Exploring the structural requirements for inhibition of the ubiquitin E3 ligase breast cancer associated protein 2 (BCA2) as a treatment for breast cancer

Article abstract of DOI:10.1021/jm901757t

The zinc-ejecting aldehyde dehydrogenase (ALDH) inhibitory drug disulfiram (DSF) was found to be a breast cancer-associated protein 2 (BCA2) inhibitor with potent antitumor activity. We herein describe our work in the synthesis and evaluation of new series of zinc-affinic molecules to explore the structural requirements for selective BCA2-inhibitory antitumor activity. An N(C - S)S - S motif was found to be required, based on selective activity in BCA2-expressing breast cancer cell lines and against recombinant BCA2 protein. Notably, the DSF analogs (3a and 3c) and dithio(peroxo)thioate compounds (5d and 5f) were found to have potent activity (submicromolar IC₅₀) in BCA2 positive MCF-7 and T47D cells but were inactive (IC₅₀ > 10 μM) in BCA2 negative MDA-MB-231 breast cancer cells and the normal breast epithelial cell line MCF10A. Testing in the isogenic BCA2 +ve MDA-MB-231/ER cell line restored antitumor activity for compounds that were inactive in the BCA2 -ve MDA-MB-231 cell line. In contrast, structurally related dithiocarbamates and benzisothiazolones (lacking the disulfide bond) were all inactive. Compounds 5d and 5f were additionally found to lack ALDH-inhibitory activity, suggestive of selective E3 ligase-inhibitory activity and worthy of further development.

Full text of DOI:10.1021/jm901757t

J. Med. Chem. 2010, 53, 2757–2765 2757
DOI: 10.1021/jm901757t
Exploring the Structural Requirements for Inhibition of the Ubiquitin E3 Ligase Breast Cancer
Associated Protein 2 (BCA2) as a Treatment for Breast Cancer
†
‡
†
‡
†
ꢀ †
Ghali Brahemi, Fathima R. Kona, Annalisa Fiasella, Daniela Buac, Jitka Soukupova, Andrea Brancale,
Angelika M. Burger,*,‡ and Andrew D. Westwell*,†
†Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB, Wales, United Kingdom, and
‡Barbara Ann Karmanos Cancer Institute, Department of Pharmacology, Wayne State University, Detroit, Michigan
Received September 1, 2009
The zinc-ejecting aldehyde dehydrogenase (ALDH) inhibitory drug disulfiram (DSF) was found to be a
breast cancer-associated protein 2 (BCA2) inhibitor with potent antitumor activity. We herein describe
our work in the synthesis and evaluation of new series of zinc-affinic molecules to explore the structural
requirements for selective BCA2-inhibitory antitumor activity. An N(CdS)S—S motif was found to be
required, based on selective activity in BCA2-expressing breast cancer cell lines and against recombinant
BCA2 protein. Notably, the DSF analogs (3a and 3c) and dithio(peroxo)thioate compounds (5d and 5f)
were found to have potent activity (submicromolar IC50) in BCA2 positive MCF-7 and T47D cells but
were inactive (IC50>10 μM) in BCA2 negative MDA-MB-231 breast cancer cells and the normal breast
epithelial cell line MCF10A. Testing in the isogenic BCA2 þve MDA-MB-231/ER cell line restored
antitumor activity for compounds that were inactive in the BCA2 -ve MDA-MB-231 cell line. In
contrast, structurally related dithiocarbamates and benzisothiazolones (lacking the disulfide bond) were
all inactive. Compounds 5d and 5f were additionally found to lack ALDH-inhibitory activity, suggestive
of selective E3 ligase-inhibitory activity and worthy of further development.
Introduction
in clinical trials for cancer.5 Other components of the ubiquitin-
proteosome system have provided additional targets for cancer
therapy, leading to the development of the 26S proteosome
inhibitor bortezomib as an approved agent for the treatment of
multiple myeloma.6
The balance between the production of new cellular pro-
teins and their targeted degradation is part of the normal
choreographed life cycle of the cell, and numerous previous
studies have elucidated the role of the ubiquitin-proteosome
system in the highly regulated degradation of >80% of cellular
proteins.1 Polyubiquitination (tagging by the small protein
ubiquitin) of target protein substrates is carried out by three
classes of enzymes, of which the diverse and abundant ubiqui-
tin E3 ligase family catalyze the final mechanistic step of
ubiquitin transfer to specific lysyl residues of target proteins
prior to proteosomal degradation.2 Ubiquitin E3 ligase bio-
logy presents a number of therapeutic targets, since deregu-
lated E3 ligase activity is known to be a feature of proliferative
diseases such as cancer.3 An example of a well-studied E3 ligase
whose deregulation has been exploited for potential therapeu-
tic benefit is murine double minute 2 protein (Mdm2a), which
targets the tumor suppressor protein p53 for destruction.4
Inhibition of the protein-protein interaction between Mdm2
and p53 for therapeutic gain is illustrated by the development
of the Nutlin class of antitumor agents currently being studied
BCA2 is an E3 ubiquitin ligase that was isolated from an
invasive breast cancer cell line, and has been shown to be
highly expressed in 56% (530/945) of invasive breast cancers,
but not in most normal tissues examined.7 Down-regulation
of BCA2 has been shown to inhibit breast cancer cell growth
and invasiveness.7 BCA2 is expressed in the cytoplasm and
nucleus, and nuclear BCA2 correlates with positive estrogen
receptor status (p<0.004). The classification of BCA2 as a
Really Interesting New Gene (RING)-finger protein8 with
ubiquitin E3 ligase activity implicates the presence of a double
Zn2þ-binding motif arranged in a cross-brace structure
(Figure 1), termed RING-finger, as being essential for ubiqui-
tin E3 ligase catalytic activity. The critical role of the Zn2þ
-
containing RINGdomain ofBCA2 hasbeen demonstrated by
point mutation of key zinc-binding cysteine residues leading
to the complete loss of enzyme activity.7,9 BCA2 (also known
as Rabring7) has been found to complex with a cytoplasmic
binding partner Rab7, a small GTPase involved in cellular
endocytosis and trafficking of oncogenic receptor tyrosine
kinases (such as epidermal growth factor receptor (EGF-R))
for destruction in the lysosome.10,11 Hence, targeting BCA2
within breast cancer cells may allow Rab7 to fulfill its func-
tion in tyrosine kinase receptor degradation, preventing the
receptor recycling and sustained mitogenic signaling knownto
contribute to the development of resistance to tyrosine kinase-
inhibitory therapeutics.10 Taken together, the data outlined
*Authors to whom correspondence should be addressed: Chemistry;
E-mail, WestwellA@cf.ac.uk. Phone: þ44 2920 875800. Biology;
E-mail, burgera@karmanos.org. Phone: þ1 313 576 8302.
