RETRACTED ARTICLE: Collateral sensitivity of multidrug-resistant cells to the orphan drug tiopronin

Article abstract of DOI:10.1021/jm2001663

A major challenge in the treatment of cancer is multidrug resistance (MDR) that develops during chemotherapy. Here we demonstrate that tiopronin (1), a thiol-substituted N-propanoylglycine derivative, was selectively toxic to a series of cell lines expressing the drug efflux pump P-glycoprotein (P-gp, ABCB1) and MRP1 (ABCC1). Treatment of MDR cells with 1 led to instability of the ABCB1 mRNA and consequently a reduction in P-gp protein, despite functional assays demonstrating that tiopronin does not interact with P-gp. Long-term exposure of P-gp-expressing cells to 1 sensitized them to doxorubicin and paclitaxel, both P-gp substrates. Treatment of MRP1-overexpressing cells with tiopronin led to a significant reduction in MRP1 protein. Synthesis and screening of analogues of tiopronin demonstrated that the thiol functional group was essential for collateral sensitivity while substitution of the amino acid backbone altered but did not destroy specificity, pointing to future development of targeted analogues. This article not subject to U.S. Copyright. Published 2011 by the American Chemical Society.

Full text of DOI:10.1021/jm2001663

ARTICLE
pubs.acs.org/jmc
Collateral Sensitivity of Multidrug-Resistant Cells to the Orphan
Drug Tiopronin
Andrew S. Goldsborough,† Misty D. Handley,† Andrꢀes E. Dulcey,‡ Kristen M. Pluchino,† Pavitra Kannan,§
Kyle R. Brimacombe,† Matthew D. Hall,† Gary Griffiths,‡ and Michael M. Gottesman*,†
†Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States
‡Imaging Probe Development Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Rockville, Maryland,
United States
§Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, United States
S Supporting Information
b
ABSTRACT: A major challenge in the treatment of cancer is multidrug
resistance (MDR) that develops during chemotherapy. Here we demon-
strate that tiopronin (1), a thiol-substituted N-propanoylglycine deriva-
tive, was selectively toxic to a series of cell lines expressing the drug efflux
pump P-glycoprotein (P-gp, ABCB1) and MRP1 (ABCC1). Treatment of
MDR cells with 1 led to instability of the ABCB1 mRNA and consequently
a reduction in P-gp protein, despite functional assays demonstrating that
tiopronin does not interact with P-gp. Long-term exposure of P-gp-
expressing cells to 1 sensitized them to doxorubicin and paclitaxel, both P-gp substrates. Treatment of MRP1-overexpressing
cells with tiopronin led to a significant reduction in MRP1 protein. Synthesis and screening of analogues of tiopronin demonstrated
that the thiol functional group was essential for collateral sensitivity while substitution of the amino acid backbone altered but did not
destroy specificity, pointing to future development of targeted analogues.
’ INTRODUCTION
from which they were selected.4 This phenomenon, termed
“collateral sensitivity” (or MDR1-selectivity),5 has been de-
monstrated using compounds such as NSC73306 (11)6 and
verapamil, which selectively kill P-gp-expressing cells and
reduce P-gp expression at subtoxic concentrations.7 This strat-
egy holds promise for overcoming or preventing clinical MDR.
Collateral sensitivity is assessed most readily in vitro by
determining the cytotoxicity (IC50) of a compound against a
parental line relative to its MDR subline.8
In exploring strategies for targeting MDR, we unexpectedly
found that the simple thiol-substituted N-propanoyl form of glycine
called tiopronin (1, N-(3-mercaptopropanoyl)glycine, Figure 1)
demonstrated collateral sensitivity. 1 is the condensation product of
glycine and thiolactic acid and is an orphan drug that has been used
over the past 30 years to treat a diverse range of pathophysiological
conditions.9 It can exert biological activity via a number of mod-
alities: (i) in chelation therapy, it scavenges toxic metal ions;10 (ii) in
a related manner, 1 can protect against oto- and nephrotoxicity in
patients receiving platinum chemotherapy;11 (iii) in redox coupling
(2RSH T RSSR + 2H+ + 2eꢀ), 1 reduces cystine disulfide bonds
and dissolves cystine stones that develop in the kidneys, ureter, and
bladder of patients suffering from cystinurea;12,13 (iv) 1 potentially
neutralizes reactive oxygen species (ROS) through its thiol, acting as
an anti-inflammatory for rheumatoid arthritis patients;14 (v) it
Chemotherapy is the front-line treatment option available for
combating metastatic and hematological malignancies. However,
multidrug resistance (MDR) conferred by the ATP binding
cassette (ABC) family of efflux transporters, including ABCB1
(MDR1, P-glycoprotein, P-gp) and MRP1 (ABCC1), presents a
significant clinical challenge to current cancer treatment, drug
design, and drug development strategies.1 The clinical signifi-
cance of expression of the drug transporter BCRP (ABCG2) in
cancer has yet to be fully determined, though it has been shown
to efflux known chemotherapeutics. MDR associated ABC
transporters reside predominately in the plasma membrane,
where they actively prevent lipophilic drugs from entering the
cell so that they do not accumulate and become cytotoxic. MDR
can be intrinsic or acquired during chemotherapy through an
increase in transporter expression and is known to negatively
affect remission rates in patients suffering from leukemia, lym-
phoma, and a variety of solid tumors.2
Strategies employed to circumvent or resolve the reduced
drug accumulation conferred by these promiscuous efflux trans-
porters include the development of inhibitors of P-gp for
adjuvant administration.3 While a number have shown promise
in vitro, they have not generally improved response to
chemotherapy.3 Therefore, alternative approaches are urgently
required to improve the efficacy of chemotherapeutic agents.
One such approach is to exploit resistance by identifying
drugs that target MDR cells over the nonresistant parental cells
Received: February 15, 2011
Published: June 09, 2011
This article not subject to U.S. Copyright.
Published 2011 by the American Chemical Society
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serves as a radioprotectant;15 (vi) it protects against the
toxicity of enediynes such as neocarzinostatin (NCS);16
(vii) it behaves as a neuroprotective agent against cerebral
ischemia by scavenging the neurotoxic aldehyde 3-amino-
propanal (currently in clinical trials).17,18
Here we demonstrate that (i) a series of human adenocarci-
noma cell lines expressing the drug efflux pump P-gp and breast
cancer cells expressing MRP1 are collaterally sensitive to 1 and
(ii) the drug has a diverse range of effects on the cells at the level
of mRNA and protein expression, though 1 is not a substrate of
P-gp. Testing of analogues of 1 revealed that (i) removing,
oxidizing, or methylating the thiol or (ii) altering the position of
the thiol abrogated the activity of 1. However, other structurally
similar thiol-bearing compounds such as N-(3-mercapto-2-
methylpropanoyl) glycine, D-penicillamine, and N-acetylcysteine
did not demonstrate significant collateral sensitivity. We also
describe the synthesis of a number of 1 analogues replacing Gly
with Ser, Val, Ala, or Phe and their efficacy against P-gp-, MRP1-,
and ABCG2-expressing cells.
’ RESULTS
The cytotoxicity of 1 (Figure 1) was assessed against parental
KB-3-1 human adenocarcinoma cells and three MDR sublines
expressing P-gp: KB-8-5, KB-8-5-11, and KB-V1 (Table 1).19
The cell lines express increasing levels of ABCB1 mRNA and
P-gp protein in the order KB-8-5 < KB-8-5-11 < KB-V1, resulting
in greater resistance to toxic substrates and increased sensitivity
to the collateral sensitivity agent 11.6 Collateral sensitivity
(abbreviated as “RR” for resistance ratio) is calculated as the
ratio of a compound’s IC50 for parental cells divided by its IC50
for MDR cells. RR > 1 indicates that the compound kills MDR
cells more effectively than parental cells, so-called collateral
Figure 1. Chemical structures of tiopronin (1, [(2-mercaptopropa-
noyl)amino]acetic acid) and analogues. 1 is the condensation product of
glycine and thiolactic acid, and a number of amino acid derivatives were
generated (2ꢀ6). Structures of S-methyl tiopronin (7), N-(3-mercapto-2-
methylpropanoyl)glycine (8), and (isobutyrylamino)acetic acid (9) are
shown. All compounds were synthesized and tested as the racemate.
