Structure Modeling of Γ-Aminobutyric Acid Transporters – Molecular Basics of Ligand Selectivity

International Journal of Biological Macromolecules 158 (2020) 1380–1389

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Structure modeling of γ-aminobutyric acid transporters – Molecular basics of ligand selectivity

Kamil Łątka, Jakub Jończyk, Marek Bajda ⁎

Jagiellonian University Medical College, Faculty of Pharmacy, Department of Physicochemical Drug Analysis, 30-688 Cracow, Medyczna 9, Poland article info abstract

Article history: γ-Aminobutyric acid transporters are responsible for regulating the GABA level in the synaptic cleft. In this way, Received 31 March 2020 they affect GABA-ergic transmission which is important for the proper functioning of the central nervous system. Received in revised form 28 April 2020 The exact structure of GABA transporters is still unknown, which hinders the design of new, potent and selective Accepted 29 April 2020 inhibitors. For these reasons, we decided to create models of all types of human gamma-aminobutyric acid trans- Available online 3 May 2020 porters. They were built based on crystal structures of related proteins from the SLC6 family using homology ﬁ Keywords: modeling methods. The reliability of the received models has been con rmed by a number of tools assessing GABA transporters the quality of protein models. To determine the ligand binding mode and indicate the amino acids responsible Structure modeling for selectivity, docking studies and molecular dynamics simulations were performed. The amino acids lining Inhibitor binding the bottom of the main binding site have a major impact on the selective ligand binding. In addition, an important element is the non-helical fragment of the transmembrane domain 10, and several amino acids within the vestibule of the transporters, which affect its volume. To check whether obtained models are suitable to distinguish active compounds from inactive ones, enrichment plots were prepared. Results suggest that our models may be useful in the search for new inhibitors of GABA transporters of the desired selectivity. © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/).

1. Introduction transporter inhibitors in the therapy appears to be large, the tiagabine (selective GAT-1 inhibitor) is solely used as a medicine [4,21]. All four γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter GATs belong to the SLC6 family of sodium-dependent membrane trans- in the central nervous system [1]. It plays many functions acting on porters. Transport of γ-aminobutyric acid is coupled with the transport + − GABAA, GABAB and GABAC receptors [2]. After being released from the of Na (and also Cl ) ions, which is a driving force for substrate trans- presynaptic axons to the synaptic cleft, it is reabsorbed by four types location against chemical gradient [22]. Obtaining the crystal structures of GABA transporters [3]. In humans, they are numbered in the follow- of the leucine (LeuT), dopamine (DAT) and serotonin (SERT) trans- ing way: GAT-1, BGT-1, GAT-2, and GAT-3, while mouse transporters porters provided information on the general structure of the proteins are designated sequentially: GAT-1, GAT-2, GAT-3 and GAT-4 [4]. from the whole SLC6 family (Fig. 1)[23–26]. Gamma-aminobutyric acid (GABA) transporters are heterogeneously The SLC6 transporters consist of 12 α-helical transmembrane do- distributed in various areas of the central nervous system (CNS) and mains, with N- and C- terminus located intracellularly. Each domain many vital organs [5]. GAT-1 and GAT-3 are expressed mostly in the contains about 20 hydrophobic amino acids. The extracellular surface CNS, however, their cellular localization is different. GAT-1 generally oc- of the transporters is formed by long EL2, EL4, and EL6 loops. The EL4 curs at presynaptic endings of neurons, while GAT-3 is present in the loop has helical fragments, which form a V-shaped structure. These membrane of glial cells in the vicinity of synapses [3,6]. BGT-1 and transporters have two different binding sites (S1 and S2). The central GAT-2 are distributed mainly in the peripheral tissues, however, their binding site (S1) is the inner ring, formed by domains TM1, TM3, TM6, expression in the brain has also been found [5]. It has been shown and TM8. The surface of the S1 pocket is lined by both polar and hydro- that enhancing GABA-ergic transmission by inhibition of GABA reup- phobic amino acid residues. It binds substrate and two sodium ions. In take can be effective in the treatment of many diseases, such as epilepsy, transporters dependent on Cl− (including transporters for GABA), the anxiety, depression, pain, and to a lesser extent also in insomnia, stroke chloride ion is accommodated close to one sodium ion. The S2 site is lo- and Alzheimer's disease [7–20]. Although the possibility of using GABA cated at the bottom of the extracellular vestibule, separated from the S1 site by the lower part of the extracellular gate created in the case of aLeuT, dDAT, hSERT and GATs by phenylalanine and tyrosine residues. ⁎ Corresponding author. The upper part of the extracellular gate is formed by polar residues of ar- E-mail address: [email protected] (M. Bajda). ginine and aspartic or glutamic acid [23,25,26].

https://doi.org/10.1016/j.ijbiomac.2020.04.263 0141-8130/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 1380–1389 1381