a Abbreviations: ABC, ATP binding cassette; ALDH, aldehyde
dehydrogenase; BAAA, bodipy-aminoacetaldehyde; BCA2, breast
cancer associated protein 2; CHX, cycloheximide; DEAB, diethylamino-
benzaldehyde; DSF, disulfiram; EGF-R, epidermal growth factor recep-
tor; Mdm2, murine double minute 2 protein; MTT, methyltetrazolium;
PIFA, phenyliodonium di(trifluoroacetate); RING, Really Interesting
New Gene.
r
2010 American Chemical Society
Published on Web 03/11/2010
pubs.acs.org/jmc

2758 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Brahemi et al.
range of potential Zn2þ ejectors including DSF, has recently
been described.17
A further “symmetrical” disulfide structurally related to
the DSF-like compounds—4,40-dithiodimorpholine (4,40-
dimorpholinedisulfide)—was synthesized in order to explore
the structural requirement of a dithioester function for
BCA2-inhibitory antitumor activity. 4,40-Dithiodimorpho-
line has previously been studied as a potential lead com-
pound for the treatment of neoplastic lesions of the cervix by
targeting zinc-binding domains of the E6 oncoprotein of
human papillomavirus.18
The carbamo(dithioperoxo)thioate series was designed as
a series of “unsymmetrical” disulfides containing a cleavable
S-S bond and the N(CdS)S—S group characteristic to
DSF. This series presented the opportunity to vary the R
and R1 groups (Figure 2) to explore SAR requirements for
BCA2-inhibitory antitumor activity. It is noteworthy that
carbamo(dithioperoxo)thioates (mixed disulfides derived
from DSF) have previously been reported as inhibitors of
human mitochondrial19 and sheep liver20 ALDH, most likely
via zinc-ejection. The registered drug status of DSF itself is
based on its inhibitory activity against ALDH.
Figure 1. Cross brace structure of the RING-H2 domain as found
in the ubiquitin E3 ligase BCA2. C, cysteine (purple); H, histidine
(green); Zn2þ, zinc ions (yellow); gray, number of other amino acids
between the cysteine and histidine motifs.
In order to further explore the structural requirements for
BCA2-mediated antitumor activity, we synthesized a diverse
series of dithiocarbamates. The dithiocarbamates bear close
structural similarities with DSF analogs, but lack the S-S
single bond, making this series an ideal candidate for explor-
ing the importance of the disulfide bond in mediating anti-
tumor activity.
Benzisothiazol-3-one derivatives have been reported to
have a zinc-ejection effect, and have been tested for antire-
troviral activity and their ability to eject Zn2þ from the HIV
nucleocapsid zinc-finger protein NCP7.21 In addition, we
were interested in the benzisothiazolone structure as a
known zinc-ejector lacking a labile S-S bond.
Synthesis of DSF Analogs (Bis(dialkylthiocarbamoyl)-
disulfides; 3a-c) and 4,40-Dithiodimorpholine (4). DSF (1) is
commercially available (Sigma-Aldrich). Other DSF ana-
logue thiuram disulfides (3a-c) were synthesized in low to
moderate yields via a one-pot reaction (General Method A,
Experimental Section, Scheme 1), by mixing the appropriate
secondary amine (2a-c) with carbon disulfide under basic
conditions (aqueous sodium hydroxide). Without further
purification, the (presumed) intermediate dithiocarbamic
acid was oxidized to the required thiuram disulfide in low
to moderate yields as previously described using either
aqueous iodine/potassium iodide22 (compounds 3a and 3b)
or hydrogen peroxide23 in acetic acid (compound 3c). More
recently, an efficient synthesis of thiuram disulfides has been
reported using carbon tetrabromide to promote reaction of
amines with carbon disulfide and subsequent disulfide bond
formation.24 Alternative methods for the chemical oxidation
of thiols to disulfides are also available, for example, the
use of bromine on hydrated silica.25 The structurally related
dithiodimorpholine (4) was synthesized as previously de-
scribed,18 via treatment of morpholine with sulfur mono-
chloride under basic conditions (Scheme 1).
above suggest that BCA2 could prove an important thera-
peutic target within the E3 ubiquitin ligase class for the future
treatment of breast cancer.
Given the crucial role of Zn2þ ions in the catalytic RING
domain of BCA2, a series of “zinc-ejecting” compounds from
the National Cancer Institute database have been screened for
their ability to inhibit BCA2 activity in BCA2-expressing
breast cancer cell lines such as MCF-7. These studies have
led to the identification of DSF (NSC25953),10,12,13 a regis-
tered drug for the treatment of alcoholism by virtue of
additional ALDH1 activity, as a potent BCA2-inhibitory
antitumor agent. In this paper, we describe the synthesis and
antitumor evaluation of four series of novel “zinc-affinic”
agents, in order to optimize selective activity against recom-
binant BCA2 and BCA2-expressing breast cancer cell lines.14
From this antitumor data, we derived SAR insight into
BCA2-inhibitory pharmacophore requirements, leading to
the identification of potent and selective BCA2-inhibitory
antitumor agents without accompanying ALDH-inhibitory
activity.
Chemistry. Rationale for Choice of Candidate Compound
Series. Four series of molecules having the potential ability
to remove Zn2þ ions from the RING domain of BCA2 were
synthesized, based on structures where the core group is
known for zinc-binding activity (Figure 2). Initial studies
focused on agents structurally related to the lead compound
DSF (bis(diethylaminothiocarbonyl)disulfide; tetraethyl-
thiuram disulfide), and derived from different secondary
amine starting materials, in order to explore the structure-
activity relationships (SAR) around DSF with respect to
BCA2-inhibitory antitumor activity.
