LAT1 activity of carboxylic acid bioisosteres: Evaluation of hydroxamic acids as substrates

Article abstract of DOI:10.1016/j.bmcl.2016.09.001

Large neutral amino acid transporter 1 (LAT1) is a solute carrier protein located primarily in the blood–brain barrier (BBB) that offers the potential to deliver drugs to the brain. It is also up-regulated in cancer cells, as part of a tumor's increased metabolic demands. Previously, amino acid prodrugs have been shown to be transported by LAT1. Carboxylic acid bioisosteres may afford prodrugs with an altered physicochemical and pharmacokinetic profile than those derived from natural amino acids, allowing for higher brain or tumor levels of drug and/or lower toxicity. The effect of replacing phenylalanine's carboxylic acid with a tetrazole, acylsulfonamide and hydroxamic acid (HA) bioisostere was examined. Compounds were tested for their ability to be LAT1 substrates using both cis-inhibition and trans-stimulation cell assays. As HA-Phe demonstrated weak substrate activity, its structure–activity relationship (SAR) was further explored by synthesis and testing of HA derivatives of other LAT1 amino acid substrates (i.e., Tyr, Leu, Ile, and Met). The potential for a false positive in the trans-stimulation assay caused by parent amino acid was evaluated by conducting compound stability experiments for both HA-Leu and the corresponding methyl ester derivative. We concluded that HA's are transported by LAT1. In addition, our results lend support to a recent account that amino acid esters are LAT1 substrates, and that hydrogen bonding may be as important as charge for interaction with the transporter binding site.

Full text of DOI:10.1016/j.bmcl.2016.09.001

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
LAT1 activity of carboxylic acid bioisosteres: Evaluation
of hydroxamic acids as substrates
Arik A. Zur a, , Huan-Chieh Chien , Evan Augustyn , Andrew Flint , Nathan Heeren , Karissa Finke ,
⇑
a
b
b
b
b
b
b
b
a
a
Christopher Hernandez , Logan Hansen , Sydney Miller , Lawrence Lin , Kathleen M. Giacomini ,
Claire Colas , Avner Schlessinger , Allen A. Thomas
Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy and Medicine, University of California San Francisco, San Francisco, CA 94158, United States
Department of Chemistry, University of Nebraska Kearney, Kearney, NE 68849, United States
Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States
Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States
c,d
c,d
b,
⇑
a
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Large neutral amino acid transporter 1 (LAT1) is a solute carrier protein located primarily in the blood–
brain barrier (BBB) that offers the potential to deliver drugs to the brain. It is also up-regulated in cancer
cells, as part of a tumor’s increased metabolic demands. Previously, amino acid prodrugs have been
shown to be transported by LAT1. Carboxylic acid bioisosteres may afford prodrugs with an altered
physicochemical and pharmacokinetic profile than those derived from natural amino acids, allowing
for higher brain or tumor levels of drug and/or lower toxicity. The effect of replacing phenylalanine’s car-
boxylic acid with a tetrazole, acylsulfonamide and hydroxamic acid (HA) bioisostere was examined.
Compounds were tested for their ability to be LAT1 substrates using both cis-inhibition and trans-stim-
ulation cell assays. As HA-Phe demonstrated weak substrate activity, its structure–activity relationship
(SAR) was further explored by synthesis and testing of HA derivatives of other LAT1 amino acid substrates
(i.e., Tyr, Leu, Ile, and Met). The potential for a false positive in the trans-stimulation assay caused by par-
ent amino acid was evaluated by conducting compound stability experiments for both HA-Leu and the
corresponding methyl ester derivative. We concluded that HA’s are transported by LAT1. In addition,
our results lend support to a recent account that amino acid esters are LAT1 substrates, and that hydrogen
bonding may be as important as charge for interaction with the transporter binding site.
Received 1 June 2016
Revised 27 August 2016
Accepted 1 September 2016
Available online xxxx
Keywords:
SLC7A5
Amino acid
Acyl sulfonamide
Tetrazole
Transporter substrate
Transporter inhibitor
Ó 2016 Elsevier Ltd. All rights reserved.