EL4 2. Results and discussion EL2 TM3 TM7 2.1. Model building and evaluation

TM1 Models of the GABA transporter were built applying homology TM8 modeling. Considering the similarity of the amino acid sequence, the best templates for building GAT models seem to be the crystal structures of dopamine and serotonin transporters (Table 1). On the other hand, EL3 the similarity between the substrates for analyzed targets and for avail- EL6 able homologous proteins with known structure is also important. In this case, transporters for leucine, which is structurally closer to GABA TM6 than dopamine and serotonin, seem to be appropriate. Considering all the above, it was decided to use all dDAT, hSERT, and aLeuT crystal structures as templates. Another challenge is the selection of a suitable conformational state of the protein. To transfer substrates from the ex- TM2 tracellular environment to the inside of the cell, SLC6 transporters change their conformation from the outward-open through the occluded to the inward-open state. Observations of DAT and SERT crystal TM11 structures [24–26,32], as well as the results of GATs mutagenesis studies [33–39], indicate that most of the inhibitors block transporters in the TM12 outward-open state. To obtain GABA transporter models that can be used to investigate the amino acids responsible for the selectivity of the ligand binding, the choice of crystals in the outward-open state is IL5 preferred. However, the transition of the transporter from one state to another is a dynamic process that can be stopped at a different moment, depending on the ligand size and its binding mode. To be sure that the TM10 optimal conformation was not omitted, we used templates in both C-terminus IL1 outward-open and occluded states. Considering all the above issues as well as the resolution of available crystals and differences in the residue conformation in the binding sites, finally, 10 templates were selected: Fig. 1. The general structure of transporters from the SLC6 family, based on the example of 4XP4, 4XP9, 4XPB (dDATs in the outward-open state), 4XPH (dDAT in Drosophila melanogaster DAT (dDAT) crystal structure (PDB code: 4XP9). the partially-occluded state), 5I6X (hSERT in the outward-open state), 2A65, 2Q6H (aLeuT-s in the occluded state), 3F3A (aLeuT in the outward-open state), 4MM7, 4MMB (LeuBAT - aLeuT with human Comparing GABA transporters and transporters with the known monoamine transporter-like pharmacology by mutating key residues crystal structure, it can be observed that their amino acid sequence, es- within the primary binding pocket). We built the models of GABA trans- pecially within transmembrane domains, is very similar [27]. This is par- porters using the Modeller program and the SWISS-MODEL server. We ticularly evident when comparing GATs with dDAT and hSERT, which took the alignment presented by Beuming [27] and this generated auto- have approximately 60% and 58% sequence similarity, respectively matically by the SWISS-MODEL, introducing minor changes (see (Table 1). The SLC6 transporters share the general structure. However, Methods section and Fig. 1S, Supporting information). In order to eval- the exact structure of GABA transporters, including their binding sites uate the quality of the generated models, various tools were used: is still unknown. This is one of the main difficulties in finding selective, QMEAN, DOPE score, Verify3D and Ramachandran plots. These tools potent inhibitors of these proteins. Literature reported the work on check a number of different parameters important from the view- modeling of the single types of GABA transporters or described the point of the correctness of built models and their similarities to ac- binding mode only for small molecule compounds [28–31]. There tual protein structures. Generally, GAT models built on dDAT and have been no comprehensive studies that present structural differences hSERT templates were higher rated than those built on aLeuT. It is affecting the selectivity of particular types of GAT. Therefore, we decided not surprising, given the higher degree of amino acid sequence sim- to build new models of four types of GAT (GAT-1, BGT-1, GAT-2, GAT-3) ilarity with GABA transporters. All of these models constructed on to fully characterize their structure, determine the binding mode of monoamine transporters have over 90% of the amino acids in the known ligands and indicate the elements responsible for the selectivity most favored regions in the Ramachandran plot (Fig. 2S, Supporting of these transporters. information), and almost have over 80% amino acids with score ≥0.2 according to Verify 3D. This indicates the high quality of these models which is also confirmed by good QMEAN and DOPE score values. Considering the last two assessment tools, models built Table 1 with SWISS-MODEL generally have higher ratings than those built The amino acid sequence identity and similarity for GABA transporters and the homolo- with the Modeller program. It can be also seen that the best models gous proteins with known structure. for each type of GABA transporter were built on 4XP9, 4XP4 and GABA aLeuT dDAT hSERT 4XPH templates (Table 1S and Fig. 2S, Supporting information). Im- transporter type portantly, this was later reflected in the docking studies. Although Identity Similarity Identity Similarity Identity Similarity [%] [%] [%] [%] [%] [%] models built on aLeuT templates were lower-rated, also in their case at least 90% of the amino acids have psi (ψ)andphi(φ)values hGAT-1 22.1 38.4 41.4 61.5 40.1 59.7 hBGT-1 23.1 39.4 41.7 59.4 39.6 58.6 in the favored range. Moreover, according to QMEAN and DOPE hGAT-2 23.1 40.1 43.1 61.1 40.4 57.7 score, the lowest-rated fragments were those that do not directly hGAT-3 21.5 40.0 41.6 58.1 40.4 58.4 participate in ligand binding, i.e. extra- and intracellular loops and aLeuT – leucine transporter from Aquifex aeolicus;dDAT– dopamine transporter from Dro- domain 12. Therefore, these models were also taken into account in sophila melanogaster;hSERT– human serotonin transporter. docking studies. 1382 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 1380–1389