The ability of compounds such as DSF (and its pyrrolidine
and piperidine analogs) to form chelation complexes with Zn2þ
has previously been reported.15,16 An alternative mechanism
for removal of Zn2þ ions from the RING domain of BCA2 is
the process of zinc-ejection, where modification of active-site
cysteine residues causes ejection of Zn2þ from the active site. In
the case of DSF and close analogues, the zinc-ejection process
appears to be favored for interactions with zinc-binding
proteins. For example, the inhibition of the histone demethyl-
ase JMJD2A by zinc-ejection, following treatment with a
Synthesis of Carbamo(dithioperoxo)thioates. A series of
carbamo(dithioperoxo)thioates (5a-j) that are structurally
related to DSF analogues were synthesized in order to extend
our understanding of SAR within the class of BCA2-inhibitory
zinc-ejectors and to further explore whether an S-S single bond
is necessary for BCA2-inhibitory antitumor activity. The one-
pot synthesis of carbamo(dithioperoxo)thioates is essentially

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2759
Figure 2. Potential zinc-binding BCA2 inhibitors.
Scheme 1a
Scheme 3a
a Reagents: (i) H2O, 5-18 h.
a Reagents: (i) CS2, NaOH (aq); (ii) I2, KI (aq) or H2O2, AcOH;
(iii) S2Cl2, NaOH, hexane/H2O, 0 °C.
Synthesis of Benzisothiazolones. The general procedure
for the synthesis of benzisothiazolones was adopted from
the previously reported method of Correa et al. (General
Method D, Experimental Section; Scheme 4).30 The first step
of the synthesis involves formation of the amide (12a-g) by
reacting methyl thiosalicylate (10) with different primary
amines (11a-g), followed by oxidative ring closure using
phenyliodonium di(trifluoroacetate) (PIFA) to give the
required benzisothiazolones (13a-g) in low to moderate
overall yield.
Scheme 2a
Biological Results
Screening of Zinc Affinic Compounds in BCA2þ and
BCA2low/- Cells. A total of 50 compounds representing the
four different classes of sulfur-based structures described
above were evaluated for antiproliferative activity by
methyltetrazolium (MTT) assay in the BCA2-positive breast
cancer cell lines MCF-7, T47D, MDA-MB-231/ER, the
BCA2-low/negative (low/-) breast cancer cell line MDA-
MB-231 and the normal breast epithelial cell line MCF10A
(Figure 3A,B). MDA-MB-231/ER is an isogenic subclone
of MDA-MB-231 generated by transfection with ERR. ERR
expression activates BCA2 expression via transcriptional
regulatory mechanisms.31 MCF10A was used as a surrogate
for assessing normal tissue toxicity of the zinc ejection
agents. A compound was considered active if the IC50
was <10 μM. We selected 10 μM as a cutoff point, because
our lead compound DSF has an IC50 in BCA2 positive cells
between 0.1 and 0.3 μM. Potential selectivity for BCA2
inhibition by a candidate compound was considered if
growth inhibition was observed in BCA2 positive cells, but
not MDA-MB-231 or MCF10A cells. None of the 29 tested
dithiocarbamates (9a-9ac) or the seven benzisothiazolones
(13a-g) showed activity in any of the breast cancer cell lines
(data not shown). All of the ten dithioperoxothioates (5a-j)
were active with IC50’s ranging from 0.15 to 2.75 μM in
BCA2þ cells, and 0.9 to >10 μM in MDA-MB-231 or
MCF10A cells (Table 1, Figure 3B). However, only 5d and
a Reagents: (i) R1SH, EtOH, reflux, 16 h.
straightforward, and is based on previous described methods
(General Method B, Experimental Section, Scheme 2).26,27
Briefly, stirring commercially available DSF (or synthetic
analogues 3a or 3c) with the appropriate thiol in ethanol
under refluxing conditions (16 h) yielded the required
carbamo(dithioperoxo)thioate product (5a-j, Scheme 2) in
low to moderate yield following purification by column
chromatography using mixtures of ethyl acetate and hexane
as eluent.
Synthesis of Dithiocarbamates. Dithiocarbamates (9a-ac)
were readily synthesized in a one-pot reaction according to
the method of Azizi et al.28,29 (General Method C, Experi-
mental Section; Scheme 3). The appropriate secondary
amine (2a, 2c, 6a-d) was mixed with carbon disulfide and
the required alkyl halide (7a-e) or Michael acceptor (8a-b)
in equimolar ratios in water (5-18 h). In some instances,
extraction of the aqueous phase using ethyl acetate gave rise
to the pure product directly after solvent evaporation,
whereas on other occasions, it was necessary to purify
products by column chromatography using mixtures of ethyl
acetate and hexane as eluent. Spectroscopic and analytical
data of dithiocarbamate products 9a-ac are recorded in
Supporting Information.

2760 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Brahemi et al.
Scheme 4a
a Reagents: (i) AlMe3, CH2Cl2, 0 °C to room temp.; (ii) PhI(OCOCF3)2, CF3CO2H, CH2Cl2.
Table 1. Activity of DSF Analogues and Carbamo(Dithioperoxo)Thioates against Human Breast Cancer and Normal Cell Linesa
mean IC50 ( SD (μM)b in cell linesc
compound
MCF-7
MDA-MB-231
MDA-MB-231/ER
T47D
MCF10A
1 (DSF)
3a
3b
3c
0.1 ( 0.01
0.03 ( 0.001
0.35 ( 0.05
0.38 ( 0.03
>10
>10
>10
0.32 ( 0.14
0.59 ( 0.12
0.29 ( 0.1
0.25 ( 0.1
>10
0.17 ( 0.03
0.27 ( 0.02
0.33 ( 0.02
0.28 ( 0.02
>10
10 ( 0.2
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
3.0 ( 0.4
>10
>10
4
5a
5b
5c
0.39 ( 0.28
0.4 ( 0.3
0.96 ( 0.03
7.4 ( 2.6
6.0 ( 2.8
>10
0.25 ( 0.1
0.3 ( 0.13
2.1 ( 0.43
0.35 ( 0.28
0.28 ( 0.17
2.75 ( 0.17
0.55 ( 0.12
0.39 ( 0.17
2.3 ( 0.22
0.25 ( 0.08
0.23 ( 0.01
0.15 ( 0.02
0.20 ( 0.02
0.23 ( 0.02
0.18 ( 0.02
0.30 ( 0.03
0.15 ( 0.01
0.35 ( 0.03
0.25 ( 0.03
1.35 ( 0.02
0.45 ( 0.25
0.43 ( 0.1
0.3 ( 0.28
0.5 ( 0.24
0.5 ( 0.2
0.65 ( 1.2
0.6 ( 0.1
1.4 ( 1.8
5d
5e
0.96 ( 0.01
>10
5f
5g
5h
5i
6.3 ( 2.6
8 ( 1.6
6.7 ( 2.5
4.7 ( 3.9
5j
a Determined by MTT assay (72 h drug exposure); see ref 42 for details. b Compounds tested in triplicate, data expressed as mean values of three independent
experiments. c Cancer cell line origin: MCF-7 (breast; BCA2 þve; ER þve), MDA-MB-231 (breast; BCA2 low/-ve; ER -ve), MDA-MB-231/ER (isogenic stable
ER transfected clone of MDA-MB-231, BCA2þve, ER þve), T47D (breast, BCA2 þve, ER þve). MCF10A, normal breast epithelial cell line.
