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Pharmacological characterization of [3H]ATPCA as a substrate for studying the functional role of the betaine/ GABA transporter 1 and the creatine transporter Anas Al-Khawaja, Anne S Haugaard, Ales Marek, Rebekka Löffler, Louise Thiesen, Monica Santiveri, Maria Damgaard, Christoffer Bundgaard, Bente Frølund, and Petrine Wellendorph ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00351 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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ACS Chemical Neuroscience

Pharmacological characterization of [3H]ATPCA as a substrate for studying the functional role of the betaine/GABA transporter 1 and the creatine transporter

Anas Al-Khawaja,† Anne S. Haugaard,† Ales Marek,‡ Rebekka Löffler,† Louise Thiesen,† Mònica Santiveri,† Maria Damgaard,† Christoffer Bundgaard,§ Bente Frølund,† and Petrine Wellendorph*,†

†

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences,

University of Copenhagen, 2100 Copenhagen, Denmark ‡

Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6,

Czech Republic, Flemingovo nam 542/2, 16610 §

Discovery Chemistry and DMPK, H. Lundbeck A/S, 2500 Valby, Denmark

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ABSTRACT The betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) is one of the four GABA transporters (GATs) involved in the termination of GABAergic neurotransmission. Although suggested to be implicated in seizure management, the exact functional importance of BGT1 in the brain is still elusive. This is partly owing to the lack of potent and selective pharmacological tool compounds that can be used to probe its function. We previously reported the identification of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA), a selective substrate for BGT1 over GAT1/GAT3, but also an agonist for GABAA receptors. With the aim of providing new functional insight into BGT1, we here present the synthesis and pharmacological characterization of the tritiated analogue, [3H]ATPCA. Using traditional uptake assays at recombinant transporters expressed in cell lines, [3H]ATPCA displayed a striking selectivity for BGT1 among the four GATs (Km and Vmax values of 21 µM and 3.6 nmol ATPCA/(min×mg protein), respectively), but was also found to be a substrate for the creatine transporter (CreaT). In experiments with mouse cortical cell cultures, we observed a Na+-dependent [3H]ATPCA uptake in neurons, but not in astrocytes. The neuronal uptake could be inhibited by GABA, ATPCA and a non-competitive BGT1-selective inhibitor, indicating functional BGT1 in neurons. In conclusion, we report [3H]ATPCA as a novel radioactive substrate for both BGT1 and CreaT. The dual activity of the radioligand makes it most suitable for use in recombinant studies...

Keywords Betaine/γ-aminobutyric acid transporter 1, BGT1, GABA uptake assay, creatine transporter, ATPCA

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INTRODUCTION The betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) is a member of the Na+/Cl-dependent solute carrier 6 (SLC6) neurotransmitter transporter gene family and one of the four transmembrane GABA transporters (GATs) identified in mammalians to date.1,2 GATs are regarded as important drug targets in neurological disorders such as epilepsy and stroke as they regulate the inhibitory GABA-mediated signalling in the adult brain by actively transporting GABA from the synapse to presynaptic neurons and surrounding glial cells.3 While this role is well-established for GAT1 (SLC6A1) and GAT3 (SLC6A11), the most abundant isoforms in the brain,1,4,5 it is generally not considered relevant for GAT2 (SLC6A13), which has a scarce neuronal and astrocytic expression pattern.5–7 Although BGT1 (SLC6A12) is expressed at much lower densities in the brain compared to GAT1 and GAT3,1 it has, nevertheless, been proposed to be involved in the regulation of GABAergic neurotransmission. Based on its reported astrocytic expression at extrasynaptic sites, BGT1 has in particular been suggested to regulate extrasynaptic levels of GABA.1,8–10 This hypothesis was further supported by a series of pharmacological studies proposing a role of BGT1 in seizure management (reviewed by Lie et al.3). The first demonstration was with the BGT1/GAT1-selective inhibitor, N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-4-(methylamino)4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF-1502, Figure 1),11 which upon peripheral administration in animal models of epilepsy showed anticonvulsive effects.12 Of particular importance was the observation that a synergistic anticonvulsive effect was achieved upon coadministration of EF-1502 with either of the two GAT1-selective inhibitors tiagabine13 or N[4,4-bis(4-fluorophenyl)-butyl]-4-amino-4,5,6,7-tetrahydrobenzo-[d]isoxazol-3-ol

