755629

research-article2018
JBXXXX10.1177/2472555218755629SLAS DISCOVERY: Advancing Life Sciences R&DBröer

Review
SLAS Discovery

Amino Acid Transporters as Disease

2018, Vol. 23(4) 303­–320
© 2018 Society for Laboratory
Automation and Screening
Modifiers and Drug Targets DOI: 10.1177/2472555218755629
https://doi.org/10.1177/2472555218755629
journals.sagepub.com/home/jbx

1
Stefan Bröer

Abstract
Amino acids perform a variety of functions in cells and organisms, particularly in the synthesis of proteins, as energy
metabolites, neurotransmitters, and precursors for many other molecules. Amino acid transport plays a key role in all
these functions. Inhibition of amino acid transport is pursued as a therapeutic strategy in several areas, such as diabetes
and related metabolic disorders, neurological disorders, cancer, and stem cell biology. The role of amino acid transporters
in these disorders and processes is well established, but the implementation of amino acid transporters as drug targets
is still in its infancy. This is at least in part due to the underdeveloped pharmacology of this group of membrane proteins.
Recent advances in structural biology, membrane protein expression, and inhibitor screening methodology will see an
increased number of improved and selective inhibitors of amino acid transporters that can serve as tool compounds for
further studies.

Keywords
solute carrier, diabetes, cancer, drug development

Introduction They are several aspects of human physiology and

Amino acids are key metabolites that are essential for cells
and organisms. They form the building blocks for the syn-
thesis of proteins, they are used as energy metabolites, and
they are or contribute to the biosynthesis of signaling mol-
ecules, such as peptide hormones and neurotransmitters.
They also contribute to the synthesis of many other mole-
cules, such as nucleotides, creatine, polyamines, glucos-
amines, and related carbohydrates, and are the principal
generators of C1 compounds.1
In view of this diversity of roles, it is not surprising that
amino acid homeostasis is highly regulated and that all cells
maintain a pool of the 20 proteinogenic amino acids at any
given time.2 Mammalian cells can synthesize nonessential
amino acids (alanine, arginine, asparagine, aspartate, cyste-
ine, glutamate, glutamine, glycine, proline, and serine), but
need to acquire essential amino acids from nutrition (histi-
dine, isoleucine, leucine, lysine, methionine, phenylalanine,
threonine, tryptophan, and valine). Cellular protein, in par-
ticular muscle protein, is the storage site for amino acids in
organisms. Most cells can survive several hours without
amino acids, inducing autophagy to degrade cellular protein
for the synthesis of essential proteins.3 Protein degradation
is combined with efficient mechanisms to recycle the gener-
ated amino acids; thus, net loss of amino acids is limited.
Transporters play a key role in the acquisition of amino
acids by the whole organism from the nutrition and in the
acquisition of amino acids by individual cells from blood
plasma.2,4,5
pathophysiology where amino acid transporters are consid-
ered disease modifiers and drug targets (Fig. 1 and Table
1).5–9 These are
-  Metabolic disorders, such as diabetes and obesity
-  Neurological disorders
- Cancer
-  Embryo development and stem cells
Three approaches are generally used to influence amino
acid homeostasis (Fig. 1). First is the blockage of transport-
ers to induce amino acid restriction or starvation. This is
used as a possible treatment to reduce the growth of cancer
cells,10–12 but also to induce protein restriction as a novel
attempt to treat diabetes.13 Second is the blocking of trans-
porters to increase the concentration of amino acids in the
interstitial space. This is often used to treat neurological dis-
orders, where amino acids such as glutamate and glycine
act as neurotransmitters.7,8,14 Third is the use of transporters
1
Research School of Biology, College of Science, The Australian National
University, Canberra, ACT, Australia
Received Oct 11, 2017, and in revised form Jan 4, 2018. Accepted for
publication Jan 5, 2018.
Corresponding Author:
Stefan Bröer, Research School of Biology, College of Science, Building
134, The Australian National University, Canberra, ACT 2601, Australia.
Email: stefan.broeer@anu.edu.au
304 SLAS Discovery 23(4)

Organism
Brain cell
Increase
extracellular
AA concentraon C
Cellular
and signalling AA absorpon

O
Organismic Malignant cell
AA absorpon Figure 1.  Current concepts of
- Protein restricon amino acid transporters as drug
to improve metabolic Cellular AA absorpon
C targets. Reducing uptake of amino
health - Reduce cell growth acids in the intestine causes protein
restriction and can improve
metabolic health. Uptake into
cells can be blocked to increase
signaling, particularly when amino
acids serve as neurotransmitters
Stem cell/Embryo or neuromodulators. Blocking
uptake of essential amino acids can
be used to reduce the growth of
malignant cells. In vitro amino acid
Cellular AA absorpon supplementation and transporter
- Opmize AA composion modulation can be used to optimize
conditions for embryo transfer and
implantation.

Table 1.  Amino Acid Transporters Covered in This Review and Their Function..