5f were active in BCA2þ, but inactive in BCA2low/- cells; 5d
was more potent than 5f (Table 1). Similarly, DSF analogues
autoubiquitination, hence the ubiquitination efficacy of such
ligases can be studied in absence of a functional substrate.7,9
Recombinant wild-type human BCA2 produced in E. coli
was used together with ATP, a rabbit E1 and the recombi-
nant human E2 UbcH5b. Compounds active toward breast
cancer cell lines expressing BCA2 (MCF-7, T47D, MDA-
MB-231/ER), but inactive in BCA2low/- MDA-MB-231
breast cancer and normal MCF10A cells (Figure 4A), plus
DSF were added to the ubiquitination reaction at concen-
trations of 5 and 50 μM (Figure 4B). Because this assay
employs highly purified, recombinant BCA2, E1 and E2
enzymes and drugs are only incubated with the enzyme
mixture for 30 min; therefore, drug concentrations higher
than those inhibiting cell growth to 50% or 100% in a 5 day
assay were used. Autoubiquitination of BCA2 was detected
with antiubiquitin antibodies. In the event of the autoubi-
quitination, a high molecular weight polyubiquitin signal is
seen (Figure 4B, 5 μM reactions) and low molecular weight
ubiquitin bands are weak or absent. In contrast, when
autoubiquitination is inhibited, ubiquitin is not attached to
the E3 ligase and shows strong bands, whereas polyubiquitin
signals are absent (Figure 4B, 50 μM reactions). All putative
BCA2 inhibitory compounds were able to inhibit the auto-
ubiquitination of recombinant BCA2 at a concentration of
50 μM, but not 5 μM (Figure 4B). Identical experiments
performed using the recombinant RING-E3 ligase Mdm2
did not show inhibition of Mdm2 autoubiquitination (data
not shown). These data suggest that our cell-based screening
system was predictive of direct BCA2 enzyme inhibition.
Effects of Zinc Ejecting Compounds 3a and 5d on BCA2
Protein Stability in Cells. To confirm that inhibition of the
(3a-c) were very active in BCA2þ cells and inactive in
low/-
BCA2
cells with the exception of 3b (Table 1). 4,40-
Dithiomorpholine (4) was inactive in both BCA2þ and
BCA2low/- cells. The most potent DSF derivative was 3a
with an IC50 in MCF-7 of 0.03 μM (Table 1, Figure 3A). On
the basis of their activity profile compounds 3a, 3c, 5d, and 5f
were considered for further biological testing in comparison
to DSF. Representative growth curves showing the antipro-
liferative activity of 3a and 5d in the BCA2-positive and
BCA2-negative cell lines are depicted in Figure 3A,B.
To test our hypothesis that the DSF analogues and
carbamo(dithioperoxo)thioates act through a zinc ejection
mechanism, MCF-7 cells were treated with compounds 1, 3a,
and 5d in the presence of zinc(II) chloride or with zinc
chloride alone (Figure 3C). When zinc chloride was added
at a range of concentrations between 600 nM and 75 μM to
MCF-7 cells exposed to the respective IC50’s of 1, 3a, and 5d
(Table 1), their growth inhibitory activity was completely
abolished (Figure 3C). Zinc chloride alone had no effect on
the growth of MCF-7 cells. These results are consistent with
our working hypothesis that the active DSF and carbamo-
(dithioperoxo)thioate analogues are causing inhibition of
BCA2-mediated cell growth via a reversible zinc ejection
mechanism.
Evaluation of Lead Compounds for Inhibition of BCA2 E3
Ligase Activity/Autoubiquitination. Lead compounds from
the cell-based screening procedure were tested for their
potential to inhibit the E3 ligase activity of BCA2. A hall-
mark of RING-finger ubiquitin E3 ligases is the capability of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2761
(Figure 5A,B, lanes 2). The addition of the protein synthesis
inhibitor cycloheximide (CHX) to 3a or 5d showed steady-
state levels (Figure 5A) or further reduction of BCA2
(Figure 5B). Importantly, when the proteasome inhibitor
N-(benzyloxycarbonyl)-leucinylleucinylleucinal (MG-132)32
was added to the zinc ejector and CHX combination or the
zinc ejector treatment alone (Figure 5A,B, lanes 4-5), BCA2
was stabilized and accumulated even in absence of protein
synthesis (Figure 5A,B, lanes 4). These studies show that
treatment with 3a or 5d leads to down-regulation of BCA2
protein, and that this process is dependent on the ubiquitin-
proteasome pathway. If proteasomal degradation of ubiqui-
tinated BCA2 protein is inhibited by 14 (MG-132), the E3
ligase accumulates (Figure 5).
Testing for ALDH Inhibition. To study whether novel
BCA2 inhibitory disulfide analogues 3a and 3c, or the
dithioperoxothioates 5d and 5f, retained the ability to inhibit
ALDH1 like DSF, we performed an ALDH1 inhibition test.
To detect ALDH1 positive cancer cells, a flow cytometry
based method was recently developed using the leukemia cell
line K562, known to have high levels of ALDH1.33 Thus, we
analyzed the ability of DSF, 3a, 3c, 5d, and 5f to inhibit the
ALDH1-positive cell fraction in K562 cells compared to a
known inhibitor of ALDH activity, diethylaminobenzalde-
hyde (DEAB) (Table 2). While DSF and 3a inhibited
ALDH1 to 57% and 79% respectively, 3c, 5d, and 5f showed
little inhibition of this enzyme. DSF analogue 3c inhibited
only a negligible fraction of ALDH1 (3.4%) and is therefore
the most selective BCA2 E3 ligase inhibitor among the series
of disulfides tested in this study. Dithio(peroxo)thioate
compounds 5d and 5f also exhibited low levels of ALDH
inhibition (9.8% and 8.6% relative to control, respectively).
