StructureâActivity Relationship, Pharmacological Characterization

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Structure-activity relationship, pharmacological characterization and molecular modeling of non-competitive inhibitors of the betaine/#-aminobutyric acid transporter 1 (BGT1). Lars Jørgensen, Anas Al-Khawaja, Stefanie Kickinger, Stine Byskov Vogensen, Jonas SkovgaardPetersen, Emil Rosenthal Jensen, Nrupa Borkar, Rebekka Löffler, Karsten K Madsen, Hans BräunerOsborne, Arne Schousboe, Gerhard F. Ecker, Petrine Wellendorph, and Rasmus Prætorius Clausen J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00924 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 9, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Structure-activity relationship, pharmacological characterization and molecular modeling of non-competitive inhibitors of the betaine/γ-aminobutyric acid transporter 1 (BGT1). Lars Jørgensena*, Anas Al-Khawajaa*, Stefanie Kickingerb*, Stine B. Vogensena*, Jonas Skovgaard-Petersena, Emil Rosenthala, Nrupa Borkara, Rebekka Löfflera, Karsten K. Madsena, Hans Bräuner-Osbornea, Arne Schousboea, Gerhard F. Eckerb, Petrine Wellendorpha, and Rasmus P. Clausena a

University of Copenhagen, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, 2100 Copenhagen, Denmark. b University of Vienna, Department of Pharmaceutical Chemistry, Althanstrasse 14, 1090 Vienna, Austria. * Contributed equally to this paper

Keywords Betaine/γ-aminobutyric acid transporter 1, BGT1, GABA transporters, non-competitive inhibition, BPDBA, computational docking, mutagenesis

ABSTRACT N-(1-Benzyl-4-piperidinyl)-2,4-dichlorobenzamide 5 (BPDBA) is a non-competitive inhibitor of the betaine/GABA transporter 1 (BGT1). We here report the synthesis and structure-activity relationship of 71 analogues. We identify 26m as a more soluble 2,4-Cl substituted 3-pyridine analogue with retained BGT1 activity and an improved off-target profile compared to 5. We performed radioligand-based uptake studies at chimeric constructs between BGT1 and GAT3, experiments with site-directed mutated transporters and computational docking in a BGT1 homology model based on the newly determined X-ray crystal structure of the human serotonin transporter (hSERT). Based on these experiments we propose a binding mode involving residues 1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 46

within TM10 in an allosteric site in BGT1 that corresponds to the allosteric binding pocket revealed by the hSERT crystal structure. Our study provides first insights into a proposed allosteric binding pocket in BGT1, which accommodates the binding site for a series of novel non-competitive inhibitors.

1 INTRODUCTION The principal inhibitory neurotransmitter in the mammalian brain is γ-aminobutyric acid (GABA).1 GABAergic neurotransmission is crucial for most CNS functions, and a number of CNS disorders are proposed to arise from malfunctions in this system.2–4 Thus, compounds that increase the GABAergic inhibitory signalling have been developed to treat various neurological diseases. One example, related to this work, is the anticonvulsant GABA-uptake inhibitor, [(R)N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid 1 (Tiagabine), Fig. 1],5 which has provided proof-of-concept for pharmacological inhibition of GABA uptake in epilepsy. In addition, pharmacological intervention with GABA uptake has also been proposed to have clinical potential in other neurological conditions, including stroke and anxiety.6–8

Four subtypes of GABA transporters (GATs) have been cloned from the mammalian brain that all belong to the solute carrier 6 (SLC6) family of sodium-dependent neurotransmitter transporters.9 According to the International Union of Basic and Clinical Pharmacology (IUPHAR), these are named GAT1, GAT2, GAT3 and BGT1 (betaine/GABA transporter 1) and will be referred to as such, regardless of the species.10 Highly selective inhibitors of GAT1 have been developed (e.g. 1)11 and successfully used for understanding the physiological function and therapeutic relevance of GAT1.12 The development of inhibitors with comparable potency and 2 ACS Paragon Plus Environment

Page 3 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


selectivity for the other GAT subtypes has, however, been difficult, which is related to the high sequence identity, especially between GAT2, GAT3 and BGT1.13 We have previously reported (RS)-4-[N-[1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl]-N-methylamino]-4,5,6,7tetrahydrobenzo[d]isoxazol-3-ol 2 (EF1502) as a mixed GAT1/BGT1 inhibitor,14 and extensive pharmacological investigation of the compound has indicated that selective inhibition of BGT1 is implicated in seizure management.15 We also reported the further development of 2 into [4,4-bis(3-methyl-thien-2-yl)-but-3-enyl]-(2-carboxy-cyclohex-2-enyl)-N-methylamine 3 (RPC425) with an improved subtype-selectivity among the GATs for BGT1.16 Some preference for BGT1 was also reported for 1-[3-(9H-carbazol-9-yl)propyl]-4-(2-methoxyphenyl)-4-piperidinol 4 (NNC 052090),17 but this compound was, however, also demonstrated to act at other targets not related to GABA.18 This has limited its use as a tool compound, and no further optimization of the compound has been described. Recently, the first BGT1 inhibitor with high selectivity among GATs and with nanomolar potency was identified. This compound, (1S,2S,5R)-5aminobicyclo[3.1.0]hexane-2-carboxylic acid, is a GABA analogue, which displays 129- and 169-fold higher potency at BGT1 (IC50 590 nM) than at GAT3 and GAT1/2, respectively.19 To allow for the identification of other lead structures, we performed a pharmacological screening of a

commercial

compound

library

and

identified

N-(1-benzyl-4-piperidinyl)-2,4-

dichlorobenzamide (BPDBA, 5) as a non-competitive inhibitor (IC50 32 µM), with selectivity for BGT1 among the GABA transporters and representing a new structural class possibly acting at an allosteric binding site.20 Furthermore, two analogues of 5, compounds 6 and 7, showed activity at both BGT1 and GAT2, while having no activity at GAT1 and GAT3.20 This represents an unusual pharmacological profile, since GAT2 and GAT3 typically display a similar pharmacology due to their high sequence identity,13 at least with regards to the orthosteric binding pocket. Thus, we found 5 to be an interesting lead structure for further development, 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 46

focusing on increasing the potency and rather poor solubility of the compound. Moreover, we aimed at exploring the so far elusive binding site for 5 in BGT1, by investigating the structureactivity relationship (SAR) and relating this to structural modeling and mutational studies. We here report our efforts through the synthesis of 71 analogues through systematic structural variations, their biological evaluation, and propose a binding site based on the SAR, uptake experiments at chimeric transporter constructs and structure-based modeling studies.

2 RESULTS AND DISCUSSION 2.1 Synthesis Most of the compounds were made from two principal routes (Scheme 1): Route I starting with acylation of the 4-amino group of 1-Boc-4-aminopiperidine, followed by removal of the Boc group and alkylation of the secondary amine in the 4-amidopiperidines; and Route II starting with alkylation of the secondary amine in 4-(N-Boc-amino)piperidine, followed by removal of the Boc group and acylation of the primary 4-amino group. Acid chlorides were employed for acylations, and alkylbromides for alkylations. Compound 14 was made by hydrolysis of tertbutyl ester 15. Esters 33a and 33b were made by acylation of 1-benzyl-4-hydroxypiperidine. 34 was made by reduction of 5 with LiAlH4.

2.2 Structure-activity relationship The structure of 5 can be divided into a dichlorobenzoic amide part (A) and a benzyl amino part (B), connected via a piperidine ring (Fig. 1). Systematic as well as simultaneous variations were introduced in each part. The structures are shown in Fig. 2 and Fig. 3, and the pharmacological activities are listed in Table 1 and 2. The compounds were tested in a [3H]GABA uptake assay using recombinant cells transiently expressing either the human (h) or mouse (m) GAT subtypes. 4 ACS Paragon Plus Environment

Page 5 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The first series of compounds had variations in the benzyl amino part (B). The benzyl group was exchanged for a range of alkyl groups (8–12) and alkyl groups capped with various functional groups such as a cyano, carboxylic acid and ester as well as phosphonate ester (13–16), but all compounds were inactive in concentrations up to 1,000 µM. In a second series, substituents were introduced at the aromatic ring of the benzyl group. Introducing a nitro (17a and 17b) or cyano (17c and 17d) group in the ortho/para position was detrimental for activity at the highest tested concentrations of 100 µM and 50 µM, respectively. Other substituents in the para-position, such as iodo (17e), carboxylic methyl ester (17f) and trifluoromethoxy (17g), also led to inactive compounds. Moving the trifluoromethoxy to the meta-position (17h), however, led to some activity at mBGT1 (IC50 160 µM). Introducing a trifluoromethyl (17i) or a fluoro moiety (17j) in this position led to compounds with potency similar to that of 5. A chloro substituent in the metaposition (17k) was not tolerated, whereas the m-methoxy substituted 17l was equipotent with 5. Methyl (17m) substituent in this position led to inactive compounds. Di-substitution in the 3- and 5-positions with methyl groups (17n) or chloro atoms (17o) gave inactive compounds, whereas methoxy groups (18) in these positions were tolerated, yielding a compound equipotent with 5 at mBGT1. A range of aromatic analogues exchanging the phenyl ring of the benzyl group with 2-, 3- and 4-pyridyl (19–21), styryl (22), naphthyl (23), quinolonyl (24) and benzoyl substituents (25) led to seemingly inactive compounds (at least for 23 and 24 when tested in concentrations up to 50 µM).