Table 1. Determination of Cytotoxicity (IC50, mM) of 1 and Collateral Sensitivity (RR) of Parental and MDR Sublines
(Indented)a
cell line
resistance mechanism
selection
cytotoxicity (IC50, mM)
RR
ref
KB-3-1
KB-8-5
7.54 ( 0.20
5.68 ( 0.48
0.35 ( 0.09
0.15 ( 0.02
0.63 ( 0.10
6.84 ( 1.27
9.61 ( 1.82
6.90 ( 1.30
8.27 ( 1.04
17.2 ( 0.33
10.11 ( 1.63
4.84 ( 0.74
9.20 ( 2.12
4.00 ( 0.26
6.43 ( 4.45
5.14 ( 1.31
5.99 ( 1.28
9.05 ( 2.76
12.27 ( 2.34
0.29 ( 0.07
0.15 ( 0.01
0.26 ( 0.04
5.34 ( 0.24
0.94 ( 0.25
5.60 ( 0.32
19
19
19
19
30
31
32
33
33
34
35
34
36
36
23
23
29
29
37
37
21
21
38
28
38
P-gp
colchicine-selected
colchicine-selected
vinblastine-selected
adriamycin-selected
arsenite-selected
1.4
33
KB-8-5-11
KB-V1
KB-A1
KAS
P-gp
P-gp
51
P-gp
14
pleiotropic
pleiotropic
1.2
0.9
CP20
cisplatin-selected
CHO
C5
P-gp
colchicine-selected
0.8
10001
10193
β-tubulin mutant
R-tubulin mutant
colcemid-selected
paclitaxel-selected
1.7
3.6
10576
NIH 3T3
G185
P-gp
ABCB1-transfected
ABCB1-transfected
adriamycin-selected
etoposide-selected
ABCC1-transfected
2.3
HeLa Tet-off
HeLa MDR Tet-off
OVCAR8
NCI/ADR-RES
MCF-7
P-gp
1.25
0.6
P-gp
VP-16
MRP1
MRP1
42.5
0.6
HEK-293 pc
HEK-293 MRP1
H460
TX50
P-gp
paclitaxel-selected
5.7
MX20
ABCG2
mitoxantrone-selected
0.95
a RR calculated as (IC50 parental)/(IC50 MDR cell line). The selection process and primary mechanism of MDR for each MDR cell line are shown.
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Figure 2. Collateral sensitivity of MDR cell lines to 1. (a) Doseꢀ
response curves of 1 against the P-gp-expressing sublines KB-V1, KB-8-
5-11, and KB-8-5 and their parental KB-3-1 human adenocarcinoma cell
line treated with 1 for 72 h. P-gp expression is KB-V1 > KB-8-5-11 >
KB-8-5, and P-gp expression corresponds with toxicity of 1. (b) MRP1-
expressing subline MCF-7/VP-16 and its parental line MCF-7. Data are
the mean ( SD from three independent experiments.
sensitivity.4 The cytotoxicity of 1 against parental KB-3-1 cells is
relatively low (7.54 ( 0.2 mM), but 1 showed increasing
cytotoxicity against the KB-8-5 (5.68 ( 0.48 mM, 1.4-fold
collaterally sensitive, hereafter termed “selective”), KB-8-5-11
(0.349 ( 0.09 mM, 13-fold selective), and KB-V1 cells (0.147 (
0.02 mM, 51-fold selective) (Table 1). Highly adriamycin-
resistant KB-A1 cells expressing P-gp also showed sensitivity to
1 (0.633 ( 0.098 mM, 14-fold selective). Doseꢀresponse curves
reveal a slight biphasic response of cells to 1, with an initial strong
response and a small population of cells surviving at higher
concentrations before all cells are killed at ∼10 mM (Figure 2a).
This phenomenon is similar to the collateral sensitivity of P-gp-
expressing Chinese hamster ovary (CHO) cells to verapamil.20
As a thiol, oxidation of 1 to its oxidized disulfide form (10,
RSH f RSSR) is possible and either 10 or 1 may be the
primary active species under physiological conditions. How-
ever, we determined that less than 10% of 1 was oxidized in
aqueous solution after 5 days of incubation (Supporting
Information Figure S1). Given that the oxidized, disulfide
form of 1 (10) is expected to be reduced in the intracellular
environment of the cell, it is unlikely that 10 is the form
entering the cell or activated in the cell to yield the active
form in vivo. Supporting this, oxidized 10 demonstrated
reduced cytotoxicity and selectivity toward KB-V1 cells
(Supporting Information Figure S2). To confirm that the
collateral sensitivity was not elicited by the introduction of
an initial pH gradient due to the carboxylic acid moiety of 1, a
pH gradient was introduced to cells by serial dilution of
acetic acid, which did not elicit selectivity or cytotoxicity
against KB cells (data not shown).
Figure 3. Effect of 1 on the transporter function of P-gp and MRP1. (a)
1 does not interfere with P-gp function. Cell lines were incubated with
the P-gp substrate rhodamine 123 (4 μM) alone (solid line) or in the
presence of 1 (20 mM, dotted line) or with the positive control P-gp
inhibitor tariquidar (200 nM, dashed line) and compared with the
fluorescence of the parental cell line KB-3-1 (black filled histogram). (b)
1 inhibits MRP1 function at high concentrations. Each cell line was
incubated with the MRP1 substrate calcein-AM (0.25 μM) alone (solid
line) or in the presence of 1 (20 mM, dotted line) or with the positive
control MRP1 inhibitor MK-571 (50 μM, dashed line) and compared
with the fluorescence of the parental cell line MCF-7 (black filled
histogram).
The cytotoxicity of 1 against a series of MDR cells was
determined to examine whether P-gp was necessary for mediating
collateral sensitivity to 1 (Table 1). Cell lines with a range of MDR
expression origins were chosen to assess activity. Relative to
parental lines, 1 did not show strong selectivity toward other
P-gp-expressing lines: the transfected murine cell line NIH 3T3
G185 (expressing human P-gp), adriamycin-selected NCI/ADR-
RES human ovarian carcinoma cells, and colchicine-selected CHO
C5 Chinese hamster ovary cells (Table 1). All showed less than
3-fold selectivity for the MDR subline, with a magnitude of
cytotoxicity similar to that of parental KB cells. These data suggest
that the expression of P-gp is not requisite for MDR cell sensitivity
to 1. As such, we examined MDR cell lines not expressing P-gp.
The KB MDR sublines KAS (selected for resistance to arsenite)
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and CP20 (selected for resistance to cisplatin), neither of
which express P-gp, did not show sensitivity to 1. We also
examined two MDR CHO cell lines (10193 and 10576) with
tubulin mutations: while 10193 (colcemid-selected) did not
show sensitivity, paclitaxel-selected 10576 cells showed modest
collateral sensitivity (RR = 3.6) compared with the parental 10001
cell line.
As others have shown that agents inducing collateral sensitiv-
ity can down-regulate P-gp expression, we carried out qRT-PCR,
Northern blotting, and Western blotting of P-gp. We used a
stably transfected tetracycline-sensitive expression system (Tet-
off) in order to separate the effects of 1 on the promoter
regulation of ABCB1from a more direct effect on the stability
of the ABCB1 mRNA.23 Following treatment of Tet-off cells with
1 (1 mM), RNA and protein were isolated and analyzed
(Figure 4). Northern analysis using RNA prepared from cells
treated for 24, 48, or 72 h demonstrated that there was a marked
reduction in the amount of ABCB1 message within 48 h but no
further reduction at 72 h (Figure 4a). No specific ABCB1 mRNA
cleavage products were evident in the Northern blot, suggesting a
mechanism of mRNA destabilization that affects the entire
message rather than a specific and discrete cleavage site. By
qRT-PCR, standardized using PMCA4 mRNA, we determined
that ABCB1 transcript was reduced by nearly 30% at 24 h and
80% at 48 h (Figure 4b). ABCB1 mRNA from KB-V1 cells was
also down-regulated by treatment with 1 (data not shown).