Discussion
During this study, we aimed to identify potent and selective
BCA2-inhibitory antitumor agents devoid of the ALDH-
inhibitory activity characteristic to DSF. Considerable pro-
gress has been made toward these goals. In terms of the
identification of potent and selective activity in BCA2-expres-
sing breast cancer cell lines, the thiuram disulfide compound
3a was found to be the most active (IC50 = 30 nM) in the
MCF-7 cell line, with no activity (IC50 > 10 μM) in the BCA2
negative MDA-MB-231 breast cancer cell line and MCF10A
normal breast epithelial cells. The activity of3ainMCF-7 cells
was found to exceed that of DSF itself (IC50 = 100 nM). The
similar activity profile of the DSF analogue 3a to DSF is
perhaps not surprising given its structural similarity; however,
it is notable that the other closely related DSF analogues were
substantially less potent and/or less selective (3b) compared to
DSF and 3a. The lack of activity for dithiodimorpholine (4) is
suggestive of the need for a dithioester function for BCA2
inhibitory activity.
Figure 3. (A) Representative growth curves for compound 3a in
BCA2-positive and BCA2-negative cell lines from Table 1. (B)
Representative growth curves for compound 5d in BCA2-positive
and BCA2-negative cell lines from Table 1. (C) Effects of com-
pounds 1, 3a, and 5d in the presence of zinc chloride. To assess
whether the addition of Zn2þ can rescue the growth inhibitory
effects of zinc ejecting compounds in BCA2-positive MCF-7 cells,
various concentrations of ZnCl2 were added together with IC50
concentrations of 1, 3a, or 5d in MCF-7 cells. The value set as 100%
is vehicle control treated MCF-7 cells without ZnCl2 and drug.
ZnCl2 did prevent growth inhibition by compounds 1, 3a, and 5d at
all concentrations tested.
Among the series of dithio(peroxo)thioates (5a-j) structu-
rally related to DSF, two compounds (5d and 5f) stand out as
having selective activity in the submicromolar IC50 range
against the BCA2-expressing breast cancercell linescompared
to BCA2-negative/low MDA-MB-231 and normal MCF10A
cells. Surprisingly, however, other dithioperoxothioate com-
pounds more closely related in structure to DSF (such as 5a
and 5e) did not display selective activity against BCA2-
expressing cancer cell lines. The selectively active compounds
5d and 5f reinforce ourhypothesis thatanN(CdS)S—Sgroup
is required for selective activity in the BCA2-expressing breast
BCA2 catalytic E3 ligase activity by zinc ejecting compounds
seen in the autoubiquitination experiments is of relevance
under physiological conditions, we performed cellular assays
with the BCA2þ MCF-7 breast cancer cell line (Figure 5). We
selected the most potent compound from each of the DSF
analogue (3a) and dithioperoxothioate series (5d). MCF-7
cells, when exposed for 16 h (approximate half-life of
BCA27) to both compounds at their IC100 concentrations,
showed a marked reduction of BCA2 protein levels

2762 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Brahemi et al.
Figure 4. (A) Western blot showing expression of endogenous BCA2 in T47D and the isogenic MDA-MB-231 parental and MDA-MB-231/
ER breast cancer cell lines. Anti-BCA2 antibodies were developed by us and used as previously described.7 β-actin was used as equal loading
control. (B) Western blot analysis of BCA2 autoubiquitination in the presence of disulfide analogues at 5 and 50 μM of drug dissolved in
dimethylsulfoxide. The membrane was probed with antiubiquitin antibodies. Shown are the ubiquitin signal (8 kD) and high molecular weight
polyubiquitinylated BCA2 (>98 kD). Polyubiquitinated proteins are typified by a high molecular weight smear.
Table 2. ALDH Inhibition in K562 Cells for Lead Compounds
ALDHþ
K562 cells (%)
compound
(15 μM)
ALDH
inhibition (%)
BAAA (positive control)
DEAB (negative control)
1 (disulfiram)
40.39 ( 2.21
0.53 ( 0.11
17.26 ( 2.21
8.3 ( 1.7
39.01 ( 0.8
36.47 ( 3.82
36.9 ( 3.15
0
98.68
57.26
79.45
3.4
3a
3c
5d
5f
9.8
8.6
Figure 5. Western blots of endogenous BCA2 protein expression in
MCF-7 breast cancer cells treated for 16 h with the BCA2 ligase
inhibitors 3a (A) and 5d (B) in the presence and absence of
the protein synthesis inhibitor CHX and the proteasome inhibitor
14. (A) MCF-7 cells treated with vehicle only (DMSO, lane 1);
compound (cpd) 3a at its 100% growth inhibitory concentration
(0.25 μM, lane 2); 3a and CHX (lane 3); 3a, CHX and 14 (lane 4);
and 3a plus 14 (lane 5). β-actin was used as equal loading control.
(B) MCF-7 cells treated with vehicle only (DMSO, lane 1); com-
pound (cpd) 5d at its 100% growth inhibitory concentration (1 μM,
lane 2); 5d and CHX (lane 3); 5d, CHX and 14 (lane 4); and 5d plus
14 (lane 5). β-actin was used as equal loading control.
vomiting, and circulatory collapse. Interestingly, studies on
the role of ALDH1 as a marker of normal and malignant
mammary stem cells associated with a poor clinical outcome34
suggest that retention of ALDH-inhibitory activity alongside
BCA2 inhibition may not necessarily be an adverse property
for the development of new anticancer agents.
By virtue of its zinc-12 and copper-35binding properties,
DSF has been shown to exhibit a number of interesting
antitumor properties. The antiproliferative and pro-apoptotic
effects of DSF have been attributed (in various cancer model
systems) to include inhibition of proteasome activity,35,36
nuclear factor kB,37 ATP binding cassette (ABC) drug trans-
porter protein activity,38,39 and angiogenesis.40 Pharmaco-
logical profiling of DSF using human tumor cell lines and
tumor cells from patients has provided further evidence of
antitumor potential.41
The literature around the antitumor activity of DSF is
strongly indicative of a diversity of molecular targets and
drug properties, mediated largely through the binding of
metal ions (zinc and copper) and the interaction of DSF with
key cysteine residues in target proteins.42 Given that the
literature on interactions of thiuram disulfides and dithioper-
oxothioates with zinc-dependent proteins is dominated by
zinc-ejection effects,17,42 we hypothesize that inhibition of
BCA2 is likely to be related to reversible zinc-ejection from
the enzyme active site via modification of key cysteine resi-
dues. This hypothesis is supported by our zinc chloride rescue
data shown in Figure 3C. Despite its rather indiscriminate
biochemical and pharmacological profile, DSF is notable
for being very well tolerated following long-term treatment
at relatively high doses (routinely 300-500 mg per day), and
for its high oral bioavailability (>80% bioavailability, with
approximately 20% of drug remaining in the body for
cancer celllines, and the cellulardatawerefurther validatedby
the ability of compounds 3a, 3c, 5d, and 5f, plus DSF, to
abolish the autoubiquitination activity of recombinant BCA2
at 50 μM (Figure 4). Moreover, for the most potent com-
pounds of each class, 3a and 5d, we could show that inhibition
of isolated enzyme activity translates into down-regulation of
endogenous BCA2 at physiologically relevant drug concen-
trations (IC100) that is dependent on its degradation through
the ubiquitin-proteasome system (Figure 5). The importance
of reversible zinc ejecting properties of 3a and 5d similar to
that of DSF (compound 1) for their mode of action, was
demonstrated by the zinc chloride rescue studies shown in
Figure 3C. When Zn2þ ions were added to 1, 3a, or 5d
concentrations that inhibit the growth of the BCA2 positive
cell line MCF-7 to 50% under physiological Zn2þ levels, their
growth inhibitory activity was abolished even at the lowest
concentration tested.
