Dithiocarbamate prodrugs activated by prostate specific antigen to target prostate cancer

Article abstract of DOI:10.1016/j.bmcl.2020.127148

Disulfiram in conjunction with copper has been shown to be a potent anticancer agent. However, disulfiram's therapeutic potential in prostate cancer is hindered by off-target effects due to its reactive and nucleophilic thiol-containing component, diethyldithiocarbamate (DTC). To minimize undesirable reactivity, we have strategically blocked the thiol moiety in DTC with a cleavable p-aminobenzyl (pAB) group linked to peptide substrates recognized by prostate specific antigen (PSA). Here we report the synthesis and evaluation in cancer cell models of two PSA-activatable prodrugs: HPD (Ac-HSSKLQL-pAB-DTC and RPD (RSSYYSL-pAB-DTC). In vitro exposure to PSA was found to trigger activation of HPD and RPD to release diethyldithiocarbamate, and both prodrugs were found to induce toxicity in prostate cancer cells, with HPD showing the most promising selectivity. With copper supplementation, the IC₅₀ of HPD was 1.4 μM in PSA-expressing LNCaP cells, and 11 μM in PC3 cells that do not express PSA. These studies demonstrate the utility of using peptide recognition handles to direct the activity of dithiocarbamate prodrugs for selective cytotoxicity of cancer cells.

Full text of DOI:10.1016/j.bmcl.2020.127148

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dithiocarbamate prodrugs activated by prostate specific antigen to target
prostate cancer
Subha Bakthavatsalama, Petpailin Wiangnaka, Daniel J. Georgeb,c, Tian Zhangb,c
,
⁎
Katherine J. Franza,
a Department of Chemistry, Duke University, Durham, NC 27708-0354 USA
b Division of Medical Oncology, Department of Medicine, Duke Cancer Institute, Duke University School of Medicine, Durham, NC 27710 USA
c Duke Cancer Institute Center of Prostate and Urologic Cancers, Duke University, Durham, NC 27710 USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Disulfiram in conjunction with copper has been shown to be a potent anticancer agent. However, disulfiram’s
therapeutic potential in prostate cancer is hindered by off-target effects due to its reactive and nucleophilic thiol-
containing component, diethyldithiocarbamate (DTC). To minimize undesirable reactivity, we have strategically
blocked the thiol moiety in DTC with a cleavable p-aminobenzyl (pAB) group linked to peptide substrates re-
cognized by prostate specific antigen (PSA). Here we report the synthesis and evaluation in cancer cell models of
two PSA-activatable prodrugs: HPD (Ac-HSSKLQL-pAB-DTC and RPD (RSSYYSL-pAB-DTC). In vitro exposure to
PSA was found to trigger activation of HPD and RPD to release diethyldithiocarbamate, and both prodrugs were
found to induce toxicity in prostate cancer cells, with HPD showing the most promising selectivity. With copper
supplementation, the IC50 of HPD was 1.4 µM in PSA-expressing LNCaP cells, and 11 µM in PC3 cells that do not
express PSA. These studies demonstrate the utility of using peptide recognition handles to direct the activity of
dithiocarbamate prodrugs for selective cytotoxicity of cancer cells.
Prostate specific antigen
Disulfiram
Diethyldithiocarbamate
Prochelator
Copper
Prostate cancer is the second most common cancer among men.1
The androgen receptor (AR) remains the major drug target for prostate
cancer.2,3 While androgen deprivation therapy can be effective at initial
stages, there are many cases where disease progresses to metastatic
cancer.4,5 Although hormonal agents targeting AR such as enzalutamide
and abiraterone acetate have improved treatment outcomes, the fre-
quent occurrence of resistance to these treatments underscores a clear
need for new prostate cancer therapies that act on targets other than
AR.
plasma of patients undergoing disulfiram treatment for alcoholism, and
in tumor tissue after disulfiram administration.13
The promising anticancer activity of disulfiram, however, also car-
ries many liabilities, as it relies on a reactive metabolite that binds non-
specifically to metal ions, reacts non-specifically with protein and other
cellular thiols, and further decomposes to form unwanted metabolites
with off-target effects.14–16 To overcome these drawbacks, we devised a
prochelator strategy that would generate the Cu(DTC)2 complex in situ
in prostate cancer tissue.17 Prochelators are masked chelators that are
activated by a trigger to release the metal-binding form specifically in
the target region.18
The avidity of prostate cancer cells to accumulate copper (Cu) has
been recognized to sensitize these cells toward cytotoxicity by com-
pounds that act via a mechanism that relies on copper.6–9 Disulfiram, a
drug commercially available as Antabuse and approved for alcohol
aversion therapy, is a principle example. In the cancer setting, the ac-
tivity of disulfiram has been attributed to its reduced version, diethyl-
dithiocarbamate (DTC), which binds Cu and leads to generation of re-
active oxygen species (ROS) and cell death.10,11 Indeed, co-
administration of Cu(II) salts dramatically enhanced the efficacy of
disulfiram to inhibit the growth of both androgen-responsive and cas-
tration-resistant prostate xenografts.12 Furthermore, the Cu(DTC)2
complex has been detected in mice fed with disulfiram, in human
Our first iteration of an enzyme-activatable DTC prodrug leveraged
gamma glutamyl transferase (GGT) as the trigger.17 The prochelator,
termed GGTDTC, selectively released DTC only in cells with measurable
GGT activity; antiproliferative efficacy also correlated with cellular
GGT activity, with 24 h IC50 values of 800 nM in prostate cancer cell
lines but over 15 μM in normal prostate epithelial cells.17
While GGT is overexpressed in many aggressive cancer cells, it is
also overexpressed in liver, especially during liver disease. To explore
alternative trigger mechanisms, in our current work we chose prostate
specific antigen (PSA) as the triggering enzyme. PSA is a serine protease
⁎ Corresponding author.
E-mail address: katherine.franz@duke.edu (K.J. Franz).
https://doi.org/10.1016/j.bmcl.2020.127148
Received 3 February 2020; Received in revised form 25 March 2020; Accepted 26 March 2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:SubhaBakthavatsalam,etal.,Bioorganic&MedicinalChemistryLetters,
https://doi.org/10.1016/j.bmcl.2020.127148