We also tested the effect of 1 on P-gp protein expression levels
by Western blotting. Tet-off cells were treated with 0.1, 1, or
10 mM 1 for 72 h prior to harvesting and Western blotting, and
band scan densities were subsequently standardized against
GAPDH. Corresponding to the reduced ABCB1 mRNA levels,
P-gp protein was reduced by 50%, 55%, and 65%, respectively,
compared with control (untreated) cells (Figure 5c). Western
blot analysis of MCF-7/VP16 cells treated with 1 (1 mM) also
showed a substantial reduction in MRP1 protein (Figure 4d).
Given that 1 down-regulates MDR1, we assessed whether
long-term treatment of KB-V1 cells with 1 (6 weeks, 5 mM)
could resensitize them to conventional chemotherapeutics. The
results shown in Figure 5 demonstrate that surviving KB-V1 cells
treated with 5 mM 1 are 5.0- and 2.0-fold more sensitive to the
P-gp substrates doxorubicin and paclitaxel, respectively. 1-treated
cells dosed with cisplatin (which is not a P-gp substrate) gave
only a 1.5-fold sensitization compared with control nontreated KB-
V1 cells. Measurement of cell surface P-gp expression demon-
strated diminished P-gp expression of cells cultured in 1 that
corresponded with the sensitization (Supporting Information
Figure S4).
Given the properties of 1 against MDR cells, we sought to
understand the molecular features of 1 essential for activity.
We first examined S-methyl tiopronin (7, Figure 1, Table 2) in
which the thiol functional group is replaced with a thioether. 7
showed no collateral sensitivity or cytotoxicity against KB-V1
cells (IC50 > 20 mM), suggesting that the thiol group of 1 is
essential for its activity. Complete loss of activity was also
found with (isobutyrylamino)acetic acid (9, Figure 1, Table 2),
where the thiol group of 1 was replaced with a methyl group
(IC50 > 20 mM).
Given that the thiol group is essential for activity of 1, we
examined whether collateral sensitivity observed in this study was
simply the result of a general property of thiols or thiol reactivity. A
number of thiol and thiol precursor compounds and disulfide
compounds were tested against KB-3-1 and KB-V1 cells. The
compounds stepronin, captopril, N-acetylcysteine, procysteine,
racecadotril, D-penicillamine, thiorphan, Cys-Gly, cystamine,
cystine, and glutathione (GSH) do not confer significant collateral
sensitivity (Table 2). Interference of intracellular GSH levels by 1
is unlikely to account for the strong collateral sensitivity of KB-V1
cells because treatment with buthionine sulfoximine, a strong
inhibitor of GSH synthesis, has no significant collateral sensitivity
A number of agents that demonstrate collateral sensitivity
against MDR cells are substrates for P-gp.4 Therefore, we
examined whether 1 interacts directly with P-gp. We assessed
this initially by testing whether 1 inhibited the efflux of the
fluorescent substrate rhodamine 123 from P-gp-expressing KB-
V1 cells (Figure 3a). At high concentrations, 1 (20 mM) had no
effect on efflux, indicating that it did not act as either an inhibitor
or a competitive substrate. As a positive control, we used the P-gp
inhibitor tariquidar (XR9576), which was found to increase
accumulation of rhodamine 123 in KB-V1 cells to levels similar
to those in the parental KB-3-1 cells. Identical results were also
found with the fluorescent P-gp substrate calcein-AM
(Supporting Information Figure S3). We also found that 1 did
not stimulate or suppress the activity of P-gp using a P-gp
ATPase assay (Promega, WI, U.S.), confirming that it was not
interacting as an inhibitor or substrate of P-gp (data not shown).
When P-gp is inhibited, MDR cells lose sensitivity to the
collateral sensitivity agent 11, indicating that functional P-gp is
required for selectivity. In contrast, inhibition of P-gp with the
inhibitors cyclosporin A or tariquidar did not affect the activity of
1 against KB-V1 cells in an MTT cell toxicity assay (data not
shown). These data collectively suggest that 1 does not interact
directly with P-gp at its usual substrate or inhibition binding sites,
and the protein does not mediate sensitivity to 1.
To assess whether 1 could elicit collateral sensitivity in cells
expressing other ABC transporters or alternatively suffer from
resistance conferred by other transporters, 1 was tested against
MRP1- and ABCG2-expressing cell lines (Table 1). MRP1-
expressing MCF-7/VP-16 cells (IC50 = 0.29 ( 0.17 mM)
selected for resistance to etoposide showed a 42.5-fold collateral
sensitivity to 1 compared with parental MCF-7 cells (IC50
=
12.27 ( 2.34 mM) (Figure 2b, Table 1). MCF7 cells showed a
biphasic response to 1 similar to that described above for KB
cells. To assess whether this collateral sensitivity was mediated by
MRP1, cytotoxicity of 1 was also assessed against HEK-293
human embryonic kidney cells transfected with ABCC1 (IC50
=
0.26 ( 0.04 mM) and plasmid control (IC50 = 0.15 ( 0.01 mM),
which showed approximately equivalent sensitivity (Table 1).21
This indicated that MRP1 was not directly mediating collateral
sensitivity, further supported by the observation that the MRP1
inhibitor MK-571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]-
phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl]-
propionic acid) did not alter sensitivity of MRP1-expressing
MCF-7/VP-16 cells (data not shown). Interestingly, while the
HEK-293 cells were not collaterally sensitive, the pc line demon-
strated an underlying sensitivity similar to that of P-gp- and
MRP1-expressing lines.
Given that thiols and anionic compounds are common
classes of MRP1 substrates,22 we examined whether 1 inter-
fered with MRP1 function. 1 (20 mM) partially inhibited the
efflux of the MRP1 substrate calcein-AM, suggesting that 1 may
be a substrate of MRP1 (Figure 3b). 1 did not show any
selective activity (RR = 0.95) toward ABCG2-expressing
H460 MX20 (IC50 = 5.6 ( 0.32 mM) cells compared with
parental H460 cells (IC50 = 5.34 ( 0.24 mM).
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Figure 4. 1 significantly down-regulates ABCB1 mRNA. (a) Northern blotting analysis of HeLa MDR Tet-off cells grown in the absence (lanes 1ꢀ3) or
presence of 1 mM 1 (lanes 4ꢀ6) for 24 h (lanes 1 and 4), 48 h (lanes 2 and 5), or 72 h (lanes 3 and 6), prior to RNA extraction and Northern blotting
with an ABCB1 cDNA biotin labeled probe. (b) Quantitative analysis of the ABCB1 mRNA extracted from HeLa MDR Tet-off cells cultured for 8, 24,
or 48 h in the absence (line with triangles) or presence of 1 mM 1 (line with filled squares) prior to RNA extraction and Taqman Q-RT-PCR analysis.
(c) Western blot of protein extracts from HeLa Tet-off MDR (P-gp expressing) cells treated with 0 mM 1 (lane 1), 0.1 mM (lane 2), 1 mM (lane 3), or
10 mM 1 (lane 4) for 24 h prior to harvesting and Western blot analysis with the anti-P-gp antibody C219. GAPDH control was used to calibrate loading.
(d) 1 strongly down-regulates the amount of MRP1 in MCF7/VP-16 cells. Cells were treated with 0 mM (lane 1), 0.1 mM (lane 2), 1 mM (lane 3), or
10 mM (lane 4) 1 for 72 h prior to harvesting, Western blotting and analysis of the ABCC1/MRP1 protein levels with the primary antibody QCRL and a
loading control anti-GAPDH antibody.