Large neutral amino acids such as tyrosine, tryptophan, leucine,
isoleucine, phenylalanine, and methionine are actively transported
across cell membranes by LAT1.1 Additionally, it transports
be inhibited by blocking LAT119–23 which is consistent with a can-
cer cell’s increased nutritional requirements. Thus, drugs that are
–6
able to mimic naturally-occurring amino acids (e.g., gabapentin)
7,8
9
24–28
amino acid-containing drugs such as gabapentin,
melphalan,
or prodrugs containing LAT1 recognition elements
may have
1
0,11
12
L-DOPA
and baclofen across the blood–brain barrier (BBB).
far-reaching utility for treating CNS diseases and cancer.
LAT1 is a sodium-independent heterodimeric membrane protein
found mainly in the brain, thymus, testis, placenta, spleen, and
skeletal muscle. Much of its appeal as a targeted drug delivery
mechanism is due to its relative high abundance at the BBB versus
Another advantage favoring LAT1 for drug delivery is that it is
relatively tolerant to substrate structural modifications.4
,29
For
example, it has been shown that in addition to hydrophobic natu-
ral
a
-amino acids, some b and
c
amino acids (e.g., gabapentin) are
2,13
8,30–32
other tissues (>100Â BBB selective).
Besides being an instru-
also transported by LAT1.
Despite some flexibility in the pre-
ment for CNS delivery, it has also been shown that LAT1 is up-reg-
sentation of the amine and carboxylic acid functional groups, it has
14
15
18
ulated in many cancer types, including prostate, esophageal,
been maintained that both of these functional groups are essential
1
6
17
4,24,33
colorectal, gastric, and non-small-cell lung cancer (NSCLC).
for transporter recognition.
The primary evidence supporting
Furthermore, it has been demonstrated that cancer growth can
a carboxylic acid requirement has been centered on observations
that replacement with esters and a closely related sulfonic acid
4
,24,29
resulted in loss of activity.
carboxylic acid bioisosteres
explored. Lately, this story has become further convoluted as
However, many of the traditional
⇑
Corresponding authors.
E-mail addresses: riko.zur@gmail.com (A.A. Zur), thomasaa@unk.edu (A.A.
34,35
have apparently not been
Thomas).
http://dx.doi.org/10.1016/j.bmcl.2016.09.001
0
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zur, A. A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.09.001

2
A. A. Zur et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Nagamori et al. reported that several carboxylic esters and a
LAT1 transport inhibitors, which may be potential substrates.
However, to more directly identify substrates, we performed a
3
3
2
6
hydroxamic acid derivative of
L-leucine were LAT1 substrates.
36
Their conclusions, based in part on a trans-stimulation assay,
trans-stimulation experiment which exploits LAT1’s alternating
4
,24,37
8,53
contradicted previous reports that esters do not bind LAT1.
Our group had been exploring carboxylic acid bioisosteres as
access mechanism
by loading cells with a radiolabeled sub-
strate followed by incubation with extracellular test compound.
The exchange efflux rate of the radiolabel in the presence of a test
compound is compared with the efflux rate in the absence of the
test compound. Compounds that are LAT1 substrates should
increase the efflux rate of the radiolabeled amino acid compared
with its efflux rate in the absence of test compound. We selected
3
8,39
LAT1 substrates, including hydroxamic acids
as part of our
40
ongoing effort to better understand LAT1 SAR prior to the recent
publication by Nagamori.32 We have been focused on modifying
the carboxylic acid rather than the amine due to the potential
metabolism and toxicity41 liabilities of the former. Besides con-
tributing to the knowledge of LAT1 SAR, replacing the carboxylic
acid functional group has the potential for altering the pharma-
3
[ H]-gabapentin as a probe substrate due to its selectivity for
8
LAT1 relative to other membrane transporters.
3
4,35
cokinetics of prodrugs
intended for LAT1 transport. Moreover,
Acyl sulfonamides 10a–b were prepared from protected amino
acids according to methodology described by Drummond.54 Tetra-
zole bioisostere 11 was synthesized from the primary amide of
Cbz-protected Phe in 3 steps using a previously published route,55
and our resulting NMR characterization was consistent with what
had previously been reported. After discovering a lack of activity
for tetrazole 11, we choose not to pursue additional amino acid
analogs. Hydroxamic acids (HA’s) were synthesized using two dif-
ferent routes. Aromatic analogs (i.e., 12a–12d) were prepared
using methodology previously described by Ahlford and Adolfs-
learning what functional groups may serve as surrogates for the
amino acid carboxyl opens up many possibilities for the design
of drugs that might benefit from this delivery mechanism.