Another series of compounds were based on variation in the dichlorobenzoic amide part (A). Exchanging the dichlorophenyl ring for an isopropyl group gave the inactive compound 26a, whereas the unsubstituted benzamide 26b displayed inhibitory activity at hBGT1 with an order 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 46

of magnitude lower potency than 5. Reintroducing the chloro substituent in the 2- and 4- position (26c and 26d, respectively) gave compounds with reduced potencies at mBGT1 compared to 5 (IC50 151 and 77 µM, respectively). Addition of a third chlorine atom in 5 gave 26e, which showed an apparent 3-fold increase in potency, giving the most potent compound in the series (IC50 11 µM). Introduction of other substituents, such as p-F (26f), o-OEt (26g) m-CF3 (26h) and the 2,3-dimethoxy substituted (26i) led to slightly increased potency (IC50 100 µM, 180 µM, 170 µM, respectively) compared to the unsubstituted 26b, but still lower potency than 5. Exchange of the phenyl ring for a 3-pyridine ring (26j) was detrimental for activity, and introduction of 2-Cl substituent at the pyridine ring (26k) was not beneficial for activity. Nevertheless, introduction of the chloro substituent in the 4-position (26l) of the 3-pyridine ring increased the potency quite dramatically (IC50 39 µM), however, at the expense of mBGT1-selectivity over mGAT2 and mGAT3. Introduction of two chlorine atoms gave the 2,4-Cl substituted 3-pyridine analogue 26m with a potency similar to 26l, but with a better subtype-selectivity for mBGT1 compared to mGAT2 and mGAT3. The 4-pyridyl analogue 26n had much weaker activity, but addition of a 3-Cl (26o) increased the activity slightly (IC50 300 µM). The 2-thienyl analogue (26p) showed activity similar to 26b.

We observed a highly improved solubility of 26m compared to 5, and this was experimentally determined to 610 µg/ml and 40 µg/ml, respectively. Compound 26m was therefore used as a new starting point for variation. Exchanging the phenyl group of the benzyl amino moiety for a 2-thienyl group (27) gave an inactive compound, whereas exchange for a 3-indolyl (28) or a benzo[d][1,3]dioxol-5-yl group (29) was tolerated, yielding equipotent compounds. The biphenyl substituted 30 turned out to be inactive. We then introduced a range of substituents in the meta position of the phenyl ring of the benzyl amino moiety. Methoxy- (31a) and ethoxy- (31b) 6 ACS Paragon Plus Environment

Page 7 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


substitution in this position was tolerated (IC50 115 and 111 µM, respectively), but 2hydroxyethoxy- (31c), nitro- (31d), methoxycarbonyl- (31e), carboxyl- (31f), carbamoyl- (31g), hydroxyl- (31h), and 4’fluorophenoxy- (31i) were all inactive. The activity of m-methoxy substituted 17l and 31a prompted further investigation. Removal of both chlorine atoms in 31a gave the inactive compound 32a, and reintroducing the 2-substituted Cl (32b) was not beneficial for potency. The 4-substituted 32c had low activity (IC50 115 µM), and as with 26l, we also started to see some activity at mGAT2 and mGAT3 (IC50 540 and 460 µM, respectively). The 4pyridyl substituted 32d and the 2-thienyl substituted 32e were inactive. Two compounds probed the exchange of the amide bond. The ester 33a had a low potency (IC50 106 µM) and displayed some activity at mGAT2 and mGAT3 (IC50 349 and 415 µM, respectively), while the dichloro analogue 33b was inactive at all GAT subtypes. Removal of the amide carbonyl oxygen in 5 gave diamino 34 that displayed reduced potency (IC50 112 µM). The profile of compounds 17i, 26e, 31a and 31b prompted us to determine the solubility of these compounds too. Compound 26e had a solubility of 40 µg/mL that is the same as 5. Compound 17i had a solubility of only 3 µg/ml, whereas 31a and 31b solubility was 240 and 130 µg/ml, respectively. Thus, 26m (610 µg/ml) was clearly the most soluble among these compounds.

2.3 Selectivity profiling of 5 and 26m at neuroreceptor and transporter panel 5 and 26m were screened for activity at 43 other neuroreceptors and transporters by the National Institute of Mental Health’s Psychoactive Drug Screening Program (NIMH-PDSP) (Supplemental Information, Table S1). 5 was found to bind to at least eight CNS targets in the nanomolar and low micromolar ranges, including the serotonin type 1D and 2B receptors, the dopamine D1, D2 and D4 receptors, the α2C adrenergic receptor and the histamine H1 receptor. Interestingly, the analogue 26m had effects at fewer off-targets. Most notably, 26m was not 7 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 46

active at the dopamine D1, D2 and D4 receptors in contrast to 5. However, 26m showed pronounced affinity in the nano- and micromolar range for sigma and serotonin type 2B receptors, respectively.

2.4 Binding site characterization We have previously demonstrated that 5 is a non-competitive inhibitor of GABA uptake at BGT1,20 which suggests that its binding site is different from the substrate binding site. In the present study, two strategies were pursued to guide mutations in hBGT1 with the aim of identifying and validating the putative binding site of 5: an experimental approach based on chimeric hBGT1/hGAT3 constructs and a computational docking study.

2.4.1 Chimeric study Chimeras between related transporters that show distinct pharmacological properties have previously been used to successfully delineate the importance of specific protein domains for ligand binding.21–23 In this study, we generated four chimeric transporter constructs consisting of varying parts of hBGT1 and hGAT3 (Fig. 4) to elucidate the binding region of 5. The hBGT1specific sequence in the N-terminus was proven necessary for functionality (data not shown), and thus, all chimeras contained hBGT1 sequence at the N-terminus and hGAT3 sequence at the Cterminus. The chimeras were created by standard overlap extension PCR24 with parts from hBGT1 and hGAT3 spliced together at positions that allowed an overlap of six identical amino acids in regions of sequence conservation. The chimeras were designated A, B, C and D with junction sites placed in the transmembrane (TM) segments 3, 6a and 8, and intracellular loop (IL) 5, respectively (Supplemental Fig. S1). Chimera A contained the least amount of hBGT1 sequence, whereas Chimera B, C and D contained progressively more and more hBGT1 8 ACS Paragon Plus Environment

Page 9 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


sequence. The chimeras were transiently expressed in tsA201 cells and tested in the [3H]GABA uptake assay.

Chimera A turned out to be non-functional, which precluded further studies using this construct. Whether the lack of function was related to dysfunctional transport properties or faulty protein translation/surface trafficking was not further investigated. On the other hand, chimeras B, C and D were functional as they were able to transport [3H]GABA. The activity for GABA at chimera B and D was in the same range as at wildtype (wt) hBGT1, whereas it was significantly lower at chimera C (Table 3). 5 had no activity at chimera B similar to wt hGAT3, while it displayed inhibitory activity at chimera C that was not significantly different from wt hBGT1. This suggested that the amino acid sequence differences between chimera B and C, i.e. TM6b–TM8a of BGT1 (Fig. 4), are necessary for the BGT1-selectivity of 5. Furthermore, the inhibitory activity of 5 at chimera D was markedly higher than wt hBGT1, implying that residues within TM8b–10 could also be involved in the region of important molecular interactions.

Examining further TM6b–TM8, we identified 19 amino acid residues in this region that differed between hBGT1 and hGAT3, according to the alignment presented in Fig. S1. These residues were mutated to the corresponding hGAT3 amino acids by site-directed mutagenesis. I386 in hBGT1 was the only residue that differed not only from hGAT3, but also from hGAT2, and was therefore mutated to the corresponding hGAT2 residue, generating hBGT1 I386F. All 20 hBGT1 single-point mutants were functional in the [3H]GABA uptake assay, implying surface expression. However, a diminished total [3H]GABA uptake was seen at hBGT1 Q299L and K310N compared to wt hBGT1. To examine whether the variable functionalities were related to differences in surface expression, we introduced a human influenza hemagglutinin (HA) epitope 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 46

tag in hBGT1 and hGAT3 that enabled us to quantify surface expression of the mutated transporters. The tag was inserted in the loop between TM3 and TM4 similar to an HA-tagged version of the dopamine transporter.25 Pharmacological characterization of HA-tagged hBGT1 and hGAT3 in the [3H]GABA uptake assay revealed that they were functional and displayed wildtype-like properties (data not shown). Using the enzyme-linked immunosorbent assay (ELISA), we could see a correlation between the level of surface expression and maximal transport capacity (data not shown). Therefore, the diminished [3H]GABA uptake by hBGT1 Q299L and K310N was inferred to be a consequence of a reduced surface expression.

