energy) score for estimating the geometric quality.26 For the ten highest ranked models, 21 22 additional quality checks were performed using the model assessment tools of the SWISS 23 24 27 29 25 MODEL server. Models with core residues showing disallowed geometry according to the 26 27 Ramachandran plot were omitted. The remaining models were visually inspected for their ability 28 29 to reflect residue proximity and accessibility data from literature.30 34 In addition, the models 30 31 32 were evaluated regarding the orientation of non conserved polar residues in TM regions. 33 34 Subsequently, the best structure was selected according to aforementioned criteria and subjected 35 36 37 to a soft minimization protocol for relaxation of the system. 38 39 40 41 42 43 BINDING SITE SAMPLING 44 45 46 47 Focused sampling of the conformational space in the putative tiagabine interaction site can be 48 49 achieved by molecular dynamics (MD) simulations. Tiagabine cannot be accommodated in the 50 51 occluded state of the transporter, as both the extracellular gate between R69 and D451 as well as 52 53 54 the upper lid of the binding side, formed by the bulky F294 side chain, are limiting the available 55 56 space 11 . In addition, preliminary MD simulations of the transporter model in the apo open to out 57 58 59 60 ACS Paragon Plus Environment 4 Page 5 of 47 Journal of Medicinal Chemistry
1 2 3 state led to rearrangement of the gating residues impeding subsequent placement of compounds 4 5 6 larger than substrates like GABA, guvacine or nipecotic acid. Thus, tiagabine was placed into the 7 8 central cavity using GLIDE 35 prior to 30 ns of molecular dynamics simulations, which was used 9 10 11 for validating and equilibrating the model. 12 13 14 Subsequently, ten representative snapshots for the last ten nanoseconds of the run were 15 16 extracted, based on maximum RMSD diversity of binding pocket residues. Thus, focused 17 18 19 sampling of the conformational space in the binding site could be achieved, using the snapshots 20 21 as input structures for subsequent docking experiments. 22 23 24 DOCKING STUDIES 25 26 27 28 The constrained GABA analogs, nipecotic acid and guvacine ( 2,3), are potent uptake inhibitors 29 30 in vitro , but are unable to penetrate the blood brain barrier.36 In addition, these compounds act 31 32 8 33 as GAT1 substrates. In contrast, tiagabine and the selective GAT 1 inhibitor SK&F 89976 A 34 35 (4,5) that contain bulky aromatic substituents are pure inhibitors that are not transported. A large 36 37 number of systematically modified derivatives of the basic tiagabine scaffold have since been 38 39 37 41 40 synthesized and tested. These derivatives contain a conformationally restricted GABA 41 42 mimetic nipecotic acid or guvacine moiety, a 4 8 atom linker and a large, mostly di aromatic 43 44 hydrophobic moiety. These analogs provide a rich data source to construct structure activity 45 46 47 relationships. 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 5 Journal of Medicinal Chemistry Page 6 of 47
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Figure 1. GABA, conformationally restricted analogs and lipophilic aromatic derivatives. 21 22 23 24 A total of 162 compounds were extracted from the literature, spanning an activity range from 25 26 low nanomolar to millimolar IC 50 values, each of them tested under comparable assay 27 28 conditions.42 Ligands exhibiting substantial activity differences linked to distinct structural 29 30 31 changes in the key regions shown in Figure 2, were selected for subsequent experimental data 32 33 guided docking.1, 43 34 35 36 An important observation was the dramatic activity loss caused by introduction of a direct link 37 38 39 between the two aromatic moieties ( 5 vs. 7). In contrast, enhanced activity had been reported for 40 41 introduction of a polar region in the linker. This is exemplified by compound pair 8 and 9, as 42 43 44 well as compound 6, being the closest available derivative to reference compound 5. In terms of 45 46 activity, extending the linker length was well tolerated, since compounds 5, 8 and 10 gave potent 47 48 inhibitors. Finally, exchange of a benzene by a pyridine leads to a dramatic loss of activity (10 49 50 51 vs. 11 ). These activity differences should also be reflected by respective differences in the 52 53 ligand/protein interaction pattern and thus aid in the prioritization of the docking poses. 54 55 56 57 58 59 60 ACS Paragon Plus Environment 6 Page 7 of 47 Journal of Medicinal Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Figure 2. Chemical structures and literature IC 50 values of ligands with key modifications in 26 27 linker length, polarity, and rigidity of the aromatic moiety. 