Solute Carrier ID Common Name Function Potential Indication

SLC1A2 GLT-1 Glutamate reuptake at the synapse ALS
SLC1A5 ASCT2 Glutamine transport, intracellular amino acid Cancer
homeostasis
SLC6A5 GlyT2 Glycine reuptake at the synapse Neuropathic pain
SLC6A9 GlyT1 Glycine homeostasis at the synapse Schizophrenia
SLC6A14 ATB0,+ General amino acid transporter Cancer
SLC6A19 B0AT1 Neutral amino acid transporter Diabetes
SLC6A20 SIT1 Proline, betaine transporter In vitro fertilization
SLC7A2 CAT2B Cationic amino acid transporter Inflammation
SLC7A5 LAT1 Neutral amino acid transporter Cancer
SLC7A9 b0,+AT Cationic amino acid transporter Unclear
SLC7A11 xCT Glutamate/cystine transporter Cancer
SLC16A10 TAT1 Aromatic amino acid transporter Autoimmune disorders
SLC38A1 SNAT1 Neutral amino acid transporter Cancer
SLC38A2 SNAT2 Neutral amino acid transporter Cancer
SLC38A3 SNAT3 Glutamine transporter Diabetes
SLC38A4 SNAT4 Neutral amino acid transporter Diabetes
SLC38A5 SNAT5 Glutamine transporter Diabetes
SLC43A1 LAT3 BCAA transporter Cancer
Bröer 305
to selectively provide amino acids to certain cells. This
approach is used less frequently but is particularly interest-
ing with regard to embryo development and stem cell biol-
ogy.15–22 The latter could be complemented by compounds
to induce transporter function or expression, but this has not
been translated to date.
Principles of Transporter Inhibition
Biomembrane transporters, including amino acid transport-
ers, are attractive drug targets for several reasons. First, they
are located at the cell surface and, similar to receptors, are
easily accessible by drugs from the bloodstream.23 Notable
exceptions are transporters in the brain, which are shielded
by the blood–brain barrier against most drugs,24–26 and
transporters in organelles, such as lysosomes or synaptic
vesicles. Second, as part of their function, amino acid trans-
porters have deep substrate binding pockets, which often
reach halfway through the membrane (Fig. 2A). The pres-
ence of a substrate binding site with a vestibule allows man-
ifold interactions and bond formation to stabilize binding of
an inhibitor.27 In contrast to enzymes, where transition-state
analogues are considered to be the best inhibitors,28 ground-
state analogues are preferred as transport inhibitors.29 The
ground state of a transporter for drug binding is the outside
open conformation.30 In contrast to the majority of enzymes
where the substrate adopts a transition state through nonco-
valent interactions, transporters form a transition state
around the substrate (induced fit), while the substrate
remains in its original conformation.31,32 The transition state
is called the occluded conformation, in which the trans-
porter protein fully encloses the substrate (Fig. 2B).33 Thus,
only compounds that are very similar to the substrate itself
fit into this cavity. Accordingly, transport inhibitors typi-
cally contain a substrate-like fragment to which a large
fragment is coupled, often containing aromatic rings.
Examples of this design are shown in Figure 3. The ratio-
nale behind this design is the wedge effect exerted by the
Figure 2.  Structure of amino
acid transporters and their
binding sites. (A) The outward-
open conformation of the leucine
transporter from Aquifex aeolicus
(LeuTAa) is shown (PDB ID 3TT1).
The cavity that leads to the
substrate binding site is visible in
the center of the molecule. (B) The
occluded state of LeuTAa (PDB ID
2A65) is shown from the side and
sliced to reveal the substrate binding
site, which encloses the substrate
(magenta) tightly. The vestibule is
visible at the top.
large aromatic fragment, which blocks the transition of the
transporter to the occluded conformation. The substrate-
like fragment ensures recognition and specificity; the wedge
fragment provides additional interactions with the trans-
porter to increase affinity and at the same time blocks adop-
tion of the transition state. This design is well suited to a
medicinal chemistry approach.34 However, transport inhibi-
tors based on substrate analogues have two potential prob-
lems. First, inhibitors may have problems binding to the
transporter in vivo in the presence of high concentrations of
substrates. The combined concentration of amino acid sub-
strates of a specific transporter in blood serum can easily
reach 1 mM, against which a competitive inhibitor has to
find its target. Second, substrate-like inhibitors may show
lack of isoform specificity. For instance, dl-threo-β-
benzoylaspartate (TBOA) inhibits all glutamate transporter
isoforms (Fig. 3).35 Allosteric inhibitors, by contrast, are
not displaced by substrates and can be isoform specific. For
instance, UCPH101 (Fig. 4A) is a specific inhibitor of the
glutamate transporter EAAT1, due to its binding between
the scaffold and transporter domain of EAAT1 (Fig. 4B),
which has a less conserved sequence than the substrate
binding site.36 Allosteric inhibitors are more likely to arise
from high-throughput screening (HTS) or specialized
screens,37 but can be further modified through medicinal
chemistry. The betain-γ-amino butyric acid (GABA) trans-
porter BGT1, for instance, can be specifically inhibited by
N-(1-benzyl-4-piperidinyl)-2,4-dichlorobenzamide (Fig.
4A), which binds to the vestibule of the transporter without
penetrating the binding site.38 Allosteric transporter inhibi-
tors have also been identified by screening cyclic peptide
libraries.39
Methods to Identify Transporter
Inhibitors
Medicinal chemistry is one of the major tools to design trans-
port inhibitors, usually starting with one of the substrates of the
306 SLAS Discovery 23(4)

Figure 3.  Examples of transport inhibitors based on substrate analogues. Three amino acid transport inhibitors are shown. The
substrate-like part of each inhibitor is highlighted in magenta. Bulky side chains (black) prevent the occlusion of the substrate by the
transporter. See references 12, 129, and 181 for the depicted inhibitors.

