International Journal of Biological Macromolecules 158 (2020) 750–772

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International Journal of Biological Macromolecules

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

γ-Aminobutyric acid transporters as relevant biological target: Their function, structure, inhibitors and role in the therapy of different diseases

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 (GABA) is a major inhibitory neurotransmitter in the nervous system. It plays a crucial role Received 3 January 2020 in many physiological processes. Upon release from the presynaptic element, it is removed from the synaptic cleft Received in revised form 17 April 2020 by reuptake due to the action of GABA transporters (GATs). GATs belong to a large SLC6 protein family whose Accepted 18 April 2020 characteristic feature is sodium-dependent relocation of neurotransmitters through the cell membrane. GABA Available online 1 May 2020 transporters are characterized in many contexts, but their spatial structure is not fully known. They are divided Keywords: into four types, which differ in occurrence and role. Herein, the special attention was paid to these transporting GABA transporters proteins. This comprehensive review presents the current knowledge about GABA transporters. Their distribu- Function tion in the body, physiological functions and possible utilization in the therapy of different diseases were fully Structure discussed. The important structural features were described based on published data, including sequence analy- Inhibitors sis, mutagenesis studies, and comparison with known SLC6 transporters for leucine (LeuT), dopamine (DAT) and Therapy serotonin (SERT). Moreover, the most important inhibitors of GABA transporters of various basic scaffolds, di- verse selectivity and potency were presented. © 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 sequentially: GAT-1, GAT-2, GAT-3, and GAT-4, while rat and human transporters are designated as follows: GAT-1, BGT-1, GAT-2, and γ-Aminobutyric acid (GABA) is the primary inhibitory neurotrans- GAT- 3. All four proteins belong to one family of sodium-dependent mitter in mammalians [1]. It is thought that GABA is prevalent in membrane transporters (SLC6) [6,8]. The SLC6 family is one of the larg- about 30% of all synapses in the brain [2]. It plays many functions, pro- est solute carrier (SLC) families, containing highly similar 20 transporter viding anticonvulsant, anxiolytic, muscle relaxing, and sedative effects. proteins. It is divided into four subclasses: monoamine, GABA, amino GABA is produced in GABAergic system neurons from glutamic acid by acid and orphan transporters, based on sequence similarity and sub- the action of glutamic acid decarboxylase (GAD). Further, GABA is col- strate specificity (Fig. 1)[9–12]. The whole family includes transporters lected in synaptic vesicles by the vesicular GABA transporter (VGAT) for norepinephrine, dopamine, serotonin, GABA, and glycine. The ma- [3]. The depolarization of presynaptic neurons stimulates the release jority of transporters for neurotransmitters contain approximately 600 of γ-aminobutyric acid from axon terminals. GABA acts as an agonist amino acid residues, ranging from 599 for GAT-1 to 632 for SERT [13]. on three types of membrane receptors: GABA-A, GABA-B and GABA-C Only two glycine transporters are exceptions and contain approxi- [4]. GABA-A and GABA-C receptors belong to the ionotropic class of re- mately 700 residues. The various types of transporters occur in the dif- ceptors and are ligand-gated chloride channels. GABA-B is a metabotro- ferent structures of the nervous system [8,12]. They are widely pic receptor, i.e. it is coupled with G-protein. Finally, GABA is disposed of distributed on neurons and less frequently on glial cells. Dysfunction in two ways: reuptake or metabolism [5]. Reuptake is dominant and oc- of GABAergic system and consequently reduction of the inhibitory effect curs through the operation of GABA transporters (GATs) [6]. On the of gamma-aminobutyric acid can lead to a variety of pathological pro- other hand, the metabolism of GABA by transaminase leads to the inac- cesses and the occurrence of diseases, such as anxiety disorders, epi- tivation of the neurotransmitter. GABA transporters are divided into lepsy, neurodegenerative disorders, schizophrenia, insomnia, motion four types, and their nomenclature is different depending on the organ- impairment or pain states [14,15]. Therefore, the ability to potentiate ism in which they occur [7]. In mice, GABA transporters are numbered the GABA effect by inhibiting the reuptake is an attractive therapeutic target. Our comprehensive review aims to gather all current knowledge about GABA transporters. The presented data contain both basic infor- ⁎ Corresponding author. mation as well as recent achievements in the GAT field. Their distribu- E-mail address: [email protected] (M. Bajda). tion in the body, functions, structure, therapeutic potential and

https://doi.org/10.1016/j.ijbiomac.2020.04.126 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) 750–772 751

Fig. 1. Phylogenetic tree for SLC6 family of sodium-neurotransmitter symporters [8]. inhibitors are described and discussed. Special attention is paid to struc- neurotransmission, the primary function of all GABA transporters is to tural aspects as they are essential for the ligand binding process. Such a bind the extracellular GABA and transport it into the cytoplasm. In this wide range of topics makes review useful for researchers interested in way, GABA transporters regulate the level of GABA at the synaptic GABA transporters: both pharmacologists and medicinal chemists cleft [16–19]. The GABA molecules that reach the GABA receptors from looking for novel active compounds. the synaptic space induce postsynaptic inhibitory potential (a phase known as the inhibition of postsynaptic current (IPSC)). The GABA con- 2. The distribution and physiological functions of GABA transporters centration continues to increase until the late IPSC decay phase. Another activity of GABAergic conduction that is influenced by GABA uptake is Gamma-aminobutyric acid (GABA) transporters are heteroge- the tonic (permanent) stimulation of GABA receptors [20]. This stimula- neously distributed in various areas of the central nervous system tion is a result of the constant maintenance of the low concentration of (CNS) and many vital organs (Fig. 2). In the context of GABAergic GABA in the extracellular space. GABA transporters capture the released