2.2. Structure of the GABA transporters model and with corresponding amino acids in BGT-1, GAT-2 and 3 models. Most of the cysteine mutants, sensitive to the sulfhydryl re- The general structure of the generated models for each type of GABA agent are those containing cysteine residues at the transporter entrance transporter is very similar to other proteins from the SLC6 family, as was and in the S2 site. On the other hand, most of the insensitive cysteine mentioned earlier (Fig. 1). Amino acid residues important from the mutants include cysteines whose side chains are directed to the trans- viewpoint of transporter function are particularly conserved. These in- porter interior, and which do not meet the extracellular environment. clude the residues that create the extra- and intracellular gates which During the transition to the occluded state, TM1b and TM6a approach regulate the access to the binding site during the transport cycle. In all towards TM3 and TM10, closing access to the binding sites and the ves- types of GABA transporters the extracellular gate consists of two pairs tibule [41]. In the cited study, reaction with the sulfhydryl reagent was of amino acids: tyrosine (140 in GAT-1, 133 in BGT-1, 129 in GAT-2, found to be inhibited in the presence of GABA, which can be explained 147 in GAT-3) and phenylalanine (294 in GAT-1, 293 in BGT-1, 288 in by shifting the balance towards the occluded and inward-open states, GAT-2, 308 in GAT-3); arginine (69 in GAT-1, 61 in BGT-1, 57 in GAT- and thus difficult access of the reagent to mentioned residues. The pres- 2, 75 in GAT-3) and aspartic acid (451 in GAT-1, 452 in BGT-1, 447 in ence of transporter inhibitor increases the sensitivity of amino acids lo- GAT-2, 467 in GAT-3). The intracellular gate is built mainly of two cated at the entrance to the transporter, which indicates that it blocks polar amino acids: arginine (44 in GAT-1, 36 in BGT-1, 32 in GAT-2, 50 the transporter in outward-open state, facilitating access to these in GAT-3) and aspartic acid (410 in GAT-1, 409 in BGT-1, 404 in GAT- amino acids. At the same time, the inhibitor by binding within S1/S2 2, 424 in GAT-3). Mutagenesis studies for GAT-1 showed that the re- sites, protected the amino acids found therein. These observations are placement of ARG69 and ASP451 with lysine and glutamic acid, respec- consistent with the results of our docking studies described later. Simi- tively, led to complete blockade of GABA transport. Similar impairment lar research as for the TM6 and TM10 was also performed for the TM8 of transporter function was observed for the mutation of the amino [39]. During the transition of the transporter from the occluded to the acids forming the intracellular gate [40]. Other conserved amino acid inward-open state the TM1a bends allowing the release of the substrate residues are those involved in binding of ions, especially the sodium and ions into the cell [41]. In this state, the internal part of TM8 lines a ones. As in the case of aLeuT, dDAT and hSERT crystal structures, also cytoplasmic accessibility path into S1 site. In the case of GAT-1, it was in models of GATs Na1 and Na2 ion complexes have octahedral and tri- showed that the presence of inhibitor (SKF100330A) reduces the sensi- gonal bipyramid geometry, respectively. Study on GAT-1 revealed that tivity of CYS399 and mutants E402C, T406C to the reaction with sulfhy- mutants N66C (amino acid residue involved in Na1 ion binding), dryl reagent. Based on our models it can be observed that the side chains S396C and D395C (amino acid residues involved in Na2 ion binding) of these residues in GAT-1 and corresponding amino acids in BGT-1, were not able to transport GABA [33,39]. In aLeuT crystals, the Na1 ion GAT-2, 3 are available to the intracellular environment only in the is coordinated i.a. by the carboxyl group of the substrate (leucine). In ad- inward-open state, which explains the protective effect of the inhibitor. dition, this group forms hydrogen bonds with GLY26 and TYR108. GABA Comparing the spatial structure and properties of the main binding transporters have a very similar structure in this fragment of the main site (S1) in all types of GABA transporters, it can be observed that in binding site (S1). Studies on GATs have shown that any modification the case of GAT-1 the S1 site is the most hydrophobic. On the other of amino acids within this area leads to severe impairment of trans- hand, in BGT-1 the central binding site is the most polar due to the ac- porter functions, which confirms that these residues are involved in cumulation of negative electrostatic potential (Figs. 2, 3). The GAT-2 binding and transfer of substrates also in the case of GABA transporters and GAT-3 transporters have very similar electrostatic properties. [31,33,34]. These differences are mainly caused by three amino acids forming the For the proper functioning of transporters, conformational changes bottom of the S1 site: TYR60, LEU300, and SER133 in hGAT-1, and the of individual amino acids and changes in the arrangement of entire do- corresponding GLU52, GLN299, and GLU126 in hBGT-1; GLU48, mains are necessary. In the outward-open state, the entrance to the LEU294, and VAL122 in hGAT-2; GLU66, LEU314, and GLU140 in binding site is formed i.a. by the external parts of TM6 and TM10. The hGAT-3 (Figs. 2B, 3). Comparing the structure of GABA and monoamine extracellular availability of these domains in GAT-1 was examined transporters (MATs), it can be observed that one of the pockets within using reaction of the cysteine mutants with sulfhydryl reagents the S1 site, where aromatic rings of DATs and SERTs inhibitors bind, is [35,38]. The sensitivity of individual cysteine residues to reagent was tighter in the case of GATs. This is due to the replacement of GLY425 generally consistent with the distribution of these residues in GAT-1 in dDAT and GLY442 in hSERT with THR400 in hGAT-1, and with

A) TM3 B) TM3

TM8 TM1 TM1 TM10

TM8

TM12

TM6 TM6 TM11 TM10

Fig. 2. A) Amino acids forming the vestibule (A) and the main binding site S1 (B) of GABA transporters. Bright navy – hGAT-1; green – hBGT-1; orange – hGAT-2; blue – hGAT-3. Sodium ions were presented as purple spheres. K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 1380–1389 1383

Fig. 3. Electrostatic potential maps for the S1 binding site of each GABA transporter type. Red color - accumulation of negative electrostatic potential; blue color - accumulation of positive electrostatic potential.