Figure 4.  Allosteric inhibitors of amino acid transporters. (A) Structures of allosteric transport inhibitors are unrelated to the
substrate. Top: UCPH101, a specific EAAT1 inhibitor. Bottom: The BGT-1 inhibitor BPDBA is thought to bind an allosteric secondary
binding site observed in some transporters of the SLC6 family. (B) The binding site of the EAAT1 inhibitor (UCPH101) was resolved
by x-ray crystallography and is located at a distance from the substrate binding site.182 The substrate is shown in the center of the
transporter, while the inhibitor is located between the translocation and scaffold domain of the transporter (lower right). The
inhibitor glues the two domains together, thereby inhibiting transport.
Bröer 307
transporter or a close analogue. The reader is referred to a com-
prehensive review of the medicinal chemistry of α-amino acids
for further information.34 To identify substrate-unrelated start-
ing points for medicinal chemistry, HTS is increasingly used in
academic environments to identify transport inhibitors. The
most frequently used techniques for transporters are briefly
described in the following.
Accumulation of Radiolabeled Substrate
Although radiolabeled compounds are less frequently used in
HTS environments due to regulatory reasons, inhibition of
radiolabeled substrate uptake is still the most robust and quan-
titative assay to study transport inhibitors. Moreover, it is the
gold standard to identify false-positive hits from other screen-
ing methods described below and to characterize the mode of
inhibition. Typically, a cell line stably expressing the trans-
porter of choice is washed with isosmotic buffer, and subse-
quently incubated with radiolabeled substrate for 5–30 min in
the presence or absence of inhibitors. After washing the cells to
remove extracellular radiolabel, the amount of radioactivity is
measured in cell extracts. This method has been used to screen
for inhibitors of glycine transporter (GlyT1)40 and neutral
amino acid transporter asc-1.41 The method can also be used in
conjunction with reconstituted protein in proteoliposomes, in
which case filtration is used to separate the proteoliposomes
from radiolabeled compounds after incubation.42
Scintillation Proximity Assay
In this assay, the radioactive substrate of a transporter is
brought into proximity with a scintillant. Weak β-particles are
strongly quenched in water; thus, only radioactive compounds
in close proximity to the scintillant generate light flashes,
which can be recorded with a normal scintillation counter.
Several variations of this assay are available, depending on
whether cell lines, membrane preparations, or reconstituted
transporters are used. Cells can be seeded out onto specific
multiwell plates with a scintillating base.43 Uptake of the radio-
labeled substrate into the cells brings the radioactivity into
proximity of the scintillant generating light emissions, which
are recorded. In a variation of this procedure, suspended cells
are brought into contact with polylysine-coated scintillation
proximity assay (SPA) beads, also resulting in an uptake-
dependent scintillation signal.41 For membrane fragments or
reconstituted protein, beads are used that are coated with poly-
lysine to bind membrane lipids. Purified transporters in deter-
gent micelles have also been characterized by this method
using copper chelate–coated beads to immobilize a tyrosine
transporter through its His tag.44
Membrane Potential–Sensitive Dyes
These can only be applied to electrogenic transporters and
require significant transport activity. Voltage-sensitive dyes
have been used for many years to study transport and ion
channels in membrane vesicles and adherent cells.45 The
dyes are membrane permeable and therefore can be washed
out. The assay has been improved by the addition of spe-
cific dyes to quench signals occurring outside the cell (U.S.
patent 6,420,183, EPO patent 0 906 572) and is available
commercially as FLIPR Membrane Potential Assay. A cell
expressing the selected transporter is required, although
expression can be endogenous or heterologous. Endogenous
transporters may interfere with the signal generated by het-
erologous expressed transporters. The assay has been suc-
cessfully used to screen for inhibitors of several amino acid
transporters, such as the neutral amino acid transporter
SLC6A19,46 glycine transporters GlyT147 and GlyT2,48 and
glutamate transporters EAAT1–3.49 In the case of SLC6A19,
several factors were important to optimize the signal. First,
heterologous expression of both SLC6A19 and its traffic
subunit TMEM27 was required for significant expression.
CHO cells were used as a host cell line because it had no
measurable Na+-dependent uptake for isoleucine, one of the
preferred substrates of SLC6A19. While a tailored platform
exists for the FLIPR assay, it can be implemented on any
suitable fluorescent plate reader.
Ion-Sensitive Dyes
Three different sodium dyes have been evaluated in prostate
cancer cell lines, namely, benzofuran isophthalate (SBFI),
CoroNa Green (Corona), and Asante NaTRIUM Green-2
(ANG-2).50 While calibration was robust in the three cho-
sen cell lines, the response of each cell line to changes in the
extracellular Na+ concentration was quite different.
Inhibition of the Na+/K+-ATPase by Ouabain caused a sig-
nificant increase of intracellular Na+ that was readily
detected by SBFI and ANG-2, but not by Corona.
Interestingly, the addition of veratridine to open Na+ chan-
nels did not result in a detectable increase of the intracellu-
lar Na+ concentration. Although principally suitable for
HTS, the author is not aware of any study that has used
Na+-sensitive dyes to screen for inhibitors of Na+ cotrans-
porters. The pH-sensitive dye BCECF has been used in
Caco-2 cells to monitor H+-peptide cotransport via the pep-
tide transporter SLC15A1.51 BCECF is a ratiometric dye
and can be preloaded as an AM ester. The ratiometric mode
of measurement is useful when BCECF is leaking out of the
cells. BCECF has not been used in combination with amino
acid transporters.
Biosensors
Glutamine transport has been analyzed in living cells using
a genetically encoded biosensor, for instance, in Cos-7 cells
transiently expressing the glutamine transporter SLC1A5.
The biosensor is based on the Escherichia coli glutamine
binding protein, glnH.52 Förster resonance energy transfer
308 SLAS Discovery 23(4)
(FRET) occurs between monomeric teal fluorescent protein
(mTFP) 1 and the yellow fluorescent protein venus. FRET-
based arginine biosensors have been generated based on the
arginine repressor/activator ahrC gene from Bacillus subti-
lis53 and through protein engineering of bacterial amino
acid binding proteins.54 A biosensor for methionine and
branched-chain amino acids (BCAAs) has been developed
using the leucine-responsive protein Lrp of Corynebacterium
glutamicum.55
Mass Spectrometry
Amino acid uptake via a transporter can be monitored by mass
spectrometry (MS) either detecting accumulation of the natu-
ral amino acid or using stable isotopes. Classical techniques,
such as gas chromatography–mass spectrometry (GC-MS) or
liquid chromatography–mass spectrometry (LC-MS), are less
suitable for large-scale inhibitor screening due to the slow
chromatography step. With the development of high-resolu-
tion mass spectrometers, complex mixtures of metabolites
from cell extracts can be analyzed in a single step or following
simple extraction. Typically, electrospray ionization or matrix-
assisted laser desorption ionization are used in combination
with a quadrupole time-of-flight (Q-TOF) mass spectrometer
to analyze liquid samples directly or after extraction using
solid-phase extraction cartridges. This methodology has been
used to identify novel inhibitors of P-glycoprotein.56 The
development of echo acoustic liquid handling by Labcyte Inc.
(San Jose, CA) will provide further improvement of sample
preparation and injection for MS.57
Docking Studies
Computational screening has become more feasible due to
the increasing number of available transporter structures.58
Several obstacles need to be overcome for computational