At the outset of our studies, we were interested in the
discovery of selective BCA2-inhibitory antitumor agents that
lacked the ALDH-inhibitory activity that is the basis for the
registered drug status of DSF in the treatment of alcoholism.
Although DSF is a safe drug, the ingestion of alcohol causes a
“hangover” effect leading to symptoms such as flushing of the
skin, accelerated heart rate, shortness of breath, nausea,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2763
1-2 weeks postingestion).42 It is likely that the most active
thiuram disulfides (3a and 3c) and dithio(peroxo)thioates (5d
and 5f) synthesized in this study will also exhibit a diverse
spectrum of antitumor activity, given their structural similar-
ity to DSF. Further studies are ongoing to further define the
antitumor potential of the lead compounds synthesized here,
and their associated stability and toxicology.
a further 30 min, hydrogen peroxide (0.37 mL of 27.5% solution,
3.0 mmol) was added dropwise while maintaining the reaction
temperature at 0 °C. n-Hexane (2.5 mL) was then added leading
to the formation of precipitate after 30 min that was collected by
filtration and washed with hexane and 2% aqueous acetic acid.
The crude product was recrystallized (toluene/hexane) to give the
product as a white solid in 39% yield. Mp 135-137 °C (lit.43
137-139 °C). 1H NMR (CDCl3) δ 3.98 (8H, m, NCH2), 2.17 (4H,
m), 2.01 (4H, m). 13C NMR (CDCl3) δ 189.31 (CdS), 57.12
(NCH2), 51.10 (NCH2), 26.75, 24.42. m/z (EIþ) 292 (Mþ), 146.
Synthesis of Dithiodimorpholine (4).18 A solution of sulfur
monochloride (3.20 mmol) in hexane (10 mL) was slowly added
to a rapidly stirred two-phase mixture of morpholine (3.20
mmol), water (20 mL), hexane (10 mL), and sodium hydroxide
(4.22 mmol) at 0 °C. The reaction mixture was then stirred at
room temperature for a further 1 h; then, the separated aqueous
layer was washed with hexane (20 mL) and the combined
organic layers washed with 1 N HCl (aq) (10 mL), then water
(10 mL). The organic layers were dried (Na2SO4), followed by
concentration in vacuo (<40 °C) and recrystallization from
n-hexane to give the product as a pale yellow solid in 65% yield.
Mp 117-118 °C (lit.18 118-120 °C). 1H NMR (CDCl3) δ 3.74
(8H, m, OCH2), 2.81 (8H, m, NCH2). 13C NMR (CDCl3) δ 67.44
(OCH2), 55.92 (NCH2). m/z (CIþ) 237 (Mþ þ 1).
Conclusions
The synthesis and evaluation of a variety of experimental
agents based on the lead compound DSF has uncovered new
antitumor agents with potent and selective antiproliferative
activity against the E3 ubiquitin ligase BCA2. Potent and
selective activity was observed in BCA2-expressing breast
cancer cell lines (e.g., compound 3a, 5d), and biochemical and
cellular studies with recombinant and endogenous BCA2,
respectively, confirmed the ability of active compounds to
inhibit the RING E3 ligase. In three cases (3c, 5d and 5f) new
BCA2-inhibitory molecules were found that lacked the ALDH-
inhibitory activity characteristic to DSF and analogues.
Experimental Section
GeneralMethod(B) for the Synthesis of Carbamo(dithioperoxo)-
thioates (5a-j). An equimolar mixture of the appropriate thiol
(9.0 mmol) and thiuram disulfide (9.0 mmol) in ethanol (20 mL)
were heated under reflux for 16 h. Following concentration of the
reaction mixture in vacuo, the pure product was obtained in low
yield after purification by column chromatography using hexane/
ethyl acetate as eluant.
Chemistry. Melting points were measured on a Griffin appa-
ratus and are uncorrected. Mass spectra were recorded on a
Bruker MicroTOF LC instrument or at the EPSRC National
Mass Spectrometry Centre (Swansea, U.K.). NMR spectra were
recorded on a Bruker AVANCE 500 MHz instrument; coupling
constants (J values) are in Hz. Merck silica gel 60 (40-60 μM)
was used for column chromatography. All commercially avail-
able starting materials were used without further purification.
Following purification, all new compounds were determined to
possess g95% purity as determined by melting point/NMR/
mass spectrometry and comparison with published data (for
previously reported compounds), or a combination of spectro-
scopic analysis and combustion analysis (% CHN tested in
duplicate) for new compounds. All compounds described were
synthesized in the laboratory except the initial lead compound
DSF, which is commercially available (Sigma-Aldrich, T1132,
>97%).
Hexyl Diethylcarbamo(dithioperoxo)thioate (5d).19 From
DSF and n-hexanethiol. Yellow oil, 9% yield. 1H NMR
(DMSO-d6) δ 4.02 (2H, q, J = 6.8 Hz, NCH2), 3.83 (2H, q,
J = 6.8 Hz, NCH2), 2.78 (2H, t, J = 6.7 Hz, SCH2), 1.59 (2H,
m), 1.40 (2H, m), 1.11-1.35 (10H, m), 0.92 (3H, t, J = 6.5 Hz,
CH3CH2CH2). 13C NMR (DMSO-d6) δ 51.08 (NCH2), 46.78
(NCH2), 37.97 (SCH2), 30.82 (CH2), 27.83 (CH2), 27.47 (CH2),
21.97 (CH2), 13.80 (CH2), 12.93 (CH3), 11.15 (CH3). m/z (CIþ)
266.1 (Mþ þ 1).