Though numerous carboxylic acid bioisosteres have been
3
4,35,42,43
described,
tetrazole, and hydroxamic acid functionalities (Table 1). These
groups were selected due to their comparable pK and/or structural
we choose to prioritize the acylsulfonamide,
a
34
similarity to the carboxylic acid functional group, and they have
4
4–46
previously demonstrated biological activity in other series.
The acylsulfonamide (or sulfonimide) has a similar geometry and
pK as a carboxylic acid, and it was successfully applied as a cys-
5
6
son. Due to problems with over-reduction of hydroxamic acids
to give primary amide (e.g., 13e) during hydrogenolysis of benzyl
protected hydroxamic acid, we used a different route to prepare
HA’s of aliphatic amino acids (Scheme 1).
a
teinyl leukotriene (LTE4) receptor antagonist that demonstrated
greater activity than the parent carboxylic acid.47 Acylsulfon-
amides were also chosen because they are convenient to synthe-
size from the corresponding carboxylic acid. Though slightly
larger than a carboxylic acid,48 tetrazoles faithfully reproduce their
Hydroxamic acids of aliphatic amino acids Leu, Ile and Met
(12e–g) were synthesized according to Scheme 1. Nucleophilic acyl
substitution with hydroxylamine on methyl esters 9 gave low
yields, but avoided having to use an amine protecting group for
Leu and Ile analogs. However, reaction of 9g with hydroxylamine
to form HA-Met gave a complex mixture that could not be purified
by recrystallization. We found that Boc protected 9g gave a cleaner
conversion to HA-Met, albeit the recrystallized yield was still rela-
tively poor (20% for steps d–e). Since our objective was to obtain
HA’s of high purity with negligible levels of parent amino acids
to avert a false positive result in our cell assay, we were generally
unconcerned about isolated yield and the potential losses resulting
a
trigonal planar shape and acidity (pK : 4.5–4.9), as the tetrazole
anion is stabilized by delocalization. The tetrazole group, which
is present in the orally active angiotensin II receptor antagonist
4
6
Losartan, has the potential to improve oral bioavailability of
resulting prodrugs relative to parent carboxylic acid. Hydroxamic
a
acids (pK : 8–9) are known primarily for their metal-chelating abil-
ities; and though dramatically less acidic than the previous two
bioisosteres, they have been reported as MAP/ERK kinase inhibitors
where they displayed similar ADME properties to carboxylic
4
4,49
acids.
However, use of hydroxamic acids could be limited by
from multiple recrystallization steps. Moreover, potentially better
50
57–60
hydrolysis to parent carboxylic acid in vivo.
methods
for preparing HA’s were not pursued, as the current
Compounds were evaluated in both cis-inhibition and trans-
routes provided satisfactory amounts of material for testing in a
relatively short time period. In contrast, the yield for substitution
with ammonia to generate leucinamide 13e was significantly
stimulation assays using HEK cells engineered to overexpress
5
1,52
human LAT1.
cis-Inhibition studies were used to identify
Table 1
3
Exchange efflux rate and uptake inhibition of [ H]-gabapentin in HEK-hLAT1 cells for carboxylic acid bioisosteres and their parent amino acids tested at 200
l
M
R
A
NH
2
Compounda
A
R
Efflux rateb
% inhibitionc
pKa1, pKa2d
1
1
1
1
a (Phe)
b (Tyr)
h (Gly)
i (Arg)
–CO
–CO
–CO
–CO
2
2
2
2
H
H
H
H
PhCH
p-HOPhCH
H
2
–
3.6 ± 0.7
2.6 ± 0.4
0.78 ± 0.2
0.75 ± 0.1
85 ± 0.6
68 ± 0.5
33 ± 3
1.8, 9.1
2
–
—
—
—
NH
PhCH
p-HOPhCH
2
(NH)CNH(CH
2
)
3
–
49 ± 2
1
0a
0b
–CONHSO
–CONHSO
2
Me
Me
2
–
0.61 ± 0.06
0.63 ± 0.03
15 ± 0.7
12 ± 0.4
1.8, 8.4
—
1
2
2
–
N
N
N
1
1
PhCH
2
–
–
0.61 ± 0.01
11 ± 1
2.5, 7.8
N
H
1
1
2a (HA-Phe)
2b (HA-Tyr)
–CONHOH
–CONHOH
PhCH
2
1.3 ± 0.1
1.0 ± 0.1
24 ± 0.5
39 ± 30
6.9, 8.0
—
p-HOPhCH
2
–
a
b
c
Cell assay data was obtained at least in triplicate. Amino acids and their corresponding derivatives possess S stereochemistry at the
a
-carbon.