S. Bakthavatsalam, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
that is expressed by prostate cells, is overexpressed in prostate cancer, is
not expressed by other normal cells in humans, and is used as a bio-
marker for prostate cancer activity.19–22
aminobenzyl alcohol. The alcohol group of pAB was then converted to
bromine to enable nucleophilic substitution to conjugate DTC. Depro-
tection of the fmoc group frees the amino group for further conjugation
to the substrate peptides. For the case of HPD, N-acetylated and alloc-
protected AcHSSK(alloc)LQ peptide was conjugated by using a peptide
coupling protocol to generate 5, which was then deprotected and pur-
ified by HPLC to afford HPD (Scheme 1). For RPD, Fmoc-RSSYYS was
coupled to 4 followed by fmoc deprotection and purification to give the
final product (Scheme S1).
The natural proteolytic substrates for PSA, a member of the kallik-
rein enzyme family,23 are semenogelin proteins SgI and SgII found in
semen.22,24 PSA is also enzymatically active in prostate tumor tissue,
but is inactivated upon binding to α1-antichymotrypsin if released into
the blood stream.25 The tumor-specific activity of PSA has been lever-
aged to develop prodrugs that have found success in pre-clinical models
to release several cytotoxic agents, including vinblastine, thapsigargin,
and doxorubicin, the latter having passed a Phase I clinical trial.26–28 In
order to create a DTC-releasing prodrug, we chose two peptide se-
quences that are known PSA substrates optimized to release a drug
upon PSA cleavage: RSSYYS and HSSKLQ.28,29 The former is derived
from chymotrypsin substrates and has been used to develop doxor-
ubicin prodrugs that demonstrated moderate selectivity for PSA-ex-
pressing vs. non-expressing cells.28,30 The HSSKLQ sequence represents
the PSA cleavage sites in SgI and SgII proteins; it has been used to
develop prodrugs of thapsigargin, doxorubicin and vinblastine.29 In this
work, we conjugated DTC via a leucine spacer and a self-immolative p-
aminobenzyl (pAB) linker to the C-terminus of these peptide sequences
to generate Arg-Ser-Ser-Tyr-Tyr-Ser-Leu-pAB-DTC and His-Ser-Ser-Lys-
Leu-Gln-Leu-pAB-DTC (RPD and HPD, respectively) as shown in Figure
1. Blocking the thiol of DTC prevents it from binding to Cu and hence is
in an inactive form, as we have shown in our previous study.17 In the
current design, PSA within tumor tissue is expected to cleave the pep-
tides at the bonds highlighted in Fig. 1, with non-specific cellular
proteases like aminopeptidases trimming the final residues to initiate
the 1,6-benzyl elimination of the pAB linker to release the DTC cargo.
The released DTC is then available to capture Cu in the tumor micro-
environment.
To check the cleavage by PSA enzyme the prodrugs HPD and RPD
were treated with 20 µg/mL of PSA enzyme. PSA is expected to cleave
between glutamine and leucine residues. The cleavage products were
monitored via HPLC, with caffeine added as an internal standard. Intact
HPD eluted at 10 min as confirmed by mass spectrometry (Fig. S3). The
relative intensity of the HPD peak eluting at 10 min decreased with
time, consistent with gradual cleavage of the peptide (Fig. 2). The
cleavage product of this reaction with HPD was confirmed to be Leu-
PAB-DTC by mass spectrometry (m/z 368 for [M+H]+ and 735 for [2M
+H]+) (Fig. S3). At least 30% of HPD remained even after 48 h
treatment with enzyme. RPD on the other hand was cleaved completely
within 8 h under these conditions. RPD was cleaved between Tyr and
Ser to release cleavage product Ser-Leu-PAB-DTC as confirmed by mass
spectrometry (m/z 477 for [M+Na]+ and m/z 909 for [2M+Na]+, Fig.
S4). These results are consistent with previous reports suggesting that
the kinetics for cleavage of HSSKLQ is slower than that of RSSYYS by
PSA enzyme.31
To test the design element that general amino peptidases in the cell
would trim the last amino acid remaining after PSA cleavage and ac-
tivate the PAB linker to release DTC, HPD and RPD were treated with a
mixture of PSA and leucine amino peptidase. DTC itself was not ob-
servable by our LC-MS conditions, but spiking the reaction mixture with
CuSO4 and analyzing by LC-MS revealed a clean peak at 14.5 min with
m/z of 358.87, consistent with the Cu(DTC)2 complex (Fig. S5). An
The peptide-DTC conjugates were synthesized following the route
summarized in Scheme 1, starting with coupling Fmoc-Leucine to p-
Fig. 1. Chemical structures of prodrugs RPD
and HPD, with products from cleavage by
PSA enzyme and subsequent amino acid
trimming by non-specific aminopeptidases.
Spontaneous 1,6 benzyl elimination of the
PAB linker releases dithiocarbamic acid,
which is then available for coordinating
copper. Wavy lines indicate the position of
cleavage by PSA.
2

