Disulfiram-based disulfides as narrow-spectrum antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2018.03.023

Sixteen disulfides derived from disulfiram (Antabuse) were evaluated as antibacterial agents. Derivatives with hydrocarbon chains of seven and eight carbons in length exhibited antibacterial activity against Gram-positive Staphylococcus, Streptococcus, En

Full text of DOI:10.1016/j.bmcl.2018.03.023

Accepted Manuscript
Disulfiram-based disulfides as narrow-spectrum antibacterial agents
Jordan G. Sheppard, Keely R. Frazier, Pushkar Saralkar, Mohammad F.
Hossain, Werner J. Geldenhuys, Timothy E. Long
PII:
S0960-894X(18)30202-6
https://doi.org/10.1016/j.bmcl.2018.03.023
BMCL 25677
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 January 2018
4 March 2018
9 March 2018
Please cite this article as: Sheppard, J.G., Frazier, K.R., Saralkar, P., Hossain, M.F., Geldenhuys, W.J., Long, T.E.,
Disulfiram-based disulfides as narrow-spectrum antibacterial agents, Bioorganic & Medicinal Chemistry Letters
(2018), doi: https://doi.org/10.1016/j.bmcl.2018.03.023
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Disulfiram-based disulfides as narrow-spectrum antibacterial agents
Jordan G. Shepparda, Keely R. Fraziera, Pushkar Saralkarb, Mohammad F. Hossaina, Werner J.
Geldenhuysb, and Timothy E. Longa,c∗
a Department of Pharmaceutical Science and Research, School of Pharmacy, Marshall University, Huntington, WV, USA
b Department of Pharmaceutical Science, School of Pharmacy, West Virginia University, Morgantown, WV, USA
c Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, USA
ABSTRACT
Sixteen disulfides derived from disulfiram (Antabuse™) were evaluated as antibacterial agents.
Derivatives with hydrocarbon chains of seven and eight carbons in length exhibited antibacterial activity
against Gram-positive Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Listeria spp. A
comparison of the cytotoxicity and microsomal stability with disulfiram further revealed that the eight
carbon chain analog was of lower toxicity to human hepatocytes and has a longer metabolic half-life. In
the final analysis, this investigation concluded that the S-octylthio derivative is a more effective growth
inhibitor of Gram-positive bacteria than disulfiram and exhibits more favorable cytotoxic and metabolic
parameters over disulfiram.
KEYWORDS
disulfiram, disulfides, antibiotic, Staphylococcus, MRSA, VISA, VRSA
∗ Corresponding author. Tel.: +1-304-696-7393; fax: +1-304-696-7309; e-mail: longt@marshall.edu

Disulfiram-based disulfides as narrow-spectrum antibacterial agents
Jordan G. Shepparda, Keely R. Fraziera, Pushkar Saralkarb, Mohammad F. Hossaina, Werner J.
Geldenhuysb, and Timothy E. Longa,b∗
a Department of Pharmaceutical Science and Research, School of Pharmacy, Marshall University, Huntington, WV, USA
b Department of Pharmaceutical Science, School of Pharmacy, West Virginia University, Morgantown, WV, USA
c Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, USA
Disulfiram (Antabuse™) is an oral prescription drug for the treatment of alcohol abuse disorder [1].
Upon absorption, disulfiram (DSF) [2] and/or its metabolites [3] inhibit aldehyde dehydrogenase (ALDH)
enzymes that oxidize acetaldehyde from ethanol metabolism into acetic acid. The inactivation of hepatic
ALDH leads to buildup of toxic acetaldehyde in the body, which manifests ‘hangover’ symptoms (e.g.,
headache, nausea) to deter alcohol consumption [4].
Figure 1. Proposed route of in vitro aldehyde dehydrogenase (ALDH) inactivation by disulfiram [2].
By chemical nature, electrophilic (δ+) DSF is readily cleaved by thiol-bearing substances such as
cysteine enzymes. The thiol-disulfide exchange reactions result in the simultaneous addition and release
of diethyldithiocarbamate (DDTC). In the case of ALDH, in vitro studies have shown that a second
cysteine residue near the addition site may cleave the labile DDTC adduct with concomitant
intramolecular disulfide bond formation (Figure 1) [2]. As a versatile inhibitor of cysteine enzymes, DSF
has been evaluated as a treatment for other clinical conditions. Recent U.S. clinical trials using repurposed
DSF in treatment include: methamphetamine dependence (NCT00731133); cocaine addiction
(NCT00395850); melanoma (NCT00256230); muscle atrophy in pancreatic cancer (NCT02671890); and
HIV infection (NCT01286259) [5]. In the area of infectious disease, we recently established that DSF
∗ Corresponding author. Tel.: +1-304-696-7393; fax: +1-304-696-7309; e-mail: longt@marshall.edu

