Leveraging γ-Glutamyl Transferase To Direct Cytotoxicity of Copper Dithiocarbamates against Prostate Cancer Cells

Article abstract of DOI:10.1002/anie.201807582

A prodrug approach is presented to direct copper-dependent cytotoxicity to prostate cancer cells. The prochelator GGTDTC requires activation by γ-glutamyl transferase (GGT) to release the metal chelator diethyldithiocarbamate from a linker that masks its thiol reactivity and metal binding properties. In vitro studies demonstrated successful masking of copper binding as well as clean liberation of the chelator by GGT. GGTDTC was stable to non-specific degradation when incubated with a series of prostate cancer and normal cell lines, with selective release of diethyldithiocarbamate only occurring in cells with measurable GGT activity. The antiproliferative efficacy of the prochelator correlated with cellular GGT activity, with 24 h inhibitory concentrations ranging from 800 nm in prostate cancer lines 22Rv1 and LNCaP to over 15 μm in normal prostate PWR-1E cells. These findings underscore a new strategy to leverage the amplified copper metabolism of prostate cancer by conditional activation of a metal-binding pharmacophore.

Full text of DOI:10.1002/anie.201807582

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Leveraging gamma-glutamyl transferase to direct cytotoxicity of
copper dithiocarbamates against prostate cancer cells
Authors: Subha Bakthavatsalam, Mark L. Sleeper, Azim Dharani,
Daniel J. George, Tian Zhang, and Katherine J. Franz
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201807582
Angew. Chem. 10.1002/ange.201807582
Link to VoR: http://dx.doi.org/10.1002/anie.201807582
http://dx.doi.org/10.1002/ange.201807582

10.1002/anie.201807582
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COMMUNICATION
Leveraging gamma-glutamyl transferase to direct cytotoxicity of
copper dithiocarbamates against prostate cancer cells
Subha Bakthavatsalam,a Mark L. Sleeper,a Azim Dharani,a Daniel J. George,b Tian Zhang,b and
Katherine J. Franza*
ABSTRACT: The avidity of prostate cancer cells for copper
sensitizes them to cytotoxicity by disulfiram, a dithiocarbamate-
containing drug approved for alcohol aversion therapy. While
disulfiram has received attention as a possible therapy for various
cancers, its lack of cancer specificity is a liability that limits this
promise. Here we present a prodrug approach to direct copper-
dependent cytotoxicity of dithiocarbamate pharmacophores to
prostate cancer cells. The prochelator GGTDTC requires activation
by gamma-glutamyl transferase (GGT) to release the metal chelator
diethyldithiocarbamate from a linker that masks the thiol reactivity
and metal binding properties of the pharmacophore prior to
activation. In vitro studies demonstrated successful masking of its
copper binding properties as well as clean liberation of the chelator
Scheme 1. Prochelator GGTDTC uses a p-amidobenzyl linker (PAB, black)
by GGT. GGTDTC was found to be stable to non-specific
degradation when incubated with a series of prostate cancer and
normal cell lines, with selective release of diethyldithiocarbamate
only occurring in cells with measurable GGT activity. The
antiproliferative efficacy of the prochelator correlated with cellular
GGT activity, with 24 h half maximal inhibitory concentrations
ranging from 800 nM in prostate cancer lines 22Rv1 and LNCaP to
over 15 µM in normal prostate PWR-1E cells. These findings
to connect a dithiocarbamate (DTC, red) pharmacophore to
a g-linked
glutamic acid. Recognition by the GGT enzyme results in transpeptidation to
release g-GluGlyGly and via 1,6-benzyl elimination of the linker, DTC that is
then available to bind copper.
metabolites that ultimately inhibit aldehyde dehydrogenase, the
eventual target that causes an aversive accumulation of
acetaldehyde in patients who consume ethanol.[5] While most of
these reactions and metabolites are counterproductive in terms
of cancer therapy, DTC’s ability to form Cu complexes is a key
facet of its anticancer properties.[6] The mechanism of Cu-
assisted disulfiram toxicity is not fully understood, although
many reports provide evidence that reactive oxygen species
underscore
a new strategy to leverage the amplified copper
metabolism of prostate cancer by conditional activation of a metal-
binding pharmacophore.