M for their ability to inhibit uptake of [ H]-gabapentin into HEK-hLAT1 cells. Data is presented as % inhibition relative to background
3
Compounds were tested at 200
Compounds were tested at 200
l
l
M for their ability to cause efflux (fmol/min) of [ H]-gabapentin from pre-loaded HEK-hLAT1 cells.
3
signal in the absence of a test compound.
d
62
values for 10a, 11, and 12a were measured at Analiza using a capillary electrophoresis technique.61
a a
pK values for Phe 1a were taken from Berg, and the pK
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A. A. Zur et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
O
R
NH
-HCl
3e: Leucinamide
2
NH
2
c
1
R
e: (CH
f: (S)-CH
g: CH
2 2
S(CH ) -
3
)
2
CHCH
2
-
O
O
d (and e for
O
3
CH (CH )CH-
2
3
R
a
R
12g only)
R
3
OH
OMe
NHOH
2
-HCl
NH
2
NH
2
-HCl
NH
12e:
1
e-g (Leu, Ile, Met)
9e-f
HA-Leu
12f: HA-Ile
2g: HA-Met
9g
b
1
Boc-9g
Scheme 1. Synthesis of compounds 13e and 12e–12g. Reagents and conditions: (a) SOCl
MeOH, 50 °C, sealed tube, 13e: 60%; (d) 50% NH
2
, MeOH, 9e: 56%, 9f: 86%, 9g: 67%; (b) Boc
OH in water, MeOH or 1,4-dioxane, 12e: 23%, 12f: 3%, 12g: 20% (2 steps); (e) 4 N HCl in 1,4-dioxane. 13e and 12e–g were
2
O, DCM, Boc-9g: 85%; (c) 7 N NH
3
in
2
purified by conversion to their HCl salts and recrystallization to >99% purity by HPLC.
better (60% recrystallized yield) than for the corresponding HA
analogs. Generally HA’s demonstrated poor solubility in both water
and organic solvents; however, we found that solubility was dra-
matically improved by conversion to the hydrochloride salt.
Of these three bioisosteres, only the hydroxamic acid 12a had
significant activity in our trans-stimulation assay relative to non-
substrates Gly and Arg (Table 1). We were surprised by this result.
We had expected the tetrazole 11 and acylsulfonamides 10a to
have been better surrogates for the acidic carboxylic acid than
to be a LAT1 substrate,32 to our knowledge this was the first time
that 13e or any amino acid primary amide has been tested for
LAT1 activity. Interestingly, 13e did not demonstrate significant
substrate activity (efflux rate: 0.69 fmol/min). This result also indi-
cated that 13e was sufficiently stable to the assay conditions so as
not to generate adequate parent Leu 1e to cause trans-stimulation.
Furthermore, it is apparent that the hydroxamic acid ‘–OH’ group
plays an important role in the observed LAT1 activity; whether that
be due to its effect on acidity, hydrogen bonding, or some other
factor is currently unclear.
1
(
2a given that the measured pKa1 values61 for the former
pKa1 = 2.5 and 1.8, respectively) were much closer to that of par-
A different story unfolded for ester 9e. In a trans-stimulation
3
2
ent amino acid Phe 1a (pKa1 = 1.8) than HA-Phe 12a (pKa1 = 6.9)
was. It is worth noting that our pKa1 values were considerably
lower than those reported for these bioisosteres when they were
present as isolated functional groups,34 which demonstrates as
experiment performed by Nagamori, both ester 9e and its parent
Leu appeared to have almost identical activity. In our hands, 9e
exhibited significantly less activity than parent Leu in both trans-
stimulation (efflux rate: 2.1 vs 3.2 fmol/min) and cis-inhibition
might be expected that the
a-amino group depresses their pK
a
as
assays (IC50: >200 lM vs 87 lM). One possible explanation for this
it would for an adjacent carboxylic acid.