inhibits the in vitro growth of methicillin-resistant Staphylococcus aureus (MRSA) at a minimum
inhibitory concentration (MIC) range of 4 – 32 µg/mL and exhibits synergism with vancomycin (VAN)
against VAN-resistant S. aureus (VRSA) [6]. The mechanism of MRSA inhibition was also attributed to
the transfer of DDTC from DSF to thiophilic substances involved in the regulation of bacterial cell
growth. Due to its labile chemical nature, we hypothesized that replacement of the DDTC component in
DSF (Figure 1) with S-alkylthio groups would increase antibacterial activity and metabolic stability.
To test this hypothesis, we first synthesized sixteen DSF-derived asymmetric disulfides (1a-p) to
deduce the relationship of structure on antibacterial activity (Scheme 1). The compounds were readily
prepared by a thiol-disulfide exchange reaction between DSF and respective thiol in DMF [7].
Purification by silica gel chromatography afforded the products as nonaromatic oils in a yield range of 32
– 67% and median yield of 53%. Spectroscopic data and physical characteristics of the compounds were
in agreement with previous findings [8].
Scheme 1. Synthesis of disulfides 1 from disulfiram via a thiol-disulfide exchange reaction.
Antibacterial testing was performed by the broth microdilution assay in 96-well plate format [9,10]. The
test agents were initially evaluated against Staphylococcus, Streptococcus, and Enterococcus spp. as our
previous research on DSF indicated that Gram-positive cocci would be susceptible [6]. Table 1 shows the
MICs of analogs 1a-p in comparison with DSF and VAN. MRSA and Staphylococcus epidermidis
exhibited the greatest overall susceptibility to the DSF analogs followed by group A Streptococcus
pyogenes (GAS), VAN-resistant Enterococcus faecium (VRE), Streptococcus pneumoniae (SP), and
group B Streptococcus agalactiae (GBS). For VISA and VRSA variants of MRSA, the S-heptyl (1i) and
S-octyl (1j) derivatives displayed equal or greater antibacterial activity than DSF and VAN.

Table 1: Susceptibility of Gram-positive cocci to disulfides 1.
speciesa | MIC (µM)
test agent
MRSA
32
16
16
>32
16
>32
16
16
4
VISA
8
VRSA
16
16
16
>32
16
>32
16
8
VISE
32
32
32
>32
32
>32
8
GAS
GBS
>32
>32
>32
>32
>32
>32
SP
>32
>32
>32
>32
>32
>32
VRE
>32
>32
>32
>32
>32
>32
32
32
32
32
32
32
>32
>32
16
1a
8
8
32
16
>32
8
1b
1c
1d
1e
1f
>32
>32
>32
32
>32
>32
32
>32
>32
8
1g
4
2
8
4
1h
4
1i
4
4
4
4
16
16
8
1j
8
8
16
32
32
32
8
16
32
>32
4
8
4
32
16
>32
>32
16
32
16
8
4
8
16
1k
16
32
32
8
32
32
1
16
16
16
2
16
8
16
32
32
8
16
32
8
1l
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
≤0.5
>32
>32
32
>32
>32
>32
1m
1n
1o
1p
disulfiram
vancomycin
4
≤0.5
≤0.5
a
methicillin-resistant Staphylococcus aureus COL (MRSA); vancomycin-intermediate resistant S. aureus ADR-217 (VISA); vancomycin-
resistant S. aureus HIP14300 (VRSA); vancomycin-intermediate Staphylococcus epidermidis NRS6 (VISE); group A Streptococcus pyogenes
H293 (GAS); group B Streptococcus agalactiae SGBS005 (GBS); Streptococcus pneumoniae TCH8431 (SP); vancomycin-resistant
Enterococcus faecium ATCC 700221 (VRE).
The data in Table 1 further reveals a distinct correlation between the length of the S-alkylthio chain and
antibacterial activity against Gram-positive cocci. Alkyl chains of seven (1i) and eight (1j) carbons were
optimal lengths for antistaphylococcal activity with a MIC of 2 – 4 µM (0.6 – 1.2 µg/mL). By comparison,
the MIC ranges of DSF and VAN were 8 – 64 µM (2.4 – 19 µg/mL) and 1 – >32 µM (1.5 – >48 µg/mL),
respectively. Short straight chain analogs of one to five carbons were less active than their longer chain
counterparts, but were more effective growth inhibitors of MRSA than DSF. Branch and cyclic carbon
chain disulfides 1d, 1f, 1m, and 1n similarly had lower activity compared to the straight chain analogs and
their respective unbranched equivalents 1c, 1e, 1g, and 1h.
Additional antibacterial testing of the compounds included the select agents Bacillus anthracis
(anthrax), Francisella tularensis (tularemia) and Yersinia pestis (plague). Gram-positive B. anthracis
exhibited the highest sensitivity to the DSF analogs followed by Gram-negative F. tularensis and Y. pestis
(Table 2). In B. anthraces, it was noteworthy that a definitive structure-activity relationship could not be
established for analogs with alkyl chains of one to eight carbons in length as seen in S. aureus. Moreover,
DSF exhibited greater overall activity for all B. anthraces strains, but not to comparator ciprofloxacin
(CIP), which was also the superior test agent against Y. pestis and F. tularensis.