The potency of GABA and 5 at all 20 hBGT1 mutants was either comparable to or higher than wt hBGT1 (Table 4). Similar results were seen for 26m at hBGT1 Q299L and I386F (Table 4). Although we did not see an attenuation of 5 or 26m activity at the hBGT1 mutants, we saw an increase in the potency of 26m at hGAT3 L314Q, corresponding to Q299L in hBGT1, compared to wt hGAT3 (Table 4). Overall, the mutational study suggested that none of the 19 residues in hBGT1 TM6b–TM8a, which differed from hGAT3, are individually responsible for the BGT1 GABA transporter subtype-selectivity of 5. However, this does not rule out that this region of BGT1 contains important determinants for the binding of 5, especially seen in the light that function, not binding, is the measurable output of the available [3H]GABA uptake assay. Therefore, perturbing the interaction with a specific residue by a mutation may not translate into a disrupted uptake inhibitory activity by 5. Additionally, single-point mutations may not be sufficient to obstruct 5 principal interactions, which could be shaped by several amino acids in combination. Furthermore, the loss of an interacting residue due to a mutation could also be masked as a result of compensatory interactions in that area. Analogously, it has been suggested that compensatory mechanisms, including backbone interactions, govern the resilience of a non10 ACS Paragon Plus Environment

Page 11 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nuclease inhibitor of reverse transcriptase against drug resistance mutations.26 Moreover, the fact that increased 5 activities are observed for several of the mutants (Table 4) further supports this notion.

In the lack of an available radioligand-based binding assay for BGT1, we therefore, expanded on the findings presented by the chimeric studies using in silico techniques to model the binding mode of 5 with the aim of identifying the molecular determinants for the selectivity of 5 for BGT1 over the other GATs.

2.4.2 Computational docking study As discussed above, the increased activity of 5 seen at chimera D compared to wt hBGT1 suggested that TM8b–10 contain residues important for the BGT1-subtype selectivity. Within this region of hBGT1, however, almost 30 residues differ from hGAT3 (Fig. S1). Therefore, we wanted to utilize a more rational approach to identify promising residues for new mutational experiments. As no crystal structure of hBGT1 has been published yet, the studies were based on a homology model.

The crystal structure of the bacterial leucine transporter (LeuT)27 has so far been the most frequently used template for modeling the outward-open state of GATs.28–31 Although the LeuT homolog shares only about 20% sequence identity with hBGT1 (Fig. S2), the highly conserved overall fold in the protein has made it a decent template for modeling GATs.9,32 The recently published crystal structures of the Drosophila dopamine transporter (dDAT)33 and the human serotonin transporter (hSERT),34 however, not only displays substantially higher sequence identity with BGT1 (47% and 43%, respectively, cf. Figure S2), but also notable structural 11 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 46

differences compared to LeuT within the regions of EL2 and TM12. It is noteworthy to add that the structure of hSERT was co-crystallized with a ligand, revealing an allosteric binding site accommodated by TMs 1, 6, 10 and 11.34 We have previously postulated that the corresponding site in hGAT3 contains the binding pocket of isatin analogues, shown to act as non-competitive inhibitors selective for GAT3 among GATs.35 Considering the non-competitive nature of 5,20 the high homology between hSERT and hBGT1, and the fact that the hSERT crystal structure has recently been used as a template to model rat GAT1,36 we regarded this crystal structure as an appropriate template for generating a hBGT1 homology model (Fig. 5A).

Cavity analysis of the TM8b–10 region of the hBGT1 model with MOE Site Finder37 suggested that only the TM10 domain is involved in a possible binding cavity positioned in the extracellular vestibule, corresponding to the allosteric pocket in the hSERT crystal structure (Fig. S3). Therefore, we hypothesized that the allosteric binding site observed in hSERT could also exist in hBGT1 and accommodate 5.

Hence, analogues of 5 were docked into the postulated allosteric binding site of hBGT1 between TM regions 1, 6, 10 and 11. Following our experimental data-guided docking approach,38–40 ten active compounds (5, 17i, 17j, 17l, 18, 26d, 26e, 26i, 26l, 26m) with IC50 values of less than 100 µM at BGT1 were selected and docked with flexible side chains using the software package GOLD.41 A total of 100 poses per compound were generated and assembled into 88 clusters according to an in-house protocol for common scaffold clustering with a root-mean-square deviation (RMSD) of less than 3 Å as a similarity threshold.40,41 Highly populated clusters that contained poses of all docked compounds were considered the most promising. Five out of the six most populated clusters (more than 50 poses) contained poses of all docked compounds and 12 ACS Paragon Plus Environment

Page 13 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were selected for further analysis (Fig. S4 and S5). Of the essence for selecting the most promising cluster was the SAR observed for compounds 17a–o and 18. Different substituents in the ortho, meta and para position of 5’s benzyl amino part (B) showed that substituents are only tolerated in the meta, but not in ortho and para position. Therefore, we concluded that the subpocket accommodating part B of the ligand provides space for meta substituents. Out of the six most populated clusters only two clusters (4 and 10) contained poses that were accommodated in such a tight sub-pocket. Cluster 4 was finally selected because its poses could explain the SAR of 17a–17l and 18. Accordingly, the 2,4-dichlorobenzoic amide ring of 5 (part A) fits into a mixed hydrophilic and lipophilic sub-pocket that is formed by T289, F293, Y453, S457, G458, I459 and S516. The dichlorobenzoic amide is stabilized by π-π interactions with Y453, hydrogen bonding of the carbonyl group with the sidechain of T289, and possible interaction of ortho and para Cl with the side chains of S457 and S516, respectively (Fig. 5B). The 1-benzyl-4-piperidinyl ring (part B) fits into the opposite part of the pocket where the benzyl ring fills a tight lipophilic sub-pocket that is formed by W60, L56, Y132, Y133, I136 and F448. This part of the pocket is connected with the orthosteric site. The meta trifluoromethyl, methoxy and dimethoxy substituents (part B) of 17i, 17l and 18 are all accommodated in the pocket by allowing W60 to move in order to form a small sub-pocket with P360 (Fig. 5B and Fig. S6). 17l’s methoxy group forms a hydrogen bond with the sidechain of R61, which replaces the interaction of the carbonyl oxygen with T289, resulting in similar activity compared to 5. The meta fluorine substituent of 17j is accommodated in a similar fashion without the need for W60 to adapt a different rotamer. Compounds 26d, 26e, 26i and 26l show an orientation in the pocket analogous to 5, but the carbonyl group undergoes hydrogen bonding with R61 instead of T289 (Fig. S6). The sub-pocket that accommodates part A of the 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 46

ligands offers enough space to accommodate orto and meta dimethoxy (26i) as well as a third chlorine in the ortho position (26e). Compound 26m shows the same binding mode as 5 and also the same activity (Fig. S6).

Y453 was selected as an interesting residue for mutational experiments, because interactions with this residue were not only observed for almost all poses in cluster 4, but also for all six highly populated clusters. Furthermore, Y453 is part of TM10, which was suggested by the chimeric studies to be involved in the BGT1 GAT-selectivity of 5. Y453 is conserved among the GATs, except for GAT3, where it corresponds to S468 (Fig. S1). Interestingly, we already proposed, in an independent study of non-competitive GAT3 selective inhibitors, GAT3 S468 to be a key residue involved in binding.35 Since 5 shows no inhibitory activity at GAT3, we expected the hBGT1 Y453S and hBGT1 Y453A mutants to show a decreased 5 inhibitory activity. On the other hand, we expected the corresponding hGAT3 S468Y mutant to introduce activity for 5 in GAT3. The three single-point mutated transporters were functional in the [3H]GABA uptake assay, but did not alter the inhibitory activities of 5 and 26m (Table 5). Also the double-mutant hBGT1 Y453S+Y454A, which addressed possible compensatory interactions by the adjacent Y454, did not lead to a disruption of the BGT1 activity of 5 or 26m (Table 5). Coleman et. al showed that the allosteric site is highly flexible.34 Therefore, the postulated loss of interactions within the double-mutant may be compensated by other residues.