Most deaths related to prostate cancer are due to metastatic
disease that progresses despite therapy. While the androgen
receptor remains the major drug target, resistance to androgen
deprivation often leads to aggressive and lethal metastatic
castration-resistant prostate cancer (mCRPC).[1] The relatively
poor prognosis and limited therapy options for patients with this
disease state underscore the need for innovative therapies
directed to targets other than the androgen receptor.
7]
mediate cell death.[4, Recently, the p-97-NPL4-UFDI protein
complex was identified as a likely target of Cu(DTC)2, ultimately
resulting in accumulation of ubiquitinated proteins and induction
of a heat shock response.[8] Furthermore, Cu(DTC)2 complexes
have been detected in mice fed DSF and in human plasma of
patients undergoing DSF treatment, validating the concept that
DSF transforms in vivo to form Cu-DTC complexes.[8]
While disulfiram has been touted as an interesting
compound for various cancers,[9] significant hurdles hinder its
implementation for cancer treatment.[10] Notably, a phase 2
clinical trial of disulfiram in mCRPC patients demonstrated
significant toxicity without improvement in efficacy.[11] The failure
of DSF in the clinical setting could be attributed to its lack of
specificity along with its unwanted metabolites.[5b] While attempts
have been made to improve formulation and drug delivery
strategies,[12] little has been done to optimize the molecule itself
for application to cancer.
We hypothesized that the efficacy of disulfiram in cancer
settings could be improved by targeting cytotoxic Cu(DTC)2
complexes selectively to cancer cells. To that end, we reasoned
that a prochelator approach that leverages a cancer-specific
activation mechanism could preferentially release DTCs in situ
to take advantage of the amplified Cu metabolism of prostate
Observations of increased copper (Cu) uptake by prostate
cancer cells have raised the intriguing concept of targeting Cu
metabolism for diagnosis and treatment.[2] The proclivity of
prostate tumors to accumulate Cu is so prominent that 64CuCl2
has shown promise as a PET tracer for imaging prostate
cancer.[3] Recent preclinical work has further shown that
castration-resistant prostate cancer cells increase expression of
several Cu trafficking proteins, a response that is accentuated
upon activation of the androgen receptor.[4] This cancer-specific
remodelling of the cell’s Cu biology creates local Cu reserves
that sensitize prostate cancer cells to cytotoxicity by disulfiram
(DSF), a known drug approved for alcohol aversion therapy.[4]
Disulfiram is a disulfide dimer that upon reduction yields
dithiocarbamate (DTC) moieties that contain reactive thiol
nucleophiles and are effective metal chelators. Further
metabolism in the liver leads to sulfoxide and sulfone
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10.1002/anie.201807582
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Scheme 2. Synthesis of GGTDTC. a) p-Aminobenzyl alcohol, HBTU, 4% NMM in DMF,
3 h, rt; b) PBr3, dry THF, 1 h, 0 °C; c) Sodium
diethyldithiocarbamate, dry ACN, 2 h, rt. Overall yield 11.6%
cancer cells for conditional cytotoxicity, while minimizing the off-
target pathways that impede disulfiram’s anticancer promise. As
a type of prodrug, prochelators are designed to hinder the metal-
binding properties of a compound until activation by a desired
stimulus initiates metal-dependent bioactivity.[13] Here, we
introduce GGTDTC as an anticancer prochelator that requires
activation by gamma-glutamyl transferase (GGT) to release DTC
from a self-immolative linker that serves to mask the thiol
reactivity and metal binding properties of DTC prior to activation
(Scheme 1).