(LU-32-

176B). Co-administration of the GAT1-selective compounds resulted in an additive anticonvulsive effect, suggesting that the seizure control obtained with EF-1502 was mediated

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through inhibition of both BGT1 and GAT1.12 Similar findings were later reported with the GAT3-selective inhibitor (S)-SNAP-511414 in combination with EF-1502, but not with tiagabine,15 and with EF-1502 and tiagabine in an in vitro study on brain slices from a rat model of epilepsy.16 Although the mechanism for the synergistic effect seen after inhibition of BGT1 and GAT1/3 is yet to be elucidated, it has been suggested to involve an increase in the extrasynaptic pool of GABA as a consequence of BGT1-inhibition, resulting in the activation of extrasynaptic GABAA receptors.10,17 While these studies provided indications for a functional role of BGT1 in epilepsy, deletion of the BGT1 gene in mice, on the other hand, failed to show an impact on seizure susceptibility.18 Although this challenges the proposed role of BGT1 in seizure management, it does not conclusively reject it either, since the expression and function of GATs, including GAT1 and GAT3, are known to be regulated in epilepsy,19–23 which could compensate for the deletion of BGT1 function from the brain. Based on the hypothesis that BGT1 expression/function is of pharmacological relevance in the mammalian CNS, more studies are needed to provide direct evidence to support or dispute the idea of a role of BGT1 in the regulation of e.g. GABA-mediated neurotransmission and in seizure management. One approach is to develop BGT1-specific tool compounds that can be used to explore the functional role of BGT1 in the CNS. We have previously reported the synthesis and pharmacological characterization of 2-amino-1,4,5,6-tetrahydropyrimidine-5carboxylic acid (ATPCA, Figure 1), a substrate-inhibitor with selectivity for BGT1 among the GATs,24 but with some agonistic activity at GABAA receptors.25 In the present study we report the synthesis and pharmacological evaluation of the tritiated analogue, [3H]ATPCA, as a novel tool to study BGT1 function.

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RESULTS AND DISCUSSION To enable functional studies on the role of BGT1 in cells and tissues in a directly quantitative and specific manner, we wanted to convert ATPCA, being the first known substrate with pronounced selectivity for BGT1 among the GATs,24 into a [3H]-labeled radioligand.

Synthesis and radiochemical stability of [3H]ATPCA We have previously reported the synthesis of ATPCA using catalytic hydrogenation over a PtO2 catalyst in concentrated aqueous hydrochloride starting from 2-aminopyrimidine-5carboxylic acid.24 To avoid formation of tritium oxide we decided to replace PtO2 by Pd/C as catalyst for tritiation. To prepare [3H]ATPCA with maximal specific activity, we screened the reaction conditions using different solvents in a pilot deuteration experiment. The solvents tried were ethanol, dichloromethane, aqueous hydrochloride, aqueous sodium hydroxide and N,N-dimethylformamide (DMF) using Pd/C as catalyst under gaseous 2H2 atmosphere. DMF was shown to be superior regarding reaction rate and enrichment of deuterium. Finally, [3H]ATPCA was synthesized using 2-aminopyrimidine-5-carboxylic acid in DMF over Pd/C [30% ] in a 3H2 (940 mbar) gaseous atmosphere (r.t., 4 h). We determined the amounts of prepared [3H]ATPCA to 290 mCi with a specific activity of 30.2 Ci/mmol and a radiochemical purity >99% after purification (HPLC). The 1H NMR confirmed less than full enrichment by tritium at the positions 4, 5 and 6 in the 1,4,5,6-tetrahydropyrimidine scaffold of ATPCA. The formulation of [3H]ATPCA was chosen as a 1 mCi/mL solution in H2OEtOH (1:1), and the radiochemical stability (radiochemical purity) was monitored (radioHPLC analysis) over time. When stored at -21 °C, the stability was determined to be 98.8% after 90 days and 97.5% after 270 days. Analogous radiochemical purity (98.6%) was

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observed when stored under similar conditions at 4°C. When stored at -196 °C for 270 days, no decomposition could be detected.