disparity may be due to the fact that the cells used by Nagamori were
different from the cells we used. Nagamori et al. used non-trans-
fected HeLa S3 cells (a cervical cancer cell line) whereas, we used
To determine whether HA’s of other LAT1 amino acid substrates
e.g., Leu, Ile, Met) were LAT1 ligands, compounds of Table 2 were
(
5
2
prepared and tested. All of the HA’s, with the exception of 12h, had
diminished activity in both our trans-stimulation and cis-inhibition
assays relative to the parent amino acids. Based on their % inhibi-
HEK-hLAT1 cells that demonstrated 8-fold higher uptake of
3
[ H]-gabapentin relative to the control cell line HEK-EV (Supple-
mentary material). LAT1 expression may have been higher in our
transfected cells and the contribution of other transporters could
differ between the two cell lines. We selected HEK cells due to their
3
tion of [ H]-gabapentin cell uptake or IC50 values, it is clear that
HA’s are weaker ligands of LAT1 than the parent amino acids.
And none of the HA’s had IC50 values below 200
6
5
l
M in our assay.
having relatively low levels of transporters, so we would expect
the observed activity in our assays to be due solely to LAT1. Our
IC50 value for 9e was consistent with earlier SAR presented by
Conversely, all of the HA’s demonstrated greater efflux rates of
3
[
H]-gabapentin from pre-loaded HEK-hLAT1 cells than did the
4
negative controls Arg and Gly. The notable exception to this trend
was HA-Gly 12h, which we did not expect to be a LAT1 substrate
by analogy to its non-substrate, parent amino acid Gly. Of the
HA’s tested, 12a and 12e–g (HA’s of Phe, Leu, Ile, Met) demon-
strated significant activity relative to the negative controls. The lar-
ger efflux rates measured for HA-Leu 12e and HA-Ile 12f relative to
HA-Phe 12a (1.5 vs 1.3 fmol/min) were juxtaposed with the activ-
ity of the parent amino acids, in which Phe 1a demonstrated a
superior efflux rate (3.6 fmol/min). It has previously been shown
Uchino that the methyl ester of phenylalanine poorly inhibited
1
4
uptake of L-[ C]-Phe into oocytes expressing LAT1. But the ostensi-
ble interpretation of the results from our trans-stimulation assay is
the same as Nagamori’s—that methyl ester 9e does appear to be a
LAT1 substrate, despite lacking an acidic carboxylic acid functional
group.
Because of our concerns and those raised by others about the
potential for a false positive result in LAT1 cell assays,37 we evalu-
ated how much parent Leu 1e would need to be present as an impu-
rity in test compounds (i.e., 9e, 12e, or 13e), either from the
synthesis or formed under the conditions of the cell assay, to result
in a significant efflux rate (>1 fmol/min) in our trans-stimulation
assay. We tested the efflux rate at concentrations ranging from
that both Leu and Ile have slightly greater LAT1 transport capacity
3,64
(V
max) values than Phe.6
Since the trans-stimulation assay relies
3
upon the kinetics of exchange between intracellular [ H]-gabapen-
tin and extracellular test compound, it is conceivable that a similar
trend for Vmax applies to the HA’s as it does to the parent amino
acids. However, considering the bounce in our assay relative to
the observed efflux rates, we cannot confidently distinguish the
4 lM up to 200 lM covering a range of Leu 1e impurity from 2%
to 100%, respectively (in relation to previous studies). The back-
ground efflux rate (0.7 ± 0.05 fmol/min) was subtracted from total
3
substrate activity of the HA’s from each other.
efflux and the net contribution to [ H]-gabapentin efflux rate is
Given the disparity in the literature4
,32,37
as to whether esters
depicted in Figure 1. The effect of increasing concentrations of Leu
are LAT1 substrates, we also tested the Leu methyl ester 9e, which
was an intermediate in the preparation of HA-Leu 12e (Scheme 1).
And to further probe the SAR for close-in derivatives of the HA’s
1e on the net LAT1 efflux rate was fitted to the Michaelis–Menten
equation (K
The K and Vmax were similar to previously reported values.
A Leu 1e concentration of 4 M did not increase the LAT1
exchange rate significantly in comparison to background signal
m
of 36.8 ± 9.8 lM and Vmax of 1.99 ± 0.19 fmol/min).