GGT is a cell surface enzyme normally localized along the
luminal surfaces of epithelial cells. It catalyzes the extracellular
cleavage of the γ-glutamyl bond of glutathione and other γ-
glutamyl amides to transfer the γ-glutamyl group to water
(hydrolysis) or to amino acid or peptide substrates
(transpeptidation).[14] As most cells cannot import glutathione,
GGT provides a key step for recycling cysteine to enable
intracellular biosynthesis of glutathione, a major component of
cellular redox homeostasis. Interestingly, many types of cancer
cells over-express GGT along their entire cell surface, and
clinical studies have found that GGT expression levels in human
tumors correlate with poor patient survival.[14a] Whereas normal
GGT-expressing cells access γ-glutamate substrates in ductal
fluids, GGT-positive tumor cells can access substrates in
interstitial fluids and in blood.[14a] This difference in location and
activity level suggests that strategies targeting GGT activity are
promising therapeutic avenues. On this front, there has been
significant work developing GGT inhibitors, γ-glutamyl drug
conjugates, and fluorescent substrates for the potential
yield the final GGTDTC after purification by HPLC.
To test our hypothesis that masking DTC impedes its ability
to bind Cu(II), we used a fluorescence competition assay in
which the fluorescence of calcein is quenched upon Cu(II)
binding. As shown in Fig. 1, addition of DTC caused an
immediate recovery of fluorescence, indicating that it readily
competes with calcein for chelating Cu(II), whereas the prodrug
GGTDTC alone did not compete with calcein for Cu(II). However,
exposure of the prochelator to the GGT enzyme resulted in
recovery of calcein fluorescence over time, indicating that
reaction with GGT releases a product capable of chelating Cu(II).
Indeed, the presence of Cu(DTC)2 as a product was confirmed
by HPLC and mass spectral analysis of solutions following
exposure of GGTDTC to GGT enzyme in the presence of Cu(II)
(Fig. S2). Together, these results validate the prochelator
concept of masking metal-chelating competency in GGTDTC
and support our molecular mechanism that GGT activation
induces DTC release.
treatment and visualization of
cancers.[15]
a variety of GGT-positive
Our design for a γ-glutamyl prodrug uses γ-glutamate (γ-Glu)
as a selective recognition and trigger moiety for prochelator
activation. In principle, this strategy leverages two different
processes to differentially target cancer cells: their
overexpression of GGT and their altered copper biology. To
maintain the required γ-glutamyl amide bond for GGT
recognition, DTC was conjugated to γ-glutamate via a self-
immolative p-aminobenzyl (PAB) linker. Once the amide bond is
hydrolyzed by GGT, 1,6 benzyl elimination of the PAB linker is
expected to release DTC specifically in the vicinity of cells
expressing GGT, where it is free to bind available metal ions
(Scheme 1). The iminoquinone methide released as an
intermediate is highly reactive and is expected to undergo
hydrolysis.[16]
GGTDTC was synthesized by conjugating commercially
available Boc-L-glutamic acid α-tertbutyl ester to p-
aminobenzylalcohol using HBTU coupling to generate
compound 2 (Scheme 2). Reaction of 2 with PBr3 converted the
alcohol functionality to bromide while simultaneously removing
the Boc and t-butyl protecting groups to give 3, which was
subsequently reacted with sodium diethyldithiocarbamate to
Figure 1. Copper-calcein competition assay showing recovery of calcein
fluorescence as a function of added chelator or prochelator in the presence of
GGT enzyme over time. Solutions contained 1 µM CuSO4, 1 µM calcein, 20
U/L of GGT enzyme, and 12.5 µM of either GGTDTC (blue) or DTC (red) in
PBS buffer pH 7.4 with 1 mM Gly-Gly; 37 °C.
To probe the reactivity of GGTDTC in comparison to the
enzyme’s natural substrate GSH, competition experiments were
conducted against the colorimetric substrate L-glutamic acid γ-
(p-nitroanilide) [E-pNA] (Fig S3). The colorimetric assay involves
monitoring p-nitroaniline release by absorbance at 405 nm upon
reaction of the probe substrate E-pNA with GGT in the presence
of an inhibiting substrate, either GGTDTC or GSH. Ki for GSH
was found to be 99 ± 7 µM under our experimental conditions
(PBS, pH 7.4, 1 mM GlyGly) and similar to prior reports (73 ± 6
µM).[17] With a Ki of 23 ± 5 µM, GGTDTC was found to have
greater affinity for GGT than glutathione. This finding shows that
GGTDTC should be preferentially cleaved over GSH. The assay
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10.1002/anie.201807582
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COMMUNICATION
Figure 2. Levels of intact GGTDTC observed by HPLC analysis of
supernatants of prostate cell lines incubated with 100 µM GGTDTC for 24 h
at 37°C in medium supplemented with 1 mM GlyGly. GGTDTC depletion
was observed over 24 h in prostate cancer 22Rv1 cells, whereas GGTDTC
levels were unchanged in 22Rv1 treated with GGT-inhibitor Acivicin or in
control prostate PWR-1E cells.