Specificity of ATPCA for BGT1 As ATPCA has not yet been evaluated for its specificity for BGT1 other than at the GABAA receptors, at which the compound has shown to be an agonist,25 we wanted to evaluate other possible targets for ATPCA that should be taken into consideration when utilizing the radioactive compound for studying native BGT1 function. Hence, we looked at targets that interact with ligands structurally related to APTCA or GABA. This included both the metabotropic GABAB receptor and transporters related to BGT1 from the SLC6 family, including the creatine transporter (CreaT), the taurine transporter (TauT), and the glycine transporters, GlyT1 or GlyT2.

For probing GABAB receptors, we tested ATPCA at two different concentrations for its ability to displace GABAB receptor binding in rat cortical homogenate using [3H]GABA as a radioligand (with concomitant saturation of GABAA receptors with isoguvacine). At 0.1 mM of ATPCA, no significant inhibition of binding was seen, whereas approximately 50% inhibition of total binding was seen at the highest tested concentration of 1 mM (Figure S1). This excludes GABAB receptors as a relevant “off target” for ATPCA.

For the SLC6 studies, radioligand-based uptake was initially assessed in Xenopus laevis oocytes. Whereas ATPCA at a concentration of 1 mM displayed no pronounced inhibition of radioligand uptake at TauT, GlyT1 or GlyT2, approximately 75% inhibition was observed for CreaT (Figure 2A). To examine this further, the activity of ATPCA was determined at CreaT

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recombinantly expressed in tsA201 cells. An uptake assay with [14C]creatine was established based on initial experiments conducted to determine the appropriate radioligand concentration and incubation time (data not shown). In this assay, ATPCA, but also the corresponding 2aminotetrahydropyridine analogue 1, a potent GABAA receptor agonist,25 and the known CreaT competitive inhibitor, 3-guanidinopropionic acid (3-GPA)26 (Figure 1), inhibited the uptake of [14C]creatine (200 nM) with IC50 values of 66, 675 and 9 µM, respectively (Figure 2B and Table 1).

Table 1. Inhibitory activities of selected compounds at CreaT in the [3H]ATPCA and [14C]creatine uptake assay. IC50 (pIC50 ± S.E.M.) (µM) 3

[ H]ATPCA uptake

[14C]Creatine uptake

ATPCA

50 (4.3 ± 0.06)

66 (4.2 ± 0.05)

1

360 (3.5 ± 0.13)

675 (3.2 ± 0.05)

3-GPA

13 (4.9 ± 0.06)

8.8 (5.1 ± 0.14)

The compounds were tested for their ability to inhibit the uptake of 30 nM [3H]ATPCA or 200 nM [14C]creatine at CreaT-expressing tsA201 cells. All experiments were performed in triplicates in three independent experiments.

[3H]ATPCA as a substrate for CreaT To further study the interaction between ATPCA and CreaT, we utilized the availability of [3H]ATPCA. Similar to the uptake assay using [14C]creatine, we incubated CreaT-expressing tsA201 cells with [3H]ATPCA (30 nM) for different time periods and saw time-dependent uptake that could be inhibited by creatine (3 mM) (Figure 2C), demonstrating that [3H]ATPCA is a substrate for CreaT. This correlates well with the reported structural features for known CreaT substrates, i.e. a carboxyl moiety and a guanidino group separated by 2–3 carbon atoms.27 Thus, not surprisingly, the ATPCA analogue, 1, where the basic part is constituted by an amidine moiety, was only weakly active at CreaT in both [14C]creatine and [3H]ATPCA uptake (Figure 2B and D, and Table 1). 7