28 29 30 Docking into ten snapshots derived from the hGAT 1 tiagabine complex was performed in a 31 32 44, 45 33 sequential ensemble like manner using GOLD, thereby allowing for minor movements of the 34 35 backbone and focused sampling of the binding site side chain orientations. The binding site was 36 37 defined within a 10 Å radius around the simulated tiagabine coordinates. Two water molecules 38 39 40 were kept optional, as they had turned out to be stably involved in the hydrogen bonding network 41 42 during the previous MD simulation. However, in the subsequent docking runs, no direct 43 44 contribution of these two water molecules to the binding of the selected ligands was observed. 45 46 47 48 Side chain orientations of possible interaction partners for polar linker compounds 6, 8, 10 and 49 50 11 were addressed individually. Conformational sampling of the binding site had been performed 51 52 with tiagabine as ligand, which lacks a corresponding electronegative moiety in the linker. 53 54 55 Hence, the full range of conformational flexibility of the R69, Y139, Y140 and S452 side chains 56 57 was explored using the internal rotamer library of the GOLD software package. 58 59 60 ACS Paragon Plus Environment 7 Journal of Medicinal Chemistry Page 8 of 47
1 2 3 For each of ligands 6 11 , 100 docking poses per snapshot were generated and ranked by 4 5 6 ChemScore, which provides parameters for a putative interaction with sodium, in analogy to 7 8 LeuT.21 However, relying on just a single scoring function bears the risk of missing relevant 9 10 11 poses, especially when no experimentally derived complexes for redocking studies are available. 12 13 Thus, all poses were re ranked using rank by rank consensus scoring that included GoldScore, 14 15 ChemPLP, London dG, GBVI and XScore scoring functions.45 50 The top ten consensus score 16 17 18 poses for each ligand were subsequently visually analyzed. 19 20 21 BINDING MODE 22 23 24 Analysis of the ten top ranked poses per ligand among the ensemble docking results clearly 25 26 27 indicated a common binding mode for tiagabine analogs (Figure 3). As already expected from 28 29 literature results, the most prominent interaction was coordination of the Na1 sodium cation by 30 31 32 the negatively charged acid moiety, which fulfills an octahedral geometry, together with side 33 34 chain atoms of N66, S295 and N327, as well as the backbone carbonyl oxygens of A61 and 35 36 S295.. The majority of observed poses showed an interaction between the positively charged 37 38 39 nipecotic acid nitrogen and the backbone of F294, while the carboxylate group interacting with 40 41 Na1 was in an equatorial conformation. In contrast, poses with the R configured carboxy group 42 43 sampled in an axial conformation tended to form an intramolecular hydrogen bond with the 44 45 46 charged nitrogen atom. While no clear preference for one of the two carboxylate orientations 47 48 could be deduced from scoring values, X ray and NMR studies of nipecotic acid indicated a 49 50 preferred equatorial configuration, which would be more pronounced by adding a bulky moiety 51 52 51, 52 53 like the biaromatic tail. In addition, Skovstrup et al. reported less stable behavior of axial 54 55 configured tiagabine poses in molecular dynamics simulations.11 Thus, the poses with the axial 56 57 58 carboxylate configuration were considered less plausible. 59 60 ACS Paragon Plus Environment 8 Page 9 of 47 Journal of Medicinal Chemistry
1 2 3 The upper boundary of the binding pocket is formed by a bent region of EL 4, extending into the 4 5 6 hydrophobic cavity with the backbone of G360 as a ’cealing beam’, hence separating it into two 7 8 pockets each of which is able to accommodate a single hydrophobic aromatic rings. As it can be 9 10 11 seen in Figure 3, the ligand transporter interaction in one hydrophobic pocket is stabilized by a 12 13 pi pi interaction with the side chain of Y139, as well as by a cavity able to accommodate a small 14 15 o substituent as present in 5 and 6. This cavity is confined by the side chains of I143 and Y140, 16 17 33 18 the latter being a residue known to be also important for ligand recognition (see Figure 3). 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Figure 3. Docking poses of tiagabine (turquoise) and analogs (orange) in ten MD snapshots of 43 44 the hGAT 1 model. Polar regions in blue, hydrophobic areas in yellow. 45 46 47 48 The second cavity is mainly shaped by the hydrophobic side chains of W68, F294 and A358 (not 49 50 shown). 51 52 53 Due to the relative torsion of the two pockets, poses of 7 tended to encounter an face initial steric 54 55 56 clash that was relieved after energy minimization of the complexes, whereas compounds with a 57 58 59 60 ACS Paragon Plus Environment 9 Journal of Medicinal Chemistry Page 10 of 47