Figure 5. Long-term culture of KB-V1 cells with 1 leads to the partial reversal of the MDR phenotype and consequent resensitization of the cells to
chemotherapeutics. Doseꢀresponse curves of KB-V1 cells that have previously been selected for growth in and cultured in the absence (line with
triangles) or presence (line with filled squares) 5 mM 1 for 6 weeks prior to the cell viability assay with the chemotherapeutics: (a) doxorubicin,
(b) paclitaxel, or (c) cisplatin. The IC50 values for each drug are shown below each graph.
activity either alone or in combination with 1 (data not shown). Of
note is N-(3-mercapto-2-methylpropanoyl)glycine (8) which is
also inactive (IC50 > 20 mM) against both KB-3-1 and KB-V1 cell
lines despite only differing from 1 by the incorporation of a
ꢀCH2ꢀ spacer to extend the thiol group away from the amide
bond (Figure 1). Neither of the two constituent components of 1,
glycine and thiolactic acid (RR = 1.05), are selective. Furthermore,
agents that react with thiols or disulfides such as dithiothreitol, β-
mercaptoethanol, N-ethylmaleimide, and DTNB (5,50-dithiobis-
(2-nitrobenzoic acid)) were not particularly selective (Table 2).
Having established that the thiol group is essential for the
activity of 1, we synthesized a number of analogues retaining the
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Table 2. Cytotoxicity of 1 and Analogues, and Sulfur Con-
taining Compounds Against Parental KB-3-1 Human Ade-
nocarcinoma Cells and the P-gp-Expressing Subline KB-V1
(IC50, mM)a
variation was allowed at the amino acid side chain position
(Figure 1). Analogues containing alanine (2), valine (3), serine
(4), and phenylalanine (5) were prepared. The methyl ester
derivative of 1 (6) was also synthesized to confirm that the acidity
of 1 was not responsible for activity of 1 and to ascertain whether
the overall negative charge of 1 was critical to activity. The
synthesis of analogues 2ꢀ6 is shown in Scheme 1. Thiolactic acid
was oxidized to 2,20-dithiobispropionic acid in 70% yield with
iodine under microwave irradiation. The bisacid was then
coupled with a series of amino acid methyl ester hydrochlorides
(2aꢀ6a) via chlorodimethoxytriazine (CDMT) activation of
2,20-dithiobispropionic acid to cleanly afford bisamide disulfides
2bꢀ6b in 77ꢀ95% yield. Disulfide reduction mediated by
tributylphosphine (Bu3P) and methyl ester saponification with
2 M sodium hydroxide (NaOH) of 2bꢀ5b was carried out in a
one-pot operation to afford the target analogues 2ꢀ5 in 42ꢀ85%
yield. Tributylphosphine mediated disulfide reduction of 6b
afforded 6, the methyl ester analogue 1, in 85% yield.
All analogues were tested against P-gp-expressing (KB-V1),
MRP1-expressing (MCF-7/VP-16), and ABCG2-expressing
(H460 MX20) cells and their respective parental cell lines
(KB-3-1, MCF-7, and H460). Analogues 2ꢀ5 demonstrated
collateral sensitivity toward P-gp- and MRP1-expressing cells
(Table 3). While all analogues were active (RR > 1), there was a
large variation in both the IC50 values and the fold-selectivity
between different drug-resistant cell line pairs. Ser-tiopronin (4)
was the most selective analogue against KB-V1 cells (RR = 74.7)
but showed minimal selectivity for the MRP1-expressing VP-16
cells (RR = 1.7). ABCG2-expressing cells were not collaterally
sensitive to 1 (RR = 0.95) or its analogues 2, 3, and 5 (RR = 1.2,
1.6, 1.56, respectively) (Table 3). Methylating the 1 carboxyl
group to produce the nonacidic ester analogue 6 resulted in a
significantly more cytotoxic compound (IC50 = 0.51 mM against
KB-3-1 versus 7.54 mM for 1) perhaps because of greater cell
entry of the neutral species. Selectivity was reduced against both
KB-V1 and VP-16 cells compared with 1, and it failed to elicit
collateral sensitivity (RR = 0.90) of ABCG2-expressing cells.
cytotoxicity (IC50, mM)
KB-3-1
KB-V1
RR
51
1
7.54 ( 0.2
>20
0.147 ( 0.02
>20
7
8
11.78 ( 0.18
>20
9.24 ( 0.23
>20
1.28
1.05
9
glutathione
thiolactic acid
glycine
>20
>20
3.34 ( 0.21
>50
3.5 ( 0.4
>50
thiol compounds
stepronin
0.86 ( 0.12
15.67 ( 0.82
29.9 ( 1.16
28 ( 3.24
0.89 ( 0.04
13.07 ( 0.51
28.8 ( 2.11
29.52 ( 3.13
1.27 ( 0.33
6.93 ( 0.11
2.84 ( 0.16
0.97
1.2
captropril
N-acetylcysteine
procysteine
racecadotril
D-penicillamine
thiorphan
1.04
0.95
2.30
0.58
1.71
2.93 ( 0.47
4.04 ( 0.37
4.87 ( 0.32
disulfide compounds
Cys-Gly
9.42 ( 0.43
0.79 ( 0.07
3.12 ( 0.49
4.23 ( 0.26
1.55 ( 0.09
2.14 ( 0.23
2.23
0.51
1.45
cystamine
cystine
disulfide/thiol reactive
dithiothreitol
N-ethyl maleimide
2.37 ( 0.24
1.89 ( 0.26
1.25
1.97
0.030 ( 0.0004
0.016 ( 0.0003
a Selectivity (RR) is calculated as the ratio of a compound’s IC50 against
parental cells divided by its IC50 against resistant cells. IC50 values are the
mean ( SD from three independent experiments. Structures of the thiol
compounds in this table are shown in Supporting Information Figure S6.
Scheme 1
’ DISCUSSION AND CONCLUSIONS
We have shown here that while not all MDR cell lines are
collaterally sensitive to 1, its cytotoxic activity is independent of
P-gp or MRP1 drug efflux transporters and may therefore be
targeting a more general feature of MDR cells that has been
acquired during drug selection. The collateral sensitivity shown by
MDR cell lines to 1 does not require expression of either P-gp or
MRP1 but does seem to require alterations associated with the
development of MDR. This is in contrast to 11, a thiosemicarba-
zone compound that requires expression and functionality of P-gp
(transfected or selected) for its selective activity.6 Cell lines such as
KB-A1, 10576, 10193, and H460/TX50 that have been selected
for resistance to a number of agents (adriamycin, paclitaxel,
colcemid, and paclitaxel, respectively) were sensitized to 1.
Thiols are known to elicit toxicity through redox cycling and
generation of reactive oxygen species (ROS), and we recently
hypothesized that the collateral sensitivity of MDR cells may be
caused by ROS.24 By testing 1 analogues (Figure 1, Table 1) and
thiol-bearing compounds (Table 2), we determined that the thiol
group is necessary for 1's activity, but a thiol functional group is
not generally sufficient for collateral sensitivity. In fact, 1 is
exquisitely sensitive to structural variation around the thiol
functional group: S-methylation (7), replacement of the thiol
racemic N-2-mercaptopropanoyl moiety but replacing the
glycine backbone of 1 in order to determine whether structural
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Table 3. Cytotoxicity (IC50 mM) of Analogues 1ꢀ6 against Parental and Drug-Resistant Cell Line Pairs: KB-3-1 Human
Adenocarcinoma Cells and the P-gp-Expressing Subline KB-V1, MCF-7 Human Breast Cancer Cells and the MRP1-Expressing
Subline MCF-7/VP-16, and the Human Large Cell Lung Cancer Cell Line H460 and its ABCG2-Expressing Subline H460/MX20a
cytotoxicity (IC50, mM)
P-gp
KB-V1
MRP1
VP-16
ABCG2
MX20
KB-3-1
RR
MCF-7
RR
H460
RR
1
2
3
4
5
6
7.54 ( 0.2
0.15 ( 0.02
0.49 ( 0.02
0.36 ( 0.10
0.24 ( 0.03
0.33 ( 0.05
0.08 ( 0.02
51.0
37.1
36.0
74.7
15.3
6.2
12.27 ( 2.34
20.2 ( 2.58
14.23 ( 3.22
1.30 ( 0.32
2.92 ( 3.13
0.22 ( 0.14
0.29 ( 0.17
0.51 ( 0.05
0.55 ( 0.03
0.76 ( 0.45
0.36 ( 0.10
0.14 ( 0.01
42.5
39.5
26.0
1.7
5.34 ( 0.24
18.38 ( 0.57
6.33 ( 0.54
>20
5.60 ( 0.32
15.38 ( 1.1
3.95 ( 0.26
>20
0.95
1.20
1.60
18.25 ( 2.61
12.77 ( 3.84
17.85 ( 0.25
4.96 ( 1.26
0.51 ( 0.26
8.0
11.32 ( 1.58
0.23 ( 0.03
7.24 ( 0.0.13
0.25 ( 0.03
1.56
0.90
1.6
a IC50 values are the mean ( SD from three independent experiments. Compounds are tiopronin (1), Ala-tiopronin (2), Val-tiopronin (3),
Ser-tiopronin (4), Phe-tiopronin (5), and tiopronin methyl ester (6).