2-Hydroxyethyl Diethylcarbamo(dithioperoxo)thioate (5f).19
From DSF and β-mercaptoethanol. Thick green oil, 12% yield.
1H NMR (DMSO-d6) δ 4.84 (1H, t, J = 6.0 Hz, OH), 3.99 (2H,
q, J = 6.8 Hz, NCH2), 3.83 (2H, q, J = 6.8 Hz, NCH2), 3.60
(2H, q, J = 6.4 Hz, CH2OH), 2.89 (2H, t, J = 6.4 Hz, SCH2),
1.25 (6H, m, 2 ꢀ CH3). 13C NMR (DMSO-d6) δ 59.15
(CH2OH), 51.18 (NCH2), 46.82 (NCH2), 40.70 (SCH2), 12.95
(CH3), 11.17 (CH3). m/z (CIþ) 226.0 (Mþ þ 1).
General Method (A) for the Synthesis of Thiuram Disulfides
(3a-b).22 A solution of sodium hydroxide (160 mg, 4.0 mmol) in
water (4 mL) was added to a mixture of secondary amine
(4.0 mmol) in water (5 mL). After stirring at room temperature
for 20 min, carbon disulfide (0.24 mL, 4.0 mmol) was added, and
the mixture was stirred for a further 90 min. A solution of iodine
in potassium iodide was prepared by adding potassium iodide
(3.19 g, 19.2 mmol) and water (8 mL) to iodine (1.02 g,
4.0 mmol). The I2/KI(aq) solution was then added dropwise to
the reaction mixture, whereupon the solution initially turned
yellow followed by formation of a precipitate. After stirring for
a further 3 h, the precipitate was collected by filtration in vacuo
and washed with excess water and 1 M aqueous sodium thio-
sulfate. The crude product was purified by recrystallization
from petroleum ether and toluene to give the required thiuram
disulfide (3a-b) as a yellow solid in low to moderate yield.
Bis(piperidinylthiocarbonyl)disulfide (3a). From piperidine.
Yield = 28%. Mp 124 °C (lit.24 124-126 °C). 1H NMR
(CDCl3) δ 4.25 (8H, m, NCH2), 1.78 (12H, m). 13C NMR
(CDCl3) δ 192.88 (CdS), 55.92 (NCH2), 45.68, 24.41. m/z
(EIþ) 320 (Mþ), 147.
General Method (C) for the Synthesis of Dithiocarbamates
(9a-ac).28,29 A mixture of secondary amine (10 mmol), carbon
disulfide (10 mmol) and alkyl halide/Michael acceptor in water
(50 mL) were stirred at room temperature for 16 h. The crude
product was extracted using ethyl acetate (3 ꢀ 50 mL), dried
(MgSO4), and concentrated in vacuo. Purification by column
chromatography using ethyl acetate/hexane as eluant gave the
crude product (9a-9ac) in moderate yield. See Supporting
Information for details of spectroscopic data.
General Method (D) for the Synthesis of Benzisothiazolones
(13a-g).30 A solution of AlMe3 (11.52 mmol, 2.0 M in toluene)
was added dropwise to a cooled (0 °C) suspension of the amine
(11.52 mmol) in dichloromethane (30 mL). When the addition
was complete, the reaction mixture was allowed to warm to
room temperature and stirring was continued for 30 min.
Methylthiosalicylate (0.7 mL, 5.76 mmol) was added and the
mixture was heated under reflux (16 h), then allowed to cool and
slowly quenched with 5% aqueous HCl (20 mL). The resulting
precipitate was removed by vacuum filtration, and the filtrate
transferred to a separating funnel, where the organic phase was
collected. The aqueous phase was extracted using dichloro-
methane (3 ꢀ 15 mL), and the organic extracts were combined
Synthesis of Bis(pyrrolidinylthiocarbonyl)disulfide (3c).23
A
solution of pyrrolidine (0.49 mL, 6.0 mmol) in THF (1 mL) was
added to a solution of sodium hydroxide (240 mg, 6.0 mmol) in
water (2 mL) with stirring at 0 °C. Carbon disulfide (0.36 mL,
6.0 mmol) was then added dropwise and the mixture stirred at
0 °C for 30 min. Crushed ice (4.5 g) and acetic acid (0.9 mL) were
then added, leading to the formation of a white precipitate. After

2764 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Brahemi et al.
and washed with a saturated solution of NaHCO3 (15 mL) and
brine (15 mL), followed by drying (Na2SO4) and concentration
in vacuo. Column chromatography (ethyl acetate/hexane) gave
the intermediate amides (12a-g), which were used in the oxida-
tive ring closure step without further purification. A solution of
phenyliodonium di(trifluoroacetate) (PIFA) (249 mg, 0.58
mmol) in CH2Cl2 (9 mL) was added at 0 °C to a solution of
the intermediate benzamide (0.39 mmol) and TFA (0.09 mL,
1.16 mmol) in CH2Cl2 (6 mL). The reaction mixture was stirred
for 1 h; then, the solvent was removed in vacuo to yield a residue
that was purified using a column chromatography (ethyl acet-
ate/hexane). See Supporting Information for details of spectro-
scopic data.
5 or 50 μM zinc ejector in 30 μL reaction volume, and the
mixtures were incubated at room temperature for 30 min before
performing the ubiquitination assay. The protein-drug solu-
tions were then mixed with 3 μL of 20 mM ATP, 1 μL ubiquitin
(1 μg, Sigma, St. Louis, MO), 1 μL E1 (20 ng, rabbit E1,
Calbiochem, San Diego, CA), and 1 μL E2 (20 ng, recombinant
human UbcH5b, Boston Biochem, Boston MA) in 50 mM Tris
HCl pH 8.0 plus 3 μL 10ꢀ reaction buffer (500 mM Tris HCl
pH 8.0, 20 mM DTT, 50 mM MgCl2) and 21 μL H2O to obtain a
final reaction volume of 30 μL. Each mixture was incubated at
30 °C for 1 h, then 10 μL 4ꢀ SDS-gel loading buffer was added
and the mixture was boiled for 3 min, followed by separation of
reactions on a 4-20% gradient SDS-PAGE gel (Novex from
Invitrogen) and immunoblotting.