CYS399, 394, 414 in hBGT-1, hGAT-2, hGAT-3, respectively, and to a These compounds are mostly derivatives of nipecotic acid (like lesser extent through replacement of VAL120 in dDAT and ILE172 in tiagabine) or guvacine with activity against hGAT-1 or hGAT-2 and hSERT with LEU136, 129, 125, 143 in hGAT-1, hBGT-1, hGAT-2, hGAT- hGAT-3 depending on their absolute configuration and the structure 3, respectively. The decrease of the volume of this pocket within the of the attached lipophilic fragments. We also took into account the main binding site of GATs was reflected in the results of the docking group of small-molecule GABA analogs that act selectively on hBGT-1 studies. transporters (Table 2S, Supporting information). We assumed that the In the case of the transporter vestibule, the non-helical fragment in carboxyl group of these ligands should interact with a sodium ion, lo- the middle of the domain 10 seems to be important from the viewpoint cated in the S1 binding site, in a similar way as the carboxyl group of leu- of selective binding of substrates and ligands (Fig. 2A). Compared to the cine (substrate) and tryptophan (inhibitor) in aLeuT crystal structures transporters with known structure, GABA transporters contain one ad- [23,44]. This is suggested by the structural similarity of both substrates ditional amino acid within this fragment. Therefore, it is difficult to - leucine and GABA, the similar structure of their binding sites in the model and there is a large diversity in its structure among generated transporters, the mutagenesis studies of GATs [31,33,45] and the fact models. Moreover, the amino acid sequence of the particular types of that some of these ligands were found to be competitive or mixed- GABA transporters differs from one another in this fragment. Its se- type inhibitors [46–48]. Initial docking allowed to select the best models quence involves SER-ALA-SER-GLY for hGAT-1, ALA-SER-SER-GLY for which for all types of GABA transporters turned out to be those built on hBGT-1, and ALA-ALA-SER-GLY for hGAT-2 and hGAT-3. In the case of 4XP4 and 4XP9 templates by SWISS-MODEL server. Then chosen com- hGAT-1, it has been demonstrated that this fragment of TM10 provides plexes were optimized in order to achieve greater coherency of the li- extra bulk that is required for rigorous gating and tight binding of the gand poses and to improve created interactions. In this way, we substrates and ions. Changing the flexibility of this loop by replacement obtained the final models of complexes for derivatives with amino of GLY457 with alanine, leucine, cysteine, and proline successively re- acid structure. For hGAT-1, hGAT-2 and hGAT-3 they were built based sulted in complete inhibition of GABA transport [42]. In the hGAT-3 on the 4XP9 template, while for BGT-1 based on 4XP4. We confirmed transporter, the space above the described loop is enlarged, compared the stability of the obtained complexes together with the key interac- to other GATs, as a result of replacing the tyrosine residue (TYR452 in tions by molecular dynamics simulations. hGAT-1, TYR453 in hBGT-1, TYR448 in hGAT-2) with serine residue In the case of the GAT-1 transporter, the carboxyl group of tiagabine (SER468). On the opposite side, near the entrance to the vestibule, forms an ionic interaction with Na+ ionaswellashydrogenbondswith hGAT-3 possesses a phenylalanine residue (PHE531) instead of the ser- GLY65 and TYR140 (Fig. 4A). The protonated nitrogen atom of the pi- ine residue found in other types of GABA transporters (SER515 in hGAT- peridine ring creates a hydrogen bond with the amide oxygen atom of 1, SER516 in hBGT-1, SER511 in hGAT-2). These two changes have a sig- the main chain of PHE294. This interaction was observed only for the nificant impact on the structure of the vestibule in hGAT-3. In the case of R-isomer of tiagabine (and its analogs) which explains its higher activity hGAT-2, the space above the main binding site is slightly enlarged by relative to S-isomer. The aromatic rings of tiagabine are located in the S2 the replacement of isoleucine with valine residue (VAL132). Interest- site, forming aromatic and hydrophobic interactions mainly with ingly, this small change is necessary for the proper functioning of the PHE294, TRP68, TYR140 and 139, as well as with ILE143. Inhibitors hGAT-2 transporters. Mutant V132I was completely unable to transport from the group of tiagabine analogs occupy both S1 and S2 sites. The GABA [31]. Another noticeable difference within the vestibule is the presented arrangement is different from that observed for monoamine presence of a lysine residue in the hGAT-1 (LYS448) instead of a gluta- transporter inhibitors which are generally located entirely at the main mine residue (GLN449 in hBGT-1, GLN444 in hGAT-2, GLN464 in binding site (S1) [24–26,32]. This can be explained by the fact that the hGAT-3). The possibility of interaction of the lysine residue with the S1 site of GABA transporters has a smaller volume in comparison with aspartic acid residue (ASP451) from the extracellular gate can change MATs. the local structure of this part of hGAT-1. Interestingly, in hGAT-1 muta- Additionally, in monoamine transporters, the aromatic rings of in- genesis studies the replacement of LYS448 with glutamine residue, to- hibitors can create beneficial π – π or CH – π interactions with the aro- gether with replacement of GLN441 with glutamic acid and ILE444 matic ring of PHE325 in dDAT or PHE341 in hSERT, which are replaced with methionine, amino acids found in hGAT-3, increased the affinity by GLN299 in hBGT-1 and by LEU300, 294, 314 in hGAT-1, hGAT-2 of hGAT-1 to β-alanine, which is the substrate of hGAT-3 [43]. and hGAT-3, respectively. These differences explain why for GABA transporters arrangement of lipophilic fragments of the ligands within 2.3. Ligand binding studies the S2 site is more probable than within the S1 site. The results described here are in line with the work of other authors [28,49]. The reli- To find the models that reflect the structure of binding site the best, ability of the presented binding mode of tiagabine was confirmed by establish the binding mode of ligands and finally indicate the amino acid 10 ns molecular dynamics simulation, during which both the position residues responsible for the selectivity of the particular types of GATs, of the entire compound and the key interactions remained very stable we performed docking studies along with molecular dynamics simula- (Fig. 5A and B). A very similar, consistent binding modes with hGAT-1 tions. Firstly, we docked a group of highly active and selective inhibitors were obtained for most analogs of tiagabine (derivatives of nipecotic of GABA transporters, with an amino-acid structure, to all built models. acid and guvacine) and also for exo-THPO derivatives in which nipecotic 1384 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 1380–1389