approaches to become competitive to HTS. First, very few
mammalian amino acid transporter structures have been
resolved, particularly in the outside open conformation, which
is the most relevant structure for inhibitor identification.36,59 As
a result, most docking studies rely on homology mod-
els.46,60,61 Second, docking studies are still relatively slow.
Even with generous computation time, only small libraries
(tens of thousands of compounds) can be screened. Larger
screens make use of a prescreen using a pharmacophore
similarity screen.62 This allows reducing very large libraries
to smaller compound libraries, but at the same time
decreases the likelihood of finding novel structures. Third,
docking studies have a strong bias toward larger molecules,
because an increase of interaction surface automatically
increases the docking score. Larger molecules often do not
comply with Lipinski’s rule of five, thereby reducing their
suitability as starting points for medicinal chemistry.63
Moreover, molecular dynamics simulations are required to
test accessibility of the compounds to the binding site.64
Thus far, only low-affinity compounds have been identified
through computational screens for amino acid transport
inhibitors, which require significant modification to
improve binding.46,60,61
Methods to Characterize Inhibitor Action in
Detail
The methods described above are frequently used to screen for
inhibitors. Further characterization of the mechanistic action of
inhibitors can require additional methods. As outlined above,
noncompetitive inhibitors can provide better specificity and
resistance against substrate competition. Electrophysiological
techniques, such as voltage-clamp recordings, are frequently
used to monitor electrogenic transporters, but on occasion can
also be used to monitor transport-associated currents in elec-
troneutral transporters. Electrophysiological techniques can be
applied to transfected cells and Xenopus laevis oocytes
expressing recombinant transporter mRNA. Rapid perfusion
techniques and the use of caged compounds that can be
released upon light exposure are particularly valuable to ana-
lyze binding events during the transport cycle. Moreover, elec-
trophysiology can be combined with fluorescent reporter
groups that are attached to mobile parts of the transporter struc-
ture. An extensive review of these methods has been compiled
by Grewer et al.65 In the case of transporters, electrophysiolog-
ical methods report currents from a large number of transport-
ers, particularly in oocytes. For reasons that are not fully
elucidated, transporters expressed in X. laevis oocytes are often
refractory to inhibition by hydrophobic compounds, thus
resulting in IC50 values that are significantly higher than in cul-
tured cells.46,66,67 If purified protein is available, proteolipo-
somes can be used to study transporter inhibition.
Proteoliposomes are obtained by detergent removal from lipo-
some–detergent–protein mixtures.42,68 Due to the curvature of
the vesicles, incorporation of protein is often asymmetric,
allowing the design of efflux or influx experiments.
More recently, single-molecule techniques have been
developed to follow transport processes in individual mol-
ecules using FRET.69 Typically, these require the introduc-
tion of cysteine residues into cys-less or cys-reduced
versions of the native transporter, which are then modified
with donor and acceptor fluorophores. These methods can
be used to study transport inhibitors. Inhibitors can provide
valuable insight for these studies because they can lock
transporters in a particular state, which is recognizable due
to the movement of fluorophores or the lack thereof.69
Role of Transporters in Nutritional
Disorders, Such as Diabetes and
Obesity
Several studies have reported an association between obe-
sity and polymorphisms in the gene encoding the general
Bröer 309
amino acid transporter SLC6A14.70–72 The contribution to
obesity of this transporter appears to be weak, with allele
frequencies showing only a few percent differences in nor-
mal and obese individuals. The result suggests only a small
influence of SLC6A14 on the obesity phenotype in general.
A mechanism has not been established but could be related
to amino acids contributing to appetite regulation in the
pituitary gland.73 Alternatively, it could provide tryptophan
for serotonin production, a neurotransmitter involved in
appetite control. Another scenario relates to the function of
SLC6A14 in fetal development.74 Reduced amino acid pro-
vision to the fetus could result in epigenetic programming,
which in turn could foster inappropriate eating behavior
later in life.
SLC6A19 is the major transporter for neutral amino
acids in the small intestine and in the proximal tubule of the
kidney.75 Elevated levels of BCAAs have been detected in
individuals with type 2 diabetes and are thought to be strong
predictors of future onset of type 2 diabetes.76–79 Mechanistically
elevated levels of BCAAs are thought to cause insulin resis-
tance over the long term by a variety of mechanisms.80,81
Leucine is an important regulator of mTORC1, which
through p70S6 kinase can cause insulin resistance by phos-
phorylation of insulin receptor substrate. Probably more
relevant is the effect of BCAA metabolites on insulin resis-
tance.76,77,79 Carnitine esters of branched-chain fatty acids
have been identified as possible causes of insulin resis-
tance79 and branched-chain keto acids that are generated
after transamination of BCAAs appears to reduce GLUT4-
mediated uptake of glucose into muscle after insulin stimu-
lation.82 On a broader scale, low-protein diets promote
general metabolic health and possibly longevity,83 and so
does methionine restriction.84 More importantly, gastric
bypass surgery has dramatic effects on insulin sensitivity
within days of surgery, long before significant weight loss
occurs.85 At least some of these effects are thought to arise
from reduced protein uptake. Together, this suggests that
reducing amino acid absorption could be an avenue to treat
type 2 diabetes and obesity. SLC6A19 is the major trans-
porter for BCAAs and methionine in the intestine, and mice
lacking this transporter show a host of responses that are
similar to those observed after gastric bypass surgery.13 The
main responses were improved glycemic control, reduced
plasma insulin, increased secretion of GLP-1 and GIP,
increased production of FGF-21, reduced plasma fatty acids
and liver triglycerides, browning of subcutaneous adipose
tissue, and increased ketone body production. FGF-21
could be an important factor of this metabolic phenotype
causing ketone body production from plasma and liver fatty
acids, and browning of adipose tissue. Humans with muta-
tions in SLC6A19 have Hartnup disorder, a largely benign
syndrome that is sometimes associated with photosensitive
skin rash.86,87 Very rarely has cerebellar ataxia been reported
in young individuals with Hartnup disorder, which may be
coincidental or caused by poor nutrition. Thus, it appears
likely that inhibition of the transporter is generally safe in
mature individuals with overnutrition.
Through the use of computational approaches and screen-
ing assays based on membrane potential–sensitive dyes, benz-
tropine was identified as an inhibitor of SLC6A19.46 Through
the use of SLC6A19 containing proteoliposomes, nimesu-
lide was identified as another inhibitor of SLC6A19.88 Both
compounds have other molecular targets, and the IC50 val-
ues are such that they can only serve as research tool
compounds.
SLC7A5 is the major transporter for BCAAs in nonepi-
thelial cells.89 It is an amino acid exchanger that can use intra-
cellular glutamine and other amino acids as an exchange
substrate to import essential amino acids.2,90 Transport exper-
iments performed in X. laevis oocytes suggest that glutamine
is a rather poor substrate of SLC7A5; however, studies in cell
lines are consistent with a significant role of glutamine as an
exchange substrate, due to its abundance in the cytosol.91 The
transporter is expressed in β cells, and leucine uptake via
SLC7A5 is important for activation of mTORC192 and also