Biology. Cell Culture. The human breast cancer cell lines
T47D, MDA-MB-231, and the human chronic myelogenous
leukemia cell line K562 were obtained from American Type
Culture Collection (Manassas, VA). The breast cancer cell line
MCF-7 and the normal mammary epithelial cell line MCF10A
were obtained from the Karmanos Cancer Institute (formerly
known as Michigan Cancer Foundation) Cell Repository.
MDA-MB-231/ER was generated by transfecting an estrogen
receptor alpha (ERR) pcDNA3 construct into parental MDA-
MB-231 following the Lipofectamine method as described by
the manufacturer (Invitrogen, Carlsbad). Stable clones were
derived by G418 selection. ERR expression was confirmed by
immunostaining.7 All cancer cell lines were cultured in RPMI
1640 medium (Invitrogen, Carlsbad, CA) containing 10% heat-
inactivated fetal bovine serum (FBS, Hyclone from Fisher
Scientific, Pittsburgh, PA). MDA-MB-231/ER was cultured in
the presence of 1 mg/mL G418 (Invitrogen, Carlsbad, CA).
MCF10A cells were grown in mammary epithelial growth
medium (MEGM Bullet Kit, Cambrex Bio Science, East
Rutherford, NJ). Cells were passaged routinely and kept at
37 °C and 5% CO2. Exponentially growing cells were used in all
experiments.
Effects of Zinc Ejecting Compounds on Endogenous BCA2
Protein Stability. Experiments were essentially performed as in
described by us previously.7 In brief, 1 ꢀ 106 MCF-7 cells were
seeded into 6-well plates and grown overnight. Compounds 3a
(IC100 = 0.25 μM) and 5d (IC100 = 1 μM) were then added at the
100% growth inhibitory concentrations for 16 h alone or in
combination with the protein synthesis inhibitor CHX (Sigma,
100 μM) and/or the proteasome inhibitor 14 (Calbiochem,
10 μM). Control cells were treated with vehicle (DMSO) for
16 h. Whole cell lysates were analyzed for BCA2 protein
expression by Western blotting.
Determination of ALDH1 Inhibition. K562 cells (human
immortalized myelogenous leukemia line) are known to express
very high levels of ALDH1. Thus, these cells were used to
analyze effects of our lead BCA2 inhibitory compounds and
DSF for the inhibition of ALDH1. The ALDH assay kit
ALDEFLUOR (StemCell Technologies, Vancouver, BC) was
used according to the manufacturers instructions. Briefly, K562
cells were suspended in ALDEFLUOR assay buffer at concen-
tration of 1 ꢀ 106 cells/mL. To prepare a substrate for ALDH1,
bodipy-aminoacetaldehyde diethyl acetal (BAAA-DA) was
activated to bodipy-aminoacetaldehyde (BAAA) in 1 N HCl.
Cells were incubated with BAAA in the presence or absence of
DEAB, an inhibitor for ALDH1 for 45 min at 37 °C. DEAB was
added at concentrations of 15 μM from a 1.5 mM stock prepared
in ethanol. Thus, we compared the effects of DEAB on
ALDH1 activity to those of our lead compounds at the same
concentration and by preparing dilutions in ethanol. Cells
were washed once with ALDEFLUOR assay buffer, and then
cellular fluorescence based on bodipy-aminoacetate con-
verted BAAA by ALDH1 was measured by BD LSR I
through channel 1 (FL1, BD Biosciences, San Jose, CA).
ALDH1þ cells were analyzed by a FL1 (horizontal) versus
side scatter (SSC) dot plots by comparing fluorescence ob-
tained from cells incubated with DEAB. The percentage of
ALDH1þ cells in the presence and absence of DEAB and
compounds was determined and the percentage of ALDH1
inhibition calculated.
MTT Assay. The MTT proliferation assay was performed to
assess at which concentration these compounds would produce
50% growth inhibition (IC50) in tumor cells in order to identify
leads that could be developed as anticancer agents. The doses
employed ranged from 0.0001 to 10 μM. Compounds requir-
ing IC50 concentrations >10 μM were considered inactive. The
MTT assay was essentially performed as previously described
by us in detail.44 For compounds found to be active (IC50
<
10 μM), three independent experiments were performed and
IC50 values determined as mean ( standard deviation (SD).
Growth curves were generated and IC50 and IC100 concentra-
tions (100% growth inhibition) were delineated from the growth
curves as reported.44 The MTT assay was further used to assess
whether the addition of ZnCl2 can rescue the growth inhibitory
activity of compounds 1, 3a, and 5d in BCA2-positive MCF-7
cells. In brief, BCA2-positive MCF-7 cells were treated with
vehicle control (growth set as 100%) and the IC50 concentra-
tions of 1, 3a, and 5d in the presence of a range of zinc chloride
concentrations between 600 nM and 75 μM for 5 days followed
by MTT assay as described above.
Western Blotting. Western blots were done for assaying the
expression of endogenous BCA2 in the breast cancer cell lines
MCF-7 and MDA-MB-231, MDA-MB-231/ER, and T47D.
Exponentially growing cells were collected to generate cell
lysates, and Western blotting was performed as described by
us previously.7
Effects of Zinc Ejecting Compounds on Recombinant BCA2
Autoubiquitination. Drug stocks (10 mM) were prepared in
DMSO and diluted to 100 μM in 1ꢀ ubiquitination assay
reaction buffer. Ubiquitination assays were essentially per-
formed as described previously by using recombinant wild-type
His-BCA2 in the presence and absence of E2 conjugating
enzyme UbcH5b extract, and with or without specific zinc
ejecting compounds.7 In brief, 10 ng recombinant His-BCA2
was used with diluted drugs to obtain a final concentration of
Acknowledgment. We acknowledge the support of the Al-
gerian Consulate (PhD studentship to GB), and the EORTC
Pharmacology and Molecular Mechanisms (PAMM) group
for a mini-grant to initiate initial chemistry studies (to ADW
and AMB). BCA2 biology and compound testing was sup-
ported by Award Number R01CA127258 from the National
Cancer Institute, Developmental Therapeutics Program
(AMB). We thank Mrs. Lin Xiong for excellent technical
support. We also acknowledge the support of the EPSRC
National Mass Spectrometry Centre, Swansea, U.K.
Supporting Information Available: Spectroscopic data for
thiuram disulfide (3b), carbamo(dithioperoxo)thioate (5a, 5b,
5c, 5e, 5g, 5h, 5i, 5j), dithiocarbamate (9a-9ac) and benzisothia-
zolone (13a-g) compounds.This material is available free of
charge via the Internet at http://pubs.acs.org.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2765
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