with a methyl group (9), or even insertion of a ꢀCH2ꢀ spacer to
extend the thiol group away from the amide bond (8) all resulted
in compounds incapable of eliciting collateral sensitivity. In
agreement with the importance of the thiol (RSH), oxidation
of 1 (Supporting Information Figure S2) to yield the disulfide 10
led to a marked reduction in selectivity toward KB-V1 cells. 1 is
known to be cell permeable,25,26 and intracellular activity (at
millimolar concentrations) has been reported for a number of
modalities.16,17,27
In contrast, structural variation of 1 by substitution of the
glycine portion of the molecule (Figure 1) for other amino acids
retained selectivity, and in the case of 4 (replacement of ꢀH with
ꢀCH2OH) selectivity increased (RR = 74.7) for KB-V1 cells.
Interestingly, conversion of the carboxyl group in 1 to a methyl
ester (6) considerably increased absolute cytotoxicity (perhaps
because of increased hydrophobicity and hence cell penetration)
but reduced its selectivity for MDR cells. This understanding
points the way to further development of analogues with increased
potency, replacing glycine in 1 for natural and non-natural amino
acids and peptides for targeted deliveryand improved efficacy. The
H460/MX20 cell line expressing ABCG2 was not particularly
sensitive to 1 or its analogues (2ꢀ6), which may indicate that cells
resistant to mitoxantrone are not sensitized to 1.
The HeLa MDR Tet-off cells, transfected with ABCB1, were
not significantly more (RR = 1.25) sensitive to 1 than the control
HeLa Tet-off cells. However, 1 does reduce ABCB1 mRNA and
consequently P-gp protein expression, both in MDR cells that
show collateral sensitivity to 1 (KB-V1) and cells that do not
(Hela MDR Tet-off), suggesting that the reduction of the ABCB1
mRNA by 1 is not a promoter-specific interaction. We do not yet
know whether 1 interacts directly with the mRNA as part of a
protein complex or initiates an unknown downstream cellular
mechanism (currently the subject of investigation). Reduction in
transporter activity can resensitize cells to P-gp substrate che-
motherapeutics such as doxorubicin and paclitaxel (Figure 5).
Treatment of cells with 0.1 mM 1 or more results in a reduction
of mRNA and consequently P-gp protein, potentially explaining
the resensitization of MDR cells to chemotherapy by 1 but not
the collateral sensitivity. 1 also reduces the amount of MRP1
protein in MCF7/VP16 cells.
against MDR cell lines) is relatively low, there is a notable lack of
serious side effects experienced by patients during treatment for
cystinuria with 1, despite daily drug dosing regimens on the order
of hundreds of milligrams to gram quantities per patient per day.
There are a number of specific clinical examples where the local
administration of drug at very high concentrations to treat MDR
malignancies is possible, such as the lumen of the bladder or the
peritoneal cavity. From the perspective of potential clinical
utilization, 1 may offer the dual advantage of targeting the
MDR cell phenotype (collateral sensitivity) at the same time as
reducing the amount of drug transporter in cells. For example, by
treatment of cancer cells with 1 prior to chemotherapy, the
amount of P-gp or MRP1 may be reduced and thereby improve
response to subsequent chemotherapy. Alternatively, co-treat-
ment could suppress the appearance of the MDR phenotype.
Work is currently underway to understand the mechanisms
behind the selectivity of 1, and further medicinal chemistry and
xenograft experiments are planned to optimize targeting of 1 to
MDR cancer cells.
’ EXPERIMENTAL SECTION
Chemicals. Tiopronin (1), D-penicillamine, captopril, thiorphan, N-
acetylcysteine (NAC), buthionine sulfoximine (BSO), cysteineꢀglycine
dipepetide, cystamine, cystine, DTT, N-ethylmaleimide, procysteine,
glutathione, glycine, and thiolactic acid were purchased from Sigma-
Aldrich (St. Louis, MO, U.S.). S-Methyl tiopronin, racecadotril and
N-(3-mercapto-2-methylpropanoyl)glycine were purchased from Tor-
onto Research Chemicals (Toronto, Canada). The tiopronin prodrug
stepronin was purchased from Sequoia Research Products (Pangbourne,
U.K.), and (isobutyrylamino)acetic acid was from Oakwood Products
(West Columbia, SC, U.S.).
Synthesis. The amino acid methyl ester hydrochlorides were
obtained from Bachem (Torrance, CA) and used as received unless
otherwise noted. Reactions were magnetically stirred under an argon
atmosphere and monitored by thin layer chromatography (TLC) with
0.25 mm Sigma-Aldrich precoated aluminum-backed silica gel plates with
fluorescent indicator. TLC visualization wasachievedusing 254 or 360 nm
UV lamp detection and/or staining with cerium molybdate (Hannesian’s
stain), phosphomolybdic acid (PMA), or potassium permanganate. Flash
column chromatography was performed on an Ana-Logix IntelliFlash 280
system, using Biotage SNAP cartridges and SNAP Samplet cartridges with
KP-Silica 60 μm. Analytical HPLC analyses were performed on an Agilent
1200 series instrument equipped with multiwavelength detectors using a
1 is an orphan drug used for many years to treat patients with
such diverse conditions as cystinuria, rheumatoid arthritis, and
metal poisoning. While the absolute cytotoxicity of 1 (high μM
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Zorbax StableBond C-18 column (4.6 mm ꢁ 50 mm, 3.5 μm) with a flow
rate of 0.5 or 1.0 mL/min. Solvent A was 0.05% trifluoroacetic acid (TFA)
in water (H2O). Solvent B was 0.05% TFA in acetonitrile (ACN), and a
linear gradient of 5% B to 95% B over 10 min was used. ESI or APCI mass
spectrometry (MS) was performed on an LC/MSD TrapXCl Agilent
Technologies instrument or on a 6130 quadrupole LC/MS Agilent
Technologies instrument equipped with a diode array detector. In each
case, purity of compounds was confirmed to be g95% by the analytical
HPLC trace displaying only a single peak during analysis. Microwave
irradiation was carriedout ina CEM Discover synthesizer with 150 W max
power. 1H and 13C NMR spectra were recorded on a Varian spectrometer
operating at 400 and 100 MHz, respectively. Chemical shifts are reported
relative to chloroform (δ 7.26), dichloromethane (δ 5.32), or deuterium
oxide (δ 4.79) for 1H NMR and chloroform (δ 77.0) or dichloromethane
(δ 54.0) for 13C NMR.
yield) as a syrup. 1H NMR (400 MHz, CDCl3): δ 7.03 (t, J = 10.4 Hz,
1H), 6.93 (bs, 1H), 4.57ꢀ4.51 (m, 2H), 3.75 (s, 3H), 3.73 (s, 3H),
3.67ꢀ3.55 (m, 2H), 2.24ꢀ2.14 (m, 2H), 1.52ꢀ1.42 (m, 6H),
1.00ꢀ0.92 (m, 12H). 13C NMR (100 MHz, CDCl3): δ 172.9, 172.6,
172.4, 172.2, 171.8(x2), 171.5, 57.6, 52.2, 49.2, 48.8, 31.1, 30.9, 19.0
(ꢁ2), 17.9 (ꢁ2), 17.8, 17.2. MS m/z = 437.2 [M + H]+.