Figure 3. Representative dose-response curves for GGTDTC with and
without supplemental CuSO4 for a) prostate cancer LNCaP and b) prostate
normal epithelial PWR-1E cell lines, as determined by a resazurin viability
assay measured at 24 h. c) GGT activity (blue) measured by E-pNA
colorimetric assay, and IC50 values of GGTDTC (red) across multiple cell
lines in FBS-free medium with 1 µM CuSO4 for 24 h. Antiproliferative activity
of GGTDTC correlates with levels of GGT enzyme activity.
also confirmed that the presence of DTC does not affect enzyme
kinetics.
To test the stability of GGTDTC and the selectivity of its
activation in human cells, an HPLC assay was performed on cell
culture supernatants after incubation with GGTDTC. Fig. 2
shows representative examples from two cell lines: 22Rv1, an
aggressive prostate cancer cell line that tested positively for
GGT activity, and PWR-1E, a normal prostate epithelial line
which tested negatively for GGT activity under these conditions
(vide infra). As shown in Fig. 2, exposure of 100 µM GGTDTC to
22Rv1 cells caused a significant loss of detectable GGTDTC.
Co-exposure with the GGT inhibitor Acivicin, however,
completely reversed this trend and GGTDTC was found to be
stable for at least 24 h. The level of intact GGTDTC was also
found to be unchanged in the GGT(-) PWR-1E cells. Together,
these results provide evidence for the selectivity of the
prochelator for GGT activation and its stability against non-
specific degradative processes in cellular environments.
In order to evaluate the potential anticancer activity of
GGTDTC, the prodrug and DSF were tested for their ability to
inhibit cell growth of prostate cancer cells 22Rv1, LNCaP, and
PC3, as well as PWR-1E prostate epithelial cells, and MCF-7
breast cancer cells. Because Cu is known to be required for
disulfiram’s antiproliferative activity, we performed checkerboard
assays to establish the Cu dependence of DSF and GGTDTC
for inhibition of cell viability under our conditions. Pilot studies in
22Rv1 cells revealed that antiproliferative activity of both DSF
and GGTDTC required as low as 300 nM Cu(II) added to the
growth medium, with 1 µM Cu providing a robust response for
both compounds (Fig S4). No difference in activity was observed
on using either CuCl2 or CuSO4; subsequent studies were
GGTDTC across these cell lines narrows but persists after 72 h
continuous exposure, with IC50 values ranging from 300 nM in
22Rv1 to 1.5 µM in PWR-1E cells. (Table S1). In contrast, DSF
was robustly effective across all cell lines, with IC50 values in the
40–150 nM range at 72 h, consistent with prior studies (Table
S1).[4] Since DSF is a dimer of DTC, the IC50 per monomer is
therefore 80–300 nM at 72 h.
In parallel to the cell viability assays, the GGT activity of
each cell line was measured by using the colorimetric test
substrate E-pNA. The prostate cancer lines 22Rv1 and LNCaP
showed high GGT activity, consistent with expectations of over-
active GGT activity in these cells (Fig 3). In contrast, MCF-7 and
PC3 revealed measurable but lower activity, while PWR-1E
showed no detectable activity at 24 h, with some activity
emerging by 72 h (Fig S5). The emergence of GGT activity over
long incubation times for these cells explains the reduction in
IC50 of GGTDTC in PWR-1E cells at 72 h (Table S1). Importantly,
the trend in GGT activity correlates with antiproliferative efficacy
of GGTDTC: the more active the cell line, the lower the IC50 for
GGTDTC.