Table 2: Antibacterial activity of disulfides 1, disulfiram (DSF), and ciprofloxacin (CIP) against select agents.
MIC (µM)
species
strain
DSF CIP
1a
4
1c
4
1g
4
1h
4
1i
1j
4
2
1
2
1o
4
4
2
4
Bacillus anthracis
Ames35
4
1
2
1
2
0.5
≤0.5
≤0.5
0.5
Sterne 34F2
UM23-1
4
2
4
4
4
4
4
2
2
1
Weybridge
Utah 112
Kim (D2)
Kuma (D7)
4
4
8
8
2
Francisella tularensis
Yersinia pestis
32
16
16
32
16
32
32
32
32
32
32
32
16
32
32
16
32
32
32
16
32
32 ≤0.5
32 ≤0.5
32 ≤0.5
To further delineate the antibacterial activity spectrum, the compounds were tested on nineteen
additional Gram-positive (n = 5) and Gram-negative (n = 14) species. Table 3 shows that the inhibitory
activity was confined to Gram-positive bacteria with Bacillus cereus exhibiting the greatest susceptibility
followed by another rod-shaped species, Listeria monocytogenes. Similar to B. anthraces, activity was not
predicated on chain length in B. cereus; however, chain lengths of seven (1i) and eight (1j) carbons were
the most effective inhibitors of L. monocytogenes as observed with S. aureus. Micrococcus luteus and
Rhodococcus erythropolis were also moderately susceptible at a MIC range of 16 – 32 µM. Conversely,
the Gram-negative species panel as a whole displayed negligible susceptibility to the analogs. Based on
the overall test results, it was concluded that DSF and analogs 1 possess similar narrow-spectrum profiles
and straight chain derivatives of seven and eight carbons in length are the most potent growth inhibitors of
Gram-positive cocci.
Table 3: Antibacterial activity of disulfides 1 against Gram-positive and Gram-negative species.
MIC (µM)
Gram-positive | -negative
strain
DSF
CIP
0.5
>32
4
1a
1
1c
1
1g
1
32
8
16
16
1h
2
16
8
16
8
1i
1j
1
1o
2
32
8
16
Bacillus cereus
Gibson 971
FS-1
Gibson
SK58
SK121
AB5075
5K33
1
4
Corynebacterium striatum
Listeria monocytogenes
Micrococcus luteus
>32 >32
16
4
16
4
>32
>32
32
32
32
16
16
16
16
16 >32
4
Rhodococcus erythropolis
Acinetobacter baumannii
Brucella neotomae
16
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
16
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
16
>32
32
>32
32
16
≤0.5
≤0.5
1
>32 >32
>32 >32
>32 >32
>32 >32
32 >32
>32 >32
>32
>32
>32
32
Burkholderia cepacia
Burkholderia multivorans
Citrobacter freundii
Escherichia coli
UCB 717
CF2
4
8
32
32 >32 >32
4_7_47CFAA
DC10B
700603
HM-752
15442
>32 >32
>32 >32
>32 >32
>32 >32
>32 >32
>32 >32
>32 >32
>32 >32
>32 >32
>32 >32
>32 >32
>32 >32
>32 >32
>32 >32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
≤0.5
≤0.5
2
≤0.5
1
≤0.5
4
≤0.5
2
Klebsiella pneumoniae
Proteus mirabilis
Pseudomonas aeruginosa
Salmonella typhi
Ty2
Shigella dysenteriae
Vibrio cholera
Newcastle 1934
TS (D4)
WA-314
P61
32
32 >32 >32
Yersinia enterocolitica
Yersinia pseudotuberculosis
>32 >32
>32 >32
>32 >32
>32 >32
≤0.5