Bisamide Disulfide 4b. The following reagents and conditions
were used: 2,20-dithiobispropionic acid (350 mg, 1.66 mmol), serine
derivative 4a (531 mg, 3.41 mmol), CDMT (599 mg, 3.41 mmol) in
EtOAc (15 mL); NMM (842 mg, 8.32 mmol) in EtOAc (10 mL); TLC
(EtOAc), 1 h. Workup B and flash column chromatography using silica
gel and EtOAc afforded 4b (501 mg, 73% yield) as a waxy solid. 1H NMR
(400 MHz, CDCl3): δ 7.43ꢀ7.34 (m, 2H), 4.74ꢀ4.63 (m, 2H),
3.87ꢀ3.81 (m, 4H), 3.80 (s, 3H), 3.78 (s, 3H), 3.72ꢀ3.67 (m, 2H),
1.46ꢀ1.41 (m, 6H). 13C NMR (100 MHz, CDCl3): δ 173.4, 172.3,
171.8, 170.9(x2), 63.3, 62.5, 62.4, 56.1, 55.1 (ꢁ2), 52.8, 17.4, 16.7, 16.4.
MS m/z = 437.3 [M + Na]+.
2,20-Dithiobispropionic Acid. Iodine (5.72 g, 22.5 mmol) was
added in portions to a solution of racemic thiolactic acid (2.39 g, 22.5
mmol) in water (H2O) (12 mL). The resulting reaction mixture was
heated to 100 °C under microwave irradiation for 30 min, after which
TLC [1:1 hexane (Hex)/ethyl acetate (EtOAc)] showed reaction
completion. The reaction was quenched by addition of a saturated
aqueous solution of sodium thiosulfate (Na2S2O3) and the mixture
extracted with EtOAc twice. The combined organic layers were washed
twice with Na2S2O3 and once with brine, dried over magnesium sulfate
(MgSO4), and concentrated. The crude mixture was purified by
recrystallization from toluene, affording 2 (1.61 g, 70% yield) as white
crystals. 1H NMR (400 MHz, CDCl3): δ 9.66 (bs, 2H), 3.57 (m, 2H),
1.50 (d, J = 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 178.8, 47.5,
46.8, 16.6. MS (m/z) = 210.1 (M + 1)+.
Bisamide Disulfide 5b. The following reagents and conditions
were used: 2,20-dithiobispropionic acid (400 mg, 1.90 mmol), phenyla-
lanine derivative 5a (841 mg, 3.90 mmol), CDMT (685 mg, 3.90 mmol)
in EtOAc (15 mL); NMM (962 mg, 9.51 mmol) in EtOAc (10 mL);
TLC (EtOAc), 1 h. Workup A and flash column chromatography using
silica gel and 35% EtOAc in hexanes afforded 5b (945 mg, 94% yield) as
a syrup. 1H NMR (400 MHz, CDCl3): δ 7.31ꢀ7.12 (m, 10H), 6.98 (bs,
1H), 6.64 (bs, 1H), 4.93ꢀ4.82 (m, 2H), 3.74ꢀ3.70 (m, 6H), 3.56ꢀ3.43
(m, 2H), 3.19ꢀ2.99 (m, 4H), 1.41ꢀ1.37 (m, 3H), 1.33ꢀ1.23 (m, 3H).
13C NMR (100 MHz, CDCl3): δ 171.9, 171.6, 171.4, 129.3, 129.2, 128.5
(ꢁ2), 127.1, 53.6, 53.4, 52.4, 50.1, 49.4, 48.8, 47.7, 37.9, 37.7 (ꢁ2), 37.6,
17.3, 16.7 (ꢁ2). MS m/z = 533.1 [M + H]+.
General Procedure for the Coupling of Bisacid Disulfide 2 with
Amino Acid Methyl Ester Hydrochlorides 2aꢀ6a. Synthesis of Com-
pounds 2bꢀ6b. A mixture of the bisacid disulfide 2,20-dithiobispro-
pionic acid (1 equiv), the amino acid methyl ester hydrochloride 2aꢀ6a
(2.05 equiv), and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) (2.05
equiv) in EtOAc was cooled to 0 °C. To this cooled mixture was added
slowly a solution of N-methylmorpholine (NMM) (5 equiv) in EtOAc.
The resulting reaction mixture was allowed to stir at 0 °C for 5 min and at
room temperature for 1ꢀ2 h while being monitored by TLC.
Workup A. Reaction mixture was diluted EtOAc and H2O. The phases
were separated, and the organic layer was washed twice with 1 M HCl,
once with brine, dried over MgSO4, and concentrated. Crude product
was purified by flash column chromatography to afford the bisamide
disulfide compound.
Bisamide Disulfide 6b. The following reagents and conditions
were used: 2,20-dithiobispropionic acid (300 mg, 1.43 mmol), glycine
derivative 6a (367 mg, 2.92 mmol), CDMT (513 mg, 2.92 mmol) in
EtOAc (10 mL); NMM (722 mg, 7.13 mmol) in EtOAc (10 mL); TLC
(3:1 EtOAc/Hex), 2 h. Workup B and flash column chromatography
using silica gel and 75% EtOAc in hexanes afforded 6b (385 mg, 77%
yield) as a syrup. 1H NMR (400 MHz, CDCl3): δ 7.16 (bs, 2H), 4.23
(dd, J = 18 Hz, 6.0 Hz, 1H), 4.10 (d, J = 5.6 Hz, 1H), 3.77 (s, 3H), 3.76 (s,
3H), 3.67 (q, J = 7.2 Hz, 1H), 3.61 (q, J = 7.2 Hz, 1H), 1.49 (d, J = 7.2 Hz,
3H), 1.45 (d, J = 7.2 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 172.4,
172.2, 171.0 (ꢁ2), 52.5, 49.6, 47.8, 41.4, 17.2, 16.7. MS m/z = 353.1
[M + H]+.
Synthesis of Analogues 2ꢀ5. A solution of the racemic bisamide
disulfide compounds 2bꢀ5b (1 equiv) in 20% H2O in tetrahydrofuran
(THF) (v/v) was thoroughly degassed by bubbling argon through the
solution for 5ꢀ10 min. Tributylphosphine (Bu3P) (3.5 equiv) was
added slowly and the resulting reaction mixture allowed to stir at room
temperature for 5 min and monitored by TLC. The reaction mixture was
diluted with ethanol (EtOH), 2 M sodium hydroxide (NaOH) added,
and stirring continued for 1 h.
Workup A. The reaction mixture was diluted with EtOH and
concentrated under reduced pressure. The residue was taken up in
H2O, extracted twice with EtOAc, and the organic layers were discarded.
The aqueous layer was acidified with 1 M HCl and extracted twice with
EtOAc. The combined organic layers were dried over MgSO4 and
concentrated to afford the 1 analogue.
Workup B. The reaction mixture was diluted with EtOH and
concentrated under reduced pressure. The residue was taken up in
H2O, extracted twice with EtOAc, and the organic layers were dis-
carded. The aqueous layer was acidified with 1 M HCl and concentrated
under reduced pressure. The solid residue was extracted with EtOH,
and the insoluble salts were filtered. The filtrate was concentrated to
afford the 1 analogue.
Workup B. Reaction mixture was filtered, and the insoluble salts were
rinsed with EtOAc. The filtrate was concentrated and the residue
purified by flash column chromatography to afford the bisamide
disulfide compound.