S. Bakthavatsalam, et al.
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N
N
OH
Br
S
S
OH
S
S
a
b
c
d
H2N
HN
HN
HN
HN
O
O
O
O
NH Fmoc
NH Fmoc
NH Fmoc
NH2
4
2
3
1
e
N
N
S
S
S
S
f
HN
HN
H
N
H
HSSK(alloc)LQ
HN
O
HSSKLQ
HPD
N
HN
O
O
O
5
Scheme 1. Synthesis of HPD, a PSA-targeted prochelator. a) FmocLeu-OH, 4% NMM in DMF, HBTU; b) Dry THF, PBr3; c) Sodium diethyldithiocarbamate, Dry THF;
d) 20% piperidine in DMF; e) AcHSSK(alloc)LQ, EDC, HOBt, DMF; f) Pd(PPh3)4, PhSiH3, DCM.
Fig. 2. % of HPD (blue) and RPD (red) remaining at different timepoints on
treatment with 20 μg/mL PSA enzyme HPLC trace at λ 254 nm of HPD for
5 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h.
isomer construct of RPD where serine in the sixth position was replaced
with D-serine was also prepared and tested in this same assay. The only
observed product was a peak eluting at 9 min with m/z of 455.06,
(DTC)2 peak was observed. This result conDfi-rSmers-Ltheuat-PaAmBi-nDoTCpe; pntoidaCsue
consistent with the PSA-cleaved product
activity is required to activate the PAB linker and release DTC.
To test the selectivity of the prodrugs in cells the cleavage of the
prodrugs in cell lysate was studied by HPLC. We chose LNCaP as a PSA-
expressing cell line and PC3 as a control cell line that does not express
PSA. The cell lysates were treated with HPD or RPD and the cleavage
was monitored over time with HPLC with caffeine as reference. RPD
was completely cleaved by LNCaP lysate within 4 h. PC3 was also able
to cleave more than 50% of the RPD prodrug within 4 h even though
Fig. 3. % Prodrugs remaining in LNCaP and PC3 cell lysate over time a) RPD
they do not express PSA enzyme. This result indicates that RPD is non-
and b) HPD.
selectively cleaved by other proteases in cells. HPD was also completely
cleaved in 4 h in the LNCaP lysate, but HPD was not cleaved in the PC3
3