1 2 3 terminal bis 5 methyl thienyl ( 5, 6) or diphenyl ( 8 10 ) group were able to bind in a 4 5 6 conformation near their global energy minimum (See Figure 4), thus explaining the activity cliff 7 8 between 5 and 7. 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Figure 4. Dihedral energy landscape of 5 methyl thiophen dihedral angles; configuration of the 35 36 docking pose of 5 is marked in yellow. Constrained aromatic system of 7 is highlighted in red. 37 38 39 The positive effect of a polar atom in the linker moiety on binding seems to be the result of 40 41 42 several factors. Transient interactions with residues in the entry path might play a significant 43 44 role, but are not reflected by the docking poses. Earlier steered MD studies of tiagabine in GAT1 45 46 by Skovstrup et al. had indicated a transient interaction with the R69 side chain upon entry in the 47 48 53 49 binding site. Hence, docking poses biased towards an interaction between this residue and one 50 51 of the electronegative linker atoms in 6, 8, 10 and 11 were generated, turning out to be possible, 52 53 but rather short lived in short MD runs due to reset forces of the basic side chain (data not 54 55 56 shown). 57 58 59 60 ACS Paragon Plus Environment 10 Page 11 of 47 Journal of Medicinal Chemistry
1 2 3 To address the relatively low activity of compound 11 , per atom contributions to G in the 4 Δ 5 54 6 binding pose were evaluated using HYDE. An unfavorable effect of the pyridine nitrogen in 7 8 the hydrophobic receptor environment was indicated (see Supporting Figure S3). In addition, 9 10 11 repulsive forces between negative partial charges of the aromatic nitrogen atom and the oxime 12 13 moiety might force the pyridine ring in a sterically unfavorable orientation. 14 15 16 Taken together, the common orientation of the compound class was in agreement with the 17 18 19 structure activity relationships of the ligand set and the topology of the extended substrate 20 21 binding pocket. 22 23 24 25 VIRTUAL SCREENING 26 27 28 In order to further probe the proposed binding mode against pharmacologically relevant chemical 29 30 space, a pharmacophore based screening strategy was applied. The 3D orientation of the main 31 32 33 tiagabine binding features was extracted from the docked complex and encoded in a 4 feature 34 35 pharmacophore model using LigandScout.55 Two hydrophobic features were placed in the 36 37 respective cavities occupied by the thiophene moieties. Groups capable of complexing sodium 38 39 40 were described by a negatively ionizable property. Finally, a positively ionizable feature was 41 42 placed on the basic nitrogen to mirror the compound's zwitterionic character (Figure 5, the model 43 44 45 is available for download at http://pharminfo.univie.ac.at). 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 11 Journal of Medicinal Chemistry Page 12 of 47
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Figure 5. Pharmacophore model of tiagabine: Hydrophobic (yellow), positive (blue) and 26 27 negative (red) ionizable features in context with the GAT1 substrate binding site. 28 29 30 The sensitivity of the pharmacophore model was validated by a decoy set generated using the 31 32 56 33 DUD E platform (http://dude.docking.org), retrieving just the compounds with known GAT 1 34 35 activity. 36 37 38 In order to test the predictive value of the model, a commercial vendor database 57, 58 consisting of 39 40 59 41 1.7 million compounds, as well as the Drugbank Index covering 1491 marketed drugs, were 42 43 screened. A total of 79 and 8 compounds, respectively, matched the pharmacophore query, and 44 45 passed the PAINS filter for frequent hitters. 60 46 47 48 49 Subsequently, the virtual hits were docked into a representative MD snapshot of the homology 50 51 model, from which the tiagabine pharmacophore model had been derived. Protein ligand 52 53 61 54 interaction fingerprints (PLIF) for the calculated poses were retrieved using MOE. The 55 56 interaction fingerprints were used to filter out compounds, which did not show an interaction 57 58 59 60 ACS Paragon Plus Environment 12 Page 13 of 47 Journal of Medicinal Chemistry