Fig. 4. Binding mode of the: A) diaryl derivatives of R-nipecotic acid and guvacine in hGAT-1; B) small molecule GABA analogs in hBGT-1; C) and D) triaryl derivatives of S-nipecotic acid in hGAT-2 and hGAT-3, respectively. acid was replaced by its bioisoster (Fig. 3S in Supporting information). group of GLN299 approached the amino group of the ligand again, Also, in this case, it was possible to explain the varied activity of isomers close enough to form a hydrogen bond mediated by the water mole- - the presented beneficial interactions with Na+, GLY65, TYR140, as well cules present between them (Fig. 5C). Moreover, this residue was in- as with PHE294, were observed only for the R isomers. volved in forming the network of interactions between GLU126 As mentioned earlier, the S1 site in the BGT-1 transporters is much (direct H-bond) and GLU52 (water-mediated H-bond), maintaining more polar compared to the one in GAT-1, which is mainly caused by re- the conformation of these residues favorable for ligand binding. Given placing TYR60 with GLU52, LEU300 with GLN299 and to a lesser extent this observation and the fact that GLN299 residue is present only in SER133 with GLU126. This explains why the strongest, selective BGT-1 hBGT-1, it appears to play a key role in the selectivity of this transporter. inhibitors are small hydrophilic compounds. Fig. 4B presents the bind- The S1 binding site for GAT-2 and GAT-3 transporters is almost iden- ing mode of such compounds. The carboxyl group of inhibitors is located tical. The only significant change concerns the replacement of VAL122 in in an analogous way as in the case of GAT-1, creating bonds with GLY57, GAT-2 with GLU140 in GAT-3. Therefore, ligands that have a high affin- TYR133 and sodium ion while the protonated amino group forms a salt ity for GAT-3 usually also have a strong effect on GAT-2. The results of bridge with GLU52 and hydrogen bond with GLN299. Interactions with docking studies show that inhibitors are located in an almost identical a sodium ion, GLY57 and GLU52 were very stable throughout 15 ns mo- way in both GAT-2 and GAT-3 models (Figs. 4C and D, 5D). Their car- lecular dynamics simulation (Fig. 5C). The hydroxyl group of the boxyl groups interact similarly as in the case of hGAT-1 and hBGT-1 in- TYR133 side chain has moved away from the ligand carboxyl group. hibitors. During molecular dynamics simulations both in hGAT-2 and However, during the simulation, the water molecules (one or two) hGAT-3 the hydrogen bond between the hydroxyl group of TYR129 formed a network of hydrogen bonds between them. Similar interac- (hGAT-2) or TYR147 (hGAT-3) and ligand became broken. In mutagen- tions were observed for the carboxyl group of tryptophan in the aLeuT esis studies on GAT-1, it was shown that the replacement of TYR140 (crystal structure PDB code: 3F3A). At the beginning of the molecular with phenylalanine led to a complete blockage of transport. However, dynamics simulation, hydrogen bond with GLN299 became also broken the inhibitor SKF100330A, which is a guvacine derivative, retained as a result of changing the conformation of this residue side chain. How- some affinity for the transporter, as measured by the ability to block ever, over the course of molecular dynamics simulation, the amide sodium-dependent charge movements [34]. This indicates that K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 1380–1389 1385

Fig. 5. A) RMSD changes for the ligands in the course of molecular dynamics. Distance changes between amino acid residues and ligand functional groups involved in key interactions, during molecular dynamics, for B) hGAT-1; C) hBGT-1; D) hGAT-2 and hGAT-3. Ligands used in the simulations: hGAT-1 – tiagabine (1); hBGT-1 - (1S,2S,5R)-5-aminobicyclo[3.1.0] hexane-2-carboxylic acid (26); hGAT-2 and hGAT-3 – DDPM-1457 (36) (numbers in brackets from Table 2S, Supporting information).

hydrogen bond with mentioned tyrosine residues, although affecting A large number of GABA transporter inhibitors have a non-amino the ligand potency, is not necessary for its binding and may partly ex- acid structure. There are i.a. derivatives of piperidin-4-ol, 4- plain the molecular dynamics results. The arrangement of the piperi- aminobutanamide, and 4-hydroxybutanamide. These compounds gen- dine fragment is different from that observed for inhibitors in hGAT-1. erally show activity on all types of GABA transporters, although depend- In the case of active S-isomers, the protonated nitrogen atom is directed ing on the substituents preferential inhibitors may be indicated [51–54] towards the bottom of the S1 site, forming an ionic interaction with (Table 2S, Supporting information). To determine their binding mode, GLU66 (hGAT-3) or GLU48 (hGAT-2). Such a preferred binding mode we docked selected representatives to each type of GABA transporter, was not observed in the case of R-isomers, which explains their lower using the induced fit docking. We took the models that proved to be activity. The stability of this interaction was confirmed in molecular dy- the best in earlier docking of amino acid compounds, i.e.: hGAT-1, 2, 3 namics simulations (Fig. 5D). It is worth mentioning that in GATs, amino models built on a 4XP9 template and hBGT-1 on a 4XP4 template, all acids located in the position of GLU66 have a large impact on GABA generated with SWISS-MODEL server. Analyzing the docking results, binding as demonstrated in mutagenesis studies [31,50]. The extensive we rejected rare poses and those in which the carbazole ring or lipophilic fragments of hGAT-2 and hGAT-3 inhibitors are arranged diphenyl fragment was located inside the polar S1 site in hBGT-1, find- within the S2 site. The three aromatic rings interact mainly with ing them unreliable just like Vogensen et al. [55]. From all complexes, TRP74 (TRP56 in hGAT-2), PHE308 (PHE288 in hGAT-2), TYR147 we selected those that appeared the most often. Then, we docked to (TYR129 in hGAT-2), TYR146 (TYR128 in hGAT-2). Interestingly, such them a larger group of compounds. Finally, we chose the models in a favorable arrangement occurred mainly in the hGAT-3 and hGAT-2. which the poses were the most coherent. The presented binding In the case of hGAT-1 and hBGT-1, the aromatic fragments of the com- modes (Fig. 6)wereconfirmed by molecular dynamics simulations pounds are directed towards the S1 site, and the nipecotic acid moiety (Fig. 7A). reaches aspartic acid and arginine from the extracellular gate, losing In the case of hBGT-1 transporters, carbazole or ethoxycarbonylo-β- the possibility of interaction with the sodium ion. This explains the carboline fragments of piperidin-4-ol derivatives are located in the ves- lack or low activity of these compounds on hGAT-1 and hBGT-1 trans- tibule (S2 site), creating hydrophobic interactions mainly with PHE293, porters. Several elements may be responsible for the difference in ligand TYR453, and TYR520 (Fig. 6A). In hGAT-3 transporters, these aromatic arrangement. The previously described ionic interaction of the proton- moieties are shifted slightly towards TM11 and EL6, which intensifies ated amine with GLU66 or GLU48 and additionally the hydrophobic in- interactions with TYR535 as well as with TYR469, TRP555, and teractions of the carbon atoms of the piperidine ring with LEU314 PHE531 (Fig. 6B). Interactions with PHE531 replace those created in (hGAT-3) or LEU294 (hGAT-2) result in an optimal conformation of hBGT-1 by TYR453. The same area was indicated by Damgaard et al. as the linker and favorable localization of the ligand's aromatic fragments. a binding site for isatin derivatives which are selective, allosteric Moreover, the volume of S2 site in the hGAT-3 transporters is increased hGAT-3 inhibitors [56]. The possibility of such an arrangement of aro- by replacement of tyrosine present in other types with a serine residue matic moieties is also confirmed by the presence of an S-citalopram (SER468), and in the case of hGAT-2 by replacement of isoleucine with molecule within this site in hSERT crystal structure (PDB code: 5I73) valine (VAL132). On the other hand, in hBGT-1 transporters, the addi- [26]. The phenyl moiety in the 4-position of the piperidine ring is ditional serine residue within the non-helical fragment of the TM10 re- rected towards the S1 site and interacts in hBGT-1 mainly with duces the space where one of the aromatic rings of the inhibitors can TYR133 (TYR147 in hGAT-3) and LEU129 (LEU143 in hGAT-3), and in bind. the case of hGAT-3 also with TYR146 and PHE308. The protonated 1386 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 1380–1389

