Nγ-Aryl glutamine analogues as probes of the ASCT2 neutral amino acid transporter binding site
Graphical abstract
Introduction
l-Glutamine is the most abundant free amino acid in mammalian blood plasma and cerebral spinal fluid.1 As such an abundant bio-molecule, l-glutamine is involved in a variety of metabolic processes.2 l-Glutamine is a major component of muscle,3, 4 and in times of surgical illness has been classified as a ‘conditionally essential’ amino acid because of the increased metabolic requirements.5 As a precursor to TCA cycle intermediates, glutamine is an important source of energy.6, 7 Glutamine acts as a vehicle for both carbon and nitrogen shuttling8 and serves as an excretion route for toxic ammonia.9 This systemic glutamine shuttling has been referred to as the glutamine cycle,3 and is also partly responsible for the pH buffering of the blood.10 Glutamine is also accepted as being a major metabolic precursor for the excitatory neurotransmitter l-glutamate11 and the inhibitory neurotransmitter GABA.12 In order for glutamine to participate in all of these roles, this small polar molecule must move throughout the body. It is this glutamine movement in which we are interested, and particularly transporter protein facilitated movement.
Neutral amino acid transporter proteins are responsible for facilitating movement of these highly polar solutes across the lipophilic cellular membrane. A number of neutral amino acid transport systems have been identified, the main transport systems being the sodium dependent systems A, ASC, N, and the sodium-independent system L.13, 14, 15 As these transport systems are responsible for the translocation of the neutral amino acids (16 of the 20 common mammalian amino acids), one may suspect there is considerable overlap of substrate specificity. One common thread of these transport systems is that they all transport l-glutamine.
Our interest focused on system ASC glutamine transport (ASC for alanine, serine, cysteine). System ASC transport has been identified as an electroneutral obligate exchanger,13 and depending on transmembrane substrate ratios, it has the ability to operate in both influx and efflux directions.16 This transporter functions through a heteroexchange process, with glutamine efflux likely coupled to the influx of the system ASC substrates alanine, serine or cysteine.11, 17 It has been proposed that the transporter ASCT2 (system ASC isoform 2) is responsible for part of the glutamine efflux from the astrocytes involved in the glutamine/glutamate cycling of glutamate neurotransmitter.11, 15, 17 The Km reported for this transport process is in the range of 24–300 μM for glutamine,18, 19 has a lower throughput (Vmax ∼ 8–15 nmol/min/mg)18, 19 than system A or N activity, and has been proposed to contribute less of the overall glutamine glial efflux than system N.7 However, it was recently reported that a neuroblastoma cell line exhibits glutamine transport primarily due to system ASC.20 Similar glutamine transport activity reported in the literature occurs in the C6 rat glioma cell line and was further characterized as activity due to ASCT2.21, 22 These observations could be the result of a general phenomenon of cancerous cells up-regulating ASCT2 mediated glutamine transport,23 and exploitation of this phenomenon poses an attractive target for cancer research.24, 25 Despite the increased interest in the ASCT2 transporter, little is known about the protein binding site and transport requirements other than the endogenous l-amino acid substrate profile (system ASC has a wide range of substrate neutral amino acids, from small to medium side chains). The goal of this study is to obtain information about the ligand-protein binding interactions and gain insight into the transport mechanism of the ASCT2 transporter.
Our study of the neutral amino acid transporter ASCT2 used the C6 rat glioma cell line as the model system. Our pharmacological analysis found the C6 cell line functionally exhibits glutamine uptake primarily by ASCT2 mediated transport (approximately 75% of total glutamine uptake, results not shown). The remaining 25% of glutamine uptake activity was consistent with system N and to a minor extent system L (these results were in the range of those reported independently).21 Uptake experiments were therefore performed in the presence of BCH (2-aminobicyclo-(2,2,1)heptane-2-carboxylic acid, a system L selective inhibitor) and at pH 6.0 (to eliminate the activity of system N) therefore isolating ASCT2 glutamine transport activity.