1 2 3 with Na1. This reduced the hit list to 13 compounds for the Enamine database available in 4 5 6 sufficient purity, and 7 compounds for DrugBank, respectively (see Figure 6). For the latter, 7 8 tiagabine, three thyroid hormones (liothyronine, levothyroxine, dextrothyroxine), two 9 10 11 angiotensin conversion enzyme (ACE) inhibitors (ramipril, perindopril), and an 12 13 antihistaminergic drug (bepotastine), were retrieved. Based on pharmacophoric fit and docking 14 15 performance (details in Table S2), bepotastine, ramipril and liothyronine were selected for 16 17 18 biological testing. 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 13 Journal of Medicinal Chemistry Page 14 of 47
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Figure 6. Compounds tested in the [ 3H] GABA uptake assay: Enamine (red) and Drugbank hits 55 56 57 (blue), reference compounds (black frame). 58 59 60 ACS Paragon Plus Environment 14 Page 15 of 47 Journal of Medicinal Chemistry
1 2 3 EXPERIMENTAL TESTING 4 5 6 7 Inhibitory potency of the selected compounds was evaluated by an uptake inhibition assay of 8 9 radiolabeled GABA in HEK cells stably expressing rGAT1. First, the IC 50 value for tiagabine (5) 10 11 12 in the test system was determined to be 0.64 ± 0.07 µM. This was about a factor of 10 higher 13 14 than the value reported for the unspecific rat synaptosome assay used by Andersen et al., but is in 15 16 accordance with data reported for mouse GAT1.37, 62 Subsequently, compounds were measured 17 18 19 at a concentration of 100 µM against 5 as standard. Diazepam (28 ) and tiagabine (5) were used 20 21 as negative and positive control. 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 7. Remaining uptake of [3H] GABA in the presence of 100 µM of the respective 48 49 compound (n=3). 50 51 52 53 As illustrated in Figure 7, one of the commercial screening compounds, 18, weakly reduced 54 55 uptake to just below 80% of saline. One DrugBank substance, 27a (liothyronine, a thyroid 56 57 hormone also known as T3), turned out to significantly inhibit radioligand uptake, which 58 59 60 ACS Paragon Plus Environment 15 Journal of Medicinal Chemistry Page 16 of 47
1 2 3 prompted the acquisition and testing of other commercially available derivatives 27b d. Among 4 5 6 those, the other bioactive hormone levothyroxine ( 27b ) showed reduced uptake, albeit weaker 7 8 than 27a . The representative of the ACE inhibitors and the antihistaminergic drug bepotastine 9 10 11 were essentially inactive. 12 13 14 The IC 50 value for liothyronine derived from a dose response curve (Figure 8) was 13 ±1.7 µM 15 16 (Figure 8), providing a direct link between reported general effects of thyroid hormones on 17 18 63 65 19 GABA uptake, and the inhibitory action of 27a on the GAT 1 subtype. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Figure 8. Inhibition curves of tiagabine (IC 50 : 0.64 ± 0.07 µM, white squares) and liothyronine 41 42 43 (IC 50 : 13.0 ±1.7 µM, black squares). 44 45 46 As illustrated in Figure 9, the pharmacophoric depiction of 27a reveals that one of the required 47 48 hydrophobic features is not, as one would expect, the aromatic ring of the 3,5 diiodophenyl 49 50 51 moiety, but rather a lipophilic iodine substituent, which could barely be deduced from chemical 52 53 similarity measures. Relying on an appropriate position of the second ring relative to the 54 55 interacting iodine atom, this type of interaction is also possible for 27b , but not for 27d . The 56 57 58 3,3',5' substituted variant of the T3 layout is missing the second substituent on the proximal ring 59 60 ACS Paragon Plus Environment 16 Page 17 of 47 Journal of Medicinal Chemistry
1 2 3 which is responsible for inducing the bioactive conformation. 66 This is in line with reported 4 5 6 structure activity relationships of thyroid hormone derivatives both at thyroid hormone receptors 7 8 and GABAergic rat brain synaptosomes.64, 67 Apparently, the steric requirements in both systems 9 10 11 are remarkably similar, relying on correct substitution pattern, stereochemistry, and degree of 12 13 lipophilicity, possibly also limiting the relative efficacy of 27c . The most remarkable difference 14 15 between the observed crystallographic hormone binding mode of 27a (PDB code 3UVV) 68 and 16 17 18 the GAT1 binding hypothesis can be found in the 4' position. The presence of the phenolic 19 20 hydroxyl group is crucial for hormone receptor binding, but not for interaction with the transport 21 22 protein. 23 24 25 26 Regarding compounds 12 to 24 , the most crucial property among those molecules seems to be 27 28 the distance between the positively ionizable group and the first occurrence of lipophilic bulk, 29 30 which is in close analogy to the linker in tiagabine analogs. Distal from the nitrogen atom (as 31 32 33 seen from the negatively ionizable group, which also can be a tetrazole moiety in a reasonable 34 35 distance 69 ), usually just one heavy atom separates the positive charge from the next aromatic 36 37 38 moiety ( 12 , 19 , 20 ), or branching position ( 13 , 21 , 24 ). Alternatively, it is part of separate ring 39 40 system not directly carrying the acidic moiety ( 14 17 , 22 ). With a slightly increased distance 41 42 between the aromatic ring and the carboxylate group, 18 displays some weak activity. 