Fig. 6. Binding mode of the piperidin-4-ol derivatives in hBGT-1 (A) and hGAT-3 (B). nitrogen atom is located between aspartic acid from the upper part of gate. In the course of molecular dynamics simulation it remains in the extracellular gate, and phenylalanine from the lower part of this close proximity to the aromatic ring of PHE293 in hBGT-1, forming a cation – π interaction, while in hGAT-3 it approaches ASP467 creating a stable ionic bond (Fig. 7C). The hydroxyl group of piperidin-4-ol derivatives is located close to serine residue from the non-helical fragment of TM10 (SER472 in hGAT-3, SER456 in hBGT-1), creating hydrogen bond. For hGAT-3 this bond is very stable during molecular dynamics simulation (Fig. 7C). In the case of hBGT-1 the initial interaction with SER456 was replaced after about 3 ns by water-mediated hydrogen bonds with SER457. In addition, this conserved water molecule formed a hydrogen bond with GLN299, resulting in a beneficial interaction network (Fig. 7B). The general arrangement of piperidin-4-ol derivatives in the hGAT-1 and hGAT-2 transporters was very similar to that described for hBGT-1 and hGAT-3. The lower activity of these compounds on hGAT-1 can be explained by replacing TYR520 from hBGT-1 with MET519, which weakened the hydrophobic interactions, and by hin- dered formation of hydrogen bonds with SER456 within TM10. Derivatives of 4-aminobutanamide and 4-hydroxybutanamide are located analogously to the piperidin-4-ol derivatives. Their aromatic fragments occupy the same sites, and the amide moiety forms a hydrogen bond with serine residue within TM10. In turn, the protonated amine in 4- position creates ionic interaction with aspartic acid from the extracellular gate, and in the case of 4-hydroxybutanamides, a hydroxyl group in 4- position creates hydrogen bond with the same residue (Fig. 4S in Supporting information). To check whether our models are suitable to distinguish active compounds from inactive ones, we prepared enrichment plots for each GABA transporter type (Fig. 8). To receive the best results we tested the models optimized for amino acid and non-amino acid derivatives both before and after 10 ns molecular dynamics. Active compounds were selected from the literature and constituted 10% of the entire database. Inactive compounds were taken from literature and were also generated on the DUD-E website. For each GABA transporter type, the entire database was finally docked to two models: one optimized for amino acid derivatives and the other optimized for non-amino acid derivatives. The highest-rated pose was selected for each active and inactive compound from the combined docking results and later taken into account in enrichment parameter calculations. The best results were obtained for hGAT-1 (Receiver Operator Characteristic area under the curve - ROC: 0.95) and hGAT-3 (ROC: 0.89). A little bit worse, but still satisfying for the hBGT-1 (ROC: 0.85) and hGAT-2 (ROC: 0.82). hGAT- 1 models enable to find about 70%, and hGAT-3 ones about 60% of the active ligands in the top 10% of all the compounds. For hBGT-1 and Fig. 7. A) RMSD changes for the ligands in the course of molecular dynamics simulations. hGAT-2 models, it is possible to select about 50% and 43% of active com- Distance changes between amino acid residues, water molecules and ligand functional pounds, respectively when the top 10% of the database was analyzed. groups involved in key interactions, during molecular dynamics, for B) hBGT-1; C) hGAT-3. Ligands used in the simulations: hBGT-1 – NNC 05-2090 (45); hGAT-3 – Such results suggest that our models may be useful in the search for NNC 05-1965 (42) (numbers in brackets from Table 2S, Supporting information). new inhibitors of GABA transporters. K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 1380–1389 1387