64
m
(Table 2), we also decided to test the structurally-related primary
l
amide Leucinamide 13e. Though ester 9e was recently reported
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4
A. A. Zur et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 2
3
Exchange efflux rate and uptake inhibition of [ H]-gabapentin in HEK-hLAT1 cells for hydroxamic acids, related carboxylic acid derivatives, and their parent amino acids
O
R
X
NH
2
Compounda
X
R
Efflux rateb
% inhibitionc
IC50 (l
M)d
1
1
a (Phe)
b (Tyr)
–OH
PhCH
p-HOPhCH –
2
2
–
3.6 ± 0.7
2.6 ± 0.4
85 ± 0.6
68 ± 0.5
—
—
1
d (Trp)
1.6 ± 0.3
79 ± 0.6
—
N
H
1
1
1
1
1
e (Leu)
f (Ile)
g (Met)
h (Gly)
i (Arg)
(CH
(S)-CH
3
)
2
CHCH
CH (CH
S(CH
2
–
3.2 ± 0.5
2.5 ± 0.1
2.4 ± 0.1
0.78 ± 0.2
0.75 ± 0.1
73 ± 0.7
—
—
33 ± 3
49 ± 2
87 ± 10
150 ± 40
180 ± 3
>200
3
2
3
)CH–
CH
H
3
2
)
2
–
NH
2
(NH)CNH(CH
)
2 3
–
—
1
1
2a (HA-Phe)
2b (HA-Tyr)
–NHOH
PhCH
p-HOPhCH –
2
2
–
1.3 ± 0.1
1.0 ± 0.1
24 ± 0.5
39 ± 30
—
—
1
2d (HA-Trp)
1.0 ± 0.1
35 ± 1
—
N
H
1
1
1
1
2e (HA-Leu)
2f (HA-Ile)
2g (HA-Met)
2h (HA-Gly)
(CH
(S)-CH
3
)
2
CHCH
CH (CH
S(CH
2
–
1.5 ± 0.1
1.5 ± 0.2
1.1 ± 0.01
0.70 ± 0.1
50 ± 1
—
—
>200
>200
>200
>200
3
2
3
)CH–
CH
H
3
2
)
2
–
38 ± 1
9
1
e (Leu ester)
3e (Leucinamide)
–OMe
–NH
(CH
3 2
) CHCH
2
–
2.1 ± 0.2
0.69 ± 0.01
—
—
>200
>200
2
a
b
c
Cell assay data was obtained at least in triplicate. Amino acids and their corresponding derivatives possess S stereochemistry at the
a
-carbon.
3
Compounds were tested at 200
Compounds were tested at 200
l
M for their ability to cause efflux (fmol/min) of [ H]-gabapentin from pre-loaded HEK-hLAT1 cells.
3
lM for their ability to inhibit uptake of [ H]-gabapentin into HEK-hLAT1 cells. Data is presented as % inhibition relative to background
signal in the absence of a test compound.
d
3
For IC50 determinations, varying concentrations of each compound were added, from 0.1 lM to 500 lM. %[ H]-gabapentin uptake at each concentration was normalized
6
6,67
relative to % inhibition by BCH
at 2 mM, which was set to 100% inhibition.
Compelled by the implications of Figure 1, we carefully scruti-
nized the purity of all of our HA’s by NMR and HPLC (Supplemen-
tary material), in particular checking for the presence of residual
parent amino acid. We recrystallized all of the HA’s at least once
to improve purity, and the amount of parent amino acid detected
by HPLC was less than 0.5%, and in most cases it was below our
limit of detection. Thus, we conclude that for the HA’s of Table 2
substrate activity was not due to parent amino acid carried over
from the synthesis.
In addition to being vigilant about purity, we also performed a
series of simple stability experiments. Thus, we exposed
1
3
C-labeled 9e and 12e (Fig. 2; synthesized using similar methods
as described for unlabeled 9e and 12e, above) to conditions to
mimic our cell assays, including incubation of compounds with
‘
buffer only’ or with ‘buffer and cells’ for various periods of time.
1
3
The resulting mixture was analyzed by C NMR (Supplementary
material).
Within the time period of our cell assay (ꢀ5 min), 13C-9e hydro-
3
13
Figure 1. Plot of net exchange efflux rate (fmol/min) of [ H]-gabapentin from pre-
lyzed to give 4% parent amino acid, whereas C-12e only gave a
marginal increase in parent C-1e relative to its initial amount
loaded HEK-hLAT1 cells versus Leu (1e) concentration (
calculated by subtracting the exchange rate without Leu (1e) (marked by a dotted
line) from the efflux rate at Leu (1e) concentrations of 4, 10, 32, 80 and 200 M.