43 44 45 46 This observation also extends to the inactive DrugBank compounds, as the nitrogen atoms of 25 47 48 and 26 are both connected to the hydrophobic part by space demanding linkers, whereas the first 49 50 aromatic ring of thyroid hormones is at a distance of two heavy atoms. 51 52 53 54 The presence of a pyridine moiety known to be unfavorable from the 10 – 11 compound pair 55 56 could further limit the potential of 25 , despite its remarkable structural similarity to the reference 57 58 59 60 ACS Paragon Plus Environment 17 Journal of Medicinal Chemistry Page 18 of 47
1 2 3 compounds. Just as for the bulk of a cyclopentane moiety attached to the polar part of 26 , this 4 5 6 might considerably inhibit optimal positioning in the binding site. 7 8 9 To further assess the degree of similarity between the compounds retrieved and the reference 10 11 12 compound tiagabine, we calculated the Tanimoto similarity values based on chemical 13 70 14 fingerprints (MACCS, FP2, FP4) derived from OpenBabel. The similarity between tiagabine 15 16 and liothyronine as well as the slightly active 18 – on basis of MACCS keys was 25.4 and 17 18 19 51.5%, respectively. Thus, the pharmacophore model performed independently from chemical 20 21 similarity. 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Figure 9. Pharmacophoric fit of 18 and 27a. Features are colored according to Figure 5. 41 42 43 44 45 46 CONCLUSIONS 47 48 49 50 With the increasing number of X ray structures available for transmembrane transporters, 51 52 structure based computational models have provided valuable insights into the molecular basis 53 54 55 of ligand transporter interaction. Within this manuscript, we propose a binding mode of the 56 57 antiepileptic drug tiagabine in GAT1, by including knowledge from ligand based studies into the 58 59 60 ACS Paragon Plus Environment 18 Page 19 of 47 Journal of Medicinal Chemistry
1 2 3 prioritization process for docking poses. Subsequent pharmacophore based virtual screening 4 5 6 followed by experimental testing further confirmed the validity of the pose, by identifying a 7 8 commonly used drug (liothyronine) as an inhibitor of GAT1. Strikingly, liothyronine has been 9 10 11 described long ago as potential GAT inhibitor without major activity on other neurotransmitter 12 63 13 reuptake systems (dopamine, serotonin, choline, aspartate) , but final experimental confirmation 14 15 for subtype GAT1 since has been lacking. Compounds with significantly higher chemical 16 17 18 similarity retrieved in a commercial vendor library all prove inactive, further implying that 19 20 selective transport inhibition of the protein can only be tackled from the side of steric feature 21 22 arrangement. 23 24 25 26 Furthermore, the results indicate that – apart from privileged tricyclic antidepressant scaffolds 27 28 known to more or less unspecifically inhibit neurotransmitter uptake 71, 72 not many drugs on the 29 30 market are likely to interact with GAT1. 31 32 33 34 EXPERIMENTAL SECTION 35 36 37 MODEL BUILDING 38 39 40 41 The GAT1 models were constructed using the crystal structure of LeuT as template which shows 42 43 highest available resolution in an open to out state of the transporter.19 When assessing the 44 45 differences between available sequence alignments of LeuT and hGAT1 (UniProt entries 46 47 48 O67854 and P30531, respectively), no differences for residues in the central binding cavity were 49 50 observed, except for a one residue gap in the middle of LeuT TM10, either placed over GAT1 51 52 G457 21 , S456 20 or A455 11 . As it has been optimized for GAT1 and also is the most recent one, 53 54 55 the alignment of Skovstrup et al. was finally chosen to build the model. Assessment of the 56 57 sequence identity between hGAT 1 and rGAT 1 the first being the effective protein of interest, 58 59 60 ACS Paragon Plus Environment 19 Journal of Medicinal Chemistry Page 20 of 47