In agreement with reported results,22 we observed that glutamate was relatively inactive at inhibiting the uptake of radiolabeled l-glutamine at pH 7.4, but was a potent inhibitor at pH 6.0. This activity is hypothesized to come from the protonation of glutamate rather than from protonation of the protein, since the activity of other substrates remained similar (Fig. 1). The protonation of glutamate at the distal carboxylate forms a neutral amino acid and resembles more closely the structure of glutamine. The effective concentration of the protonated form of 1 mM glutamate at pH 6.0 is 30 μM (based on the calculated pKa of 4.57 for the distal acid group using ACD 6.0 software), implying that the protonated form of glutamate has a very high affinity for the ASCT2 binding site. d/l-Homocysteine (–SH pKa = 10.52) was found to be a more potent inhibitor of glutamine uptake. at pH 6.0 than l-glutamine (amide–NH pKa = 16.52). These observations suggest that the distal group of the ligand may participate in a hydrogen bond donation that enhances the binding affinity of the ligand dramatically, and the more acidic the proton (without being fully ionized), the stronger the hydrogen bond. This hypothesized hydrogen bonding interaction may be a way to not only enhance the potency of the synthetic ligands, but to impart selectivity for the ASCT2 transporter over the other glutamine transporters.
Therefore, a key design feature of the synthetic analogues included a component to alter the pKa of the glutamine amide NH to test the H-bonding hypothesis. It was anticipated, in support of this hypothesis, that lowering the pKa of the amide NH would increase binding affinity to the ASCT2 transporter. In order to affect the acidity of the amide NH of l-glutamine, electron withdrawing and electron donating groups could be positioned such that the electronic effects could influence the stability of the resulting negative charge forming on the nitrogen. If substituents are placed directly on the nitrogen, effects other than electronic (i.e., steric, H-bonding, lipophilic, hydrophilic) may be the cause of activity changes. We decided to use the N-phenyl substitution as the template for altering the pKa of the NH. By using this substitution, the conjugated ring allows more direct substituent effects on the nitrogen electronic density while not influencing the steric environment in the immediate NH vicinity.
Another important design feature of the synthetic analogues is the three-dimensional positioning of the functional groups. Since little is known about the 3-D requirements of binding to the ASCT2 transporter binding site, an acyclic template was decided upon. This would impart little conformational bias between the different N-phenyl substituted analogues and allow more direct analysis of the electronic effects of the substituents. Since glutamine has a slightly greater affinity for the ASCT2 transporter than asparagine,20 the glutamine length template was used. Therefore, the synthetic analogue design to test the H-bond hypothesis was established as the substituted γ-N-phenyl l-glutamine derivative illustrated in Figure 2.
Section snippets
Results and discussion
The synthetic scheme to obtain the N-phenyl analogues used l-glutamic acid as the chiral starting material, following a reported sequence shown in Scheme 1.26 Nitrogen protection of glutamic acid with benzyl chloroformate resulted in the diacid carbamate 1. In order to differentiate the two carboxylic acid groups, the selective conversion to the azalactone 2 was performed in high yield. The resulting monoacid 2 was treated with thionyl chloride in refluxing methylene chloride to yield the acid
Conclusion
Based on initial characterization, the ASCT2 H-bond hypothesis was proposed. Through the use of glutamine analogue probes, this hypothesis was supported and an additional bonding hypothesis was generated: a lipophilic pocket is in the binding site. These hypotheses are summarized in the graphic in Figure 7, illustrating a preliminary binding site map for the ASCT2 neutral amino acid transporter. Also through these experiments, a commercially available potent inhibitor of the ASCT2 transporter
General
l-[3,43H(N)]-Glutamine was purchased from Perkin–Elmer and l-γ-nitrophenyl glutamyl anilide was purchased from ICN. d/l-Homocysteine, l-glutamate, anilines, and Cbz-chloride were purchased from ACROS Chemicals. NMR spectra were obtained on a Varian 400 MHz instrument using D2O with acetonitrile in D2O (δ 1.93 for proton, δ 1.3 for carbon) as the external standard, or DMSO with acetonitrile in DMSO as the external standard. Optical rotations were taken on a Perkin–Elmer polarimeter. HRMS were
Acknowledgements
We would like to give a special thanks to Whitney Widhalm for all her efforts in helping the project along, Erin M. O’Brien for her help with cell culture, Professor Richard Bridges, Director of the Center for Structural and Functional Neuroscience, for his input and suggestions, Professor John Gerdes, Director of the Molecular Computational Core Facility, and the Department of Chemistry, The University of Montana and NSF for the use of the NMR instrument. This research was made possible by NIH
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