Fig. 2. Anatomical and cellular localization of GABA transporters [12,21–25]. 752 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772 neurotransmitter until its concentration reaches the level that is neces- for the neuronal release [18]. In this way, GAT-3 limits the prolonged re- sary to stimulate the primary pool of receptors. Maintaining a constant lease of GABA by neurons. GAT-3 was also found expressed on astro- tonic inhibitory potential facilitates reducing the ascending excitatory cytes distant from GABAergic terminals and occasionally on stimuli and lowering the pulsation resulting from continuous depolari- neighboring axon terminals near the excitatory synapses, where GABA zation. The diverse distribution of different GABA transporters in the uptake by GAT-3 could limit the action of GABA on distant GABA recep- brain, in combination with their different affinities to GABA, results in tors [41,56]. an individualized response of neurons to the release of the Differences in the cellular distribution of GAT-1 and GAT-3 allow neurotransmitter. both these transporters to perform different roles in synaptic function. GAT-1 limits the escape of GABA from the synaptic cleft, while GAT-3 2.1. GAT-1 and GAT-3 controls the tonic inhibition by mediating the basic concentration of GABA [6,57]. Both transporters complement each other's functions by The distribution and function of the GAT-1 and GAT-3 transporters regulating different signaling pathways mediated by GABA released are related to each other. Both GAT-1 and GAT-3 are expressed mostly via vesicular and nonvesicular mechanisms or during low-frequency in the CNS [25–32]. Distribution of GAT-1 is strongly related to the local- and sustained neuronal activity [58,59]. The amplitude and duration of ization of GABAergic neurons [17,18]. Presynaptic localization of GAT-1 the release of GABA into the synaptic cleft affect the ability of GABA was proved using in situ hybridization and immunocytochemistry in transporters to alter the receptor activation. GABA uptake exerts only GABAergic neurons [27,33–35]. The highest level of GAT-1 expression a small effect on the IPSC profile after a brief increase of transmitter con- was detected in the cerebral cortex in symmetrical synaptic axon termi- centration, but it significantly regulates the recruitment of neighboring nals in the rat brain [26,36]. Experiments on the cerebral cortex of synapses [60–63]. GABA transporters limit the escape of GABA beyond humans and monkeys yielded very similar results [27]. The results of active synapses and thus control the local specificity of GABAergic trans- Western blot analysis, immunohistochemistry, and electron microscopy mission [6]. together demonstrated the presence of GAT-1 in the suprachiasmatic Some reports suggest that both GAT-1 and GAT-3 have the ability to hypothalamic nucleus of adult rats [37]. In addition to neurons, studies release GABA toward the extracellular space (reverse mode) [64–67]. on the mRNA expression and immunoreactivity of GAT-1 have shown Not considering the pathological conditions, it seems that this action its presence in astrocytes distributed in the cerebral cortex, retina, hip- (reverse mode) contributes to the maintenance of the tonic GABA-A pocampus, cerebellum, and thalamus of the rat brain receptor-mediated conductance [64,68–70]. [26,31,32,36,38,39]. Apart from the astrocytes surrounding GABAergic It's worth mentioning that recent studies have also provided evi- synapses, astrocytic GAT-1 was also found in the brain structures that dence of GAT-1 expression on the antigen (Ag) activated T cells [21]. are distant from GABAergic terminals near the excitatory synapses GAT-1 has been reported to modulate the production of cytokines and [17,19,40,41]. GAT-1 immunoreactivity was detected in the immature the proliferation of T cells [71]. A study on GAT-1−/− mice showed a and mature oligodendrocytes of Sprague-Dawley albino rats [42]. Un- higher expression of proinflammatory cytokines compared to wild- like mostly neuronal distributed GAT-1, GAT-3 was highly expressed type (WT) mice [72]. This is one of many examples showing how in the brain glial cells, retina and in the astrocytes (around axons, much we can still learn about the function and distribution of GABA around the nerve cell bodies, and near the dendrites of symmetrical transporters. and asymmetrical synapses) [6,43]. Further examination confirmed the expression of GAT-3 in many areas of the brain of adult rats, includ- 2.2. BGT-1 ing the following: cerebral cortex, where GAT-3 was the second most expressed GABA transporter; astrocytic cells in the thalamus; cerebel- BGT-1 expression has been demonstrated in many mammalian tis- lum glial cells (Bergman cells) that were enveloping the Purkinje axon sues [12,23,73,74]. However, the distribution of BGT-1 transporters in terminals devoid of GAT-1 transporters on the presynaptic membrane; tissues varies to a large extent depending on the species. For example, and deep cerebellar nuclei [6,32,38,44,45]. In the brains of mammals, in mice, the expression of BGT-1 mRNA was found in the kidneys, cere- such as cats, monkeys, and humans, the presence of GAT-3 was found bellum, cerebral cortex, brainstem, and in the liver. On the other hand, additionally in oligodendrocytes [30,46]. Some studies described the in the rat, species that is closely related to mice, the transcripts of this presence of GAT-3 in neurons, but the co-expression of heat shock pro- transporter were identified only in the liver. In the case of dogs, BGT-1 tein 70 in all GAT-3-positive neurons did not allow an explicit confirma- expression was found only in the kidneys [23,36]. In humans, BGT-1 is tion of this location in physiological conditions [47,48]. mostly expressed in the liver, kidneys and to a smaller extent in the Both transporters also occur in structures related to vision. The ret- brain and on pellets [22]. ina and optic nerve were found to exhibit a brain-like distribution of Lower specificity for transported substances and the presence on GAT-1, mainly in neurons including amacrine, displaced amacrine and many different organs mean that BGT-1 participates in many more pro- interplexiform cells, ganglion cells, and Müller cells [39,49,50]. High ex- cesses than GAT-1 or GAT-3. pression of GAT-3 has been shown in the retina in many works, but The high concentration of BGT-1 protein in the liver is connected these have also indicated its presence in other elements that innervate with the significant role of this transporter in betaine uptake into hepa- the eyes including the optic nerve glial cells, amacrine cells, cells in tocytes where betaine participates in the methionine cycle. It acts as a the ganglion cell layer, and Müller cells [43,51–54]. methyl donor in the conversion process of plasma homocysteine to me- Reuptake of GABA into presynaptic neurons (forward mode) is the thionine [75]. In the kidneys, BGT-1 expression was restricted to the main function of GAT-1. About 80% of the released neurotransmitter is basolateral membranes in the medulla. A high concentration of BGT-1 taken up again by this transporter [55]. was found at the tip of the renal papilla and the thick, ascending limbs Uptake by GAT-3, which is the main transporter found on astrocytes, of Henle loop [23]. BGT-1, present in the renal medulla, is involved in is the second most important way to limit the amount of released GABA. preventing osmotic stress. In the medullary nephron segments, BGT-1 The role of astrocytes in the insulation of synapses and the participation accumulates betaine from the vascular system, where it acts as an of astrocytes, highly expressing GAT-3, in the modulation of GABA were osmolyte [23]. supported by the studies that focused on GAT-3 expression and deter- The total concentration of BGT-1 in the CNS was found to be lower mined the distribution of GABA transporters [6,25,28,43]. It has been than in the other organs. Of all the areas in the CNS, leptomeninges pre- shown that after uptake into the astrocytes, GABA molecules are metab- sented the highest expression of BGT-1 [16,23,76,77]. A fairly high level olized by GABA transaminase into succinic semialdehyde. For this rea- of BGT-1 in this structure, suggests that BGT-1 performs a physiologi- son, GABA, transported into astrocytes, is not immediately available cally relevant function [12]. The presence of BGT-1 in the brain has K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772 753 been strongly linked with osmoregulation [77–79]. BGT-1 might con- GABAergic synapses [24,96,97,101]. On the other hand, taurine defi- tribute to volume regulation in the CNS due to its ability to transport be- ciency leads to abnormalities related to brain development, retinal deg- taine [73,78,80]. However, the other osmolyte transporters SMIT and radation and other pathologies [93]. Further examination showed that TAUT are much more widespread in brain structures [23,80]. Among taurine may function as an osmolyte in the nervous system [102]. It is all the brain osmolytes, betaine occurs at the lowest concentration believed that taurine contributes to the subsequent regulatory volume [81]. Experiments on animals with either salt loading or water depriva- decrease (RVD) of both neurons and astrocytes [103]. So it seems that tion showed that no change occurred in the brain levels of betaine another role for GAT-2, as for BGT-1, may be maintaining homeostasis [23,80,82]. Moreover, only insignificant changes in BGT-1 mRNA were within the CNS [24,96,97,101]. found in the brain during acute and chronic salt loading [83]. All these results indicate the rather negligible contribution of BGT-1 in volume 3. Structure of transporters regulation in the CNS. In addition to leptomeninges in the brain, BGT- 1 was identified mostly in astrocytes, but some studies described BGT- The primary structure of the membrane transporters of GABA, in 1 expression in neurons (especially in the pyramidal neurons) both humans and other organisms (mice, rats), was studied many [78,79,84,85]. The distribution of BGT-1 mRNA in the brain was not years ago by cloning these proteins using molecular biology methods. found to correlate with GABAergic pathways, which is in line with the The analyses of the amino acid sequence and mutagenesis data allowed results of the experiments on BGT-1 −/− mice that suggest a minimal predicting the secondary structure of these proteins and identifying the impact of BGT-1 on the inactivation of GABA [17,23,86]. On the other amino acids that are critical for their activity as transporters hand, GABA uptake inhibitor EF1502, which inhibits both GAT-1 and [77,96,104,105]. However, their exact spatial structure is still unknown. BGT-1, has been described as exerting a synergistic effect with the Fortunately, the analysis of crystallographic data of the other trans- GAT-1 inhibitor tiagabine as an anticonvulsant [87,88], which can be porters belonging to the SLC6 family (transporters of leucine, dopamine, interpreted as evidence for the functional role of BGT-1 in control of ab- serotonin) and the attempts to align their sequences with the sequences normal neuronal activity. It could be assumed that BGT-1 is involved in of GABA transporters [13,106] enabled predicting the general spatial the transport of GABA diffused from synaptic regions [89,90]. In contra- structure of these proteins. This allowed the indication of elements dictionwiththatfindings, BGT-1 is expressed at a low level and also has that play a main role in their functioning (Fig. 3). alowaffinity to GABA [77,91]. A comparison of WT and BGT-1 −/− mice models showed no difference in seizure threshold [86]. In this 3.1. Primary structure light, the role of BGT-1 in the inactivation of GABA is at least ambiguous, and some studies have focused on the less-obvious functions of BGT-1. GAT-1 derived from rats was the first transporter, both among GABA BGT-1 expression was also demonstrated in human platelets and transporters and in general of all SLC6 family proteins, for which the found involved in the regulation of the clotting process [22]. Some amino acid sequence was established. It consists of 599 amino acids. tested GABA analogs were found to successfully inhibit the clothing sig- For the sequence of this transporter (as well as the sequences of BGT- nal transduction by limiting the conversion of prostaglandins H2 to 1, GAT-2, and GAT-3), 12 transmembrane domains with N-andC- thromboxane A2. It shows the participation of BGT-1 and GABA in the terminus located inside the cell were detected. Three main kinase C - regulation of the aggregation process [22]. phosphorylation sites are located intracellularly (Ser24, Thr46, and Ser562 for GAT-1). On the large extracellular loop between domains 3 2.3. GAT-2 and 4, three glycosylation sites were found [96,107]. The human GAT- 1 transporter also contains 599 amino acids, and its sequence has high Along with BGT-1, GAT-2 is another GABA transporter distributed homology with that of the rat transporter. The sequences of human mainly in the peripheral tissues. Immunocytochemistry studies have and rat GAT-1 differ only in 17 residues, with the majority of these pres- confirmed the highest expression of this protein in the plasma mem- ent on the N-andC-terminus. The sequence within the 12 potential branes of hepatocytes and the proximal tubules in the renal cortex transmembrane domains is almost identical in the transporter of both [24,25,92]. This is associated with the GAT-2 ability to transport β- the species [104]. Mouse GAT-1 transporter shows very high homology alanine and taurine [25,93]. Due to these findings, GAT-2 was proposed to the GAT-1 transporter of human and rat and also exhibits almost to play a nutritional role [45,94]. identical pharmacological properties [108]. A lesser amount of GAT-2 was found in the leptomeninges and some The GAT-2 transporter has 602 amino acids. A comparison of the se- of the colocalized blood vessels and in other brain structures, such as the quence of GAT-2 with that of other transporters showed a 69% identity granular layer of the cerebellum, choroid plexus, and brain ependyma with BGT-1 and 51% identity with GAT-1 [25]. GAT-3 transporter has has been confirmed in some studies [24,45,94]. In addition, GAT-2 was also been cloned. Human GAT-3 consists of 632 amino acids, whereas shown to be expressed by epithelial, glial, and neuronal cells [95]. Ex- mice GAT-3 consists of 627 amino acids. A comparison of the sequence amination of the cell cultures of type 1 and O-2A/type 2 astrocytes con- of these transporters with that of other transporters showed 70% iden- firmed the expression of GAT-2 mRNA [96,97]. The presence of GAT-2 tity with GAT-2, 65% with BGT-1, and 53% with GAT-1 [25,28,109]. was also confirmed in the elements that innervate the eyes, such as Among the membrane transporters of GABA, the previously mentioned optic nerve, pigment, and ciliary body epithelia of the retina and Müller BGT-1 transporter has an affinity to betaine in addition to GABA. It con- cells (in the last case only in bullfrog) [43,53,54,98]. sists of 614 amino acids [78,105]. The presence of GAT-2 in the CNS is also thought to be associated with the distribution of β-alanine and taurine [25,93]. β-Alanine may 3.2. Spatial structure act as a “false transmitter,” replacing GABA and activate GABA-A or GABA-C receptor [99]. Therefore, reuptake of β-alanine through GAT-2 Analysis of the crystal structures of the representatives of SLC6 may protect against the unwanted activation of the GABA-A and transporters along with mutagenesis studies on GABA transporters GABA-C receptors. Taurine, just like β-alanine can activate the GABA-A allowed for the prediction of the three-dimensional structure of these receptors. It also showed the ability to binds to the metabotropic transporters. So far, three transporters have been investigated using GABA-B receptor [100]. In one study, GAT-2 −/− mice showed a 50% X-ray methods: the transporter of leucine (LeuT) derived from Aquifex reduction in the liver level of taurine and a 20% increase in the brain aeolicus [106,110,111], the transporter of dopamine (DAT) from Dro- level of taurine. This observation gives grounds for stating that GAT-2, sophila melanogaster [112,113], and human serotonin transporter present in the brain, is responsible for the removal of taurine outside (SERT) [114,115]. All these proteins are built of 12 transmembrane do- the CNS [24]. These findings emphasize the role of GAT-2 in protecting mains, with the N- and C-terminus located intracellularly (Fig. 4). The 754 K. Łą k ta./ItrainlJunlo ilgclMcooeue 5 22)750 (2020) 158 Macromolecules Biological of Journal International / al. et tka – 772

Fig. 3. Alignment of the amino acid sequences of GABA transporters with those of the other proteins of the SLC6 family having a known spatial structure. This comparison was based on the data presented by Beuming et al. [13]. N-andC-terminus were omitted. Amino acids involved in ligand binding are marked with blue arrows ( ), and those involved in sodium ions binding with purple circles ( ) and in chloride ions binding with blue circles ( ). Amino acids that form the extracellular gate are marked with green triangles ( ), while those that form an intracellular gate with orange triangles ( ). Abbreviations: aLeuT – leucine transporter from Aquifex aeolicus;dDAT– dopamine transporter from Drosophila melanogaster;hSERT– human serotonin transporter; hGAT-1, hBGT-1, hGAT-2, and hGAT-3 – human GABA transporters. K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772 755

Fig. 4. The general structure of the transporters of dopamine (A) (PDB: 4M48) and leucine (B) (PDB: 3F3A). The same colors indicate the structural elements that are common in both transporters.