stimulates the release of insulin as an allosteric activator of
glutamate dehydrogenase.93 Inhibition of SLC7A5 using
BCH reduces mTORC1 activation and insulin release.
However, BCH has also been reported to stimulate insulin
release through the same mechanism as leucine.94
Amino acids play an important role in the modulation of
insulin release from pancreatic β cells,94,95 and transporters
are involved in this process.92 It is thought that amino acids
stimulate insulin secretion through ATP production inside
mitochondria. Glutamine and glutamate, in particular, are
readily metabolized inside mitochondria to produce ATP.
Leucine, in addition, acts as an allosteric inducer of gluta-
mate dehydrogenase, which provides intermediates for the
tricarboxylic acid (TCA) cycle derived from glutamine. As a
result, a combination of leucine and glutamine potently
increases insulin secretion.94 Glutamine can enter β cells via
the glutamine transporter SLC38A3,96 while leucine is trans-
ported by SLC7A5 (see above). Expression of glutamate
transporters is too low to support significant metabolism.97
Glutamine uptake via SLC38A5 supports α-cell prolifer-
ation. Glucagon is produced in α cells of the pancreas acting,
among other cells, on hepatocytes to induce gluconeogene-
sis. Glutamine and alanine are the main precursors in the
early hours of fasting. In diabetes, elevated levels of fasting
glucose are observed, suggesting an overstimulation of glu-
coneogenesis. However, attempts to reduce glucagon signal-
ing cause hyperglucagonemia and α-cell hyperplasia.98
Mice with a liver-specific lack of the glucagon receptor
show a 3- to 7-fold increased proliferation of α cells, sug-
gesting a factor that is released by hepatocytes to induce
α-cell proliferation.99 Systematic metabolomic analysis
revealed that l-glutamine was the hepatocyte factor, act-
ing through mTOR signaling and the FoxP transcription
310 SLAS Discovery 23(4)
factor.21,22 Expression of SLC38A5 was upregulated in α
cells from glucagon receptor knockout mice, while it was
barely detectable in wild-type mice. Blocking glucagon
signaling in the liver, by contrast, reduced the expression
of amino acid transporters SLC38A3, SLC38A4, and
SLC7A2, which mediate the import of amino acids for
gluconeogenesis.22
As an overall strategy, it is conceivable that insulin
secretion and hepatic glucose output can be modulated by
blocking amino acid transporters. This could be combined
with transport inhibitors that cause methionine restriction.
Role of Amino Acid Transporters in
Neurological Disorders
GABA, glutamate, glycine, d-serine, and glutamine are
neuroactive amino acids, the modulation of which can be
exploited to treat several neurological pathologies.
Glycine transporters GlyT1 (SLC6A9) and GlyT2
(SLC6A5) are the major transporters for the neurotransmitter
glycine in the brain. Pain is a normal response to injury or
inflammation and is mediated by nociceptive neurons.8,14
Acute adaptive changes to these circuits also lead to hyper-
sensitivity that serves a protective function during healing.
However, nerve injury in the peripheral or central nervous
system can lead to chronic pain or hypersensitivity that
persists beyond the healing process. Glycine and GABA are
the major inhibitory neurotransmitters in the nervous system
and are released to dampen excitatory neurotransmission,
which is used to transmit signals to the brain, including pain.
Physiologically, neurotransmitters are rapidly removed
from the synaptic cleft to terminate the signal. It is thought
that chronic pain is caused by a reduction of glycinergic
inhibition in the spinal cord, where glycine transporters are
particularly abundant.8 A broad variety of glycine transport
inhibitors have been generated, for both GlyT1 and GlyT2.14
Inhibitors based on sarcosine (N-methyl-glycine) are selec-
tive for GlyT1. However, GlyT1 is widely expressed in
astrocytes of the nervous system, and its inhibition enhances
excitatory neurotransmission by increasing extracellular
glycine as a co-agonist at NMDA receptors.9 GlyT2 inhibi-
tors ALX1393 and ORG25543 were shown in a mouse
model of neuropathic pain to have antiallodynia effects,100
that is, preventing an innocuous stimulus from becoming
painful.
Schizophrenia is a devastating neuropsychiatric disorder
characterized by positive symptoms (i.e., symptoms that
healthy people do not experience, such as delusions and
hallucinations), negative symptoms (i.e., deficits compared
with healthy individuals, such as lack of emotion and moti-
vation), and cognitive deficits.9,101 The cause of this disease
remains unclear, but reduced activity of NMDA receptors
may be involved in the negative symptoms. NMDA recep-
tors are allosterically modulated by d-serine and glycine,
and their extracellular levels are controlled by specific
transporters. In the case of glycine, GlyT1 is thought to
control glycine levels in the synaptic cleft.9 The main
transporter for d-serine is the neutral amino acid trans-
porter asc-1;102 smaller contributions are mediated by
amino acid transporters SLC1A4 and SLC1A5.103 In both
cases, blocking the uptake of glycine or d-serine would
increase the concentration of these allosteric modulators
in the synaptic cleft, thereby enhancing NMDA receptor
activity. Surprisingly, inhibitors of asc-1 decreased extra-
cellular d-serine levels, suggesting that this transporter is
rather involved in the efflux of d-serine from astro-
cytes.104,105 While modulation of d-serine levels in schizo-
phrenia requires more research, GlyT1 inhibitor bitopertin
has been used in clinical trials (up to clinical trial phase 3).9
Unfortunately, the results were inconclusive and no separa-
tion between placebo and bitopertin-treated patient groups
was observed.106 Bitopertin was well tolerated, overcoming
concerns that complete inhibition of GlyT1 could result in
severe motor and respiratory deficits, as observed in GlyT1
knockout mice.107
Despite years of intensive research on glutamate trans-
porters in the brain, a promising pharmacological approach
has yet to emerge. Glutamate transporters are essential for
the removal of the excitatory neurotransmitter glutamate
from the synaptic cleft. Modulation of glutamatergic neuro-
transmission is involved in many neurological and psychi-
atric diseases.7 It is thought that elevated levels of glutamate
are neurotoxic due to overactivation of glutamate receptors,
which in turn causes excessive calcium influx into neurons,
resulting in cell damage and death. As a result, pharmaco-
logical approaches using glutamate transporters rely on
compounds that enhance glutamate transport rather than
inhibiting it. In a broad screen, Rothstein et al. identified
β-lactam antibiotics as compounds that increase transcrip-
tion of the gene encoding glutamate transporter EAAT2
(SLC1A2).108 Activators of EAAT2 translation were identi-
fied by using an enzyme-linked immunosorbent assay
(ELISA) in combination with antibodies that bind to the
C-terminus of EAAT2.109 In the used cell line, EAAT2
expression was driven by a cytomegalovirus (CMV) pro-
moter to increase the likelihood of identifying translational
activators. In amyotrophic lateral sclerosis (ALS), motor
neurons gradually degenerate, resulting in muscle atrophy,
weakness, and eventually respiratory failure and death. The
etiology of neurodegeneration in ALS is not fully under-
stood, but glutamate excitotoxicity may contribute to the
disease progression.110 A clinical trial was conducted to
evaluate whether enhanced expression of EAAT2 by ceftriax-
one would delay the functional decline in patients with ALS.110
It appears that ceftriaxone showed some beneficial effects in
earlier stages of the disease, but no differences were observed
in overall survival. The translational activator 3-([(2-methyl-
phenyl)methyl]thio)-6-(2-pyridinyl)-pyridazine was able to
Bröer 311
protect neurons from excitotoxicity and delayed functional
decline in an animal model of ALS.111
Role of Amino Acid Transporters in
Cancer
Alteration of metabolism is almost universally observed in
cancer cell lines.112 These adaptations are governed by the
needs of an anabolic cell to generate sufficient biomass to