Solid line represents a non-linear regression fit of the data to Michaelis–Menten
kinetics.
l
M). Net efflux rate was
13
(
0.8% vs 0.4%, respectively) that was likely within the variability
l
of NMR peak integration. To take the experiment further, we incu-
bated compounds with cells for an hour at 37 °C. This resulted in a
moderate increase in the amount of C-1e (12%) from ester C-9e,
but only a nominal amount (1%) arising from HA-Leu C-12e
hydrolysis. Even after incubation of HA-Leu C-12e with cells for
5 h at 37 °C, only 3% C-1e was observed in the C NMR spectrum.
1
3
13
13
(
p value = 0.146), and higher concentrations were required to
3
13
facilitate the exchange of [ H]-gabapentin. In fact, more than 10%
of a parent Leu 1e impurity would be required to fully account for
HA-Leu 12e’s LAT1 activation (1.5 ± 0.1 fmol/min). Nevertheless,
contamination of test compounds with parent amino acid may
result in increased efflux ratio, and this should be examined care-
fully when performing trans-stimulation assays.
1
3
13
1
3
Due to C NMR analysis requirements, these stability experi-
ments had to be performed with 1 mg of compound/well, which
was ꢀ25–50Â more compound than typically used in our cell
assays, done at 200 lM concentration. Consequently, we can’t rule
Please cite this article in press as: Zur, A. A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.09.001

A. A. Zur et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
O
O
O
13C
13C NHOH
13C OH
OR
choline buffer (pH 7.4) only
or plus HEK-hLAT1 cells
OMe
NH
2
NH
2
NH
2
13C-9e
13C-12e
13C-1e
Figure 2. Carbon-13 labeled leucine derivatives [13C]-9e (methyl ester) and [13C]-12e (HA-Leu) used in stability experiments to assess the amount of parent leucine that
could form under the conditions of the LAT1 cell assays.
Figure 3. Predicted binding mode of LAT1 with leucine and its analogs. Predicted pose of the known substrate leucine 1e is shown in cyan sticks, and the two leucine analogs
including (A) the recently discovered substrate HA-Leu 12e and (B) non-ligand Leucinamide 13e are illustrated with orange and green sticks, respectively. The hydrogen
bonds between LAT1 binding site residues and the leucine analogs are shown as dashed lines, including 1e (yellow), 12e (black), and 13e (black).
out the possibility of enzyme saturation at this higher concentra-
tion, and that either 9e and/or 12e might be undergoing intracellu-
lar enzymatic cleavage as the assay is normally conducted.
However, it was previously shown68 that leucinamide 13e is more
sensitive to enzymatic hydrolysis than the leucine methyl ester
backbone atoms of Ile63 and Gly67 in a manner similar to that of
LAT1 amino acid ligands such as leucine (Fig. 3A). We postulate
that maintaining these hydrogen bonds is important for activity,
and that this is the primary reason hydroxamic acids are LAT1 sub-
strates. Interestingly, docking of non-ligand leucinamide 13e
70
1
2e. So, if enzyme-catalyzed formation of parent Leu was problem-
(Fig. 3B) using two different docking programs (i.e., FRED and
7
1
atic, it seems likely that leucinamide would also have given a false
positive; yet, 13e lacked activity in our trans-stimulation assay.
Upon relating these stability results to Figure 1, it appears that
the concentration of parent Leu 1e within the timeframe of our cell
Glide SP; Supplementary material) does not rule out that 13e is
a LAT1 ligand. We therefore estimated the binding energies of
Leu (1e), Leu methyl ester (9e), HA-Leu (12e), and leucinamide
(13e) to LAT1 using Molecular Mechanism Generalized Born Sur-
face Area (MMGBSA) calculated by Prime (Schrödinger suite).72
assay from either methyl ester 9e or HA-Leu 12e (ꢀ8 and 2
respectively) does not explain our trans-stimulation assay results
2.1 and 1.5 fmol/min, respectively). For the observed activity to
be solely due to parent amino acid, Figure 1 suggests that Leu
would have to be present at ꢀ80 and ꢀ30 M concentrations for
e and 12e, respectively, and all within the 3 min of the assay.