1 2 3 the second the one used in cell assays stated 100% sequence identity for the observed core 4 5 6 region and 97.9% for the whole modeled sequence (see Supporting Figure S4). 7 8 9 The crystal structure of LeuT retrieved from the PDB ( www.pdb.org ,73 , accession code 3F3A) 10 11 12 was mutated in silico at position 290 using MOE with a serine side chain orientation 13 14 corresponding to the former glutamic acid. A chlorine ion was placed at the coordinates of the 15 16 previous center of the E290 side chain, then further optimized according to interaction potential 17 18 19 calculations, giving Cl as probe. Tethering the backbone, S290 and its surrounding residues 20 21 were carefully energy minimized for final optimization of the local coordinates. 22 23 24 The models were built using Modeller9v8 in the automodel class, including water molecules, the 25 26 27 chloride ion and two co bound sodium ions as non protein atoms. A disulfide bridge was defined 28 29 between C164 and C173 using a Modeller patch command. 100 models were generated using 30 31 32 very thorough VTFM (variable target function method) optimization, as provided in Modeller. 33 34 Output models were ranked according to DOPE score and the top ten were further assessed by 35 36 the SWISS MODEL server. According to PROCHECK results and Ramachandran plots, models 37 38 39 with disallowed backbone geometries in transmembrane regions were omitted. Hydrogen atom 40 41 assignment and soft energy minimization of the raw models was performed within MOE using 42 43 LigX and the Charmm27 all atom force field, otherwise with default settings.74 44 45 46 75, 76 47 Molecular Dynamics simulations were performed in GROMACS 4.5.3. The selected 48 49 complex was inserted into a pre equilibrated and solvated POPC membrane by applying the 50 51 program g_membed, 77 using the GROMOS 53A6 united atom force field 78 and periodic 52 53 54 boundary conditions. All simulations were performed at 310 K. The system was neutralized by 55 56 adding sodium and chlorine ions to a final salt concentration of 150 mM. Gradually, position 57 58 59 60 ACS Paragon Plus Environment 20 Page 21 of 47 Journal of Medicinal Chemistry
1 2 3 restraints on the main complex were reduced from 500 kJ mol 1 nm 2 (500 ps) to 250 kJ mol 1 4 5 2 6 nm (500 ps). After an equilibration phase without restraints, a fully stable system was achieved 7 8 after 20 ns. Between ns 21 30 of the production run, the frames were clustered according to 9 10 11 RMSD of residues within a radius of 7 Å around the ligand. 10 most diverse snapshots were 12 13 extracted and energy minimized. Two water molecules were kept in the binding site. One 14 15 showed a stable H bond with Y60 (89% occupancy; distance ≤ 3.5 Å; angle ≤ 60 degrees), 16 17 18 another one was directly attached and showed an almost equally stable H bond interaction. 19 20 21 LIGAND PREPARATION 22 23 24 Molecules used for docking were drawn in MOE and processed with CORINA.79 Protonation 25 26 27 states were sampled according to possible states in a physiological pH range of 7.2 +/ 0.2 using 28 29 LigPrep.80 These states were cross checked with the major microspecies calculated by the 30 31 32 ChemAxon web tool chemicalize.org. 33 34 35 DOCKING 36 37 38 Primary placement of tiagabine was done using GLIDE in standard precision (SP) mode using 39 40 41 default settings. The receptor grid was defined around binding site residues 60 63, 64 66, 136, 42 43 140, 294 297, 300, 396 and 400. 44 45 46 Docking and GoldScore / ChemPLP rescoring in the ten MD snapshots were performed with 47 48 49 GOLD 5.0.1, using ChemScore as primary scoring function. Early termination was disabled, 50 51 keeping the 100 best solutions per ligand and snapshot. Two water molecules were set to 'spin' 52 53 54 and 'toggle'. All other settings were set to default. External rescoring was performed using 55 56 57 58 59 60 ACS Paragon Plus Environment 21 Journal of Medicinal Chemistry Page 22 of 47
1 2 3 XScore (XScore) and MOE (London dG, GBVI). Consensus scores were calculated by summing 4 5 6 up indices assigned according to respective ranks within a scoring function. 7 8 9 Secondary docking runs for investigating side chain orientations for specific interactions with 10 11 12 polar linker moieties were performed by a) constraining residues Y139, Y140 and S456 to the 13 14 internal library of allowed rotamers in GOLD and b) for investigating interactions as reported by 15 16 Skovstrup et. al. 2012, likewise rotamer rotations of R69 and F294 were allowed, but with an 17 18 19 additional distance constraint of 1.5 to 3.5 Å (spring constant 5) between the R69 guanidine 20 21 function and the polar linker moiety. 22 23 24 Determination of the potential energy landscape for different dihedral angle configurations was 25 26 81 27 performed with GAUSSIAN 09. After initial geometry optimization with HF/3 21G 28 29 implemented in the software package, configurations with an increment of 15° were calculated 30 31 82, 83 32 using the M06 2X hybrid functional and the 6 31G* basis set. 33 34 35 SCREENING 36 37 38 Pharmacophore models were built using LigandScout 3.0.55, 84, 85 39 40 41 42 For assembling the customized decoy library, 50 decoys per active compound ( 5 11 ) were 43 44 compiled in SMILES format using the DUD E platform (dude.docking.org). The non redundant 45 46 compounds with similar physico chemical properties but dissimilar 2 D topology for each input 47 48 49 line were extracted from the ZINC database. The retrieved set of decoys and the active 50 51 compounds as SMILES were assembled and processed to a LigandScout screening database 52 53 54 using the maximum number of possible conformers. 55 56 57 58 59 60 ACS Paragon Plus Environment 22 Page 23 of 47 Journal of Medicinal Chemistry