Although the mutational studies were unsuccessful in delineating the specific residues involved in the BGT1 subtype selectivity of 5, the postulated binding hypothesis within the suggested allosteric site was able to explain the SAR of selected BGT1-active 5 analogues. Thus, the attachment of a methylenedioxy group to the benzamide ring (part A) as in 6 is tolerated (IC50 of 14 ACS Paragon Plus Environment

Page 15 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


42 µM) since the sub-pocket that contains part A offers enough space to accommodate the additional residue. Attaching a naphthamide (7) slightly increases activity (IC50 15 µM), because the additional conjugated double bonds not only fit nicely into the pocket, but can also undergo π-π-stacking with Y453. Exchanging the dichlorobenzene ring with propyl deprives 26a of its activity, because the π-π interaction with Y453 is lost. Once the phenyl ring is reintroduced (26b), weak activity (IC50 290 µM) is regained. Sequential attachment of chlorine in the ortho and para position gradually increases activity, as shown in the series of the analogues 26c, 26d and 26e (IC50 151, 77 and 11 µM, respectively), because the chlorine substituents in the ortho and para position not only increase the van der Waals interaction by nicely filling the pocket, but can also interact with the side chains of S516 and T512. Exchanging the chlorine substituents with ortho and meta methoxy (26i), ortho ethoxy (26g) or meta trifluoromethyl (26h) is tolerated by a slight decrease of activity (IC50 91, 180 and 170 µM, respectively), because the sub-pocket offers enough space to accommodate these residues, but no additional interactions are formed. Displacing the phenyl ring of 5 with 3-pyridine gives 26m, which shows the same activity (IC50 37 µM) as 5 because of its identical binding mode. Detaching 2-Cl from 26m gives 26l, which shows similar activity (IC50 39 µM), but selectivity is lost, which is also observed for 32c and 26d. Surprisingly, the attachment of only one chlorine atom in the ortho position yields the inactive compound 26k (cf. 26c). We hypothesize that the possible interaction of ortho chlorine with S457 might trigger selectivity. Although S457 is conserved among the GATs, it is part of the three amino acid motif ASS457, which corresponds to AAS452, AAS472 and SAS456 in GAT1, GAT2 and GAT3, respectively. S457 in BGT1 resembles a one-amino acid insertion in the middle of an unwound region in TM10, which is unique for the GATs and the highly related taurine and creatine transporter.42 The selectivity drop of 26l, 26d and 32c might be linked to this three-amino acid motif, as for 26l and 32c activity is introduced for GAT2 and GAT3 that share 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 46

the same motif.

8–16 lack the benzyl ring of part B and therefore, do not fully occupy the lipophilic pocket and thus, show no activity. 17a–17o contain different substituents in the ortho, para, and meta position. Residues D452, Y132, Y133 and W60 of the tight lipophilic sub-pocket that accommodates part B sterically hinder the attachment of functional groups in the ortho and para position, which could explain the lack of activity of 17a–17b, 17e–17g. Lipophilic meta substituents are tolerated with decreasing volume (17h–17j, 17l, 31a–b, IC50 160, 51, 34, 26, 115 and 11 µM, respectively), as they can be accommodated in the lipophilic sub-pocket due to the conformational flexibility of W60. Methoxy and ethoxy substituents are also tolerated in the 26m analogues series, as shown for 31a and 31b (IC50 115 and 111 µM, respectively). Polar substituents in the meta position are not tolerated in the lipophilic sub-pocket, which explains the inactivity of 31c–h. However, one has to note, that the inactivity of 17m–o, which contain small lipophilic substituents in the meta position, cannot be explained by the binding hypothesis. 19– 21 show no activity because of the introduction of a nitrogen atom into the benzylamine ring, which might not be tolerated in the highly lipophilic sub-pocket. Actually, the same should account for indolyl 28, which, however, shows activity (IC50 58 µM). Inactive compounds 20–24 contain sterically demanding substituents instead of the benzylamine part, which may lead to steric clashes in the lipophilic sub-pocket.

3 CONCLUSION Within this study, we have designed and tested 71 analogues of 5 for their inhibitory profile at the four GATs. Because of their non-competitive nature, and in analogy to the recently resolved structure of hSERT, we propose a new allosteric pocket in BGT1 as a possible binding cavity. 16 ACS Paragon Plus Environment

Page 17 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Computational docking studies of a series of 10 analogues of 5, supported by chimeric experiments, postulated residues within TM10 as important for the BGT1-selectivity among the GATs. Unfortunately, efforts to validate the binding hypothesis by single- and double-point mutations were unsuccessful, possibly due to the inherent flexibility of transporter proteins and the lack of an established BGT1 binding assay that can directly assess how inhibitor binding is affected by mutations. Nevertheless, the postulated binding hypothesis allows to explain key elements of the SAR observed in the data set, and thus represents a valid starting point for further studies. Ideally, such studies would be strongly aided by the availability of a BGT1 inhibitorradioligand, which would permit testing of the binding hypotheses directly. Furthermore, the 2,4Cl substituted 3-pyridine derivative, 26m, exhibits improved solubility and specificity when compared to the original lead structure 5, proposing this analogue as a better tool compound than BDBPA for studying BGT1. In conclusion, our study provides the first insights into a proposed allosteric binding pocket in BGT1, which accommodates the binding site for a series of novel non-competitive inhibitors, and could prove to be instrumental in terms of designing new and better tool compounds for further studying the physiological function and therapeutic relevance of BGT1.

4 EXPERIMENTAL SECTION Materials Dulbecco's Modified Eagle Medium (DMEM) with GlutaMAX-I, Dulbecco's Phosphate Buffered Saline (DPBS), fetal bovine serum (FBS), penicillin-streptomycin (P/S), trypsin and Hank's Balanced Salt Solution (HBSS) were purchased from Life Technologies (Paisley, UK). PolyFect Transfection Reagent was purchased from Qiagen (West Sussex, UK). Poly-D-lysine (PDL), HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) and GABA were 17 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 46

purchased from Sigma-Aldrich (St. Louis, MO, USA). [2,3-3H(N)]GABA (35.0 Ci/mmol) and MicroScint-20 were purchased from PerkinElmer (Boston, MA, USA). Blasticidin-S was purchased from InvivoGen (San Diego, CA, USA).

Molecular biology Generation of hBGT1/hGAT3 chimeric transporters. The four chimeric constructs were generated using standard molecular biology techniques and a standard overlap extension PCR strategy 24; first amplification of the first part of the chimera (PCR-1), then amplification of the second part of the chimera (PCR-2) and finally an overlap PCR connecting the two pieces. The junction sites for chimera A, B, C and D were positioned in TM3 (129-134), TM6a (289-294), TM8 (392-397) and IL5a (480-485), respectively (hBGT1 amino acid numbering, Fig. S1). The sequences of the external and internal primers for chimera A, B, C and D with the BGT1 Nterminus and the GAT3 C-terminus (Fig. S1) are given below.

External primers: BGT1

forward

primer,

5’-gccatggacgggaaggtggc-3’;

GAT3

reverse

primer,

5’-

gccatggacgggaaggtggc-3’.

Internal primers: Chimera A, forward primer, 5’-atttgaatgtctactacatcatcatcctggcatgg-3’; reverse primer: 5’gccaggatgatgatgtagtagacattcaaatatgactcg-3’.

Chimera

B,

forward

primer:

5’-

acccagatcttcttctcctatgccatttgcctgg-3’; reverse primer, 5’-tggcataggagaagaagatctgggtgcccgcatcc3’. Chimera C, forward primer, 5’-ttcctagggctggacagccagtttgtgtgtgtgg-3’; reverse primer, 5’acacacacaaactggctgtccagccctagg-3’.

Chimera

D,

forward

primer,

5’18

ACS Paragon Plus Environment

Page 19 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


gaccgtttctatgacaacattgaagacatgattggctacc-3’; reverse primer, 5’-gtcttcaatgttgtcatagaaacggtcc-3’.

The PCR strategy is exemplified for chimera A as follows: PCR-1: BGT1 forward primer and chimera A reverse primer with hBGT1 subcloned into the pcDNA5/FRT vector43 as template. PCR-2: Chimera A forward primer and GAT3 reverse primer with hGAT3 (pcDNA5/FRT)43 as template. PCR-overlap: BGT1 forward primer and GAT3 reverse primer with PCR-1 and PCR-2 as template. Subsequently, the final PCR product was inserted into the mammalian pcDNA5/FRT vector according to the protocol of the manufacturer (pcDNA5/FRT/V5-His TOPO® TA Expression Kit, Invitrogen, Paisley, UK).

Epitope tagging of hBGT1 and hGAT3. To enable evaluation of surface expression of mutated BGT1 and GAT3 constructs using ELISA, we inserted an epitope tag accessible from the extracellular side of the plasma membrane. Inspired by the work of Sorkina and co-authors,25 where a human influenza hemagglutinin (HA) peptide tag (YPYDVPDYA) was inserted in the extracellular loop between TM3 and TM4 in the human dopamine transporter (hDAT), we inserted an HA tag in hBGT1 and hGAT3 subcloned into the pcDNA5/FRT vector43 in positions corresponding to the location in hDAT. Using overlap extension PCR, the HA tags were introduced after predicted N-glycosylation sites between TM3 and EL2 in hBGT1 and hGAT3 (as predicted using the CBS NetNGlyc 1.0 server).44 In hBGT1, the HA tag was inserted immediately after S173 with the deletion of the subsequent 6 amino acids (GAGTVT), while it was placed immediately after N190 in hGAT3 with the deletion of the subsequent 7 amino acids (YSHVSLQ) (Fig. S1).

The final PCR product was inserted into the FRT/pcDNA5 vector per the manufacturer’s 19 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 46

instructions (pcDNA5/FRT/V5-His TOPO® TA Expression Kit, Invitrogen).

Site-directed mutagenesis of hBGT1 and hGAT3. The HA-tagged hBGT1 Y453S, Y453A, Y453S+Y454A and hGAT3 S468Y constructs were obtained from GenScript (Piscataway, NJ, USA). The other mutations were introduced in-house into either HA-tagged hBGT1 or untagged hGAT3 using the QuikChange II Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA).