produce new cells. Protein forms the majority of new cell
mass, and cancer cells therefore show enhanced rates of
amino acid uptake. In addition, amino acids are used for a
wide variety of metabolic purposes in cancer cells. One of
the earliest approaches to combat cancer relied on antifo-
lates, which are essential to provide one-carbon compounds
to metabolic pathways.113 One-carbon compounds are par-
ticularly important for the synthesis of nucleobases.
5-Fluorouracil is an inhibitor of thymidylate synthase,
which converts nucleotide dUMP to dTMP for DNA bio-
synthesis. In the process, N5,N10-methylene tetrahydrofolate
is converted to dihydrofolate. Dihydrofolate is recycled in
two steps involving reduction and recharging of a C1 com-
pound, which is derived from serine-to-glycine conversion
by serine hydroxymethyl transferase. Thus, serine is an
important metabolite for cancer cells, which can be gener-
ated from the glycolytic intermediate 3-phosphoglycerate.
Serine depletion causes growth arrest in several cancer cell
lines through its role in C1 metabolism, but in addition, ser-
ine depletion increases respiration and production of reac-
tive oxygen species, by reducing the allosteric activation of
pyruvate kinase PKM2.114 Equally important in cancer cells
is the amino acid glutamine, which contributes to the bio-
synthesis of biomolecules in several ways. First, it provides
nitrogen from its amino group for nucleobase biosynthesis,
Second, it is used to synthesize aspartate (also required for
nucleobase biosynthesis) and pyruvate in a pathway called
glutaminolysis, a linearized version of the TCA cycle used
in rapidly proliferating cells.115,116 Through this pathway,
glutamine can sustain anaplerosis of the TCA cycle, which
provides citrate, for example, for fatty acid biosynthesis.
Third, glutamine is essential for the hexosamine pathway
that produces aminated carbohydrates for the purposes of
glycosylation, the amino group being derived from gluta-
mine.117 Fourth, glutamine is an important precursor of glu-
tathione biosynthesis.118 Differentiated cells often produce
glutamine to dispose of ammonia from amino acid metabo-
lism, while cancer cells become glutamine dependent.
Many cancer cells cease to grow when glutamine is removed
from the media.119 The glutamine dependence is a conse-
quence of the multiple uses of glutamine in the pathways
mentioned above. Consequently, glutamine transport has
been considered a target for cancer therapy, but the approach
is not straightforward due to the multitude of glutamine
transporters in mammalian cells.6,10,120,121 Mammalian cells,
in general, can only synthesize the nonessential amino
acids, while essential amino acids must be acquired from
nutrition or blood supply in the case of cancer cells.
SLC1A5 is an amino acid exchanger for small neutral
amino acids, including serine, threonine, and glutamine.122 It
is upregulated in many cancer cells and functionally the dom-
inant glutamine transporter in most cancer cell lines.122
Silencing of SLC1A5 reduces cell growth in some cancer cell
lines123–126 but not in others.121,126 Related to this, SLC1A5
silencing reduces mTORC1 activity in some cell lines90,123
but not in others.121 Pharmacological studies have been ham-
pered by the poor selectivity of initially published inhibitors
and by the fact that most of them are competitive binders and
are easily displaced by amino acids in media.60,121,127,128
Potentially more powerful and selective inhibitors have been
published more recently (Fig. 3).129 The mechanism by which
blockage of SLC1A5 reduces tumor growth is still unclear.
As an exchanger, SLC1A5 cannot mediate net uptake of
amino acids, but it can quickly restore depleted amino acids
at the expense of abundant amino acids.121 It appears likely
that in cell lines where blocking SLC1A5 reduces mTORC1
activity, reduced growth is observed, while in cells where
mTORC1 signaling is unaffected, growth persists. Silencing
of SLC1A5 induces amino acid restriction and activation of
the GCN2/ATF4 pathway, which in turn upregulates other
amino acid transporters.121,126 The variability in the response
of cancer cell lines to blockage of SLC1A5 is also illustrated
by the antitumor efficacy evaluation of a monoclonal anti-
body against SLC1A5.130 Due to the lack of potent inhibitors,
clinical trials or pharmacological experiments in animals
have not been reported. SLC1A5 knockout mice are viable
and fertile,131 suggesting that blocking of ASCT2 in tumor
cells is unlikely to affect differentiated cells. Immune cell
populations were found to be normal even when proliferating
intensively.131
SLC7A5 is the major transporter for many essential
amino acids in cancer cells.11,122,132 Like SLC1A5, it is an
amino acid exchanger, which can mediate the uptake of
essential amino acids (BCAAs, aromatic amino acids) at the
expense of nonessential amino acids. Moreover, it is over-
expressed in many cancer cell lines,11,12,122,133 and high
expression is often correlated with poor clinical progno-
sis.134 Genetic deletion of LAT1 in human colon adenocar-
cinoma cells significantly reduced cell proliferation, the
formation of tumor spheroids, and the volume of tumor
xenografts.135 Similar to the deletion of SLC1A5, deletion
of SLC7A5 induced an amino acid stress response, as evi-
denced by phosphorylation of eIF2α and GCN2 and tran-
scription of ATF4. This has also been observed in other
cancer cell lines.136 It is well established that SLC7A5
forms a covalent heteromeric complex with CD98, which is
required for trafficking to the plasma membrane,137,138 but is
not involved in the transport process itself.139 However,
genetic deletion of CD98 in human colon adenocarcinoma
312 SLAS Discovery 23(4)
cells had a much milder phenotype than deletion of
SLC7A5, although transport activity was reduced by
90%.135 Blocking the residual transport activity with the
SLC7A5 inhibitor JPH203 completely abolished growth of
the CD98 knockout cells. Reduced growth was accompa-
nied by reduced mTORC1 activity. Inhibitors for SLC7A5
have been developed and reduce tumor growth in vivo12,140
or in vitro.141–143 No human clinical trials have been
reported. A potential concern is the role of SLC7A5 as the
major transporter for essential amino acids in the blood–
brain barrier.144 Global knockout of SLC7A5 and CD98 in
mice has been reported as being embryonically lethal.145,146
SLC7A11 encodes a glutamate/cystine exchanger, which
plays an important role in providing cysteine for the synthe-
sis of glutathione in a variety of cell types.147 Triple-negative
breast cancer cell lines that were highly glutamine depen-
dent also showed elevated expression of SLC7A11, cystine
uptake, and glutamate secretion. SLC7A11 expression is
responsible for the methionine dependency of cancer cell
growth observed in some breast cancer cell lines.148 In these
cells, cystine uptake reduces the use of methionine as a pre-
cursor for cysteine. SLC7A11 is highly regulated in cancer
cells, being downregulated by oncogenic PI3K148 and also
by overexpression of mutated p53.149 Downregulation of
SLC7A11 renders cells more susceptible to cell death
caused by reactive oxygen species, such as ferroptosis.149
The anti-inflammatory pro-drug sulfasalazine, an inhibitor
of SLC7A11, reduced tumor size in xenografts.118 While
sulfasalazine is not used as a drug to treat cancer due to its
chemical instability, it provides proof of concept that target-
ing xCT could be a therapeutic strategy in certain subgroups
of cancer, particularly in combination with anticancer drugs
that increase oxidative stress.150
SLC6A14 is a Na+- and Cl–-dependent amino acid trans-
porter, which accepts all amino acids, except aspartate and glu-
tamate. The transporter is expressed in selected tissues, such as
lung, trachea, salivary gland, pituitary, distal ileum, and colon.
Due to its tissue-specific expression, it is not generally upregu-
lated in cancer but is found in colon cancer,151 cervical can-
cer,152 estrogen receptor–positive breast cancer,153 and
pancreatic cancer.120 The tumor-promoting role of SLC6A14
has been documented in estrogen receptor–positive breast can-