cases additionally due to a favorable conformation of the non-helical fragment of domain 10. Non-amino acid compounds are arranged along with the vestibule of the GABA transporters. Carbazole, benzhydryl moiety and analogous fragments are located near the entrance to the vestibule, in the hydrophobic space between TM10 and TM11. The second aromatic group of these compounds is directed towards the S1 site, reaching tyrosine from the extracellular gate. The hydroxyl or amide groups of the ligands form hydrogen bonds with serine residues within the non-helical part of TM10. In turn, protonated nitrogen atoms create ionic interactions with aspartic acid from the upper part of the extracellular gate or the cation-π interaction with phenylalanine from the lower part of the extracellular gate. In addition to deter- mining the ligand binding mode and indicating the amino acid residues responsible for selectivity, we also prepared enrichment plots for all types of GATs. We obtained very good results, which indicate that the built models can be used to screen databases in search of new active compounds of the desired selectivity.

4. Methods

4.1. Homology modeling

The protein sequences for human GAT-1, BGT-1, GAT-2, and GAT-3 Fig. 8. Enrichment plots for each type of GABA transporters. were obtained in FASTA format from the UniProt database. A total of 10 crystal structures of aLeuT (PDB code: 2A65, 2Q6H, 3F3A, 4MM7, 3. Conclusions 4MMB), dDAT (PDB code: 4XP4, 4XP9, 4XPB, 4XPH), and hSERT (PDB code: 5I6X) were used as templates. The models were generated in GABA transporters are interesting therapeutic targets. Designing the Modeller 9.18 program and the SWISS-MODEL server. N-andC- new, selective GAT inhibitors is difficult due to a lack of their exact struc- termini were omitted because of the low homology. In the case of Mod- ture, in particular their binding sites. Based on homologous proteins eller, the models were built using the alignment presented by Beuming from the SCL6 family we built models for each type of the GABA trans- [27] with minor adjustments based on our observations and studies of porters. Models built on dDAT templates, especially on 4XP4 and 4XP9 the other groups [28,42] (Fig. 1S, Supporting information). The same crystal structures, proved to be the highest rated by tools checking the alignment was used to build models on aLeuT templates in the SWISS- quality of protein models and their similarity to actual protein struc- MODEL. In the case of dDAT and hSERT templates, models were created tures i.e. QMEAN, DOPE score, Verify3D. This was later reflected in based on alignment generated automatically by the SWISS-MODEL. The docking studies and molecular dynamic simulations. These studies only change was introduced within the non-helical fragment of TM10: showed that amino acids lining the bottom of the main binding site SER456 from hSERT was aligned with SER456 (hGAT-1), SER457 (S1) have the largest contribution to the selective binding of ligands (hBGT-1), SER452 (hGAT-2) and SER472 (hGAT-3). This alignment with an amino acid structure. In the case of GAT-1 transporters, this was consistent with that used in the Modeller program, with slight dif- site is the most hydrophobic, due to the presence of TYR60, LEU300 ferences within EL2, EL4 and the loop between TM8 and TM9 (Fig. 1S, and SER133 residues. The nipecotic acid fragment of tiagabine (and Supporting information). Ten models for each template were generated other analogs) is arranged in a way that allows the formation of hydro- by the automodel class in the Modeller. Cysteine residues forming the phobic interactions between the carbon atoms of the piperidine ring disulfide bridge within EL2 were defined. A high optimization level and side chains of TYR60 and LEU300. The protonated nitrogen atom was set. Heteroatoms were excluded. Sodium and chloride ions were of the inhibitor forms a hydrogen bond with the main chain of transferred directly from templates. In the case of models built on PHE294. In the case of hGAT-2 and hGAT-3 transporters, the replace- LeuT templates, which lack chloride ions, these ions were transferred ment of the tyrosine residue with the glutamic acid residue (GLU48 in from the DAT template after the alignment of fragments involved in hGAT-2, GLU66 in hGAT-3) forces a different orientation of the nipecotic their binding. The same was done for sodium ions in models built on acid fragment. The protonated amine is directed towards the bottom of the 5I6X template (lack of one of the sodium ions). Template and the binding site and forms an ionic interaction with the mentioned model structures were aligned using the PyMOL program. For each tem- glutamic acid residue. Such favorable interactions were observed in plate one the best model generated by the SWISS-MODEL and one the hGAT-1 only for R isomer of nipecotic acid, and in hGAT-2 and 3 only best model (according to DOPEscore) built in the Modeller program, for S isomers of nipecotic acid derivatives, which explains the various passed for further assessment and docking studies. This resulted in a activities of enantiomers towards different types of the transporter. In pool of 20 various models for each type of GABA transporter. Their qual- the case of hBGT-1, the S1 site is the most polar, which is associated ity was evaluated using the DOPE score, QMEAN, Verify 3D and with the presence of GLU52, GLN299, and GLU126 residues. It is not sur- Ramachandran plot (Table 1S, Supporting information). prising then that small, hydrophilic molecules like (1S,2S,5R)-5- aminobicyclo[3.1.0]hexane-2-carboxylic acid are currently the most po- 4.2. Docking studies tent inhibitors of these transporters. GLN299 residue, present only in hBGT-1, appears to play a key role in the selectivity of this transporter. Ligands for all dockings were prepared in the LigPrep module. Ioni- Lipophilic moieties of hGAT-1, 2 and 3 inhibitors are located at the S2 zation states were generated at physiological pH (7.4 ± 0.2) using the binding site. Large triaryl moieties bind selectively to hGAT-2 and 3 as Epik program. Additionally, pKa for ligands with more than one basic a consequence of different orientation of nipecotic acid fragment and in- group was calculated with the Marvin program. All predicted for phys- creased volume of S2. In hGAT-2 this is due to the replacement of isoleu- iological pH ionization states were used in docking studies. The optimi- cine