1 2 3 The DrugBank database was downloaded from the web site www.drugbank.ca and consisted of 4 5 6 1,491 entries (version of June 2013). Enamine Advanced and HTS screening collections were 7 8 obtained from the download site at www.enamine.net (n= 1,719,682; version 032013). 9 10 11 Counterions were removed using the Software MOE. LigandScout command line modules 12 13 idbgen and iscreen were used for conformation generation and for performing the 14 15 pharmacophore screening. Training set, decoy compilation and DrugBank compounds were 16 17 18 prepared using OMEGA best settings (max. 500 conformations), Enamine Advanced and HTS 19 20 databases were compiled with OMEGA fast settings (max. 25 conformations) 21 22 (www.eyesopen.com, 86 88 ). 23 24 25 26 Path based (FP2) and substructure based (MACCS, FP4) similarity fingerprinting was performed 27 28 using OpenBabel 2.3.1. 29 30 31 32 POSE FILTERING 33 34 60 35 Primary checking for pan assay interference (PAINS) compounds was done by uploading 36 37 retrieved virtual screening hits to the PAINS remover web service (available at 38 39 40 http://cbligand.org/PAINS), returning no suspicious compounds. Protein Ligand interaction 41 42 fingerprints of docked virtual hit compounds were calculated in MOE. Poses missing the bin for 43 44 Na1 interaction were removed. 45 46 47 48 PHARMACOLOGICAL TESTING 49 50 51 Screening compounds were purchased from Enamine (Enamine Ltd., Riga, Latvia), 52 53 54 Fluka/Sigma Aldrich (Sigma Aldrich Co., Saint Louis, MO) and AvaChem (AvaChem 55 56 57 58 59 60 ACS Paragon Plus Environment 23 Journal of Medicinal Chemistry Page 24 of 47
1 2 3 Scientific, San Antonio, TX), all with a purity 95% (see Supporting Table S4). Tiagabine was 4 ≥ 5 6 obtained from Sanofi Synthelabo, Montpellier, France. 7 8 9 Cell lines of HEK293 cells stably expressing YrGAT 1 were generated as described elsewhere. 8 10 11 4 12 Cloned cells (~4 x10 cells / well) were seeded and grown at 37° on poly (D) lysine coated 13 14 standard plasticware 24 hours in advance. 15 16 17 Uptake of [3H] GABA into was measured in the presence of 100 µM of the compounds, while 18 19 20 unspecific uptake was defined as uptake in the presence of 100 µM tiagabine. For IC 50 21 22 determination tested concentrations were: 5:0.001, 0.01, 0.1, 0.3, 1, 10, 100 µM; 27a: 0.01, 0.1, 23 24 3 25 0.3, 1, 3, 10, 30, 100 µM. After 3 minutes preincubation, [ H] GABA (35 Ci/mmol, 26 27 PerkinElmer, Boston, MA) in a final concentration of 0.015 µM was added. Uptake was stopped 28 29 by adding ice cold Krebs HEPES buffer (10 mM HEPES adjusted to pH 7.4 with 35.9 mM solid 30 31 32 NaOH, 120 mM NaCl, 3 mM KCl, 2 mM CaCl 2, 2 mM MgSO 4 and 2 mM D glucose as 33 34 supplement). Cells were lysed with 1% SDS (sodium dodecyl sulfate) solution, taken up in 2 mL 35 36 scintillation cocktail (Rotiszint® Eco Plus, Carl Roth GmbH, Karlsruhe, Germany) and counted 37 38 39 in a standard liquid scintillation counter (Packard TriCarb 2300TR, Packard Instruments). At 40 41 least three independent experiments per compound were performed, each in triplicates. Data 42 43 analysis was performed by nonlinear regression using Prism 6.01.89 44 45 46 47 ASSOCIATED CONTENT 48 49 50 Supporting Information 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 24 Page 25 of 47 Journal of Medicinal Chemistry