Bisamide Disulfide 2b. The following reagents and conditions
were used: 2,20-dithiobispropionic acid (400 mg, 1.90 mmol), alanine
derivative 2a (544 mg, 3.90 mmol), CDMT (685 mg, 3.90 mmol) in
EtOAc (15 mL); NMM (962 mg, 9.51 mmol) in EtOAc (10 mL); TLC
(1:1 Hex/EtOAc), 2 h. Workup A and flash column chromatography
using silica gel and 75% EtOAc in hexanes afforded 2b (714 mg, 95%
yield) as a syrup. 1H NMR (400 MHz, CDCl3): δ 7.16 (t, J = 9.6 Hz,
1H), 6.80 (bs, 1H), 4.65ꢀ4.58 (m, 2H), 3.76 (s, 6H), 3.63ꢀ3.52 (m,
2H), 1.54ꢀ1.41 (m, 12H). 13C NMR (100 MHz, CDCl3): δ 174.3,
173.5, 173.2, 171.6, 171.5, 52.2, 49.6, 48.7, 48.5, 48.2, 18.2, 18.1, 18.0,
17.9, 17.2, 16.7. MS m/z = 381.1 [M + H]+.
Bisamide Disulfide 3b. The following reagents and conditions
were used: 2,20-dithiobispropionic acid (400 mg, 1.90 mmol), valine
derivative 3a (654 mg, 3.90 mmol), CDMT (685 mg, 3.90 mmol) in
EtOAc (15 mL); NMM (962 mg, 9.51 mmol) in EtOAc (10 mL); TLC
(1:1 Hex/EtOAc), 2 h. Workup A and flash column chromatography
using silica gel and 40% EtOAc in hexanes afforded 3b (803 mg, 94%
Ala-tiopronin (2). The following reagents and conditions were
used: bisamide disulfide 2b (700 mg, 1.84 mmol), 20% H2O in THF
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(v/v, 10 mL), Bu3P (1.30 g, 6.44 mmol); TLC (1:1 Hex/EtOAc), 5 min;
EtOH (4 mL), 2 M NaOH (5 mL), 1 h. Workup A was used to afford
Ala-tiopronin analogue 2 (271 mg, 42% yield) as a fluffy solid. 1H NMR
(400 MHz, D2O): δ 4.35 (q, J = 7.2 Hz, 1H), 3.67ꢀ3.58 (m, 1H),
1.49ꢀ1.42 (m, 6H). 13C NMR (100 MHz, D2O): δ 176.4 (ꢁ2), 48.8,
36.2, 20.4, 15.9. MS m/z = 178.1 [M + H]+.
Val-tiopronin (3). The following reagents and conditions were
used: bisamide disulfide 3b (800 mg, 1.83 mmol), 20% H2O in THF
(v/v, 10 mL), Bu3P (1.30 g, 6.44 mmol); TLC (1:1 Hex/EtOAc),
5 min; EtOH (5 mL), 2 M NaOH (5 mL), 1 h. Workup A was used to
afford Val-tiopronin analogue 3 (577 mg, 77% yield) as a white solid.
1H NMR (400 MHz, CD2Cl2): δ 6.86 (d, J = 8.0 Hz, 1H), 4.45 (td, J =
8.4, 4.8 Hz, 1H), 3.52ꢀ3.47 (m, 1H), 2.26ꢀ2.20 (m, 1H), 2.18ꢀ2.14
(m, 1H), 1.52 (d, J = 7.2 Hz, 3H), 0.97ꢀ0.94 (m, 6H). 13C NMR (100
MHz, CD2Cl2): δ 174.4, 173.4, 57.3, 37.9, 30.8, 21.8, 18.8, 17.3. MS
m/z = 206.1 [M + H]+.
U.S.) and supplemented as described above. Resistant cell lines were
additionally cultured in the following cytotoxic drugs to maintain
transporter expression: KB-8-5, colchicine (10 ng/mL); KB-8-5-11,
colchicine (100 ng/mL); KB-V1, vinblastine (1 μg/mL); KB-A1,
adriamycin (1 μg/mL); NIH-3T3 G185, colchicine (60 ng/mL);
H460/TX50, paclitaxel (50 ng/mL); MCF-7/VP16, etoposide
(4 μM); H460/MX20, mitoxantrone (20 nM).19,28 The parental
Clontech Hela “Tet-off” and the HeLa “MDR Tet-off” (American Type
Culture Collection, Manassas, VA, U.S.) derived cell lines were grown in
high glucose (DMEM) medium supplemented with 10% tetracycline
free fetal bovine serum and 5 mM L-glutamine, 50 units/mL penicillin,
and 50 μg/mL streptomycin from Life Technologies (Carlsbad, CA, U.
S.). The medium of the HeLa MDR Tet-off cell line was additionally
supplemented with colchicine (20 ng/mL) to maintain P-gp expression.
MTT Cytotoxicity Assay. Cytotoxicity was measured with a
colorimetric viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) (Molecular Probes, Eugene, OR) as
previously described.29 Cells (5000 cells per well of a 96-well plate) were
allowed to attach for 24 h. Stock solutions of compounds (3 M) were
prepared in H2O and then 2-fold serially diluted in medium to give a
range of final tissue culture concentrations of 20 mM to 78 μM. After
72 h, cell viability was examined. Cytotoxicity (IC50) was defined as the
drug concentration that reduced cell viability to 50% of the untreated
control. Resistance ratios (RR) are also reported for each cell line pair,
determined by dividing the IC50 of the parental cell line by that of the
transporter-expressing cell line. RR > 1 indicates that the MDR cell
population is collaterally sensitive to the tested drug, while RR < 1
indicates that the MDR cells are resistant to the drug relative to the
parental cell line.4
Ser-tiopronin (4). The following reagents and conditions were
used: bisamide disulfide 4b (500 mg, 1.21 mmol), 20% H2O in THF
(v/v, 8 mL), Bu3P (858 mg, 4.24 mmol); TLC (EtOAc), 5 min; EtOH
(3 mL), 2 M NaOH (3 mL), 1 h. Workup B was used to afford Ser-
1
tiopronin analogue 4 (305 mg, 65% yield) as a white solid. H NMR
(400 MHz, D2O): δ 4.58ꢀ4.52 (m, 1H), 4.04ꢀ3.97 (m, 1H),
3.96ꢀ3.91 (m, 1H), 3.72ꢀ3.67 (m, 1H), 1.51ꢀ1.49 (m, 3H). 13C
NMR (100 MHz, D2O): δ 176.7, 173.3, 60.8, 54.9, 36.4, 20.5. MS m/z =
194.1 [M + H]+.
Phe-tiopronin (5). The following reagents and conditions were
used: bisamide disulfide 5b (460 mg, 0.863 mmol), 20% H2O in THF
(v/v, 6 mL), Bu3P (611 mg, 3.02 mmol); TLC (1:1 Hex/EtOAc), 10
min; EtOH (2 mL), 2 M NaOH (2 mL), 1 h. Workup A was used to
afford Phe-tiopronin analogue 5 (371 mg, 85% yield) as a thick syrup. 1H
NMR (400 MHz, CDCl3): δ 7.61 (bs, 1H), 7.31ꢀ7.23 (m, 3H),
7.18ꢀ7.14 (m, 2H), 6.93ꢀ6.86 (m, 1H), 4.86ꢀ4.79 (m, 1H),
3.43ꢀ3.39 (m, 1H), 3.26 (ddd, J = 14.4, 5.6, 2.8 Hz, 1H), 3.12 (dd,
J = 14.0, 6.4 Hz, 1H), 1.96 (d, J = 8.0 Hz, 1H), 1.46 (d, J = 7.2 Hz, 3H).
13C NMR (100 MHz, CDCl3): δ 174.6, 173.3, 135.5, 129.4, 128.6, 127.3
(ꢁ2), 53.3, 37.9, 37.2, 21.8 (ꢁ2). MS m/z = 254.1 [M + H]+.