first ten domains form the main core of transporters, which is responsi- In LeuT, leucine (substrate) and tryptophan (inhibitor), which are ble for the binding of ligand, sodium and chloride ions. Domains 1–5 located at S1 site, interact via the carboxyl group with the sodium ion, overlap with domains 6–10 by a pseudo-twofold axis in the membrane the amide nitrogen of leucine 25 and glycine 26 (TM1), and in the plane [106,112,114]. The extracellular surface of the transporters, avail- case of leucine, with the hydroxyl group of tyrosine 108 (TM3) (Fig. 5 able to the extracellular fluid, is formed primarily by the following: long AandB)[106,110]. Research on the domain TM1 of GAT-1 demon- EL2 loop, connecting the domains TM3 and TM4; EL4 loop, connecting strated that the G63C (corresponding to G24 in LeuT) and G65C (corre- TM7 and TM8 domains; and EL6 loop connecting TM11 and TM12 do- sponding to G26 in LeuT) mutants were not able to transport GABA, mains. In the eukaryotic transporters of serotonin and dopamine, the whereas the L64C (corresponding to L25 in LeuT) mutant showed EL2 and EL6 loops are much longer than in the bacterial leucine trans- only little activity. Interestingly, in the latter case, the presence of porter. The EL2 and EL4 loops possess helical fragments, which in the GABA, as well as the compound SKF100330A (GAT-1 inhibitor), case of EL4 form a V-shaped structure. It was demonstrated that EL4 protected cysteine 25 from reacting with the methanethiosulfonate re- and EL6 are, to some extent, responsible for the binding of GABA, al- agent [119]. In another study, it was shown that the replacement of gly- though it is not clear whether the underlying mechanism is direct ligand cine 63 to aspartic acid, serine, or alanine also blocked GABA transport binding or change in the protein conformation [116]. [120]. Similar results were obtained in the case of GAT-2. Mutations of The surface of contact with the cytoplasm mainly consists of the fol- G51A and G51L (corresponding to G63 in GAT-1) led to complete inhi- lowing: IL1 loop connecting TM2 and TM3 domains; IL5 loop connecting bition of GABA transport [121]. These results confirm that both in LeuT, TM10 and TM11 domains; and in the case of DAT and SERT also a helix DAT, and SERT, and in GABA transporters, these residues are involved in at the C-terminus. Monoamine transporters have a kink in the middle of the binding of substrates and inhibitors. Tyrosine 108 is also preserved the domain TM12 along their length in contrast to leucine transporter. in all the transporters of the SLC6 family. A study showed that in GAT- Extra- and intracellular loops are fragments in which the sequences of 1, replacement of tyrosine 140 (108 in LeuT) successively by serine, transporters differ the most. However, some common elements, such phenylalanine, and tryptophan led to the complete blockage of GABA as the disulfide bridge within EL2 (Cys148 and Cys157 in DAT, Cys200, transport [122]. This indicates that the hydroxyl group of tyrosine is and Cys209 in SERT, absent in LeuT) can be found. This loop also con- also involved in substrate binding. tains several N-linked glycosylation sites [106,112,114]. Studies on The protonated amino group of leucine and tryptophan was found to GAT-1 showed that faulty N-glycosylation (by mutation of Asn176, interact with oxygen from the carbonyl group of the main chain of ala- Asn181, or Asn184) decreased the stability of the protein and its trans- nine 22 (TM1), phenylalanine 253, and threonine 254 (TM6), as well as location toward the cell membrane. Blocking of the glycosylation also with the hydroxyl group of serine 256 (TM6) [106,110]. The nonhelical led to a reduction in GABA uptake, which was probably caused by a de- fragment of TM6 was stabilized by the preserved residues of glutamic crease in affinity to sodium ions [117,118]. acid 62 and glutamic acid 419 (numbering for LeuT), enabling the Domains TM1 and TM6, characterized by the highest homology proper activity of the transporter. In a study on GAT-1, it was shown among the representatives of the SLC6 family, have a non-helical frag- that the substitution of glutamic acid 101 by glycine, alanine, or even ment present in the middle of their length. They form numerous inter- glutamine led to a complete blockage of the transporter activity. A mu- actions with ligands and ions. The non-helical fragments divide these tant containing aspartic acid at this position showed only b2% of the ini- domains into TM1a and TM1b and TM6a and TM6b, respectively. In ad- tial activity [123]. The introduction of glutamine in the position of dition, the domains TM3 and TM8 surrounding the non-helical frag- Glu467 had little effect on GABA transport, but the E467K mutant exhib- ments of TM1 and TM6 possess amino acids playing a pivotal role in ited significantly lesser activity than WT [124]. The aliphatic side chain transport. These four domains form the main substrate-binding site of leucine and the indole ring of tryptophan are located in the hydro- (S1) [106,112,114]. Above this site, there is the vestibule, including an phobic pocket, created mainly by the side chains of valine 104, tyrosine additional binding site (S2). These binding sites are separated from 108, phenylalanine 253 and 259, and isoleucine 359 (Fig. 5)[106,110]. each other by an extracellular gate. The intracellular gate regulates ac- Comparing the central binding site (S1) in leucine transporter with cess to the S1 from the cytoplasmic side. the binding site in monoamine transporters (DAT, SERT), it can be 756 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772

Fig. 5. Binding mode of leucine (substrate) in the occluded state of LeuT (PDB: 2A65) (A), tryptophan (inhibitor) in the outward-open state of LeuT (PDB: 3F3A)(B),3,4- dichlorophenethylamine (analog of the substrate) in the partially occluded state of DAT (PDB: 4XPH) (C), and amitriptyline (inhibitor) in the outward-open state of DAT (PDB: 4M48) (D). Sodium ions are shown as purple spheres, chloride ions as green spheres, and water as red spheres.

seen that the main difference between them is the replacement of gly- these transporters bind to this subsite. In addition to the hydrophobic cine at position 24 in LeuT by aspartic acid in SERT and DAT. The car- residues, in the dopamine transporter there are also aspartic acid 121 boxyl group of aspartic acid in monoamine transporters occupies the and serine 422 residues, which are responsible for the binding of polar same place as the carboxyl group of leucine (substrate) in LeuT, en- groups (e.g., dopamine hydroxyl groups). It is worth noting that abling ionic and hydrogen interactions with the amine group of sub- among leucine transporters, the region corresponding to subsite B is strates and inhibitors of those transporters, as well as the binding of considerably reduced. It is caused by the presence of the side chain of sodium ion (Fig. 5 C and D) [112–114]. isoleucine 359 in the position of glycine 425 from DAT (442 from The binding site in the transporters of dopamine and serotonin is SERT) [113,114]. The same is observed in the case of GABA transporters, often divided into three subsites: A, B, and C [113,114,125]. In the dopa- where threonine (GAT-1) or cysteine (BGT-1, GAT-2, GAT-3) is present mine transporter, subsite A includes the aspartic acid 46 (Asp98 in in this position. SERT) mentioned above, as well as phenylalanine 43 (Tyr95 in SERT), Subsite C includes phenylalanine 319 (335 in SERT), isoleucine 483 glycine 322 (338 in SERT), and serine 421 (438 in SERT). Studies on do- (valine 501 in SERT), and alanine 479 (threonine 497 in SERT) and main TM1 in GAT-1 showed that the replacement of tyrosine 60 (Phe43 reaches up to the aspartic acid 475 residue (glutamic acid 493 in in DAT) by glutamic acid or threonine led to the complete blockage of SERT) located in the extracellular vestibule [114,125]. The vestibule is GABA transport [120]. In a study on GAT-2, the E48A mutant displayed formed by the TM1b, TM3, TM6a, and TM10 domains and a fragment about 50% of the WT activity, whereas the E48L and E48Y mutants of the EL4 loop. It includes the S2 site, to which some inhibitors (alloste- showed only about 10% of the activity [121]. Subsite A has been ric inhibitors) bind [110,111,114,127]. In a study on GAT-1, it was shown to display significant variation within the SLC6 family and deter- shown that the tryptophan 68 (TM1b) and isoleucine 143 (TM3) resi- mine the substrate specificity of the subtypes of GABA transporters dues are close to each other. A similar situation can be observed in the [126]. Subsite B is composed of tyrosine 124 (176 in SERT), valine 120 case of DAT and SERT crystals for the corresponding amino acids (isoleucine 172 in SERT), phenylalanine 325 (341 in SERT), and alanine [128]. A mutagenesis study on GAT-1 showed that conversion of the 117 (169 in SERT). The hydrophobic rings of substrates and inhibitors of three amino acids lying within the domain