The sequences of the chimeric, HA-tagged and mutated constructs were confirmed by DNA sequencing (GATC Biotech AG, Konstanz, Baden-Württemberg, Germany), and all primers were obtained from TAG Copenhagen (Copenhagen, Denmark).

Pharmacology Cell culture and transfection. DMEM with GlutaMAX-I supplemented with 10% FBS and 1% P/S was used as growth medium for the tsA201 cells and for the four human embryonic kidney 293 (HEK293) cell lines each stably expressing one of the four mGAT subtypes.45 mGATHEK293 cells were selected with blasticidin-S (5 µg/mL). tsA201 cells were transfected with DNA constructs encoding the four hGATs subcloned into the pcDNA5/FRT vector

43

using

PolyFect according to the manufacturer’s instructions (Qiagen, West Sussex, UK) with one minor modification (40 µL PolyFect instead of 80 µL in a 10 cm culture dish). All cell cultures were propagated at 37 °C in a humidified atmosphere of 95% O2 and 5% CO2.

[3H]GABA uptake assay. The assay was performed as previously described.46 Cells expressing recombinant wildtype or mutated GAT constructs were plated into PDL-coated white 96-well 20 ACS Paragon Plus Environment

Page 21 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


polystyrene cell culture microplates (PerkinElmer). The growth medium was removed approximately 24 hours after, and the cells were washed once with assay buffer (HBSS supplemented with 20 mM HEPES, 1 mM CaCl2 and 1 mM MgCl2, pH 7.4). Then, assay buffer supplemented with 30 nM [3H]GABA and various concentrations of test compound was added to each well. After three minutes of incubation at 37 °C, the cells were washed three times with icecold assay buffer, and MicroScint-20 scintillation liquid was added to each well. The plate was shaken for at least one hour, and the radioactivity was subsequently measured for three minutes in a TopCount NXT Microplate Scintillation & Luminescence Counter (PerkinElmer).

ELISA. Approximately 16 hours after transfection, tsA201 cells were plated into PDL-coated clear 24-well tissue culture plates (PerkinElmer). One day after, the cells were washed with icecold assay buffer (PBS supplemented with 1 mM CaCl2) and subsequently fixed with 4% paraformaldehyde (12 minutes on ice). The subsequent steps were performed at room temperature. Cells were washed twice with assay buffer and incubated with blocking solution (3% dry milk in 50 mM Tris-HCl supplemented with 1 mM CaCl2, pH 7.5) for 20 minutes. Cells were then incubated with mouse Anti-HA antibody (HA1.1 monoclonal antibody; Nordic BioSite, Copenhagen, Denmark; or Covance, San Diego, Ca, USA) diluted 1:1,000 in blocking solution. Next, the cells were washed three times with assay buffer and incubated with blocking solution for 20 minutes followed by 45 minutes of incubation with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Invitrogen) diluted 1:400 in blocking solution. Finally, cells were washed three times with assay buffer, and transporter expression was quantified using the 3,3’,5,5’-tetramethylbenzidine (TMB) liquid substrate system (SigmaAldrich) according to the protocol of the manufacturer. Absorbance was measured at 450 nm in an EnSpire 2300 Multilabel Reader (PerkinElmer). HA-tagged hBGT1 was used as a positive 21 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 46

control, while untagged wt hBGT1 or hGAT3 were used as negative controls. All experiments were performed in triplicate or quadruplicate measurements in at least three independent experiments.

Data analysis. Data were analyzed and statistically evaluated in GraphPad Prism 5.0a or 7.02 (GraphPad Software, San Diego, CA, USA). Concentration-response curves generated from the uptake assay were fitted by non-linear regression using the equation for sigmoidal concentrationresponse with variable slope: Y=Bottom+(Top-Bottom)/(1+10^((logIC50-X)×HillSlope)), where Y is the response, X is the logarithm of the concentration, Top and Bottom are the plateaus in same units as Y, logIC50 is the concentration giving a response halfway between Bottom and Top, and the HillSlope is the steepness of the curve. Unpaired Student’s t-test and One-way ANOVA followed by Dunnett’s multiple comparison test were performed when appropriate and indicated in the figure captions. Data are presented as means ± S.E.M. of at least three independent pooled experiments performed in triplicates.

Chemistry All starting materials were obtained from commercial sources and used without further purification. Dry column vacuum chromatography (DCVC) was performed using Fischer Silica 60A (20-45 µm). Reverse phase chromatography was performed using Supelco Discovery® DSC-C8. NMR spectra were recorded on a 300/75 MHz Varian spectrometer at room temperature using the solvent indicated below. Chemical shifts (δ) are quoted in ppm relative to residual solvent peaks. MS data was recorded using electrospray ionization liquid chromatography-mass spectrometry (ESI-LC/MS) on an Agilent 6410 Triple Quadrupole Mass Spectrometer instrument coupled to an Agilent 1200 HPLC system using a C18 reverse phase 22 ACS Paragon Plus Environment

Page 23 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


column (Agilent XDB-C18, 4.6 ´ 50 mm, 1.8 mm) with a linear gradient of the binary solvent system of H2O/MeCN/formic acid (A: 95:5:0.1 and B: 5:95:0.043) with a flow rate of 1 mL/min. During analysis, evaporative light scattering (ELS) traces were obtained with a Sedere Sedex 85 Light Scattering Detector used for purity evaluation. Elemental analysis was performed at the University of Vienna and are within ±0.4 of the theoretical values if not stated otherwise. Melting points were determined in an open capillary tube and are uncorrected. Final compounds are more than 95% according to elemental analysis and NMR. The experimental procedures for synthesizing all compounds can be found in the supporting information. N-(1-Benzylpiperidin-4-yl)-2,6-dichloronicotinamide (26m) 4-Amino-1-benzylpiperidine (0.50 mL, 2.45 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (0.44 mL, 3.18 mmol) was added. 2,6-Dichlorpyridine-3-carbonyl chloride (619 mg, 2.94 mmol) was added and the mixture was stirred at room temperature for 1 hour. The reaction mixture was filtered through a plug of silica, eluting with ethyl acetate. The solvent was evaporated. The residue was re-crystallized from ethyl acetate and heptane yielding white crystals of 26m (766 mg, 2.10 mmol, 86 %). 1H NMR (CDCl3): δ 8.11 (d, J = 8.0 Hz, 1 H), 7.40 (d, J = 8.0 Hz, 1 H), 7.37-7.27 (m, 5 H), 6.43 (d, J = 7.7 Hz, 1 H), 4.14-4.00 (m, 1 H), 3.57 (s, 1 H), 2.90-2.84 (m, 2 H), 2.31-2.21 (m, 2 H), 2.12-2.05 (m, 2 H), 1.74-1.58 (m, 2 H). 13C NMR (CDCl3): δ 162.7, 151.3, 146.0, 141.9, 138.1, 129.8, 128.9, 128.1, 126.9, 123.3, 63.0, 51.9, 47.6, 31.9. Mp 134-135 °C. Anal. Calcd for C18H19Cl2N3O: C, H, N. LC/MS: Calcd for C18H19Cl2N3O: 396.1; found: 397.2 [M+H]+, 395,3 [M-H]-.

Computational Methods

23 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 46

Homology modeling. The PDB crystal structure (PDB code 5I73) of hSERT with a cocrystallized escitalopram in the allosteric site was selected as template for homology modeling.34 The sequence alignment was conducted with the online tool PROMALS3D (http://prodata.swmed.edu/promals3d/promals3d.php).47 Models were generated with Modeller 9.16,48 ranked according the the DOPE score49, and checked with Ramachandran plots and the online service ProQM (http://www.bioinfo.ifm.liu.se/ProQM/index.php?about=proqm, Fig. S7 and S8).50 Special attention was paid to modeling the insertion in TM10 where no structural template is available. The top ranked models were selected to undergo a loop refinement with Modeller 9.16 allowing the two residues before and after the insertion to be independently modeled from the template. The models with the refined TM10 were ranked again according to DOPE score, and the top 10 were visually inspected. The model with the most frequently occurring TM10 fold was selected as a final model. The authors will release the atomic coordinates upon article publication.

Docking. Flexible sidechain docking (W60, R61, Q290, F293, D452, Y453) was performed with GOLD 5.2.2.41 The binding site was determined by superposing the hBGT1 model onto the hSERT crystal structure with MOE 2016.0837 and selection of the corresponding residues in the vicinity of the allosteric escitalopram ligand (4.5 Å). Protein and ligand preparations was performed with PrepWiz and LigPrep, respectively, in the Schrödinger Suit 2015-2.51,52 100 poses per ligand were generated and hierarchically clustered according to an in-house protocol for common scaffold clustering with a maximal distance of 3 Å within one cluster.40 Poses were analyzed and visualized with MOE 2016.0837. The top most scored poses of the most populated clusters were considered as most promising.


Page 25 of 46


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 46

Corresponding authors: Chemistry: Associate Professor Rasmus P. Clausen Tel.: +45 3533 6566 E-mail address: [email protected] Pharmacology: Associate Professor Petrine Wellendorph Tel.: +45 3533 6397 E-mail address: [email protected] Modeling: Professor Gerhard F. Ecker Tel. +431 4277 55110 E-mail address: [email protected]

FUNDING SOURCES This work was supported by the Lundbeck Foundation (grant R133-A12270), the Austrian Science Fund (grants # F03502 and W1232), the Drug Research Academy of the University of Copenhagen, and the A.P. Møller Foundation for the Advancement of Medical Sciences.