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CreaT is found both at the luminal and abluminal sides of the brain capillary endothelial cells, where it is responsible for the active transport of creatine into the brain.28 We therefore speculated that CreaT could mediate the active transport of ATPCA, which, due to its hydrophilic and charged nature, would not otherwise be expected to pass the blood-brain barrier (BBB). Interestingly, BBB penetration experiments in mice showed that ATPCA enters the brain to a limited extent (Supporting Information). This was evident from the fact that unbound concentrations of ATPCA were much higher in plasma compared to brain throughout the course of the study. Inherently, the exposure-based approach to assess brain penetration of poorly distributed compounds is hampered by experimental factors, such as potential contamination from drug residing in brain capillaries. Still, however, the unbound distribution ratio of 8% observed at the terminal time point does not completely rule out a potential brain penetrance. The obvious lack of brain penetration of the related analogue 1 (Supporting Information) along with its weak potency at this transporter (Table 1), further supports the proposition that CreaT is implicated in the brain uptake of ATPCA, yet the low amounts accumulated in the brain imply that efflux mechanisms may also play a role.28,29

Having explored the activity of ATPCA at some of the most obvious possible targets outside of the GAT subfamily, we next turned to the primary goal of the study, namely to evaluate the pharmacological properties of [3H]ATPCA at BGT1 and its utility for examining BGT1 function.

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Pharmacological characterization of [3H]ATPCA at human recombinant transporters At BGT1 stably expressed in Chinese hamster ovary (CHO) Flp-InTM cells, [3H]ATPCA (20 nM) showed time-, temperature- and Na+-dependent uptake (Figure 3A and B). These are known characteristics for active transport through the Na+/Cl--dependent neurotransmitter transporters of the SLC6 family.2,30 The uptake was linear for up to ~10 minutes at 37 °C (Figure 3A), and similar uptake characteristics were seen with a radioligand concentration of 100 nM (data not shown). The following uptake competition experiments with [3H]ATPCA as a radiolabelled substrate were therefore performed similarly to the [3H]GABA uptake assay described previously,31 i.e. 3 minutes of incubation with 30 nM of radioligand at 37 °C. Under these conditions, prominent [3H]ATPCA uptake was measured at BGT1, whereas this was marginal at GAT2 and, importantly, absent at GAT1 and GAT3 (Figure 3C). For comparison, the CPM values obtained with uptake of [3H]GABA (30 nM) at BGT1 were approximately 30% lower than with [3H]ATPCA (30 nM) (data not shown). The [3H]ATPCA uptake at BGT1 was saturable and followed Michaelis-Menten kinetics with Km and Vmax values of 21 µM and 3.6 nmol ATPCA/(min×mg protein), respectively (Figure 3D and Table 2). While the maximum uptake rate of ATPCA was close to what we have reported previously for GABA (2.6 nmol GABA/(min×mg protein)), the Km value was approximately 3 times lower, emphasizing a higher affinity of ATPCA for BGT1 uptake compared to GABA.24

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Table 2. Experimentally determined Michaelis-Menten kinetics parameters of ATPCA at BGT1, and GABA for reference.

ATPCA GABA

a

Km

Vmax ± S.E.M.

(pKm ± S.E.M.) (µM)

(nmol compound/(min×mg protein)

21 (4.7 ± 0.07)

3.6 ± 1.08

58 (4.3 ± 0.10) *

2.6 ± 0.67 ns

The experiment was performed by determining the uptake velocity of ATPCA in the [3H]ATPCA uptake assay at BGT1-expressing CHO Flp-InTM cells. Four independent experiments were performed. The pKm and Vmax values of GABA were compared to those of ATPCA (no significance (ns) P>0.05, * P

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