with valine residue (VAL132), while in hGAT-3 because of the zation of ligands was carried out with the OPLS_2005 force field. For replacement of tyrosine with a serine residue (SER468), and in both ligands described in the literature, the determined absolute 1388 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 1380–1389 configuration was retained. In the case of compounds with unknown 2 fs and the total duration of 10 ns. CHARMM36m force field was ap- configuration, all possible stereoisomers were generated. plied. The interval for both energy and trajectories recording was Models were prepared in the Protein Preparation Wizard using de- 10 ps. The results were analyzed with the VMD program. fault settings. In the case of models with the closed lower part of the extracellular gate, it was opened by changing the conformation of the 4.4. Enrichment plots phenylalanine which is a part of this gate (hGAT-1: PHE294; hBGT-1: PHE293; hGAT-2: PHE288; hGAT-3: PHE308). This allowed ligands to A group of active and inactive compounds for each type of GABA access the S1 binding site. Models in which this conformational change transporter was prepared. Active compounds constituted 10% of the caused steric hindrance with TM10 were rejected. whole database and were obtained from the literature data. Inactive In order to choose the best models for further optimization, initial compounds were partly taken from literature and partly generated in docking was done in the KNIME program using Schrödinger's Glide En- the DUD-E website. The entire group of compounds was docked to fi semble Docking node. The grid center was de ned by TYR140 and both models optimized for amino acid derivatives and models opti- PHE294 residues for the hGAT-1 model based on the 4XP9 template, mized for non-amino acid derivatives (described in Docking studies sec- generated in SWISS-MODEL. The superimposition of the other models tion) as well as to those models after 10 ns MD simulation. Both to this one enabled the use of one grid center in all initial docking. standard and extra precision, as well as different grid sizes, were used Inner box size was 15 Å × 15 Å × 15 Å. Amino acid derivatives with rel- to find optimal settings. The model optimization level and settings giv- atively high activity and selectivity were docked using standard preci- ing the best results in enrichment was presented in Table 3S in sion. Five poses per ligand were written out. The obtained poses were Supporting information. The enrichment calculations took into account assessed in terms of their convergence and the possibility of forming in- the highest-rated pose of each compound among the combined results teractions between the carboxyl group of the ligands and sodium ion of two dockings (to model optimized for amino acid derivatives and within the S1 site, analogous to those observed for the leucine and tryp- model optimized for non-amino acid derivatives). In the case of chiral tophan in aLeuT crystal structures. For all types of transporters, the best compounds (both active and inactive) with the unknown activity of in- results were obtained for models generated in the SWISS-MODEL, based dividual enantiomers, the enantiomer with the higher docking score on 4XP4 and 4XP9 templates. The selected complexes were then opti- was taken into account. The same was done for compounds occurring mized using Prime Minimize protocol in which the ligand and side in several ionization states. chains of amino acids located at different distances around it have been optimized. After each optimization, ligands were redocked to eval- uate the improvement of the results. Redocking was performed in the Author statement Glide program, using the centroid of the optimized ligand as the grid Łą center. The grid size, as in initial docking, was 15 Å × 15 Å × 15 Å. This Kamil tka: Conceptualization, Investigation, Writing - Original ń allowed to receive the final hGAT-1 model (SWISS-MODEL, 4XP9). In Draft, Writing - Review & Editing. Jakub Jo czyk: Investigation. the case of hBGT-1, hGAT-2, and hGAT-3 complexes additional optimi- Marek Bajda: Conceptualization, Investigation, Writing - Original zation of the ligand and atoms within 7 Å around it was performed Draft, Writing - Review & Editing, Supervision, Funding acquisition. using Refine Protein-Ligand Complex protocol. After that, the ligands were redocked and the optimization procedure was repeated. As a re- Declaration of competing interest sult, the final models hBGT-1 (SWISS-MODEL, 4XP4), hGAT-2 and hGAT-3 (both based on the 4XP9 template and generated in SWISS- The authors declare that they have no known competing financial MODEL) were obtained. interests or personal relationships that could have appeared to influ- In order to find the binding mode for non-amino acid derivatives, the ence the work reported in this paper. Induced Fit Docking procedure was applied. Selected derivatives of piperidin-4-ol, 4-aminobutanamide, and 4-hydroxybutanamide were Acknowledgments docked to the best models (before optimizations) chosen in the previ- ous step, using extended sampling protocol. The box center was defined The project was financially supported by the National Science Cen- by residues PHE294, TYR140, TYR452, ARG69 in hGAT-1, and by the cor- tre, Poland (grant no. 2016/23/D/NZ7/01172) and Jagiellonian Univer- responding amino acids in hBGT-1, hGAT-2, and hGAT-3. The box size sity Medical College (grant no. N42/DBS/000015). was 10 Å × 10 Å × 10 Å. Sodium and chloride ions were skipped during Prime refinement. Among the generated complexes the best ones were Appendix A. Supplementary data selected. Next, a group of ligands was docked to each of them using Glide (grid center: centroid of the ligand from complex; grid size: 15 Supplementary data to this article can be found online at https://doi. Å × 15 Å × 15 Å). Results were evaluated based on pose convergence, org/10.1016/j.ijbiomac.2020.04.263. created interactions and values of the docking score. This allowed choosing models the best optimized for non-amino acid derivatives. References In all described above calculations the OPLS_2005 force field was applied. [1] K. Gale, GABA and epilepsy: basic concepts from preclinical research, Epilepsia 33 (Suppl. 5) (1992) S3–12. [2] J. Bormann, The ‘ABC’ of GABA receptors, Trends Pharmacol. Sci. 21 (2000) 16–19. 4.3. Molecular dynamics [3] A. 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