ACKNOWLEDGMENTS Mark Svan and Kristian K. Thomsen are acknowledged for preparing some of the compounds and Christa Ancher Marvig for determining solubility. Furthermore, we acknowledge Margot Ernst for providing her guidance and expertise for generating and selecting the homology model. The authors would like to thank Bolette Kragholm for generating BGT1/GAT3 chimeric constructs and Maja Michelle Hansen for generating BGT1 mutants.

ABBREVIATIONS 26 ACS Paragon Plus Environment

Page 27 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Betaine/γ-aminobutyric

acid

transporter

1,

BGT1;

N-(1-benzyl-4-piperidinyl)-2,4-

dichlorobenzamide, BPDBA; human serotonin transporter, hSERT; transmembrane, TM; domains γ-aminobutyric acid, GABA; GABA transporters, GATs.

ASSOCIATED CONTENT Supporting Information. Experimental data for compounds (8 -34) and additional tables and figures, molecular formula strings and three dimensional coordinates of the homology model of BGT-1. This material is available free of charge via the Internet at http://pubs.acs.org



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 46

(1) Mody, I.; Pearce, R. A. Diversity of Inhibitory Neurotransmission through GABAA Receptors. Trends Neurosci. 2004, 27 (9), 569–575. (2) Ting Wong, C. G.; Guin Ting Wong, C.; Bottiglieri, T.; Carter Snead, O. GABA, γ-Hydroxybutyric Acid, and Neurological Disease. Ann. Neurol. 2003, 54 (S6), S3–S12. (3) Fatemi, S. H.; Hossein Fatemi, S.; Stary, J. M.; Earle, J. A.; Araghi-Niknam, M.; Eagan, E. GABAergic Dysfunction in Schizophrenia and Mood Disorders as Reflected by Decreased Levels of Glutamic Acid Decarboxylase 65 and 67 kDa and Reelin Proteins in Cerebellum. Schizophr. Res. 2005, 72 (2-3), 109–122. (4) Inan, M.; Petros, T. J.; Anderson, S. A. Losing Your Inhibition: Linking Cortical GABAergic Interneurons to Schizophrenia. Neurobiol. Dis. 2013, 53, 36–48. (5) Rowley, N. M.; Madsen, K. K.; Schousboe, A.; Steve White, H. Glutamate and GABA Synthesis, Release, Transport and Metabolism as Targets for Seizure Control. Neurochem. Int. 2012, 61 (4), 546–558. (6) Clarkson, A. N.; Huang, B. S.; Macisaac, S. E.; Mody, I.; Carmichael, S. T. Reducing Excessive GABA-Mediated Tonic Inhibition Promotes Functional Recovery after Stroke. Nature 2010, 468 (7321), 305–309. (7) Schwartz, T. L.; Nihalani, N. Tiagabine in Anxiety Disorders. Expert Opin. Pharmacother. 2006, 7 (14), 1977–1987. (8) Lie, M.; Al-Khawaja, A.; Damgaard, M.; Haugaard, A. S.; Schousboe, A.; Clarkson, A. N.; Wellendorph, P. Glial GABA Transporters as Modulators of Inhibitory Signalling in Epilepsy and Stroke. In Glial Amino Acid Transporters; Ortega, A., Schousboe, A., Eds.; Springer; p in press. (9) Kristensen, A. S.; Andersen, J.; Jørgensen, T. N.; Sørensen, L.; Eriksen, J.; Loland, C. J.; Strømgaard, K.; Gether, U. SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation. Pharmacol. Rev. 2011, 63 (3), 585–640. (10) Alexander, S. P.; Kelly, E.; Marrion, N.; Peters, J. A.; Benson, H. E.; Faccenda, E.; Pawson, A. J.; Sharman, J. L.; Southan, C.; Davies, J. A.; CGTP Collaborators. The Concise Guide to


Page 29 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


PHARMACOLOGY 2015/16: Transporters. Br. J. Pharmacol. 2015, 172 (24), 6110–6202. (11) Nielsen, E. B.; Suzdak, P. D.; Andersen, K. E.; Knutsen, L. J.; Sonnewald, U.; Braestrup, C. Characterization of Tiagabine (NO-328), a New Potent and Selective GABA Uptake Inhibitor. Eur. J. Pharmacol. 1991, 196 (3), 257–266. (12) Sałat, K.; Kulig, K. GABA Transporters as Targets for New Drugs. Future Med. Chem. 2011, 3 (2), 211–222. (13) Damgaard, M.; Haugaard, A. S.; Kickinger, S.; Al-Khawaja, A.; Lie, M.; Ecker, G. F.; Clausen, R. P.; Frølund, B. Development of Non-GAT1-Selective Inhibitors: Challenges and Achievements. In Glial Amino Acid Transporters; Ortega, A., Schousboe, A., Eds.; Springer: Cham, 2017, pp 315-332. (14) Clausen, R. P.; Moltzen, E. K.; Perregaard, J.; Lenz, S. M.; Sanchez, C.; Falch, E.; Frølund, B.; Bolvig, T.; Sarup, A.; Larsson, O. M.; Schousboe, A.; Krogsgaard-Larsen, P. Selective Inhibitors of GABA Uptake: Synthesis and Molecular Pharmacology of 4-N-Methylamino-4,5,6,7Tetrahydrobenzo[d]isoxazol-3-Ol Analogues. Bioorg. Med. Chem. 2005, 13 (3), 895–908. (15) White, H. S.; Watson, W. P.; Hansen, S. L.; Slough, S.; Perregaard, J.; Sarup, A.; Bolvig, T.; Petersen, G.; Larsson, O. M.; Clausen, R. P.; Frølund, B.; Falch, E.; Krogsgaard-Larsen, P.; Schousboe, A. First Demonstration of a Functional Role for Central Nervous System Betaine/{gamma}-Aminobutyric Acid Transporter (mGAT2) Based on Synergistic Anticonvulsant Action among Inhibitors of mGAT1 and mGAT2. J. Pharmacol. Exp. Ther. 2005, 312 (2), 866–874. (16) Vogensen, S. B.; Jørgensen, L.; Madsen, K. K.; Borkar, N.; Wellendorph, P.; Skovgaard-Petersen, J.; Schousboe, A.; White, H. S.; Krogsgaard-Larsen, P.; Clausen, R. P. Selective mGAT2 (BGT-1) GABA Uptake Inhibitors: Design, Synthesis, and Pharmacological Characterization. J. Med. Chem. 2013, 56 (5), 2160–2164. (17) Thomsen, C.; Sørensen, P. O.; Egebjerg, J. 1-(3-(9H-Carbazol-9-Yl)-1-Propyl)-4-(2Methoxyphenyl)-4-Piperidinol, a Novel Subtype Selective Inhibitor of the Mouse Type II GABATransporter. Br. J. Pharmacol. 1997, 120 (6), 983–985. (18) Dalby, N. O.; Thomsen, C.; Fink-Jensen, A.; Lundbeck, J.; Søkilde, B.; Man, C. M.; Sørensen, P. O.;



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 46

Meldrum, B. Anticonvulsant Properties of Two GABA Uptake Inhibitors NNC 05-2045 and NNC 05-2090, Not Acting Preferentially on GAT-1. Epilepsy Res. 1997, 28 (1), 51–61. (19) Kobayashi, T.; Suemasa, A.; Igawa, A.; Ide, S.; Fukuda, H.; Abe, H.; Arisawa, M.; Minami, M.; Shuto, S. Conformationally Restricted GABA with Bicyclo[3.1.0]hexane Backbone as the First Highly Selective BGT-1 Inhibitor. ACS Med. Chem. Lett. 2014, 5 (8), 889–893. (20) Kragholm, B.; Kvist, T.; Madsen, K. K.; Jørgensen, L.; Vogensen, S. B.; Schousboe, A.; Clausen, R. P.; Jensen, A. A.; Bräuner-Osborne, H. Discovery of a Subtype Selective Inhibitor of the Human betaine/GABA Transporter 1 (BGT-1) with a Non-Competitive Pharmacological Profile. Biochem. Pharmacol. 2013, 86 (4), 521–528. (21) Barker, E. L.; Perlman, M. A.; Adkins, E. M.; Houlihan, W. J.; Pristupa, Z. B.; Niznik, H. B.; Blakely, R. D. High Affinity Recognition of Serotonin Transporter Antagonists Defined by SpeciesScanning Mutagenesis: An Aromatic Residue in Transmembrane Domain I Dictates SpeciesSelective Recognition of Citalopram and Mazindol. J. Biol. Chem. 1998, 273 (31), 19459–19468. (22) Barker, E. L.; Kimmel, H. L.; Blakely, R. D. Chimeric Human and Rat Serotonin Transporters Reveal Domains Involved in Recognition of Transporter Ligands. Mol. Pharmacol. 1994, 46 (5), 799–807. (23) Buck, K. J.; Amara, S. G. Structural Domains of Catecholamine Transporter Chimeras Involved in Selective Inhibition by Antidepressants and Psychomotor Stimulants. Mol. Pharmacol. 1995, 48 (6), 1030–1037. (24) Horton, R. M.; Hunt, H. D.; Ho, S. N.; Pullen, J. K.; Pease, L. R. Engineering Hybrid Genes without the Use of Restriction Enzymes: Gene Splicing by Overlap Extension. Gene 1989, 77 (1), 61–68. (25) Sorkina, T.; Miranda, M.; Dionne, K. R.; Hoover, B. R.; Zahniser, N. R.; Sorkin, A. RNA Interference Screen Reveals an Essential Role of Nedd4-2 in Dopamine Transporter Ubiquitination and Endocytosis. J. Neurosci. 2006, 26 (31), 8195–8205. (26) Ren, J.; Nichols, C.; Bird, L. E.; Fujiwara, T.; Sugimoto, H.; Stuart, D. I.; Stammers, D. K. Binding of the Second Generation Non-Nucleoside Inhibitor S-1153 to HIV-1 Reverse Transcriptase