TM10 and the EL5 loop to K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772 757 those found in GAT-3 (Q441E, I444M, K448Q) increased the inhibitory Sodium ions, and in most cases chloride ions (except LeuT), are nec- effect of β-alanine on GAT-1 mutant, leading to decreased GABA trans- essary for the transport of substrates by the SLC6 proteins, including port. On the other hand, mutation of GAT-3 in the opposite direction re- GABA transporters [134–137]. Sodium ions play a key role in binding duced the effect of β-alanine on GABA transport. These observations the substrates and stabilizing the domains of the transporters indicated that these amino acids are involved in the initial binding of [106,112–114]. It is not surprising that the amino acids involved in the substrates [116]. Another study on the extracellular availability of binding of sodium ions are almost identical within the SLC6 family TM6a in GAT-1 was focused on the sensitivity of cysteine mutants to [13,106]. In LeuT, leucine-binding sodium ion (Na1) interacts with oxy- [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) re- gen from the carbonyl group of the alanine 22 main chain, oxygen from agent. Results showed that the orientation of the amino acid residues the side chain of asparagine 27 and 286 as well as threonine 254 (serine in this transporter was similar to the corresponding amino acids in the in DAT, SERT, and GABA transporters), the carboxyl group of leucine known DAT, SERT, and LeuT crystals. Reaction with the MTSET reagent (substrate), and in the case of dopamine transporter, the carboxyl was found to be increased in the presence of sodium ions (shifting the group of aspartic acid 46, directly and through the water molecule. balance toward the outward-open state, which increases the access to This complex has an octahedral geometry [6,12,24]. A study investigat- amino acids lining the entrance to site S1) and inhibited in the presence ing the GAT-1 transporter showed that conversion of asparagine 66 (27 of GABA (shifting the balance toward the occluded and inward-open in LeuT) to cysteine led to complete blockage of the sodium-dependent states). The SKF100330A inhibitor protected the S295C mutant (by oc- GABA transport [119]. The second sodium ion (Na2) is coordinated by cupying the S1 site), and to a lesser extent the T290C mutant (within oxygen from the carbonyl group of the main chains of glycine 20, valine the S2 site, probably due to the close proximity to the aromatic rings 23 (isoleucine in GABA transporters), and alanine 351 (leucine in DAT, of the inhibitor). However, it did not protect the mutants D287C and SERT, and GABA transporters) and the hydroxyl groups of serine 355 L286C (these residues lie at the entrance to the vestibule and are ex- and threonine 354 (aspartic acid in DAT, SERT, and GABA transporters). posed in the outward-open state) [129]. Similar study analyzed the ex- This complex has a trigonal bipyramid geometry [106,112]. In a study tracellular parts of the TM10 and showed that, as observed with the on GAT-1, it was found the mutants S396C (355 in LeuT) and D395C known crystals from the SLC6 family of proteins, this domain is available (threonine 354 in LeuT) were not able to transport GABA [138]. In gen- to the extracellular environment [130]. In GABA transporters, in the eral, leucine transport is independent of Cl− ions. In the case of other middle of the length of the domain TM10, near glycine 457 (numbering transporters, these ions play a role in transport, probably by balancing for GAT-1), there is one additional amino acid. It has been demonstrated the charge of the sodium ions [137,139]. In monoamine transporters, that this amino acid provided extra bulk that was required for rigorous the chloride ion is coordinated by two serine residues (320 and 356 in gating and tight binding of the substrates and ions. The glycine 457 res- DAT; 336 and 372 in SERT), a glutamine residue (316 in DAT; 332 in idue was found to be particularly important for the proper arrangement SERT), and a tyrosine residue (69 in DAT; 121 in SERT). This complex of the nonhelical fragment of TM10, since its successive replacement by has a tetrahedral geometry [112–114]. alanine, leucine, cysteine, or proline led to complete inhibition of trans- port. In the case of the G457A mutant, the activity was partially restored 4. The mechanism of transport by the deletion of neighboring amino acids (serine 456 or to a lesser ex- tent methionine 458), which indicated that this fragment should have Transporters of GABA and other proteins from the SLC6 family bind an appropriate size to enable the proper transport of GABA [131]. the substrates and the ions located outside the cell and then transfer The extracellular gate consists of two pairs of amino acids: tyrosine them through the membrane to the inside. For GAT-1, GAT-2, and (108 in LeuT, 124 in DAT) and phenylalanine (253 in LeuT, 319 in GAT-3 the transport stoichiometry is Na+:Cl−:GABA = 2:1:1, while DAT); arginine (30 in LeuT, 52 in DAT) and aspartic acid (404 in LeuT, for BGT-1 3:2 (or 1):1 [36]. In order to transport ions and substrates, 475 in DAT). The last two form ionic interactions with each other many residues must undergo conformational changes, as well as the when the gate is closed. Arginine forms an additional cation–π interac- changes in the arrangement of whole domains, must appear. Working tion with phenylalanine 253 (LeuT) [106,112,114]. In studies on GAT-1, transporters go through three main stages. The outward-open state pro- arginine 69 was found to be crucial for the activity of this transporter vides access to the main binding site from the extracellular environ- [119,124]. The replacement of arginine 69 by six different amino acids, ment, allowing to bind the substrate and ions. In closed (occluded) including histidine and lysine (having a positive charge as arginine), state access to the binding site is blocked from both the extra- and intra- led to a twofold decrease in GAT-1 activity [124]. Apart from that, the re- cellular side. Inward-open state of transporter provides access to the placement of phenylalanine 294 (253 in LeuT) by cysteine led to the in- binding site from the cytoplasmic side, allowing the release of the sub- hibition or severe impairment of transport [129,132]. Interestingly, the strate and ions into the cell (Fig. 6). The occluded state is often distin- conversion of phenylalanine to tyrosine did not lead to any change in guished into two temporary states: outward-occluded and inward- the activity of the transporter. The F294A and F294I mutants exhibited occluded. The outward-occluded state occurs immediately after closing b10% of WT activity, whereas the F294G mutant showed completely the extracellular gate when access to the extracellular vestibule is still no activity [132]. These findings prove that the aromatic ring is neces- possible. In contrast, a state just before the opening of the intracellular sary for the proper functioning of the extracellular gate. gate, when the intracellular fragments of domains are moving away From the intracellular side, access to the S1 binding site is blocked by from each other and the extracellular ones are close to each other, the TM1a, TM6b, and TM8 domains. As in the case of the extracellular blocking access to both S1 and S2 sites is called inward-occluded state gate, there is a pair of amino acids in the intracellular gate, arginine (5 [8]. in LeuT) and aspartic acid (369 in LeuT), forming ionic interactions As already mentioned, there are currently no crystal structures pre- with each other. Arginine 5 also forms hydrogen bonds with serine senting GABA transporters in particular conformational states. How- 267 and cation–π interaction with tyrosine 268 (numbering for LeuT). ever, the similarity of these transporters to the others from the SLC6 In the proximity to these amino acids, tryptophan 8 is present, whose family with a known structure allows us to state that the conformational indole ring stabilizes the conformation of the domains TM1a and changes accompanying the transport are analogous in all mentioned TM6b. The residues that form the intracellular gate are preserved in all transporters. Currently, crystal structures for DAT, SERT, and LeuT in the representatives of the SLC6 family [106]. In the case of GAT-1, the the outward-open state, DAT in the partially occluded state, and LeuT substitution of arginine 44 (corresponding to arginine 5 in LeuT) by ly- in the occluded state are available. So far, only one transporter (LeuT) sine was found to lead to a significant decrease in the activity of the in the inward-open state has been crystallized. For the LeuT crystal in transporter (about 15% of WT activity). The mutant R44H was also the outward-open state, the distance between tyrosine 108 in TM8 less active than WT [133]. and phenylalanine 253 in TM6a (the amino acids forming the 758 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772