With a potential application as a treatment for resistant staphylococcal infections, analog 1j was further
evaluated for synergistic potential in comparison with DSF. The MICs of different VAN-1j and VAN-
DSF concentration combinations were determined using the checkerboard microdilution assay in 96-well
plate format [11,12]. Isobologram analysis revealed that analog 1j and DSF lowered the MIC of VAN in
MRSA, VISA, and VISE by comparable additive effects (Table 3) [13]. In VRSA, a synergistic effect
was observed for both analog 1j and DSF. From these studies it was concluded that disulfide 1j and DSF
can similarly lower the MIC of VAN and, therefore, both may have therapeutic utility as antibiotic
adjuvants for staphylococcal infections with reduced VAN susceptibility.
Table 3: Results of in vitro synergy studies.
MIC (µg/mL)a
VANb/DSFc
ΣFICd
VANb/DSFc
0.63 (+)
0.75 (+)
<0.16 (++)
1 (+)
strain
VAN
DSF
8
4
16
4
VANb/1jc
1/0.25
0.5/0.25
1/1
VANb/1jc
0.75 (+)
0.56 (+)
0.5 (++)
0.75 (+)
1j
2
0.5
2
MRSA COL
2
4
1/1
2/1
8/2
4/2
VISA AR-217
VRSA HIP14300
VISE NRS53
>128
8
2
2/1
a vancomycin: VAN; disulfiram: DSF
b lowest MIC of VAN in combination with DSF or 1j
c lowest MIC of DSF or 1j in combination with VAN
d lowest ΣFIC measurement; synergy (++) ≤ 0.5; additive (+) 0.5 < to ≤ 1; indifferent ( ) 1< to ≤ 4; antagonism (-) > 4 [12]
The investigation also compared the cytotoxicity of the analogs with DSF in human liver HepG2
carcinoma cells using the MTT assay [14]. The dose-response curve in Figure 2 revealed that the half-
maximal inhibitory concentrations (IC50) of analogs 1c (51 µM), 1h (51 µM), 1j (50 µM), and 1k (55
µM) were above DSF (38 µM), thereby indicating lower cytotoxicity. The >13:1 ratio of S. aureus MIC
(4 µM) to IC50 further suggests that compound 1j is selectively toxic to the bacterium. The apparent
selectivity was partially attributed to the higher glutathione content in mammalian cells [15]. The
thiophilic tripeptide, which is found in low abundance in Gram-positive bacteria, has been shown to
inactivate disulfide-based antibacterials through a thiol-disulfide exchange reaction [6,14,16].
150
1c
100
1i
1j
50
0
1k
DSF
0.001 0.01 0.1
1
10 100 1000
concentration ( M)
Figure 2. Dose-response curve for liver HepG2 cells.

Additional studies of analog 1j included comparisons of microsomal stability and physiochemical
properties to DSF (Table 4). Measurement of the in vitro metabolic stability using pooled rat liver
microsomes indicated that the elimination half-life (t ) and intrinsic clearance (CLint) was longer for the
½
DSF analog [17,18]. A comparison of the physiochemical chemical properties also revealed that both
DSF and derivative 1j are hydrophobic compounds. The higher clogP and lower molecular polar surface
area (PSA) for disulfide 1j suggest that replacement of the DDTC component in DSF with the more
lipophilic S-octylthio group may confer better tissue and membranes penetration [19]. This marked
difference with DSF could partially account for the increased susceptibility of S. aureus to analog 1j if
cell entry is required of both agents to inhibit growth.
Table 4: Microsomal stability and calculated physiochemical properties.
DSF
296.5
0.86
3.82
121
1j
293.6
0.78
6.53
85.9
11
MW (g/mol)
pKaa
clogPa
PSA (Å2)a
rotatable bondsa
7
t (min)b
0.75
1848
61.2
22.6
½
CLint (µL/min/mg protein)c
a calculated value [20,21]
b elimination half-life in pooled rat liver microsomes
c intrinsic body clearance
In the final analysis, S-alkylthio analogs of DSF exhibited up to eight times greater antibacterial activity
compared to DSF. The activity spectrum of the analogs was similar to DSF with Gram-positive
Staphylococcus and Bacillus spp. exhibiting the highest level of susceptibility. Analogs with S-heptylthio
(1i) and S-octylthio (1j) groups were found to be the most effective inhibitors of MRSA growth and
retained their potency against VISA and VRSA. The select antibacterial activity was partly attributed to
the low abundance of redox-buffering glutathione in the cytoplasm of Gram-positive bacteria [14].
Glutathione has been shown to inactivate disulfide-based antibacterial agents [6,14,16] and the higher
abundance in Gram-negative bacteria may account for the Gram-type selectivity. This investigation also
considered that the outer membrane barrier in Gram-negative bacteria could be a factor; however, the data
from Table 3 indicated that increasing the lipophilic property of the compounds, which would facilitate
cell membrane permeation, did not effect antibacterial activity in Gram-negative bacteria. Other factors
that would account for the Gram-type selectivity, but were not investigated during the study, are the
existence of potential pharmacological targets and cellular pathways required for antibacterial action.
This research further resolved that disulfide 1j can lower the MIC of VAN in VISA and VISE,
suggesting possible therapeutic utility as an antibiotic adjuvant in VAN-intermediate infections.
Preliminary assessment of cytotoxicity and microsomal stability in comparison with DSF also confirmed