Page 31 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Involves Extensive Main Chain Hydrogen Bonding. J. Biol. Chem. 2000, 275 (19), 14316–14320. (27) Singh, S. K.; Piscitelli, C. L.; Yamashita, A.; Gouaux, E. A Competitive Inhibitor Traps LeuT in an Open-to-Out Conformation. Science 2008, 322 (5908), 1655–1661. (28) Wein, T.; Petrera, M.; Allmendinger, L.; Höfner, G.; Pabel, J.; Wanner, K. T. Different Binding Modes of Small and Large Binders of GAT1. ChemMedChem 2016, 11 (5), 509–518. (29) Schlessinger, A.; Wittwer, M. B.; Dahlin, A.; Khuri, N.; Bonomi, M.; Fan, H.; Giacomini, K. M.; Sali, A. High Selectivity of the -Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-Based Approach. J. Biol. Chem. 2012, 287 (45), 37745–37756. (30) Jurik, A.; Zdrazil, B.; Holy, M.; Stockner, T.; Sitte, H. H.; Ecker, G. F. A Binding Mode Hypothesis of Tiagabine Confirms Liothyronine Effect on γ-Aminobutyric Acid Transporter 1 (GAT1). J. Med. Chem. 2015, 58 (5), 2149–2158. (31) Skovstrup, S.; Taboureau, O.; Bräuner-Osborne, H.; Jørgensen, F. S. Homology Modelling of the GABA Transporter and Analysis of Tiagabine Binding. ChemMedChem 2010, 5 (7), 986–1000. (32) Loland, C. J. The Use of LeuT as a Model in Elucidating Binding Sites for Substrates and Inhibitors in Neurotransmitter Transporters. Biochim. Biophys. Acta (BBA) - General Subjects 2015, 1850 (3), 500–510. (33) Penmatsa, A.; Wang, K. H.; Gouaux, E. X-Ray Structure of Dopamine Transporter Elucidates Antidepressant Mechanism. Nature 2013, 503 (7474), 85–90. (34) Coleman, J. A.; Green, E. M.; Gouaux, E. X-Ray Structures and Mechanism of the Human Serotonin Transporter. Nature 2016, 532 (7599), 334–339. (35) Damgaard, M.; Al-Khawaja, A.; Vogensen, S. B.; Jurik, A.; Sijm, M.; Lie, M. E. K.; Bæk, M. I.; Rosenthal, E.; Jensen, A. A.; Ecker, G. F.; Frølund, B.; Wellendorph, P.; Clausen, R. P. Identification of the First Highly Subtype-Selective Inhibitor of Human GABA Transporter GAT3. ACS Chem. Neurosci. 2015, 6 (9), 1591–1599. (36) Dayan, O.; Nagarajan, A.; Shah, R.; Ben-Yona, A.; Forrest, L. R.; Kanner, B. I. An Extra Amino Acid Residue in Transmembrane Domain 10 of the γ-Aminobutyric Acid (GABA) Transporter



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 46

GAT-1 Is Required for Efficient Ion-Coupled Transport. J. Biol. Chem. 2017, 292 (13), 5418–5428. (37) Chemical Computing Group ULC. Molecular Operating Environment (MOE); 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2017. (38) Sarker, S.; Weissensteiner, R.; Steiner, I.; Sitte, H. H.; Ecker, G. F.; Freissmuth, M.; Sucic, S. The High-Affinity Binding Site for Tricyclic Antidepressants Resides in the Outer Vestibule of the Serotonin Transporter. Mol. Pharmacol. 2010, 78 (6), 1026–1035. (39) Klepsch, F.; Chiba, P.; Ecker, G. F. Exhaustive Sampling of Docking Poses Reveals Binding Hypotheses for Propafenone Type Inhibitors of P-Glycoprotein. PLoS Comput. Biol. 2011, 7 (5), e1002036. (40) Richter, L.; de Graaf, C.; Sieghart, W.; Varagic, Z.; Mörzinger, M.; de Esch, I. J. P.; Ecker, G. F.; Ernst, M. Diazepam-Bound GABAA Receptor Models Identify New Benzodiazepine Binding-Site Ligands. Nat. Chem. Biol. 2012, 8 (5), 455–464. (41) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. Development and Validation of a Genetic Algorithm for Flexible Docking. J. Mol. Biol. 1997, 267 (3), 727–748. (42) Vogensen, S. B.; Jørgensen, L.; Madsen, K. K.; Jurik, A.; Borkar, N.; Rosatelli, E.; Nielsen, B.; Ecker, G. F.; Schousboe, A.; Clausen, R. P. Structure Activity Relationship of Selective GABA Uptake Inhibitors. Bioorg. Med. Chem. 2015, 23 (10), 2480–2488. (43) Kvist, T.; Christiansen, B.; Jensen, A. A.; Bräuner-Osborne, H. The Four Human GammaAminobutyric Acid (GABA) Transporters: Pharmacological Characterization and Validation of a Highly Efficient Screening Assay. Comb. Chem. High Throughput Screen. 2009, 12 (3), 241–249. (44) Julenius, K. NetCGlyc 1.0: Prediction of Mammalian C-Mannosylation Sites. Glycobiology 2007, 17 (8), 868–876. (45) White, H. S.; Sarup, A.; Bolvig, T.; Kristensen, A. S.; Petersen, G.; Nelson, N.; Pickering, D. S.; Larsson, O. M.; Frølund, B.; Krogsgaard-Larsen, P.; Schousboe, A. Correlation between Anticonvulsant Activity and Inhibitory Action on Glial Gamma-Aminobutyric Acid Uptake of the Highly Selective Mouse Gamma-Aminobutyric Acid Transporter 1 Inhibitor 3-Hydroxy-4-Amino-


Page 33 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4,5,6,7-Tetrahydro-1,2-Benzisoxazole and Its N-Alkylated Analogs. J. Pharmacol. Exp. Ther. 2002, 302 (2), 636–644. (46) Christiansen, B.; Meinild, A.-K.; Jensen, A. A.; Braüner-Osborne, H. Cloning and Characterization of a Functional Human Gamma-Aminobutyric Acid (GABA) Transporter, Human GAT-2. J. Biol. Chem. 2007, 282 (27), 19331–19341. (47) Pei, J.; Kim, B.-H.; Grishin, N. V. PROMALS3D: A Tool for Multiple Protein Sequence and Structure Alignments. Nucleic Acids Res. 2008, 36 (7), 2295–2300. (48) Webb, B.; Sali, A. Comparative Protein Structure Modeling Using MODELLER. Curr. Protoc. Bioinf. 2014, 47, 5.6.1–32. (49) Shen, M.-Y.; Sali, A. Statistical Potential for Assessment and Prediction of Protein Structures. Protein Sci. 2006, 15 (11), 2507–2524. (50) Ray, A.; Lindahl, E.; Wallner, B. Model Quality Assessment for Membrane Proteins. Bioinformatics 2010, 26 (24), 3067–3074. (51) Schrödinger Release 2015-2: LigPrep, Schrödinger, LLC, New York, NY, 2015. (52) Schrödinger Release 2017-2: Schrödinger Suite 2017-2 Protein Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, 2016; Impact, Schrödinger, LLC, New York, NY, 2016; Prime, Schrödinger, LLC, New York, NY, 2017. (53) Yamashita, A.; Singh, S. K.; Kawate, T.; Jin, Y.; Gouaux, E. Crystal Structure of a Bacterial Homologue of Na+/Cl--Dependent Neurotransmitter Transporters. Nature 2005, 437 (7056), 215– 223.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 46

FIGURE LEGENDS

Fig. 1. Chemical structures of selected GABA uptake inhibitors.

Fig. 2. Chemical structures 8–25 employed in this study.

Fig. 3. Chemical structures of 26–34 employed in this study.