Fig. 6. Conformational changes that occur during transport by proteins from the SLC6 family. For clarity, TM11, TM12, ions, and substrates are not shown.

extracellular gate) is equal to 7.2 Å. In the occluded state, this distance is the transporter in the outward-open state, closing access from the cyto- found to be only 4.2 Å. This change is due to the shift of the domains plasmic side to cysteine 399 [141]. Similar results were observed while TM6b, TM1b, and TM2 toward the domains TM3 and TM10 [110]. A sig- examining the entire TM8 domain, which confirmed that its fragment nificant difference can be noticed in the arrangement of phenylalanine located closer to the intracellular side is the path of access to the S1 319 (253 in LeuT) in the outward-open and occluded states of the dopa- site in an inward-open state [138]. In addition to the deflection of the mine transporter. The residue rotates by about 90° (around Cα – Cβ domain TM1a, many other conformational changes were found. Domain bond) toward tyrosine 124 (108 in LeuT), closing access to the S1 site TM6b deviated from the binding center by about 17°. In the extracellu- [113]. In crystal structures of the leucine transporter, the shift of the ar- lar parts of transporters, the domains TM1b and TM6a bent toward the ginine residue 30 can be observed to restore salt bridge (mediated by domains TM11 and TM10, hindering access to the S2 binding site. This water molecules) with the aspartic acid 404 (Fig. 6)[103,116]. The phe- site was then completely separated from the external environment by nylalanine residue 259 (325 in DAT), located below the phenylalanine the EL4 loop. This loop pressed between the TM1b and TM7 domains 253 (319 in DAT), also exhibits high mobility, changing its position dur- on one side and between the TM3 and TM8 domains and the EL2 loop ing the binding of substrates and inhibitors. It forms hydrophobic inter- on the other side. These conformational changes led to the formation actions with the aromatic rings of the bound compounds [113]. of a number of hydrophobic interactions, as well as a hydrogen bond be- In the outward-open and occluded states, sodium ions are firmly tween aspartic acid 401 in TM10 and alanine 319 in EL4 [140]. Studies bound in the S1 site through numerous coordination interactions. To- on GAT-1 showed that the deletion of amino acids within EL4 led to gether with the small shifts of these ions, occurring during the transition the complete inhibition of GABA transport. However, the replacement to occluded state, domains containing residues that are involved in the of individual amino acids did not have such a drastic impact on the ac- binding of these ions also shift. It is suggested that the sodium ions tivity of the transporter [142]. The correct conformation of this loop is keep the intracellular gate closed by transferring the interactions be- important for the transport of the substrate. This was also confirmed tween the intracellular parts of the domains TM1, TM2, TM6, and by the observation that the introduction of the binding site for Zn2+ TM7, which change their conformations during the transport of sub- ions (through mutations T349H/E370H or T349H/Q374C) led to the in- strate, and the domains that form the core (TM3, TM4, TM8, TM9), hibition of transport in the presence of these ions [143]. In another which are stable. This is confirmed by the fact that the inward-open study, different sensitivities of cysteine residues introduced into the state of the leucine transporter crystal was achieved by the replacement EL4 loop to MTSET reagent were demonstrated. This effect was depen- of threonine 354 and serine 355 (which bind Na2 ion) by valine and al- dent on the presence of the substrate (GABA – transporter switched to anine, respectively, as well as the replacement of tyrosine 268 (which is the occluded and inward-open state) and inhibitor (SKF100330A – a part of the intracellular gate) by alanine [140]. transporter was stabilized in the outward-open conformation) [144]. Analyzing the difference in the spatial structure of LeuT transporter Along with changes in the external part of the transporter, on the cyto- in the outward-open and inward-open states, it can be observed that plasmic side, a disruption was observed in the network of interactions the most extensive changes occur in the arrangement of the domain within the intracellular gate. Bonds between arginine 5, tryptophan 8 TM1a. In the inward-open state, this domain is bent by 45° in relation (N-terminus) and serine 267, tyrosine 268 (TM6b), glutamine 361, to its position in the outward-open state. In the case of GAT-1, it was ob- and aspartic acid 369 (TM8) were found to be broken. The separation served that the presence of substrate (GABA) increases the sensitivity of of TM5, TM7, and TM1a domains from one another resulted in the cysteine 399 (TM8, just below the site S1) to the 2-aminoethyl breaking of the interactions between the amino acids constituting methanethiosulfonate reagent (MTSEA), while the presence of inhibitor them. All these changes led to the opening of the intracellular gate, SKF100330A reduces this effect. This situation can be explained by the which gave access to the S1 binding site from inside and allowed the re- fact that in the presence of GABA, the transporter switches to the lease of the substrate and ions [140]. It was shown that in GAT-1, the in- inward-open state by a hinge-like movement the domain TM1a, and teraction between the extra- and intracellular gates was crucial for the thus exposing cysteine 399. The inhibitor, on the other hand, blocks proper transport of GABA. When the function of the extracellular gate K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772 759 was compromised (by the D451E mutation), the transporter was tiagabine were also found in GAT-1-knockout mice [158]. The mecha- inhibited in the outward-open state, while the mutation within the in- nisms behind these effects are considered to be associated with the dif- tracellular gate (D410E) abolished the GABA transport, inhibiting the ferent paths that GABA takes after being transported into neurons or transporter in the inward-open conformation. The mutant with impair- glial cells. Generally, GABA, transported inside the neuron via GAT-1, ment in both extra- and intracellular gates was active, probably due to is captured by the vesicular GABA transporter which supplies the pool the restoration of the balance between the described conformational of neurotransmitter for the release into the synapse. The neurotransmit- states [145]. ter absorbed by astrocytes is metabolized sequentially by GABA trans- aminase and succinic semialdehyde dehydrogenase into succinic acid, 5. GABA transporters as a therapeutic target which is converted to α-ketoglutarate in the Krebs cycle. α- Ketoglutarate is in turn a substrate for transaminase and reductase The elements of the GABAergic system are distributed throughout and gets converted into glutamine which goes back to the neuron the body, in particular within the CNS [6,12,17]. The GABAergic conduc- [159]. The metabolism reduces the GABA pool directly available to the tion co-controls the course of many physiological processes. GABA neuron [160]. This lowering of the available GABA by blockage of its transporters are one of the key elements of the GABAergic system. transport to the interior of the neuron by tiagabine can trigger an epi- Therefore, the substances regulating the function of GABA transporters leptic seizure. For these reasons, the possibility of using the inhibitors may have applications in the therapy of many diseases. Transporters of other subtypes of transporters, as well as the inhibitors that act of GABA are a fascinating biological target due to their diverse functions nonselectively on several subtypes of GABA transporters, for the treat- and heterogeneous distribution. So far, the anticonvulsant activity of ment of epilepsy is investigated. Compound SNAP-5114 has the ability many GAT inhibitors has been demonstrated in the studies to inhibit the GAT-2 and GAT-3 transporters. After its administration [5,146,147]. The blockage of the GABA transporters after the administra- in rats, the GABA concentration in thalamic structures was effectively el- tion of inhibitors allows a high concentration of GABA to be maintained, evated without causing any changes in the hippocampus. SNAP-5114 which extends the IPSC phase. Prolongation of the IPSC phase can in- also effectively protected against clonic and tonic seizures in a mouse hibit a group of neurons that fire in an abnormal, excessive way [6,36]. model of sound-induced epilepsy. However, in the case of attacks Tiagabine with GAT-1 inhibitory properties is the first drug approved caused by electric shock and PTZ, the anticonvulsant effect was not by the Food and Drug Administration (FDA) [148,149]. Originally used achieved [154]. Another nonselective GAT inhibitor, NNC 05–2045, act- in the treatment of partial seizures, it is now prescribed off-label for ing through BGT-1 and GAT-3 (and to a minimal extent on other trans- the treatment of anxiety disorders and panic disorder, as well as the porters) was found to cause an increase in GABA levels both within the treatment of neuropathic pain [150]. Tiagabine is also used for stroke, hippocampus and the thalamus. NNC 05–2045 effectively counteracted a condition during which a massive release of many excitatory neuro- the tonic and clonic seizures induced by sound as well as the tonic at- transmitters occurs. Attempts to control the inhibitory effect of the tacks caused by electric shock or PTZ [154,161]. Another structural ana- GABA through its transporters have been made to use this strategy for log, NNC 05–2090, which is a nonselective inhibitor of BGT-1, GAT-1, counteracting the effects of stroke excitotoxicity. The imbalance in the and GAT-3, showed a very similar profile of anticonvulsant efficacy. stimulating and inhibiting neurotransmitters associated with diseases, NNC 05–2090 reducing the severity and duration of seizures after stim- such as Alzheimer's disease and sleep disorders encourages to utilize ulation with the amygdala of the rats [161]. The EF1502 compound, the GAT inhibitors in therapy. Transporters located outside the nervous which inhibits both GAT-1 and BGT-1 transporters, has a broad antiep- system are also considered as a biological target in the treatment of im- ileptic profile in general and was found to be effective against partial sei- munodeficiency and lymphocyte differentiation disorders. Most of the zures in many animal models [87,162]. In combination with tiagabine or current research focuses on the best-known transporter of GABA, SNAP-5114, it has been shown to exert a synergistic effect in mice with GAT-1; however, new reports on the importance and function of other sound-induced epilepsy. Such results are reflected in the location of transporters, such as BGT-1, GAT-2, and GAT-3, show that these may transporters where these compounds act. GAT-3 transporters, which also have enormous therapeutic significance in the future. are blocked by SNAP-5114, are located directly at the synapse, where the GAT-1 transporters are also located. Therefore, the combination of 5.1. Epilepsy tiagabine with SNAP-5114 induces only an additive effect [163]. In turn, BGT-1 transporters are located at a considerable distance from A disturbance in the balance between the activity of the stimulatory the synapse, and so it is not surprising that the combination of both and inhibitory systems in CNS is the main reason for the occurrence of tiagabine and SNAP-5114 with EF1502 produces a synergistic effect. In- epileptic seizures [151,152]. The GABAergic system plays a major inhib- terestingly, despite the effective action of the compounds acting itory function in the nervous system [153]. Drugs that are currently used through BGT-1, a seizure threshold was found unaltered in knockout in the treatment of epilepsy in many cases affect the elements of this mice [86]. All these examples demonstrate the large possibilities of system. Phenobarbital, lorazepam, diazepam, or clonazepam act on re- using the inhibitors of GABA transporter in the treatment of various ceptors for GABA, and vigabatrin inhibits the enzyme responsible for types of epilepsy, as well as the need for further research in this direc- the degradation of GABA. Tiagabine influences reuptake via transporters tion [164]. of GABA [149,152]. As mentioned above, tiagabine is the only registered and currently used drug that works on GABA uptake through the GAT-1 5.2. Depression and anxiety transporter [148–150]. It is effective against partial-onset seizures [36]. Experiments, carried out on rats, showed a significant increase in GABA The dysfunction of the GABAergic system is also one of the causes of concentration within the thalamus and hippocampus after administra- anxiety and depression [165,166]. In studies on humans and various an- tion of tiagabine. The drug was effective in inhibiting tonic as well as imal models, it was observed that the stimulation of GABA receptors clonic seizures in a mouse model of epilepsy induced by sounds generally produced anxiolytic activity, while the antagonists produced (14 kHz, 111 dB for 30 s). It also effectively prevented the tonic attacks anxiogenic-like effects [167]. Benzodiazepines, which have been used caused by the administration of pentetrazol (PTZ). However, tiagabine for many years in anxiolytic therapy, are allosteric modulators of was ineffective against tonic seizures induced by electric shock [154]. GABA-A receptors. These compounds can effectively and quickly reduce Selective GAT-1 inhibitors also cause several side effects, including rest- anxiety in patients suffering from panic attacks and many other anxiety lessness, sedation, and psychotic episodes [90,155,156]. Tiagabine may states. However, benzodiazepines are not free of side effects. Prolonged also have a seizure-initiating effect, particularly “absence” seizures use of benzodiazepines leads to the development of tolerance and ad- [157]. Remarkably, disorders caused by the undesirable effects of diction. Due to these reasons, they cannot be used chronically. 760 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772