that the S-octylthio analog was of lower toxicity to human hepatocytes and had a longer half-life, a
parameter with implications on dosing frequency. Future research will focus on defining the mechanisms
of action and resistance development for disulfide-based antibacterials. In addition, pharmacokinetic-
pharmacodynamic (PK-PD) studies will be performed in vivo to determine their viability as antibiotic
adjuvants in VAN therapy. The PK studies will be used to guide dosing with a regimen that accounts for
the influence of tissue glutathione on the disulfide concentration and gives a preferred trough VAN
concentration of 4 to 5 times the MIC [22] with the disulfide for the test pathogen (e.g., VISA).
Acknowledgements
This research was supported by the Marshall University School of Pharmacy FRS Grant Program. The
authors also thank the Marshall University Department of Chemistry for use of the NMR facility. Bacteria
were acquired from the American Type Culture Collection, FDA-CDC Antimicrobial Resistance Isolate
Bank, and the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution
by BEI Resources, NIAID, NIH.
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Eppendorf tube. The mixtures were incubated at 37°C for 0, 5, 10, 15, 30, and 60 mins with mild
shaking. The reaction was terminated after each specified time point by placing it in an ice bath and
adding 500 µl of cold MeCN. The incubation mixtures were then centrifuged for 10 mins at 3000 rpm
and an aliquot of each supernatant transferred for analysis. Test compounds are quantified using
LC/MS/MS, and its metabolic half-life in the in vitro test system is derived from percent remaining
vs. time data. The LC-MS/MS setup consisting of Agilent 6490 triple quadrupole mass spectrometer
equipped with an Agilent 1260 Infinity HPLC unit. Positive ESI LC-MS/MS was done using MRM
transitions 297.06 > 214.8 and 294.14>148.1 for DSF and disulfide analog 1j, respectively.
Separation was performed on Agilent Eclipse plus C18 column eluted with MeOH-water gradient
acidified with 0.1% formic acid.
19. Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple KD. Molecular properties that
influence the oral bioavailability of drug candidates. J Med Chem. 2002;45(12):2615-23.
20. Calculated using Advanced Chemistry Development (ACD/Labs) Software V11.02.
21. Calculated from ChemDraw Ultra Software 8.0.
22. Hidayat LK, Hsu DI, Quist R, Shriner KA, Wong-beringer A. High-dose vancomycin therapy for
methicillin-resistant Staphylococcus aureus infections: efficacy and toxicity. Arch Intern Med.
2006;166(19):2138-44.

Disulfiram-based disulfides as narrow-spectrum antibacterial agents
Jordan G. Shepparda, Keely R. Fraziera, Pushkar Saralkarb, Mohammad F. Hossaina, Werner J.
Geldenhuysb, and Timothy E. Longa,c∗
a Department of Pharmaceutical Science and Research, School of Pharmacy, Marshall University, Huntington, WV, USA
b Department of Pharmaceutical Science, School of Pharmacy, West Virginia University, Morgantown, WV, USA
c Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, USA
∗ Corresponding author. Tel.: +1-304-696-7393; fax: +1-304-696-7309; e-mail: longt@marshall.edu