Fig. 4. Schematic representation of the topology of wildtype hBGT1 and the four chimeric transporters between hBGT1 (blue) and hGAT3 (red). The topology is based on the structures of the hSERT,34 dDAT,33, and LeuT53 crystals. All chimeric constructs contained BGT1 in the Nterminal and GAT3 in the C-terminal parts. The position of the junction sites is stated in brackets. TM, transmembrane; IL, intracellular loop; EL, extracellular loop.

Fig. 5. Homology model of hBGT1 and the binding pose of 5. A. The homology model of hBGT1 based on the hSERT crystal structure with an overview of the proposed binding location of 5. B. Binding pose of 5 depicted with the molecular surface of the pocket. Hydrophilic areas are depicted in blue, and lipophilic areas are depicted in yellow. C. Binding pose of 5 depicted as sticks. Poses of the other docked compounds (17i, 17j, 17l, 18, 26d, 26e, 26i, 26l and 26m) are depicted in Fig. S6.


Page 35 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURES

Fig. 1.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 46

Fig. 2.


Page 37 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 3.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 46

Fig. 4.


Page 39 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 5.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Route I

Boc N

Page 40 of 46

Route II

NH2

Boc NH

HN

a

b O R1

HN

N

NH

NH2

R2 b

a O

O R2

R1

R1 N

N

NH

NH

R2 d

c 15

5

14

34

a N

OH

33a, 33b

Scheme 1. Reagents and conditions: a) 1. RCOCl, NEt3, DCM, RT, 1 hr; 2. TFA, DCM, RT, 1 hr or HCl, dioxane, RT, 1 hr b) R-Br, NEt3, DCM, RT, 1 hr, recryst., or R-Br, K2CO3, DMF, RT, 1 hr, recryst., or R-CHO, NaB(OOCCH3)H, THF, RT, 16 hrs, recryst. c) HCl (aq.), 80ºC, 24 hrs, recryst. d) LiAlH4, THF, reflux, 16 hrs, recryst.


Page 41 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TABLES Table 1. The potencies of compounds 1–25 determined in the [3H]GABA uptake assay at recombinant GATs. Results from mouse (red numbers) and human (italic numbers) transporter subtypes are shown in the same column. IC50 (pIC50±S.E.M.) (µM) mBGT1 mGAT2 mGAT3 hBGT1 hGAT2 hGAT3 1a 0.8 300 >300 800 2b 7 26 >300 >300 3c 307 45 >300 286 4d 19 1.4 41 15 >200 32 (4.50±0.13) >200 >200 5 >100e 18 (4.87±0.12) >100e >100e e 6 >100 42 63 >100 7e >100 15 23 >100 >1,000 >1,000 >1,000 >1,000 8 >1,000 >1,000 >1,000 >1,000 9 >1,000 >1,000 >1,000 >1,000 10 >1,000 >1,000 >1,000 >1,000 11 >1,000 >1,000 >1,000 >1,000 12 >1,000 >1,000 >1,000 >1,000 13 >1,000 >1,000 >1,000 >1,000 14 >1,000 >1,000 >1,000 >1,000 15 >1,000 >1,000 >1,000 >1,000 16 >100 >100 >100 >100 17a >100 >100 >100 >100 17b >50 >50 >50 >50 17c >50 >50 >50 >50 17d >100 >100 >100 >100 17e >100 >100 >100 >100 17f >1,000 >1,000 >1,000 >1,000 17g >1,000 160 (3.90±0.08) >1,000 >1,000 17h >1,000 51 (4.29±0.10) >1,000 810 (3.09±0.22) 17i >1,000 34 (4.47±0.07) >1,000 >1,000 17j >300 >300 >300 >300 17k >200 26 (4.59±0.15) >200 >200 17l >300 >300 >300 >300 17m >300 >300 >300 >300 17n >300 >300 >300 >300 17o >1,000 30 (4.52±0.05) 1,200 (2.92±0.35) 640 (3.19±0.19) 18 >1,000 >1,000 >1,000 >1,000 19 >1,000 >1,000 >1,000 >1,000 20 >1,000 >1,000 >1,000 >1,000 21 >100 >100 >100 >100 22 >50 >50 >50 >50 23 >50 >50 >50 >50 24 >100 >100 >100 >100 25 The compounds were examined for their ability to inhibit the uptake of 30 nM [3H]GABA in tsA201 cells Compounds

mGAT1 hGAT1

transiently expressing the respective construct. Ki values from a) ref. 15, b) ref. 14, c) ref 16, d) ref. 17, e) ref. 20.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 46

Table 2. The potencies of compounds 5–7 and 26–34 determined in the [3H]GABA uptake assay at recombinant GATs. Results from mouse (red numbers) and human (italic numbers) transporter subtypes are shown in the same column. IC50 (pIC50±S.E.M.) (µM) Compounds mGAT1 mBGT1 mGAT2 mGAT3 hGAT1 hBGT1 hGAT2 hGAT3 >200 32 (4.50±0.13) >200 >200 5 >100e 18 (4.87±0.12) >100e >100e f 6 >100 42 63 >100 7f >100 15 23 >100 >1,000 >1,000 >1,000 >1,000 26a >1,000 290 (3.53±0.06) >1,000 >1,000 26b >400 151 (3.82±0.08) >400 >400 26c >1,000 77 (4.11±0.12) 590 (3.23±0.14) >1,000 26d >1,000 11 (4.96±0.05) 1,700 (2.77±0.24) 1,000 (3.00±0.23) 26e >300 100 (4.00±0.07) >300 >300 26f >500 180 (3.74±0.02) >500 >500 26g >500 170 (3.77±0.07) >500 >500 26h >500 91 (4.04±0.05) >500 >500 26i >1,000 >1,000 >1,000 >1,000 26j >1,000 1,650 (2.78±0.08) >1,000 >1,000 26k >1,000 39 (4.41±0.06) 150 (3.82±0.08) 550 (3.26±0.12) 26l >400 37 (4.43±0.09) >400 >400 26m >1,000 44 (4.39±0.10) 479 (3.3±0.07) >1,000 >1,000 1,150 (2.94±0.08) >1,000 >1,000 26n >1,000 300 (3.52±0.07) >1,000 >1,000 26o >400 179 (3.75±0.06) >400 >400 26p >300 >300 >300 >300 27 >300 58 (4.24±0.02) >300 >300 28 >300 60 (4.22±0.07) >300 >300 29 >300 >300 >300 >300 30 >300 115 (3.94±0.03) >300 >300 31a >300 111 (3.98±0.11) >300 >300 31b >300 >300 >300 >300 31c >300 >300 >300 >300 31d >300 >300 >300 >300 31e >300 >300 >300 >300 31f >300 >300 >300 >300 31g >300 >300 >300 >300 31h >300 >300 >300 >300 31i >1,000 >1,000 >1,000 >1,000 32a >1,000 1,670 (2.78±0.09) >1,000 >1,000 32b >1,000 115 (3.94±0.07) 540 (3.27±0.07) 460 (3.34±0.06) 32c >1,000 >1,000 >1,000 >1,000 32d >400 390 (3.41±0.07) >400 >400 32e >1,000 106 (3.97±0.06) 349 (3.46±0.05) 415 (3.28±0.05) 33a >1,000 >1,000 >1,000 >1,000 33b >400 112 (3.95±0.10) >400 >400 34 The compounds were examined for their ability to inhibit the uptake of 30 nM [3H]GABA in tsA201 cells transiently expressing the respective construct. Ki values from a) ref. 20.


Page 43 of 46

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. Potencies of GABA, 5 and 26m in the [3H]GABA uptake assay at wt hBGT1, wt hGAT3 and at four chimeras between hBGT1 and hGAT3 (Fig. 3). IC50 (pIC50±S.E.M.) (µM) Construct GABA 5 26m hBGT1 (wt) 44 (4.39±0.10) 25 (4.75±0.12) 18 (4.87±0.12) >100 >1,000 hGAT3 (wt) 3 (5.48±0.19)* Chimera A Chimera B >300 >1,000 39 (4.56±0.26)ns N.D. Chimera C 58 (4.60±0.06)ns 181 (3.78±0.10)** ns Chimera D N.D. 9 (5.41±0.40) 2 (5.79±0.10)**** The compounds were examined for their ability to inhibit the uptake of 30 nM [3H]GABA in tsA201 cells transiently expressing the respective construct. All experiments were performed in triplicates in at least two independent experiments. Chimera A was not functional in the [3H]GABA uptake assay. N.D., not determined. The pIC50 values at the chimeras were compared to the wild type (wt) hBGT1 (One-way ANOVA followed by Dunnett’s multiple comparison test, no significance (ns) P>0.05, * P0.05, * P1,000 1 (6.00±0.04) The compounds were examined for their ability to inhibit the uptake of 30 nM [3H]GABA in tsA201 cells transiently expressing the respective construct. All experiments were performed in triplicates in at least three independent experiments. The pIC50 values at the mutants were compared to the wild type (wt) transporter (One-way ANOVA followed by Dunnett’s multiple comparison test for the BGT1 mutants and unpaired Student’s t-test for the GAT3 mutant, no significance (ns) P>0.05, * P

StructureâActivity Relationship, Pharmacological Characterization

Recommend Documents

StructureâActivity Relationship, Pharmacological Characterization