cer using α-methyl-tryptophan, a small-molecule blocker of
the transporter.154 SLC6A14 knockout mice, when crossed
with the polyoma middle T oncoprotein mouse breast cancer
model, showed delays in the development of mammary
tumors.155 More advanced inhibitors are required to evaluate
this target in cancer treatment.
SLC43A1 is a Na+-independent transporter for large neu-
tral amino acids.156 In contrast to SLC7A5, which operates as
an amino acid exchanger, SLC43A1 mediates facilitative dif-
fusion of its substrates.157 The gene is overexpressed in pros-
tate cancer, together with SLC7A5. Silencing of SLC43A1
reduced mTORC1 signaling and cell growth in LNCaP cells,
but less so in PC-3 cells, which appear to rely more on
SLC7A5 activity.136 In prostate cancer cells, SLC7A5 and
SLC43A1 were regulated in a coordinated fashion. High
expression of SLC43A1 is accompanied by low expression
of SLC7A5 and vice versa. The expression of SLC43A1 is
regulated by androgen receptor binding, while SLC7A5 is
regulated by an amino acid response element that increases
transcription after amino acid depletion.136
SLC38A1 and SLC38A2 are Na+-dependent transporters
for small and medium-sized neutral amino acids. When suf-
ficient amino acids are present, SLC38A2 is not found at
the plasma membrane, but its expression increases mani-
fold after amino acid depletion.121,158 The expression is
tightly regulated at the transcriptional level,159 mRNA
level,160 and protein level.161,162 Importantly, SLC38A1 and
SLC38A2 are involved in providing glutamine for glutami-
nolysis.121 The pharmacology of SLC38A1 and A2 is ill-
developed. The proline analogue N-methylamino-isobutyric
acid is frequently used as an SLC38A1/2 inhibitor and
reduces growth of osteosarcoma cells.121 Silencing of
SLC38A1 also reduced the growth of colon cancer cell lines
and pancreas cancer cell lines.163,164
These results suggest that amino acid transporters have a
significant potential in cancer therapy, but the field lacks,
with a few exceptions, highly specific inhibitors that could
be used in animal models and clinical trials.
Role of Amino Acid Transporters in
Inflammation and Immune Disorders
Arginine is the precursor for the generation of nitric oxide.
The inducible cationic amino acid transporter 2 (SLC7A2,
splice form CAT2B) provides arginine for nitric oxide syn-
thase, particularly in macrophages.165 Nitric oxide is thought
to contribute to the pathology of inflammatory bowel dis-
ease and Crohn’s disease. Provision of arginine has shown
beneficial effects in animal models of colonic inflamma-
tion.166 The beneficial effects were lost in mice lacking
SLC7A2.167 Consistently, SLC7A2 knockout mice develop
chronic inflammation in the lungs, potentially because of
reduced NO production in macrophages.168
SLC7A5 is the major transporter for BCAAs in activated
lymphocytes.145 A global knockout of SLC7A5 is embry-
onically lethal, but a CD4-driven cre-lox SLC7A5 knock-
out results in normal animal and normal immune cell
number in unchallenged animals.145 However, immunologi-
cal activation of T cells was largely abolished in SLC7A5
knockout cells. Deletion of SLC7A5 not only prevented
activation of mTORC1, but also interfered with metabolic
reprogramming, such as upregulation of glucose and gluta-
mine transport.145
SLC1A5 is an amino acid exchanger that is used to har-
monize amino acid levels within rapidly proliferating cells.
As a result, it is highly expressed in cancer cells, but also in
Bröer 313
Development of
transcriptional/
translational/
allosteric Improved
Mutaon activator amino acid
associated balance
with under
disease metabolic
phenotype
stress
condions
Figure 5.  Future strategies to modulate metabolic diseases. Lack of transporter activity is associated with metabolic diseases.
Enhancing the activity of the transporter protects the cell against metabolic stress.
rapidly proliferating immune cell populations. Nakaya
et al.169 reported that SLC1A5 knockout animals had attenu-
ated inflammatory T-cell responses. Particularly, interferon-γ-
producing T-helper cells were significantly reduced.
Masle-Farquhar et al., by contrast, reported normal B- and
T-cell development and normal B-cell function, suggesting a
limited effect of this transporter on immune function.131
SLC16A10 is the major transporter for aromatic amino
acids in many cell types.170 The transporter mediates facili-
tated diffusion of aromatic amino acids and may therefore
serve as a route for uptake (e.g., in the liver, where metabo-
lism of aromatic amino acids occurs) or efflux (e.g., in epi-
thelial cells, where aromatic amino acids are released across
the basolateral membrane). NOD (nonobese diabetic) mice
are a widely used model of type 1 diabetes, an autoimmune
disorder, which results in the destruction of β cells by lym-
phocytes infiltrating the islets of the pancreas.171 NOD mice
spontaneously develop type 1 diabetes at an elevated rate,
with an onset at an age of more than 100 days. Transposon-
mediated inactivation of SLC16A10 increased the suscepti-
bility to T1D in NOD mice.172 The transporter is highly
expressed in macrophages, which play an important role in
antigen presentation and the release of pro-inflammatory
cytokines. The mechanism by which inactivation of
SLC16A10 increases the incidence of T1D remains to be
elucidated.
Role of Amino Acid Transporters in
Embryo Development and Stem Cells
In vitro fertilization is an important process in research (mouse
embryos), animal husbandry, and human reproduction.
Significant efforts have been made to optimizie media for
embryo development in vitro. While embryos develop at phys-
iological osmolarity in vivo, in vitro development beyond the
two-cell stage requires reduced osmolarity. The two-cell block
can, however, be overcome in normal osmolarity by the addi-
tion of osmolytes, such as glycine, betaine, proline, β-alanine,
and hypotaurine,19 which are accumulated by amino acid
Robust
amino acid Disease
homeostasis prevention
transporters. Amino acid transporters are highly regulated dur-
ing early embryo development.16,173 For instance, GLYT1 is
essential for the regulation of oocyte size and is activated as the
oocyte undergoes meiosis,19 transporting the osmolyte glycine.
The osmolyte betaine is transported by SLC6A20, which is
upregulated in one- and two-cell embryos.20 Cationic amino
acids are recruited through SLC7A9, while neutral amino
acids are taken up by SLC7A5.174 The sodium neutral amino
acid transporter SLC38A2 is expressed in pluripotent stem
cells, and its activity is required for cell differentiation by accu-
mulating l-proline.175 Competitive inhibition of SLC38A2 by
high concentrations of glycine and alanine in the oviductal
fluid may prevent development before implantation in the
uterus.16 Future research in this area is projected to focus on
modulation of epigenetic signatures,176 which could be
achieved by media supplements, but also by activation or inhi-
bition of amino acid transporters. Embryonic stem cells, for
instance, acquire epigenetic changes associated with the for-
mation of early primitive ectoderm-like cells, which are
induced by l-proline.175,177
Amino acid supplementation and modulation of amino
acid transport could be used to optimize the growth and dif-
ferentiation of stem cells and to optimize conditions for in
vitro fertilized oocytes.
Conclusion and Future Outlook
Amino acid transporters are often found at the beginning of
metabolic pathways and influence a wide variety of biological
processes, in particular, cell proliferation, but also cell signal-
ing and nutrient homeostasis. Thus far, very few amino acid
transport inhibitors have found their way into clinical trials.
However, this is largely the result of the limited development
of high-affinity and selective inhibitors. With improvements of
tools to express membrane transporters and HTS being increas-
ingly available in academic environments, this is likely to
change in the near future. Solute carriers are considered an
underdeveloped area of drug development.178 In addition to
current approaches presented in this review, several strategies
314 SLAS Discovery 23(4)