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DTC pharmacophores bind extracellular Cu or interact with Cu already
incorporated into the cell. Ongoing efforts to differentiate these me-
chanistic details are important for informing this promising strategy to
overcome limitations of current prostate cancer therapies.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
The authors acknowledge support from the Duke Cancer Institute
P30 Cancer Center Support Grant (NIH CA014236),
a Foerster-
Bernstein Postdoctoral Fellowship for S.B., and a scholarship from the
Royal Government of Thailand‘s Development and Promotion of
Science and Technology Talents Project for P.W.
Fig. 4. IC50 in µM of HPD and RPD in LNCaP (blue) and PC3 (red) cell lines.
Inset: Representative plot of % viability vs prodrug concentration. The plot
presented is for HPD in LNCaP (circle) and PC3 cell lines (square).
Appendix A. Supplementary data
lysate even up to 48 h (Figs. 3 and S6–S9). Monitoring the cleavage in
lysates shows that HPD is more selective toward PSA-expressing cells
and is stable against nonselective proteases.
Supplementary data (all experimental details and supporting data
figures) to this article can be found online at https://doi.org/10.1016/j.
bmcl.2020.127148.
The anticancer activity of the prodrugs was evaluated in LNCaP and
PC3 cell lines. The cells were treated with HPD or RPD and 1 µM CuSO4
since copper is essential for cytotoxicity induced by DTC (Fig. S10).12
Inhibition of cell proliferation was monitored at 24 h using the re-
sazurin assay. RPD displayed an IC50 of 0.77 µM in LNCaP cells and
1.8 µM in PC3 cells (Fig. 4). The D-Ser variant of RPD did not show any
considerable antiproliferative activity at the concentrations tested (Fig.
S10). These data confirm that neither the whole prodrug construct nor
this level of supplemental Cu is toxic to these cells, and support the
conclusion that toxicity is induced upon prodrug cleavage and sub-
sequent interaction with Cu. Based on the cell lysate studies and above
data we conclude that RPD is non-selectively cleaved by cellular pro-
teases. Since RSSYYS peptide was derived from a chymotrypsin sub-
strate it might be sensitive to other chymotrypsin-like serine proteases.
On the other hand, HPD displayed an IC50 of 1.4 µM in LNCaP and
11.2 µM in PC3 cells. While RPD demonstrated a lower IC50, HPD
demonstrated selectivity consistent with the lysate studies. With this
approach we have successfully developed a PSA targeting prochelator
of DTC.
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