Therefore, the search for other groups of compounds that can improve direct activation of the GABA receptors. In a study conducted on rats the functioning of the GABAergic system is ongoing. GABA reuptake in- using microdialysis, the GABA level in the areas of the thalamus respon- hibitors are one of the groups of compounds intensively developed in sible for modifying pain sensation increased twofold after the system- this direction [168]. The first evidence for the possible use of the inhib- atic administration of tiagabine at a dose of 30 mg/kg body weight itors of GABA transporter in the treatment of anxiety and depression [177]. Tiagabine also significantly increased the pain threshold in the disorders was provided by the studies on mice lacking the gene for paw pressure test. This compound proved its effectiveness in two GAT-1 [169,170]. In the open-field test, commonly used for the anxiety other tests performed on rats and mice using different pain stimuli: model, mice lacking GAT-1 transporters were observed to spend more chemical – abdominal contraction test, carried out after intraperitoneal time in the middle of the field and more likely to enter that area com- administration of acetic acid, and thermal – hot plate test. In the per- pared to WT mice, which indicated the reduced level of anxiety in formed assays, the analgesic activity of tiagabine was completely these animals. Other tests, such as the light-dark emergency test and blocked by the administration of a GABA-B receptor antagonist (CGP the elevated plus maze test also showed a reduced level of anxiety in 35348). This effect indicated that the increase in GABA concentration these mice [169]. These models have also been tested for depressive- in the synaptic space after the administration of tiagabine led to in- like behaviors using the forced swim test and the tail suspension test. creased activation of GABA-B receptors and induced an antinociceptive In both tests, mice lacking GAT-1 transporters showed significantly effect [177,178]. In the formalin test carried out on mice, the analgesic shortened immobility, which indicated a lower level of depression com- effect of tiagabine was tested in both the acute and delayed pain phases. pared to WT mice. Additionally, it was found that the level of corticoste- Administration of tiagabine 5 min before the test led to a dose- rone in these mice was significantly lower, which indicated that the dependent reduction in the behavior, indicating the occurrence of enhancement of GABAergic transmission by inhibition of GABA reup- pain in both phases [177,179,180]. In addition, the GABA-A receptor an- take affected the mental function through modulating the activity of tagonist, bicuculline, has been shown to have the ability to block the the hypothalamic-pituitary-adrenal (HPA) axis [170,171]. A study antinociceptive activity of tiagabine against the acute phase of pain, showed significant downregulation of GAT-3 in cLH (congenital help- and the GABA-B receptor antagonist (saclofen) produced the same ef- less) rats used as model animals of depression behaviors. Among the an- fect during both the acute and delayed phases of pain. Experiments imals with reduced expression of GABA transporter, the same with another GAT-1 inhibitor, NO-711, conducted on rats showed a sim- behavioral changes were noted as in knockout animals. This finding ilar analgesic effect as that observed after tiagabine administration. NO- strengthens the hypothesis that impaired glial functions leads to or 711 caused a reduction in the release of glutamic acid and aspartic acid, co-occurs in depression [172]. The antidepressant and anxiolytic effects and its activity was blocked by the antagonists of both GABA-A and of GAT inhibitors have also been confirmed in tiagabine-administered GABA-B receptors [181]. Nonselective inhibitors of GABA transporters mice. In both the tests involving single and repeated administration, a also showed analgesic properties. BM 130, BM 131, and (S)-SNAP- significant reduction in the symptoms of anxiety and depression was 5114 dose-dependently diminished the nocifensive response in the observed in animals [168]. Besides that, the plasma level of corticoste- writhing test. On the other hand, none of the tested compounds showed rone was reduced after the administration of tiagabine. There was also a statistically significant effect in the hot plate assay [173]. In the forma- a decrease in the expression of the gene coding for corticotropin- lin hind paw test, the antinociceptive activity of BM 131 and BM 130 releasing factor in the hypothalamus and the genes of the hypothalamic was observed. The ED50 value determined for BM 131 in the first steroid receptors. These data indicate that inhibition of these trans- phase of the test was similar to that reported for morphine. In the sec- porters led to a reduction in the activity of the HPA system. A similar ef- ond phase of the test, a highly significant antinociceptive activity was fect was noted for compounds that also interact with other GABA observed for both compounds, BM 131 and BM 130. (S)-SNAP-5114 transporters. BM 130, a nonselective inhibitor of GAT-1 and BGT-1, did not demonstrate any antinociceptive activity in the formalin hind showed a statistically significant antidepressant-like effect in the forced paw test [173]. In addition to decreasing acute pain, GABA reuptake in- swim test in mice. The antidepressant efficacy of another compound hibitors also reduced chronic, neuropathic pain. This effect was ob- from the same series, BM 131, was lower than that of BM 130 but similar served both during animal studies and in clinical trials in humans to that of (S)-SNAP-5114. In the four-plate test, the investigated com- [179,182,183]. Neuropathic pain is a serious health problem. It is often pounds displayed an anxiolytic-like effect. Both compounds, BM 130 accompanied by allodynia (pain occurring with physiological stimuli) and BM 131 showed similar anxiolytic-like efficacy and produced stron- and paresthesia/dysesthesia (sensory disorders). In rats with neuro- ger effects than (S)-SNAP-5114 [173]. pathic pain induced by nerve palsy, GAT-1 transporters were found to be overexpressed. It led to an increased GABA uptake and an increased 5.3. Pain pain sensation. These findings indicate that the inhibitors of these trans- porters may be potentially used in the treatment of neuropathic pain GABAergic neurons are found in practically all areas of the brain, in- [184]. In a study on the allodynia mouse model induced by dynorphin, cluding the thalamus, associated with the modulation of pain sensations it was demonstrated that the systemic administration of tiagabine re- reaching the sensory part of the cerebral cortex. Besides, GABA- duced allodynia in a dose-dependent manner [179]. Sciatic nerve liga- releasing nerve fibers are present in the neighborhood of spinothalamic tion was induced in rats (chronic constriction injury (CCI)) to mimic tract neurons, intervertebral neurons, and afferent fibers terminating in the human conditions of neuropathy. CCI markedly increased mechan- the posterior corners of the spinal cord (structures that are responsible ical and thermal allodynia and spontaneous pain behavior in the studied for the conduction of pain sensations) [174,175]. This arrangement indi- animals. Treatment with NO-711, another selective GAT-1 inhibitor, sig- cates the important role of the GABAergic system in the modulation of nificantly reduced all the tested pain behaviors [185]. In another study, pain not only at the level of the thalamus but also at the lower levels NO-711 prevented the development of paclitaxel-induced thermal of the nervous system. A study on rats showed that the stimulation of hyperalgesia and cold allodynia in mice [186]. In a 4-week pilot study both GABA-A and GABA-B receptors present in the dorsal part of the spi- involving 17 patients, the beneficial effects of tiagabine were demon- nal cord led to a reduction in pain level [176]. This effect resulted from strated. There was a significant reduction in the sensation of general the changes in the release of transmitters responsible for the conduction pain, surface pain, acute pain, and burning and sensitivity of the skin, of pain stimuli, such as glutamic and aspartic acids, together called ex- as well as the feeling of coldness. However, there was no reduction in citatory amino acids, after the activation of the GABA receptors. GABA the feeling of dull, deep pain. These effects were observed even at low reuptake inhibitors appear to be promising drug candidates capable of doses of the drug (4–8 mg), whereas higher doses caused severe side ef- inhibiting the transmission of pain by increasing the level of GABA with- fects without any improvement of the analgesic effect [183]. The activity out any potential adverse effects and limitations associated with the of tiagabine on chronic pain has been demonstrated in another study K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772 761 conducted on a group of 46 patients. During the 3-month study, patients should lead to a better understanding of the role of GABAergic transport were given 15 mg of drug per day in two separate doses. Tiagabine re- in stroke and related diseases. duced the intensity of pain experienced by the patients and significantly improved their sleep quality [182]. After the first successes of limiting 5.5. Alzheimer's disease neuropathic pain with GAT-1 inhibitors, tests were carried out on com- pounds acting through other GABA transporters. The ability of the BGT- Alzheimer's disease (AD) is the most common cause of dementia. 1 inhibitor NNC 05–2090 to overcome neuropathic pain was studied in a Due to its complex etiology, AD remains an incurable disease with the mouse model of sciatic nerve ligation in comparison with SKF89976A, a only available treatment being essentially symptomatic. New findings selective GAT-1 inhibitor, and (S)-SNAP-5114, an inhibitor with high ac- indicate that many promising targets may be considered as a starting tivity against GAT-3 [187]. NNC 05–2090 showed the strongest point in the search for effective AD treatment. Among them, we can antiallodynic action among all the tested compounds when adminis- find the proteins related to neuroinflammation and dysregulation of tered either intrathecally or intravenously. (S)-SNAP-5114 showed no the neurotransmitter management systems [205]. The GABAergic sys- effect on allodynia in the tested animals. The antiallodynic effect of tem is also known to be involved in the modulation of memory and NNC 05–2090 is questionable due to the results of the study on CHO learning [169]. One of the first evidence of possible disruptions of the cells, which revealed that the tested BGT-1 inhibitor caused the inhibi- GABAergic system in AD came from the clinical reports showing an in- tion of serotonin, noradrenaline, and dopamine transporters with simi- creased frequency of anxiety and epileptic seizures in AD patients lar strength [187]. [206,207]. Many different studies have shown that GABA neurotrans- mission is progressively decreased in AD [208,209]. Further investiga- 5.4. Stroke tions showed the disproportionate GAT expression in AD. Studies on postmortem brain tissue derived from the hippocampus, entorhinal GABA also plays a crucial role in cerebral ischemia [188]. cortex, and superior temporal gyrus showed a significant increase in Excitotoxicity caused by the increased release of glutamate and other BGT-1 expression in the dentate gyrus, the stratum oriens of the CA2 stimulating neurotransmitters is one of the critical mechanisms of neu- and CA3 regions, and the superior temporal gyrus. The changes were as- ronal injury that occurs following stroke [189]. Overactivation of post- sociated with a significant decrease in GAT-1 expression in the entorhi- synaptic glutamate receptors initiates a cascade of events that may nal cortex and the superior temporal gyrus. GAT-3 immunoreactivity lead to the dysfunction and degeneration of neurons. As the main inhib- was decreased in the stratum pyramidale of the CA1 and CA3 regions, itory neurotransmitter of the CNS, GABA acts by reducing the glutamate the subiculum, and the entorhinal cortex [210]. Further evidence came release induced by depolarization and ischemia [190,191]. Through from the results of animal studies. For instance, GAT-1−/− mice exhib- GABA-A and GABA-B receptors, GABA can trigger the hyperpolarization ited impaired fear memory in passive avoidance and contextual fear of neurons and counteract the initiating events in the cascade of ische- conditioning tests [211]. In the Morris water maze test, the spatial learn- mic biochemical changes [192,193]. Activation of GABA-ergic transmis- ing and memory ability of GAT-1−/− mice was also found to be im- sion during the acute phase of stroke can induce neuroprotective effects paired [169]. These results have drawn the attention of scientists such as hypothermia, reduction of respiratory rate, preservation of glu- toward GABA transporters located outside the neurons. Two aspects cose, and reduction of acidosis. Each of these increases the chance of have been taken into account: participation of astrocytic GAT in the survival of the nerve cells during ischemia [194,195]. The beneficial ef- very process of memorization and changes in the behavior of GAT dur- fect of GAT inhibitors in reducing the effects of stroke has only been ob- ing neuroinflammation. In the 5xFAD model of AD, a high GABA content served in the acute phase. The use of tiagabine in gerbil stroke models was noted in the AD-reactive astrocytes. Increased tonic current in resulted in a reduction of infarct volume and general neuroprotection 5xFAD animals was associated with memory deficit. After the adminis- [196–200]. The role of other GATs in ischemia is not fully understood. tration of NO-711, a large tonic current was induced in both 5xFAD and An unselective inhibitor of GAT-2 and GAT-3, (S)-SNAP-5114, led to in- WT animals. In contrast, SNAP-5114 caused no significant change in creased mortality in the photothrombotic (PT) stroke mice model [200]. tonic current in WT animals, but in 5xFAD animals, the compound re- However, the role of GAT-3 and post-stroke changes in its expression duced the tonic current retrieving the impairment of long-term poten- and function are still objects of research. Following the photothrombotic tiation and memory deficit [65]. This and other studies provided stroke, observations of the motor cortex showed a reduction in the level evidence that through GAT-3, the GABA current is reversed in reactive of GAT-3 protein that persisted for around 42 days after stroke [201]. astrocytes during neuroinflammation [66]. Since the expression level There is some evidence that after stroke, GAT-3 transports GABA in of GAT-3 has been found to be significantly increased in AD-reactive as- the reverse direction, out from the astrocytes. In studies on astrocytes trocytes in 5xFAD animals as well as in AD patients, GAT-3 may serve as of rats and humans, glutamatergic excitability led to tonic GABA inhibi- a novel drug target for developing a therapy to treat memory deficit tion through the reversal transport by GAT-3 [202,203]. Stroke and caused by an increased tonic current in AD patients [65]. As mentioned many other neurological diseases lead to the development of neuroin- earlier, only strong and selective compounds affecting individual astro- flammation, and astrocytes are involved in the response regulation. In cyte transporters of GABA can be effective for targeting the astrocytic several disease animal models, the development of neuroinflammation GABA transporters to combat AD. led to astrocytes activation and reversed GAT-3 transport [65,66]. In a study in which GABAergic transport was investigated in conjunction 5.6. Sleep disorders with neuroinflammation in models of multiple sclerosis (MS), GAT-2 expression was found to be increased. A study showed that in the cell Until today, most treatment approaches used for insomnia target culture of macrophages taken from MS patients and human macro- GABAergic transmission. The drugs first used to treat insomnia were phage lineage cells stimulated with interferon-γ, the GAT-2 expression barbiturates. These drugs increase the binding of GABA to GABA-A re- was induced. Use of the synthetic allopregnanolone analog, ganaxolone ceptors [65]. Benzodiazepines were the next generation of hypnotics (GNX), repressed GAT-2 expression in activated macrophages. In vivo widely used for insomnia because their anxiolytic and hypnotic proper- GNX treatment reduced GAT-2 expression in the spinal cord which in ties were comparable to barbiturates, and their toxicity was much lower turn correlated with improved neurobehavioral outcomes and reduced [212,213]. The third generation of hypnotics was named as the Z-drugs neuroinflammation [204]. Reverse GABA transport and neuroinflamma- because of their brand names zolpidem, zaleplon, and zopiclone tion occurring during the stroke and other neuro-damaging diseases are [214,215]. Despite the structural differences between benzodiazepines closely related, but further studies on this subject are needed. Applica- and Z-drugs, both these generations of hypnotics bind to the same site tion of novel, more selective, brain-permeable, and potent inhibitors of the GABA-A receptors [214]. This property of benzodiazepines and 762 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772