CONCLUSIONS
The data communicated herein show that the dithiocarbamate-
releasing prochelator GGTDTC is activated by GGT both in vitro
and in select cancer cells, with Cu-dependent cytotoxicity
correlating with the cells’ level of GGT enzyme activity.
Interestingly, disulfiram itself must undergo reduction prior to
conducted in medium supplemented with
1
µM CuSO4.
formation of Cu complexes,
a process that may involve
Representative dose-response curves for GGTDTC with and
without supplemental Cu are shown in Fig. 3a for LNCaP cells
and 3b for PWR-1E cells, with IC50 values for all cell lines in the
Cu-supplemented condition shown in Fig. 3c. The data in Fig. 3
were collected at 24 h, where the difference in the
antiproliferative activity of GGTDTC across the cell lines is
already evident, with IC50 values ranging from 800 nM in 22Rv1
and LNCaP cancer cell lines to over 15 µM in normal prostate
PWR-1E cells. The difference in antiproliferative activity of
additional redox steps relevant to its cytotoxic mechanism of
6b]
action.[6a,
GGTDTC, on the other hand, releases
dithiocarbamate in situ directly available for metal coordination,
making it an interesting tool to investigate further the molecular
mechanisms underlying the Cu-dependent cytotoxicity of these
kinds of agents. While the efficacy of GGTDTC awaits in vivo
validation, the current data substantiate the concept that using
an activatable targeting group to mask metal-binding
functionality of a pharmacophore like dithiocarbamate provides a
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10.1002/anie.201807582
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COMMUNICATION
Mattova, C. Driessen, Q. P. Dou, J. Olsen, M. Hajduch, B.
Cvek, R. J. Deshaies, J. Bartek, Nature 2017, 552, 194-199.
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promising strategy for engendering disease specificity for metal-
interacting drugs. A potential complication while translating
GGTDTC in vivo could be presented by GGT expressed in the
liver. We note that the versatile synthetic strategy reported here
for GGTDTC can be readily modified to target different cancers
or diseases with their own unique biomarkers, an objective we
are pursuing.
[9]
ACKNOWLEDGEMENTS
We gratefully acknowledge support from the National Science
Foundation (NSF CHE-1152054), the Duke Cancer Institute P30
Cancer Center Support Grant (NIH CA014236), and a Foerster-
Bernstein postdoctoral fellowship for S.B.
[10] a)S. Mohapatra, M. R. Sahoo, N. Rath, Gen Hosp Psychiatry
2015, 37, 97.e95-97.e96; b)A. M. G. Farci, S. Piras, M. Murgia,
A. Chessa, A. Restivo, G. L. Gessa, R. Agabio, Eat Behav
2015, 16, 84-87; c)K. K. Kumar, S. Bondade, F. A. Sattar, N.
Singh, Indian J Psychol Med 2016, 38, 344-347; d)A. T. Tran,
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Armstrong, M. A. Rudek, S. Yegnasubramanian, M. A.
Carducci, Prostate Cancer Prostatic Dis 2013, 16, 357-361.
[12] a)H. Fasehee, R. Dinarvand, A. Ghavamzadeh, M. Esfandyari-
Keywords: prochelator • copper • γ-glutamyl transpeptidase •
dithiocarbamate • cancer
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10.1002/anie.201807582
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
COMMUNICATION
Subha
Bakthavatsalam, Mark
L. Sleeper, Azim
Dharani, Daniel J.
George, Tian Zhang,
and Katherine J.
Franz*
Page No. – Page No.
Leveraging gamma-
glutamyl transferase
to direct cytotoxicity
Chelator in disguise: Dithiocarbamate, a metal ion chelator is masked for its
ability to bind to Cu(II) with a γ-glutamyl transpeptidase (GGT) enzyme trigger.
Activated by GGT overexpressed in cancer cells, the chelator is unveiled to form
cytotoxic Cu complexes in situ selectively in GGT-expressing cancer cells.
of
copper
dithiocarbamates
against
prostate
cancer cells
This article is protected by copyright. All rights reserved.