Stress Response
(alternative
transcripts) Restored Growth
amino acid Migration
homeostasis Metastasis
Block of a
single
amino acid
transporter Amino acid
Well- imbalance
nourished Nutrient
tumor cell stress Figure 6.  Future strategies
combining two stress factors to
reduce cell growth. A significant
problem using amino acid
Inhibitors of
Breakdown transporters as drug targets is
Stress response Stagnation
redundancy. Blocking the adaptive
Pathways of cellular Apoptosis
homeostasis stress response or inducing an
Stress inducers independent stress can overcome
metabolic adaptation.

are emerging as new opportunities for targeting amino acid Funding

transport. One such strategy is augmentation (Fig. 5). Whole-
genome sequencing is increasingly used to identify mutations,
including those affecting amino acid transporters associated
with disease phenotypes. These mutations are most likely inac-
tivating transporters, and as a result, transport activators rather
than inhibitors will be required. For instance, mutation in
the putative monocarboxylate transporter Slc16a11 was
identified as being associated with the development of type
2 diabetes in Mexican populations.179 This provides evi-
dence that an activator of Slc16a11 could be used to treat or
prevent type 2 diabetes.180 Transport activators can be iden-
tified at three levels, namely, transcriptional, translational,
and allosteric. Transcriptional and translational activators
have been successfully identified for amino acid transporters,
as outlined above.108,109 Another strategy is combination (Fig.
6). Amino acid transport is a tempting target for tumor chemo-
therapy, but a better understanding of amino acid transporter
redundancy and plasticity will be required. Tumor cells often
escape inhibition of amino acid transporters through upregula-
tion of alternative transporters via the GCN2/ATF4 path-
way.121,136 This pathway reduces conventional Cap-dependent
translation and, at the same time, activates transcripts with
complex secondary structures.2 As outlined above, transla-
tional activators have been identified that appear to weaken
complex secondary structures occurring in mRNA mole-
cules.109 It appears feasible to identify compounds that stabilize
secondary structures, thereby inhibiting translation. Combining
amino acid transporter inhibition with inhibition of rescue
mechanisms could open new chemotherapeutic approaches.
Declaration of Conflicting Interests
The author declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
The author disclosed receipt of the following financial support for
the research, authorship, and/or publication of this article:
Research in the laboratory of the author is supported by grants of
the National Health and Medical Research Council ID 1128442
and 1105857 and the Australian Research Council DP180101702.
ORCID iD
Stefan Bröer https://orcid.org/0000-0002-8040-1634
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