Z-drugs demonstrates that GABA transmission plays a key role in sleep Asignificant breakthrough came with the discovery of nipecotic acid and sleep pathologies, such as insomnia. Tiagabine, used in the treat- and guvacine (5,6), which are cyclic GABA analogs, as the inhibitors of ment of epilepsy, often induced sedation as a side effect during treat- GABA transporters. The R-isomer of nipecotic acid was found to be ment [216]. This finding led to the use of tiagabine to trigger the much more active than the S-isomer. It showed high activity against sedative or hypnotic effect [217]. However, the role of GAT in sleep- GAT-1, significantly lower activity against GAT-2 and GAT-3, and was wake regulation is unclear. GAT-1−/− mice were found to exhibit a re- practically devoid of activity against BGT-1 transporters. Guvacine duction of EEG power in low frequencies, which corresponded to the showed a similar activity profile; however, it exhibited a slightly slow-wave activity (SWA) during nonrapid eye movement (NREM) lower affinity to GAT-1 [227]. Despite the relatively high activity, these sleep. The length of the SWA periods corresponded to the changes in compounds were not able to penetrate the CNS due to their high polar- the duration and intensity of wakefulness and sleep. Rapid eye move- ity, which limits their use in therapy. This problem was solved by ment (REM) sleep of knockout mice was elevated both during the attaching a lipophilic fragment to the nitrogen atom of nipecotic acid light and dark periods, while NREM sleep was reduced during the and guvacine. The derivatives thus formed currently serve as the largest light period only [218,219]. In healthy adults, tiagabine administration group of GABA uptake inhibitors (Table 2). led to an increased SWA as well as improved sleep efficiency [220]. In The first lipophilic derivatives of nipecotic acid and guvacine, which studies on both adults and elderly patients with primary insomnia, showed high activity, contained the 4,4-diphenyl-3-butenyl moiety (7 – tiagabine increased SWA in a dose-dependent manner. Nevertheless, SKF89976A, 8 – SKF100330A). Compounds with a longer, shorter, or in both cases, the sleep latency or the number of awakenings was unaf- saturated carbon linker were inactive [228]. In the following years, a fected [217,221–223]. This limited hypnotic effect and the occurrence of large group of derivatives based on SKF89976A and SKF100330A were specific side effects significantly limit the possibility of using tiagabine synthesized and tested. They acted as selective inhibitors of GAT-1 in the treatment of insomnia. Another selective GAT-1 inhibitor, NO- transporters. The R-isomers of these compounds (as in the case of 711, caused a marked enhancement of EEG activity in mice in the fre- nipecotic acid) showed much higher activity. The first modifications quency ranges of 1.5–6.75 Hz during the NREM sleep with a signifi- carried out in these compounds involved the conversion of the but-3- cantly shortened sleep latency of NREM sleep and with increased enyl chain to methoxyethyl chain and the introduction of halogen- duration of NREM sleep and increased number of NREM sleep episodes. substituted phenyl rings or tricyclic or heterocyclic group (9). Among On the other hand, a high dose of NO-711 induced epilepsy-like EEG ac- these compounds, one derivative with a high affinity to GAT-1 (9 -Cl tivity [224]. The hypnotic effectiveness of compounds that interact via 966) entered the first phase of clinical trials; however, it was withdrawn other GABA transporters has not been determined yet. However, bear- due to strong side effects [234]. The introduction of methyl moiety in ing in mind that the side effects that occur after blockage of GAT-3 or position 2 (with respect relative to the linker) in one or both the aro- BGT-1 in relation to GAT-1 inhibition are insignificant, these compounds matic or heteroaromatic rings resulted in a series of compounds with can be very promising in the treatment of insomnia and other sleep an increased activity relative to the initial structures (SKF89976A and disorders. SKF100330A). One of them, (R)-1-[4,4-bis(3-methyl-2-thienyl)-3- butenyl]-3-piperidinecarboxylic acid (10 – tiagabine) progressed through all phases of clinical trials and is the only GABA reuptake inhib- 6. GABA transporter inhibitors itor currently used in the treatment of epilepsy [235]. A further increase in activity was achieved by introducing an oxime As an endogenous ligand, GABA (1) has a similar affinity to GAT-1, (11 – NO-711) or a vinyl ether moiety within the linker [236]. The next GAT-2, and GAT-3 and a slightly lower affinity to BGT-1 transporters step in the development of diaryl derivatives of nipecotic acid and (Table 1). The latter is also capable of transporting betaine (2). It was guvacine involved changing the connection of the aromatic fragment demonstrated that GABA analog with a shorter carbon chain – β- with the linker from symmetrical to asymmetrical. Among the first de- alanine (3) – is preferentially transported by GAT-2 and GAT-3 [225]. veloped derivatives, high activity was shown by the compounds with Later, in the search for other compounds with high affinity and selectiv- 1,2-bis(2-fluorophenyl)ethylidene and 2-(benzyl)phenyl moiety at- ity for these transporters, among the small compounds with the struc- tached to the oxime and ether fragment, respectively [237]. In another ture of amino acid, isoserine (4) was identified [226]. study, a vinyl ether linker was used, to which an unsubstituted or a

Table 1 GABA and its small-molecule analogs.

Compound pIC50 Reference hGAT-1 hBGT-1 hGAT-2 hGAT-3 mGAT-1 mGAT-2 mGAT-3 mGAT-4

1 (GABA) 5.00 4.59 4.96 5.00 [225] 2 (Betaine) b2.00 3.23 b2.52 b2.00 [225] 3 (β-Alanine) 2.30 3.18 4.38 4.44 [225] 4 (Isoserine) 2.60 3.64 5.37 5.23 [225] 5 (R-Nipecotic acid) 5.07 3.28 4.71 4.79 [227] 5 (S-Nipecotic acid) 4.13 3.12 3.71 3.51 [227] 6 (Guvacine) 4.87 3.31 4.59 4.59 [227]

Activity determined for mouse transporters are shown in italics. K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772 763

Table 2 Diaryl derivatives of nipecotic acid and guvacine.

Compound pIC50 Reference hGAT-1 hBGT-1 hGAT-2 hGAT-3 mGAT-1 mGAT-2 mGAT-3 mGAT-4

7 (SKF89976A) 6.16 3.43 3.71 3.56 [227] 8 (SKF 100330A) 6.70b –––[228] 9 (Cl 966) 6.59 3.52 2.89c 3.48 [229] 10 (Tiagabine) 6.88 52%a 64%a 73%a [227] 11 (NO-711) 6.83 3.20 3.62 3.07 [227] 12 6.52 71.1%a 4.55 4.31 [230] 13 7.43 73%a 91%a 4.20 [231] 14 7.72 71%a 4.79 4.38 [232] 15 (DDPM-2571) 8.27 4.31 4.35 4.07 [233]

Activity determined for mouse transporters are shown in italics. a remaining GABA uptake (%) at 100 μM concentration of the tested compound. b Measured for rat brain synaptosomes. c Measured for rat GAT-2.

fluorine-substituted 2-(benzyl)phenyl (12) or 2-(benzoyl) phenyl moi- Modifications that were made in the structure of diaryl inhibitors of ety was added. Derivatives were synthesized in both E and Z configura- GABA transporters also involved the amino acid fragment. One of them tions, of which the latter showed significantly higher activity. Generally, was the conversion of nipecotic acid to its bioisostere exo-THPO. N- compounds with the 2-(benzyl)phenyl fragment were found to be more methyl-exo-THPO is a weak, preferential inhibitor of GAT-1 (16) potent, among which the most active was derivative 12, as shown in (Table 3). Attachment of symmetrical diaryl fragment increased the af- Table 2 [230]. A further increase in potency was achieved by replacing finity of this compound to GAT-1 and the affinity of some derivatives the five-atom-long ether spacer with a but-3-enyl or but-3-ynyl moiety (with fragment as in the case of compounds 7 and 8)toBGT-1,although and introducing a 2′,4′-dichloro(1,1′-biphenyl)-2-yl moiety (13, 14) to a lesser extent. In the case of compound EF1502 (17), the difference [231,232]. The same fragment is present in currently the most active in- in activity between the R- and S-isomer was demonstrated. R-EF1502 hibitor DDPM-2571(15), which was identified during the screening of showed a medium-high affinity to GAT-1 and a medium affinity to oxime derivatives of guvacine [233]. BGT-1, whereas the S-isomer showed no affinity to GAT-1 but the

Table 3 Derivatives with a modified amino acid fragment.

Compound pIC50 Reference hGAT-1 hBGT-1 hGAT-2 hGAT-3 mGAT-1 mGAT-2 mGAT-3 mGAT-4

16 (N-Me-exo-THPO) 3.35 b2.52 b2.52 b2.52 [239] 17 (R-EF1502) 5.40 4.66 b3.82 b3.82 [239] 17 (S-EF1502) 3.92 4.47 b3.82 b3.82 [239] 18 6.46a ––4.58a [240] 19 5.67 58%b 4.37 59%b [241] 20 3.25 4.67 3.03 3.54 [242]

Activity determined for mouse transporters are shown in italics. a Measured for bovine GABA transporters. b remaining GABA uptake (%) at 100 μM concentration of the tested compound. 764 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772

Table 4 Derivatives with a triaryl moiety showing high activity toward GAT-2 and GAT-3.

Compound pIC50 Reference hGAT-1 hBGT-1 hGAT-2 hGAT-3 mGAT-1 mGAT-2 mGAT-3 mGAT-4

21 (SNAP-5114) 4.07 56%a 5.29 5.71 [243] 22 (DDPM-859) 4.19 4.12 4.85 5.78 [243] 23 (DDPM-1457) 4.40 4.42 5.47 5.87 [243] 24 4.55 53%a 4.99 5.50 [244] 25 4.85 4.63 5.55 5.54 [244] 26 68%a 56%a 4.77 5.24 [241]

Activity determined for mouse transporters are shown in italics. a remaining GABA uptake (%) at 100 μM concentration of the tested compound. moderate affinity to BGT-1, which made it a preferential inhibitor of derivatives with a comparable, high activity and increased chemical sta- these transporters [238,239]. bility compared to SNAP-5114 [243]. Further modifications of the com- Some compounds with pyrrolidine-2-yl-acetic acid fragment and pound DDPM-1457 involved the introduction of other aromatic groups their 2-substituted derivatives (18, 19) also exhibited high activity within the triaryl fragment. Among the obtained derivatives, relatively [240,241]. Among many other amino acid derivatives, the compound high activity and selectivity were shown by the derivatives in which with (1R,2S)-2-aminocyclohexane-1-carboxylic acid fragment (20)is one of the methoxyphenyl rings was replaced with a benzodioxol-5-yl worth mentioning. Although it is only moderately active, it has a rela- or benzothiophen-5-yl moiety (24, 25). The compounds with tris- tively high preference for BGT-1 [242]. benzothiophen-5-yl and tris-benzodioxol-5-yl moieties were signifi- While most of the diaryl derivatives of nipecotic acid and its analogs cantly less active, whereas the compound with the tris-benzofuran-5- are selective or preferential inhibitors of GAT-1 transporters, the deriv- yl fragment had activity at a pIC50 level N 5.00. However, they were all atives with a larger lipophilic fragment were found to be selective/pref- nonselective [244]. The derivatives with an alkene linker described erential against GAT-2 and GAT-3 transporters (Table 4). abovewerefoundtobetrans-isomers. Cis-Isomers proved to be less ac- Interestingly, derivatives of nipecotic acid with three phenyl rings tive in most of the cases except a compound with an additional methy- attached to the end of the ether linker retained their selectivity against lene group between the triphenyl group and the double bond, which

GAT-1. The addition of methoxy groups in the para position of each of showed high activity toward GAT-1 (pIC50 = 6.00) while maintaining these rings changed the activity of these derivatives toward GAT-2 moderate affinity toward GAT-3 (pIC50 =4.82)[245]. Similar to the and GAT-3 (21 – SNAP-5114). Moreover, among them, the S-isomers diaryl derivatives, the nipecotic acid in the triaryl compounds can be ef- were much more active, in contrast to the previously described GAT-1 fectively replaced with pyrrolidine-2-yl-acetic acid fragment and its 2- inhibitors [229]. Addition of a methyl group to one of the substituted derivatives (26)[240,241]. methoxyphenyl rings (22 – DDPM-859) or replacement of the ethoxy Recently, a library of nipecotic acid derivatives with substitution at spacer with a propenyl moiety (23 – DDPM-1457) resulted in the 5-position, containing a hydrazone moiety in the linker, was

Table 5 5-Substituted nipecotic acid derivatives with hydrazone moiety.