lM,
Though the predicted
for 1e, 12e, and 13e, respectively, correlated with experimental
data, the predicted
bind for 9e (À52 kcal/mol) did not. As a large
component of the calculated binding energies was due to electro-
static interactions (i.e.,
D
Gbind values of À79, À64, and À58 kcal/mol
(
D
G
l
9
D
G
Coulomb of À63, À53 and À47 kcal/mol
Our stability experiments indicated that 610% of these levels were
for 1e, 12e and 13e, respectively), it is possible that our calcula-
tions are underestimating other interactions (e.g., dipole–dipole)
with LAT1 that exist for ester 9e which account for its observed
activity.
actually present, supporting the notion that the observed exchange
3
efflux of [ H]-gabapentin (Table 2) was mostly caused by the test
compounds themselves. Even so, it is probable that a small fraction
of the activity was due to Leu, particularly for the less stable
methyl ester 9e.
Based on our results and those recently reported by Nagamori,32
it appears that the previous view that LAT1 substrates must possess
an acidic functional group needs to be revised. As most of the
Though the atomic structure of the human LAT1 is not known,
we have developed a homology model based on a structure of a
related transporter, the arginine-agmatine transporter AdiC from
4
,37,73
earlier conclusions
were based on inhibition experiments
(e.g., cis-inhibition or rat brain perfusion) at fixed concentrations
of test compound, it is possible that those assays were not sensitive
enough to detect substrates such as esters with weaker
interactions with LAT1. The trans-stimulation assay may be more
sensitive to identify weak ligands, as it is based on the exchange
5
1,69
Escherichia coli.
This model has been recently refined using
4
0
newly characterized ligands and improved LAT1/AdiC alignment.
The LAT1 model helped rationalize the amino acid selectivity
among amino acid transporters, and virtual screening against this
model followed by experimental testing identified previously
unknown LAT1 ligands.51 Docking of 12e against our LAT1 model
suggests that hydroxamic acids establish hydrogen bonds with
3
of pre-loaded substrates (e.g., [ H]-gabapentin) only for test
40
compounds that employ LAT1 to cross a cell membrane, rather
than inhibition potency.
Please cite this article in press as: Zur, A. A.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.09.001

6
A. A. Zur et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Though we do not currently have an explanation for why tetra-
zoles and acylsulfonamides lacked activity, our data point toward
LAT1 binding being less sensitive to the pK of the carboxylic acid
20. Rosilio, C.; Nebout, M.; Imbert, V.; Griessinger, E.; Neffati, Z.; Benadiba, J.;
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Y.; Masuko, T. Biochem. Biophys. Res. Commun. 2011, 406, 649.
a
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2. Shennan, D. B.; Thomson, J. Oncol. Rep. 2008, 20, 885.
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oxygens of an amino acid carboxylic acid are likely involved in
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E.A., A.F., K.F., L.H., and S.M. all thank the University of Nebraska
at Kearney (UNK) University Research Fellows (URF) Program for
financial support. E.A. also thanks the UNK Summer Student
Research Program (SSRP). This work was supported by the UNK
Research Services Council (RSC) and the Nebraska EPSCoR Under-
graduate Research Experiences (URE) Program (to A.A.T.). We
appreciate OpenEye Scientific Software Inc. for granting us access
to its high-performance molecular modeling applications through
its academic license program. This work was supported in
part by the National Institutes of Health grant R01 GM108911
30. Jandeleit, B.; Fischer, W.-N.; Koller, K. J. WO2015117147, 2015.
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A. Abstract of Papers, 50th Midwest Regional Meeting of the American Chemical
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MWRM279.
(
to A.S. and C.C.), by the Department of Defense grant
W81XWH-15-1-0539 (to A.S. and C.C.), and by the National
Institutes of Health’s National Institute of General Medical Sciences
grant U01 GM61390 (to A.A.Z., H.C.C., L.L. and K.M.G.).
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9. Thomas, A. A.; Augustyn, E.; Finke, K.; Hansen, L. M.; Heeren, N.; Miller, S.; Zur,
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the American Chemical Society, St. Joseph, MO, October 2015; MWRM3.
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Supplementary data (full experimental details, compound char-
acterization data, IC50 curves, stability data and model refinement
and ligand docking description) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.
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