Tiopronin-Me Ester (6). A solution of bisamide disulfide 6b (380
mg, 1.08 mmol) in 20% H2O in THF (v/v, 10 mL) was thoroughly
degassed by bubbling argon through the solution for 10 min. Bu3P (763
mg, 3.77 mmol) was added slowly and the resulting reaction mixture
stirred for 10 min under argon. TLC (EtOAc) showed complete
reduction. Reaction mixture was concentrated under reduced pressure,
and the residue was subjected to flash column chromatography using
silica gel and 1:1 Hex/EtOAc to afford the tiopronin-Me ester analogue
6 (332 mg, 85% yield) as a thick syrup. 1H NMR (400 MHz, D2O): δ
4.05 (s, 1H), 3.79 (s, 3H), 3.68 (q, J = 7.2 Hz, 1H), 1.51 (d, J = 6.8 Hz,
3H). 13C NMR (100 MHz, D2O): δ 177.2, 171.9, 52.7, 41.3, 36.4, 20.6.
MS m/z = 178.1 [M + H]+.
Cell Lines. The cell lines used were the following: the human
cervical epithelial adenocarcinoma cell line KB-3-1 (a HeLa derivative)
and its P-gp-expressing MDR sublines KB-A1, KB-V1, KB-8-5-11, and
KB-8-5; the human breast cancer cell line MCF-7 and its MRP1-
expressing MDR subline MCF-7/VP16; the human lung carcinoma cell
line H460 and its ABCG2-expressing MDR subline H460/MX20 and
P-gp-expressing variant H460/TX50; NIH-3T3 murine fibroblast cells
and its mutant human P-gp-expressing variant NIH-3T3 G185; OV-
CAR8 human ovarian carcinoma cells and its P-gp-expressing variant
NCI/ADR-RES. All cell lines were grown at 37 °C in 5% CO2 and
cultured as follows. The KB, NIH-3T3, and MCF-7 lines were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum, 5 mM L-glutamine, 50 units/mL penicillin, and
50 μg/mL streptomycin, all obtained from Life Technologies (Carlsbad,
CA, U.S.). The H460 lines were cultured in Roswell Park Memorial
Institute (RPMI) 1640 medium from Life Technologies (Carlsbad, CA,
ABCB1 and ABCC1 mRNA Analysis. 1 ꢁ106 MDR Tet-off cells
were seeded for 24 h before treating (in the absence of doxycycline)
either with or without 1 (1 mM) for 8, 24, or 48 h. Cells were then
trypsinized, rinsed with PBS three times, and the total RNA was purified
using an RNeasy mini kit according to the manufacturer’s instructions
(Qiagen, Germantown, MD, U.S.). First strand cDNA was prepared
from 1 μg of total RNA using the high capacity cDNA reverse
transcription kit (Applied Biosciences, Foster City, CA, U.S.) followed
by PCR analysis using either ABCB1 (Hs00184491_m1) or the PMCA4
control (Hs00608058_m1) Taqman probe sets from TaqMan Universal
PCR Master Mix and loaded on an ABI Prism 7900 HT sequence
detection system according to the manufacturer’s instructions (Applied
Biosciences, Foster City, CA, U.S.). The percentage of ABCB1 mRNA
remaining was calculated from the PCR crossing point threshold (Ct)
values and the total amount of RNA adjusted using the RNA control.
Northern Blotting. The pTM1 plasmid containing the ABCB1
cDNA was digested with XhoI and NcoI restriction enzymes, and
following agarose gel electrophoresis, the bands were purified using a
QIAquick gel extraction kit (Qiagen, Germantown, MD, U.S.). The
cDNA was biotinylated using the Brightstar psoralen-biotin kit
(Ambion, Austin, TX, U.S.). An amount of 2 μg of total RNA from
MDR Tet-off cells was resolved on a nondenaturing 1% TAE agarose gel.
Equal loading of RNA in the gel was confirmed by ethidium bromide
staining showing 18S and 28S rRNA bands (Supporting Information
Figure S5). Northern blots were performed using NorthernMax mem-
branes and detection was performed with BrightStar BioDetect accord-
ing to the manufacturer's instructions (Ambion, Austin, TX, U.S.).
Western Blot Analysis. The expression of ABC transporters in
eachcelllinewas visualizedbyWestern blotanalysis. Protein sampleswere
prepared and run on a gel as described by Brimacombe et al.29 In brief,
lysed cells were incubated in sodium dodecyl sulfate (5ꢁ) buffer, loaded
onto a 3ꢀ8% NuPAGE Novex Tris-acetate gel (Invitrogen Corp.,
Carlsbad, CA, U.S.), and transferred to nitrocellulose membranes. Dry
blots were blocked in 20% milk for 30 min at 21 °C. Blots were then
probed for expression of P-gp, MRP1, and ABCG2 protein using the three
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primary antibodies C219 (1:10,000), QCLR (1:5,000), and BXP-21
(1:10,000), respectively, for 60 min at 21 °C, washed 3 ꢁ 10 min,
immunoprobed with the secondary antibody ImmunoPure goat anti-
mouse IgG peroxidase-conjugated (GAMP, 1:10000, Pierce Biotechnol-
ogy, Rockford, IL, U.S.) for 60 min at 21 °C, and washed again. Each blot
was immunoprobed for glyceraldehyde 3-phosphate dehydrogenase
(GAPDH, Ambion, Austin, TX, U.S.) as a loading control.
Resensitization Assay. KB-V1 cells were grown in 1 at 0.1 mM
and then 1 mM and finally 5 mM over 6 weeks. In parallel, control KB-
V1 cells were grown for the same period of time in the absence of
selecting agent (vinblastine). Cells were then tested for rensensitization
to doxorubicin, paclitaxel, or cisplatin using a standard MTT cell
viability assay.
Inhibition of Transporter Function. Since transporter sub-
strates (at high concentrations) can inhibit ABC transporter function
as competitive substrates, we determined whether 1 could inhibit the
function of P-gp. We measured inhibitory activity by the uptake of
rhodamine 123 as described previously.29 For each condition, 2 ꢁ 105
cells were suspended in 1 mL of Iscove’s modified Dulbecco’s medium
(IMDM) supplemented with 5% fetal bovine serum. The cells were first
pretreated with a P-gp inhibitor (positive control, 200 nM tariquidar), 1
(20 mM), or medium (negative control) for 10 min in a 37 °C water
bath. Cells were isolated by centrifugation, resuspended in 1 mL of
IMDM, and incubated with rhodamine 123 (4 μM). Cells were then
incubated in the dark for 45 min in a 37 °C water bath, centrifuged,
resuspended in 300 μL of 0.1% bovine serum albumin in 1ꢁ PBS, and
kept on ice until analysis. For each cell treatment fluorescence intensity
(cellular uptake of fluorescent substrate) was recorded for a total of
10 000 cells using a FACS Calibur flow cytometer (Becton Dickinson
Biosciences, San Jose, CA, U.S.). FACS data were analyzed using FlowJo
software (Tree Star, Inc., Ashland, OR, U.S.).
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’ ASSOCIATED CONTENT
S
Supporting Information. Assessment of 10 (Figures S1
b
and S2), calcein-AM P-gp efflux assay supporting the Rh123 data
presented in Figure 3a (Figure S3), evidence for down-regulation
of P-gp in long-term culture with 1 (Figure S4), ethidium
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available free of charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: +1 301 496 1530. Fax: +1 301 402 0450. E-mail: gottesmm
@mail.nih.gov.
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’ ACKNOWLEDGMENT
This research was supported by the Intramural Research
Program of the National Institutes of Health, National Cancer
Institute, and National Heart, Lung, and Blood Institute.
We thank George Leiman for editorial assistance. We thank
King-Leung Fung for the gift of LLC-PK1 cells.
’ ABBREVIATIONS USED
ABC, ATP-binding cassette; GSH, glutathione; MDR, multidrug
resistance; RR, resistance ratio
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