Compound pIC50 Reference hGAT-1 hBGT-1 hGAT-2 hGAT-3 mGAT-1 mGAT-2 mGAT-3 mGAT-4

27 4.26 4.28 5.11 5.27 [246] 28 75%a 72%a 5.18 4.99 [246] 29 4.64 64%a 4.48 4.12 [247]

Activity determined for mouse transporters are shown in italics. a remaining GABA uptake (%) at 100 μM concentration of the tested compound. K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772 765

Table 6 Preferential GAT-2 inhibitors with imidazole structure.

Compound pIC50 Reference hGAT-1 hBGT-1 hGAT-2 hGAT-3 mGAT-1 mGAT-2 mGAT-3 mGAT-4

30 3.21 3.99 4.76 4.33 [248] 31 62.8%a 3.28 4.54 3.51 [248] 32 73.7%a 77.3%a 3.71 47.2%a [248] 33 4.90 4.54 5.13 4.99 [248]

Activity determined for mouse transporters are shown in italics. a remaining GABA uptake (%) at 100 μM concentration of the tested compound.

synthesized and tested (Table 5). Among these compounds, two with an dependent on their conformation. It was demonstrated that only the activity profile similar to the previously described selective GAT-2 and (2S,3R) isomer of compound 36, containing the trans-cyclopropyl frag- GAT-3 inhibitors were identified (27, 28)[246]. Compound 29 exhibited ment, is active. In the case of compound 37,thesyn-form, in which only moderate activity to GAT-1, but it is worth noting because unlike both the cyclopropyl fragment and the carboxyl group are present tiagabine (which is a competitive inhibitor), it was proved to be an allo- above the surface of the cyclopentane ring, is active. This compound is steric inhibitor [247]. currently the most potent and highly selective among the inhibitors of GABA reuptake inhibitors with the amino acid structure also involve BGT-1 transporters [250,251]. the compounds in which the amino group was included in the imidaz- All the above-described derivatives have an amino acid or an amino ole ring (Table 6). acid-like structure. The second large group of GABA uptake inhibitors is 1H-Imidazol-4-yl-acetic (30) and 3-(1H-imidazol-2-yl)propanoic devoid of the acidic moiety. Some of them are the small-molecule deriv- acids (31) are the preferential inhibitors of GAT-2 transporters with atives of isatin, which are moderately potent and currently the most se- moderate activity. (2E)-3-(1H-Imidazol-2-yl)prop-2-enoic acid (32) lective among the ligands of GAT-3 (38, 39)(Table 8). In kinetic studies, was significantly less active. Attachment of a lipophilic fragment to these compounds were found to be noncompetitive inhibitors [252]. these structures showed an increase in potency, but only in a few Other nonamino acid-based compounds are very diverse in struc- cases. The most active was compound 33. However, it did not retain ture, although some elements could be found in common. All these mol- its high selectivity toward GAT-2. The 3-(1H-imidazol-2-yl)propanoic ecules contain lipophilic groups located at the ends, between which acid derivative with the lipophilic fragment as in SNAP-5114, which is there are an amino group and acceptor of hydrogen bonds (Fig. 7, not surprising, showed an increased activity toward GAT-3 (pIC50 = Table 9). The first group of compounds with such a structure is 5.10). In turn, derivatives of this acid and 1H-imidazol-4-yl-acetic acid piperidin-4-ol derivatives. Compound with 3-(carbazol-9-yl)propyl with a 1-(4-(9-(4-methoxyphenyl)-9H-fluoren-9-yl)oxy)butyl frag- fragment in 1-position and ortho-methoxyphenyl fragment in 4- ment, were selective inhibitors of BGT-1, although they showed only position of the piperidine ring is a preferential inhibitor of BGT-1 (40). moderate activity [248]. The conversion of the ortho-methoxyphenyl fragment to para- Among the small, hydrophilic compounds with amino acid struc- methoxyphenyl or para-chlorophenyl increased the affinity of the com- ture, selective inhibitors of BGT-1 transporters have been identified pounds to the GAT-2 and GAT-3 transporters. An even greater effect on (Table 7). A medium-high potency was observed for the derivatives these transporters was found after the introduction of a 3-(3- with a fragment of tetrahydropyridine and tetrahydropyrimidine (34, ethoxycarbonylo-β-carbolin)propyl fragment, which made the com- 35). Interestingly, their aromatic analogs were utterly inactive [249]. pounds nonselective ligands (41)[253]. Another group includes derivatives based on the structure of GABA, The next group of compounds is the 2-substituted derivatives of 4- conformationally restricted with a cyclopropane ring, or a bicyclo[3.1.0] aminobutanamides and 4-hydroxybutanamides. They are moderately hexane fragment (36, 37). The activity of these compounds is strictly active, nonselective inhibitors. In some cases, the activity is shifted

Table 7 BGT-1 inhibitors with amino acid structure.

Compound pIC50 Reference hGAT-1 hBGT-1 hGAT-2 hGAT-3 mGAT-1 mGAT-2 mGAT-3 mGAT-4

34 b4.00 5.0 b4.00 b4.00 [249] 35 b3.00 5.6 4.4 4.0 [249] 36 b4.00 5.26 4.43 4.86 [250] 37 b4.00 6.23 b4.00 4.12 [251] 766 K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772

Table 8 Selective and noncompetitive GAT-3 inhibitors.

Compound pIC50 Reference hGAT-1 hBGT-1 hGAT-2 hGAT-3 mGAT-1 mGAT-2 mGAT-3 mGAT-4

38 b3.00 3.21 3.74 5.20 [252] 39 3.07 3.64 4.24 5.09 [252]

toward a particular type of transporters. Generally, 4-aminobutanamide methyl-4,4-diphenylbut-3-en-1-amine moiety in 2-position had a ben- derivatives are more potent than the 4-hydroxybutanamide ones, eficial effect on activity in both hydroxy- and amino- derivatives. In the which is particularly evident for BGT-1 (42). However, in the case of case of amine derivatives, such an effect was observed for 4- GAT-3, and to a lesser extent in GAT-1, the activity of some 4- (diphenylmethylidene)piperidine, 4-(diphenylmethyl)piperidine, and hydroxybutanamides derivatives (43)iscomparabletoorevenhigher 4,4-diphenylbut-3-en-1-amine (only for BGT-1) fragments. The influ- than that of 4-aminobutanamides ones. It was observed that the N- ence of the substitution in a benzyl group on the activity was also

Fig. 7. The general structure of nonamino acid GABA reuptake inhibitors with two lipophilic fragments, together with examples of compounds from particular groups. K. Łątka et al. / International Journal of Biological Macromolecules 158 (2020) 750–772 767

Table 9 three-dimensional structure of GABA transporters remains unknown. The activity of the compounds presented in Fig. 7. Neither crystal structure nor NMR structure has been obtained so far.

Compound pIC50 Reference Therefore, information about ligand binding mode comes from molecu- lar modeling studies based on the similarity of GATs and leucine, dopa- hGAT-1 hBGT-1 hGAT-2 hGAT-3 mGAT-1 mGAT-2 mGAT-3 mGAT-4 mine and serotonin transporters. Understanding the mechanism of GATs' action and the process of ligand binding may contribute to the de- 40 (NNC 05-2090) 4.72a 5.85a 4.39a 4.82a [253] 41 (NNC 05-1965) 4.64a 5.59a 5.00a 5.55a [253] velopment of new more effective inhibitors of GABA reuptake. More- 42 4.86 5.23 4.96 4.99 [257] over, the knowledge of the difference between various GAT types may 43 4.72 4.90 5.14 5.00 [256] lead to the design and synthesis of novel ligands of the desired selectiv- b 44 65.7% 4.99 4.64 4.58 [258] ity. Nowadays, finding very potent inhibitors of BGT-1, GAT-2 or GAT-3 45 70.7%b 77.1%b 5.09 4.53 [258] is still a considerable challenge for many research groups. Summing up, 46 (BPDBA) b4.00 4.87 b4.00 b4.00 [259] 47 b4.00 4.82 4.64 b4.00 [259] GABA transporters and other SLC6 family members are being broadly 48 4.36 4.89 5.02 5.11 [227] studied to provide new findings to the scientist community. Each dis- 49 4.48 4.84 5.11 4.83 [227] covery is a step forward to give the patients a chance of better and Activity determined for mouse transporters are shown in italics. more effective treatment. a pKi values calculated on the basis of Ki given in the reference. b μ remaining GABA uptake (%) at 100 M concentration of the tested compound. Acknowledgments observed. In the case of BGT-1, preferred substitution was ortho-chloro The project was financially supported by the National Science Cen- and to a lesser extent, para-methyl; for GAT-2, ortho-andpara-chloro; ter, Poland (grant no. 2016/23/D/NZ7/01172) and Jagiellonian Univer- and for GAT-3 mainly para-methyl and ortho-chloro. [254–257]. sity Medical College (grant no. N42/DBS/000015). 1,5-Disubstituted tetrazole derivatives represent a similar general structure. The function of the hydrogen bond acceptor is fulfilled by Declaration of competing interest the nitrogen atoms of the aromatic ring. In one series of compounds, a 2,4,4-trimethylpentan-2-yl group is present in 1-position instead of The authors declare no conflict of interest. the aromatic ring, which in the case of some derivatives exerts a favor- able effect on the activity of the compounds. An example is the most ac- References tive compound 45, which has been proved to be a moderately potent, [1] K. Gale, GABA and epilepsy: basic concepts from preclinical research, Epilepsia 33 selective inhibitor of GAT-2. Interestingly, the substitution of N-(3,3- (Suppl.5)(1992)S3–12. diphenylpropane)aminomethyl moiety by N-(3,3-diphenylprop-2- [2] G.E. Fagg, A.C. 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