1 2 3 Additional tables and figures illustrating model selection criteria, backbone RMSD of the MD 4 5 6 production run as well as compound inhibition and purity data. This material is available free of 7 8 charge via the Internet at http://pubs.acs.org. 9 10 11 AUTHOR INFORMATION 12 13 14 Corresponding Author 15 16 Phone: +43 4277 55110. Fax: +43 4277 9551. E mail: [email protected] 17 18 19 Notes 20 21 22 The authors declare no competing financial interest. 23 24 25 ACKNOWLEDGMENTS 26 27 The computational results presented have in part been achieved using the Vienna Scientific 28 29 30 Cluster (VSC). We thank Peter Wolschann for his support with quantum chemical calculations, 31 32 Martin Kratzel for kindly providing D and L Thyroxine samples, and Christina Dangl for 33 34 additional HPLC analyses. We acknowledge financial support provided by the Austrian Science 35 36 37 Fund, grants F35 and W1232. We are grateful to Inte:Ligand and OpenEye for providing us with 38 39 licenses for their software packages. 40 41 42 ABBREVIATIONS 43 44 GABA R, GABA receptor subtype A; GAT, GABA transporter; HEPES, (4 (2 hydroxyethyl) 45 A 46 47 1 piperazineethanesulfonic acid ); MACCS, molecular access system; NSS, 48 49 neurotransmitter:sodium symporter; PAINS, pan assay interference compounds. 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 25 Journal of Medicinal Chemistry Page 26 of 47
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1 2 3 87. Hawkins, P. C.; Skillman, A. G.; Warren, G. L.; Ellingson, B. A.; Stahl, M. T. Conformer 4 5 6 generation with OMEGA: algorithm and validation using high quality structures from the Protein 7 8 Databank and Cambridge Structural Database. J. Chem. Inf. Model. 2010, 50 , 572 584. 9 10 11 88. Hawkins, P. C.; Nicholls, A. Conformer generation with OMEGA: learning from the data 12 13 set and the analysis of failures. J. Chem. Inf. Model. 2012, 52 , 2919 2936. 14 15 89. Prism version 6.01 , GraphPad Software, San Diego, CA: 2012. 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 37 Journal of Medicinal Chemistry Page 38 of 47
1 2 3 for Table of Contents use only 4 5 6 7 8 A binding mode hypothesis of tiagabine confirms liothyronine effect on γ aminobutyric 9 10 11 acid transporter 1 (GAT1) 12 13 1 1 2 2 2 1 14 Andreas Jurik , Barbara Zdrazil , Marion Holy , Thomas Stockner , Harald H. Sitte , Gerhard F. Ecker* 15 16 1 17 University of Vienna, Department of Pharmaceutical Chemistry, Althanstraße 14, A 1090 Vienna, Austria. 18 19 2Medical University of Vienna, Institute of Pharmacology, Center of Biomolecular Medicine and Pharmacology, 20 21 Währingerstraße 13a, A 1090 Vienna, Austria. 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment 38 Page 39 of 47 Journal of Medicinal Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 GABA, conformationally restricted analogs and lipophilic aromatic derivatives. 27 65x37mm (300 x 300 DPI) 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Journal of Medicinal Chemistry Page 40 of 47
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Chemical structures and literature IC50 values of ligands with key modifications in linker length, polarity, 28 and rigidity of the aromatic moiety.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Docking poses of tiagabine (turquoise) and analogs (orange) in ten MD snapshots of the hGAT-1 model. 38 Polar regions in blue, hydrophobic areas in yellow. 39 40 63x57mm (300 x 300 DPI) 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Journal of Medicinal Chemistry Page 42 of 47
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Dihedral energy landscape of 5-methyl-thiophen dihedral angles; configuration of the 21 docking pose of 5 is marked in yellow. Constrained aromatic system of 7 is highlighted in red. 22 Calculations were performed with GAUSSIAN 09. 53 23 66x25mm (300 x 300 DPI) 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 43 of 47 Journal of Medicinal Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 Pharmacophore model of tiagabine: Hydrophobic (yellow), positive (blue) and negative (red) ionizable features in context with the GAT1 substrate binding site. 39 77x71mm (300 x 300 DPI) 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Journal of Medicinal Chemistry Page 44 of 47
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Compounds tested in the [ 3H]-GABA uptake assay: Enamine (red) and Drugbank hits (blue), reference 46 compounds (black frame). 47 177x203mm (300 x 300 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 45 of 47 Journal of Medicinal Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Remaining uptake of [ H] GABA in the presence of 100 µM of the respective compound (n=3). 25 90x45mm (300 x 300 DPI) 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Journal of Medicinal Chemistry Page 46 of 47
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Inhibition curves of liothyronine (IC : 13.0 ±1.7 µM, black squares) and tiagabine 34 50 (IC 50 : 0.64 ± 0.07 µM, white squares) 35 66x52mm (600 x 600 DPI) 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment Page 47 of 47 Journal of Medicinal Chemistry
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 Pharmacophoric fit of 18 and 27a . Features are colored according to Figure 5. 32 54x38mm (300 x 300 DPI) 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment