International Journal of Antimicrobial Agents 47 (2016) 36–47

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International Journal of Antimicrobial Agents

journal homepage: http://www.elsevier.com/locate/ijantimicag

Ammonium salts of carbamodithioic acid as potent vaginal

trichomonacides and fungicides
Bhavana Kushwaha a,1 , Dhanaraju Mandalapu b,1 , Veenu Bala b,1 , Lokesh Kumar a ,
Aastha Pandey a , Deepti Pandey a , Santosh Kumar Yadav a , Pratiksha Singh c , P.K. Shukla c ,
Jagdamba P. Maikhuri a , Satya N. Sankhwar d , Vishnu L. Sharma b , Gopal Gupta a,∗
a
Division of Endocrinology, CSIR – Central Drug Research Institute, Lucknow 226 031, India
b
Division of Medicinal and Process Chemistry, CSIR – Central Drug Research Institute, Lucknow 226 031, India
c
Division of Microbiology, CSIR – Central Drug Research Institute, Lucknow 226 031, India
d
Department of Urology, King George’s Medical University, Lucknow 226 003, India

a r t i c l e i n f o a b s t r a c t

Article history: Chemical attenuation of the reactive oxygen species (ROS)-sensitive anaerobes Trichomonas vaginalis,
Received 10 September 2015 which is the most prevalent non-viral sexually transmitted infection, and two often coexisting vagi-
Accepted 22 October 2015 nal infections, namely Candida albicans and Staphylococcus aureus, which are opportunistic reproductive
tract infections, was attempted with novel ammonium salts of carbamodithioic acid through inhibition
Keywords: of free thiols. In vitro and in vivo efficacies of the designed compounds were evaluated as topical vagi-
Trichomonas vaginalis
nal microbicides. Five compounds showed exceptional activity against drug-resistant and -susceptible
Candida albicans
strains with negligible toxicity to host (HeLa) cells in vitro in comparison with the standard vaginal micro-
Staphylococcus aureus
Metronidazole
bicide nonoxynol-9 (N-9), without disturbing the normal vaginal flora (i.e. Lactobacillus). The compounds
HeLa significantly inhibited the cytopathic effects of Trichomonas on HeLa cells in vitro with efficacies compa-
Lactobacillus rable with metronidazole (MTZ); however, their efficacy to rescue host cells from co-infection (protozoal
and fungal) was greater than that of MTZ. The compounds inhibited ␤-haemolysis of red blood cells
caused by Trichomonas and were found to be active in vivo in the mouse subcutaneous abscess assay.
Some compounds rapidly immobilized human sperm. A mechanism involving inhibition of free thiols
and consequently the cysteine proteases of T. vaginalis by the new compounds has been proposed. Thus,
a unique scaffold of antimicrobial agents has been discovered that warrants further investigation for
development as contraceptive vaginal microbicides.
© 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

1. Introduction [6]. Hence, physical and/or chemical shielding of the genital

Worldwide, Trichomonas vaginalis is the most prevalent non-
viral sexually transmitted infection (STI) [1] that promotes the
acquisition and transmission of viral STIs such as human immun-
odeficiency virus (HIV), human papillomavirus (HPV) and herpes
simplex virus (HSV) [2,3]. Trichomoniasis in women is also associ-
ated with vaginitis, endometritis, adnexitis, pyosalpinx, infertility,
preterm delivery, bacterial vaginosis and risk of cervical cancer
[4], whilst in men it has been implicated in the pathogenesis
of prostate cancer [5]. Trichomoniasis is often asymptomatic
(especially in males) and therefore goes unreported, resulting
in persistent spread of T. vaginalis through heterosexual contact
∗ Corresponding author. Tel.: +91 522 277 2450x4391; fax: +91 522 277 1941.
E-mail addresses: g gupta@cdri.res.in, ggupta.cdri@gmail.com (G. Gupta).
1
These authors contributed equally to this work.
tract during sexual intercourse becomes desirable. Unfortunately,
condom use is grossly limited by the users’ personal prefer-
ences, and the US Food and Drug Administration (FDA)-approved
metronidazole (MTZ) has limited efficacy when used vaginally.
On the other hand, 5-nitroimidazole class compounds are prone
to drug resistance. The first case of drug resistance was reported
with MTZ within 2 years of its introduction [7], and more than
100 cases were reported by 2003 [8]. Similarly, vulvovaginal
candidiasis, caused by Candida albicans, has a very high preva-
lence in sexually active women, with a significant number of
drug-resistant cases [9]. This infection often co-exists with T.
vaginalis, resulting in increased morbidity [10]. Since women
disproportionately bear the long-term consequences of STIs
[11], highly effectual molecules (preferably non-nitroimidazoles)
against T. vaginalis and associated yeast infections need to
be discovered for potential use as ‘women controlled’ topical
microbicides.

http://dx.doi.org/10.1016/j.ijantimicag.2015.10.022
0924-8579/© 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
 B. Kushwaha et al. / International Journal of Antimicrobial Agents 47 (2016) 36–47
We previously reported a unique molecular series that acted
by its exceptional affinity towards thiols present on Trichomonas
[6], suggesting thiols as a vulnerable target for directing top-
ical antitrichomonal agents for prophylaxis during unprotected
sex. Since chiefly anaerobic cells are susceptible to agents deplet-
ing thiols, which are meant for reactive oxygen species (ROS)
buffering, the new compounds were also tested against some
pathogenic bacteria and fungi that infect the female vaginal tract.
Here we report some novel molecules with much improved scaf-
folds for better efficacy and safety over the previous drug designs.
An attempt has been made to mechanistically substantiate the
potential of these molecules as antitrichomonal agents suitable for
topical use.
2. Materials and methods
2.1. Chemical synthesis
All of the new compounds were designed and synthesized in-
house by the medicinal chemist authors (DM, VB and VLS). The
synthesis schemes and chemical characterization details are avail-
able as Supplementary data.
2.2. Trichomonas vaginalis cultures and trichomonacidal assays
Trichomonads were cultured axenically in trypticase–yeast
extract–maltose (TYM) (pH 6.8) (Sigma–Aldrich, St Louis, MO) sup-
plemented with 10% heat-inactivated foetal bovine serum (FBS)
(GibcoTM ; Life Technologies–Thermo Fisher Scientific, Waltham,
MA), 2% vitamin mixture (Sigma–Aldrich), 100 U of penicillin/mL
and 100 ␮g/mL streptomycin at 37 ◦ C under partial anaerobic con-
ditions (i.e. with ca. 0.5 mL of air trapped above the medium). Drug
susceptibility assays were conducted as detailed previously [6].
Briefly, the parasites were incubated under partial anaerobic con-
ditions at 37 ◦ C in culture medium containing serially diluted test
compounds or MTZ (Sigma–Aldrich) in 48-well Corning culture
plates (Corning Inc., Corning, NY). Dimethyl sulphoxide (DMSO)
0.05% (Sigma–Aldrich) in culture medium (the highest concentra-
tion of DMSO in test wells) was used as vehicle in the control
wells. Cell viability was checked after 48 h by trypan blue exclu-
sion assay (Sigma–Aldrich) and the minimum concentration of
test agent at which all cells were found dead was considered its
minimum inhibitory concentration (MIC). 100% eradication was
confirmed by transferring 100 ␮L of the suspension to a 15 mL
tube with fresh medium and recording the growth at 37 ◦ C. MTZ,
which is the most widely used drug against T. vaginalis, was used
as the reference standard. All experiments were repeated three
times.
2.3. Microbial cultures, and antifungal and antibacterial assays
The fungi C. albicans and Candida glabrata were maintained
in RPMI 1640 (Sigma–Aldrich) buffered with MOPS [3-(N-
morpholino)propanesulfonic acid] (Sigma–Aldrich) and were
incubated at 35 ◦ C. Staphylococcus aureus ATCC 25923 was grown
in Mueller–Hinton broth (DifcoTM ; BD, Franklin Lakes, NJ) and was
incubated at 37 ◦ C. Susceptibility assays were performed by the
standard broth microdilution method as per Clinical and Laboratory
Standards Institute (CLSI) guidelines. The maximum concentration
tested was 50 mg/L and the inoculum load in each test well was in
the range of 1–5 × 103 cells. Plates were incubated for 24–48 h for
yeasts and 24 h for bacteria at 37 ◦ C for determination of the MIC
[12].
37
2.4. Cervical epithelial (HeLa) and fibroblast (L929) cell cultures
and cytotoxicity assays
HeLa and L929 cells procured from the National Centre for Cell
Science (NCCS, Pune, India) and the American Type Culture Collec-
tion (ATCC, Manassas, VA), respectively, were grown in Dulbecco’s
Modified Eagle Medium (DMEM) (Sigma–Aldrich) supplemented
with FBS (10%) and antibiotics (penicillin/streptomycin mixture,
100 U/mL) (Sigma–Aldrich) [6]. The cytotoxicity of compounds
was tested by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide] assay (Sigma–Aldrich) [13]. L929 cells were
also fixed and stained with Giemsa for 30 min at 37 ◦ C for light
microscopy.
2.5. Compatibility of commensal vaginal flora (Lactobacillus)
with new compounds
Lactobacillus jensenii ATCC 25258 and a cocktail of Lacto-
bacillus spp. (Lactobacillus acidophilus, Lactobacillus rhamnosus,
Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei and
Lactobacillus fermentum; LactogutTM , Medispan Ltd., Chennai, India)
were grown in 6% Rogosa SL broth medium (HiMedia, Mumbai,
India) containing 0.132% acetic acid at 37 ◦ C. The effect of test
compounds on L. jensenii was determined as described previously
[6].
2.6. Spermicidal assay
Freshly ejaculated human semen samples received from healthy
volunteers with their prior informed consent was analyzed for
sperm count and motility. Samples with >65 million/mL sperm
count, >70% motility and normal sperm morphology were used to
determine the spermicidal minimum effective concentration (MEC)
of the test compounds in vitro [6]. This study was approved by
the Institutional Animal Ethics Committee of CSIR – Central Drug
Research Institute (Lucknow, India).
2.7. Flow cytometric analysis of Trichomonas vaginalis viability
Trichomonas vaginalis incubated with test compounds at their
MICs for 24 h were pelleted, washed and re-suspended in
medium containing 25 ␮g/mL propidium iodide (Sigma–Aldrich)
for 10 min and the viability was evaluated on a flow cytome-
ter (FACSCaliburTM ; BD Biosciences, Singapore) equipped with an
argon laser (488 nm) for excitation [14].
2.8. Inhibition of cytopathic effects of Trichomonas vaginalis and
Candida albicans
HeLa and L929 cells were grown separately on eight-chambered
slides (BD) for 24 h at 37 ◦ C, were washed and were then infected
with pathogen(s) in interaction medium DMEM:TYM (2:1) con-
taining test compounds at trichomonacidal MICs in co-cultures
of HeLa and T. vaginalis, and at anticandidal MICs in co-cultures
of L929, T. vaginalis and C. albicans. Following incubation, slides
were fixed with methanol. The cytopathic effects of parasites
on the host cells were observed by Giemsa staining under a
microscope.
2.9. Inhibition of the ˇ-haemolytic activity of Trichomonas
vaginalis by the test compounds
An equal number of freshly washed rat erythrocytes were
mixed with 2 × 106 trophozoites in 2.5 mL of Hank’s Balanced Salt
Solution (Sigma–Aldrich) with or without the test compounds
38 B. Kushwaha et al. / International Journal of Antimicrobial Agents 47 (2016) 36–47

Table 1
Trichomonacidal activity of compounds 15, 16, 18, 24 and 26 against MTZ-susceptible and -resistant strains, cytotoxicity towards cervical epithelial (HeLa) cells and
compatibility with vaginal microflora (Lactobacillus) of novel compounds.

Compound Structure Antitrichomonal MIC (mg/L) Spermicidal Mean Mean compatibility (IC50 )
activity cytotoxicity in (mg/L) with:
(MEC) HeLa cells
(mg/L) (IC50 ) (mg/L)

MTZ-susceptible strain MTZ-resistant strains Lactobacillus Lactobacillus

jensenii strains a

ATCC 33592 ATCC 50143 ATCC 30238

15 1.56 6.25 7.8 1160 >1000 539.9 2327.58

16 1.56 6.25 15.6 1170 >1000 >936 >4000

18 1.56 6.25 7.8 61.5 >1000 >936 >4000

24 3.12 12.5 15.6 234 >1000 574.3 2192

26 1.56 12.5 31.2 >10 000 >1000 424.3 1725

MTZ 3.25 50.06 62.5 >10 000 >1000 684.6 >4000

N-9 43.27 46.60 50.0 150.0 33.2 32.07 52.68

MTZ, metronidazole; MIC, minimum inhibitory concentration; MEC, minimum effective concentration; IC50 , concentration inhibiting cell viability by 50%; N-9, nonoxynol-9.
a
Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei and Lactobacillus fermentum.

Table 2
Bactericidal activity of new test compounds against drug-susceptible and -resistant strains of Staphylococcus aureus.

Compound MIC (mg/L)

ATCC 25923 ATCC 700699 (meticillin-resistant) ATCC 29213 ATCC 33592 (gentamicin-resistant)

15 12.5 12.5 12.5 12.5

16 6.25 6.25 6.25 6.25
18 12.5 12.5 25 12.5
24 6.25 3.12 6.25 6.25
26 12.5 25 50 25
Gentamicin 0.78 >50 1.56 >50
Norfloxacin 0.78 >50 1.56 0.78

MIC, minimum inhibitory concentration.

Table 3
Antifungal activity of test compounds against seven strains of Candida albicans and two strains of Candida glabrata.

Compound MIC (mg/L)

C. albicans strains C. glabrata strains

Patient isolate ATCC 10231.3 ATCC 14053.4 ATCC 60193.5 ATCC 66027.6 ATCC 90028.7 MTCC-183.8 ATCC-MYA-2950.10 ATCC 2001.11

15 12.5 25 12.5 25 12.5 25 25 25 50

16 6.25 12.5 6.25 12.5 6.25 12.5 6.25 12.5 12.5
18 12.5 25 25 50 12.5 25 25 25 50
24 6.25 12.5 6.25 12.5 6.25 6.25 6.25 12.5 12.5
26 12.5 25 12.5 25 12.5 12.5 12.5 6.25 6.25
Fluconazole 1 1 0.12 0.5 0.5 0.5 8 2 1.4

MIC, minimum inhibitory concentration.

at their respective MICs for 24 h. Negative control tubes con-
tained only red blood cells (RBCs) to estimate spontaneous
haemolysis and the value was subtracted from the experimen-
tal tubes. Following incubation, cultures were centrifuged and
the absorbance of the supernatant was measured at 412 nm in a
spectrophotometer.
2.10. Qualitative assessment of free thiols on Trichomonas
vaginalis
The effect of test compounds on free thiols of trichomonads
was examined and imaged [15] using a fluorescence microscope
(Nikon Eclipse 80i microscope; Nikon Corp., Tokyo, Japan) after
 B. Kushwaha et al. / International Journal of Antimicrobial Agents 47 (2016) 36–47 39

Fig. 1. (A–G) Representative cytograms of propidium iodide-labelled Trichomonas vaginalis cells pre-treated with metronidazole or compounds 15, 16, 18, 24 and 26 for 24 h
at their trichomonacidal MIC (1.56–3.12 mg/L). R1 = % dead cells. (H) Statistical data of percent viable Trichomonas in the presence of metronidazole or compounds 15, 16, 18,
24 and 26. Mean ± standard error of three independent experiments. ***Significant difference from control (P < 0.001). MIC, minimum inhibitory concentration.

labelling with a fluorometric thiol detector (Cayman Chemical Co.,
Ann Arbour, MI). Trichomonas vaginalis treated with vehicle or test
compounds at the MIC for 24 h at 37 ◦ C were pelleted at 700 × g
for 10 min at 4 ◦ C, were washed and 50 ␮L of fluorescent thiol
detector (pre-diluted 100× with dilution buffer) was added. After
incubation for 5 min in the dark, a drop was taken on a glass
slide, covered with a coverslip and imaged on a Nikon Eclipse 80i
microscope equipped with epifluorescence illumination using the
UV-1A filter (Nikon Corp.). The exposure time was the same for all
samples.
2.11. Cysteine protease activity
Trichomonas vaginalis cells (2 × 106 ) were treated with test
compounds at the MIC for 24 h and were pelleted, washed and
suspended in lysis buffer [50 mM Tris–HCl, 0.25 M sucrose, 25 mM
KCl, 5 mM MgCl2 and 5 mM ethylene diamine tetra-acetic acid
(EDTA), pH 7.5; Sigma–Aldrich] and lysed by freezing (−70 ◦ C)
and thawing (30 ◦ C). The lysates were separated on 10% sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
co-polymerised with gelatin (0.2%) at 100 V. SDS was removed
by incubation in 2.5% Triton-X100 for activating proteinases,
which was assessed by development of bands in incubation buffer
(Sigma–Aldrich) for 4 h at room temperature and staining with
0.25% Coomassie blue (Sigma–Aldrich) and destaining; 1 mM TLCK
(tosyl-l-lysyl-chloromethane hydrochloride) (Sigma–Aldrich) was
used for comparison [16].
2.12. Subcutaneous abscess assay for in vivo antitrichomonal
efficacy
The mouse subcutaneous abscess assay of Krieger et al. [17]
was used. Six-week old mice were inoculated subcutaneously
with T. vaginalis (50 ␮L of 2 × 106 organisms/mL) into the left
hind flank. Control animals were injected with sterile saline. The
abscess/lesion was determined by palpation 7 days after injection
and was measured daily thereafter. Infected animals were then
treated subcutaneously with compounds at 5.0 ␮g in 50 ␮L of saline
daily for 7 days. The area was calculated as ␲r2 . MTZ was used as
a positive control. Control injections of sterile saline did not result
in abscess formation. The assay was approved by the Institutional
Animal Ethics Committee.
40 B. Kushwaha et al. / International Journal of Antimicrobial Agents 47 (2016) 36–47

Fig. 2. (A–D) Light microscopy images of cell viability of fibroblast (L929) cell line in the presence of compounds 16, 24 and 26 at the anticandidal MIC (6.25 mg/L) using
Giemsa staining. (E) Histogram of percentage cell viability of fibroblast (L929) cells in the presence of compounds 16, 24 and 26 in a dose-dependent manner using the MTT
assay. MIC, minimum inhibitory concentration.

3. Results summarized in Table 1. Compared with MTZ, compound 18 had

3.1. Antitrichomonal activity and selectivity/safety ratio
A total of 29 compounds were designed and synthesized for
antimicrobial activity (Supplementary Table-1). Among these, the
antitrichomonal activities (MICs of 1.56–3.12 mg/L) of 11 com-
pounds (15–18, 20–24, 26 and 27) were comparable with that of
MTZ (MIC = 3.25 mg/L) against drug-susceptible strains and were
also much more effective than MTZ against drug-resistant T. vagi-
nalis (MICNEW-COMPOUNDS = 6.25–25.0 mg/L vs. MICMTZ = 50.0 mg/L).
Of these compounds, five with the most promising activity (15, 16,
18, 24 and 26) were selected for further study and the results are
the best activity profile against drug-susceptible and -resistant Tri-
chomonas (2.99- and 11.52-fold), followed by compound 16 (2.85-
and 10.96-fold), compound 15 (2.82- and 10.84-fold), compound
26 (3.19- and 6.13-fold) and compound 24 (1.42- and 5.48-fold).
Nonoxynol-9 (N-9) was the least active, with 0.27- and 3.87-
fold activity against drug-susceptible and -resistant Trichomonas
in comparison with MTZ (Table 1). The selected compounds were
also found to be extremely safe towards human cervical (HeLa)
cells [concentration inhibiting cell viability by 50% (IC50 ) = 3350 to
>4000 mg/L] and were highly compatible with the normal vaginal
flora (i.e. Lactobacillus) (IC50 = 1775 to >4000 mg/L). Compounds 15,
16, 18 and 26 were least toxic and were comparable with MTZ in
 B. Kushwaha et al. / International Journal of Antimicrobial Agents 47 (2016) 36–47 41

Fig. 3. (A) Control cervical epithelial (HeLa) cells. (B–G) HeLa cells infected with Trichomonas vaginalis (T.v) for 24 h in the presence of vehicle (0.05% DMSO in culture medium)
(B), metronidazole (MTZ) (3.12 mg/L) (C) and compounds 15, 16, 18 and 26 (1.56 mg/L) (D–G, respectively). Magnification bar = 10 ␮m. DMSO, dimethyl sulphoxide.

safety towards HeLa in vitro. Compounds 16 and 18 were most
compatible with Lactobacilli (similar to MTZ), whilst compounds
15, 24 and 26 were marginally less compatible. N-9 was the least
compatible both with HeLa and Lactobacillus (Table 1).
3.2. Antibacterial activity
The five selected compounds exhibited moderate activity
(MIC = 3.12–50 mg/L) against antibiotic-susceptible as well as
meticillin- and gentamicin-resistant strains of S. aureus. Com-
pounds 16 and 24 exhibited MICs of 6.25 mg/L and 3.12 mg/L,
respectively, against the meticillin-resistant strain and an MIC
of 6.25 mg/L against the gentamicin-resistant strain, in compari-
son with that of gentamicin (MIC ≥ 50 mg/L against both strains)
and norfloxacin (MIC ≥ 50 mg/L against meticillin-resistant strain).
The test compounds were >2–16 times more active than gen-
tamicin and norfloxacin against meticillin-resistant strains and
>2–8 times more active than gentamicin against gentamicin-
resistant S. aureus. The compounds did not differentiate between
antibiotic-susceptible and -resistant strains of bacteria, indicating
their different mode of action (Table 2).
3.3. Antifungal activity
The selected compounds displayed moderate activity against
different Candida strains (MIC = 6.25–50 mg/L) in comparison with
fluconazole (MIC = 0.12–8.0 mg/L). The test compounds were evalu-
ated against seven strains of C. albicans and two strains of C. glabrata.
42 B. Kushwaha et al. / International Journal of Antimicrobial Agents 47 (2016) 36–47

Fig. 4. (A) Control fibroblast (L929) cells. (B–D) L929 infected with Trichomonas vaginalis (B), Candida albicans (C) or both (D) for 24 h in the presence of vehicle. (E–H) L929
co-infected with T. vaginalis and C. albicans and incubated in the presence of 3.12 mg/L metronidazole (E) or compounds 15, 18 and 26 (1.56 mg/L) (F–H, respectively) for 24 h.

Compounds 16 and 24 exhibited appreciable anticandidal activity
against different strains of the two species (MIC = 6.25–12.5 mg/L),
whilst the other three compounds were only moderately active.
All compounds were less active than the standard drug fluconazole
against Candida species/strains, except against MTCC-183.8 strain
where compounds 16 and 24 were more active than fluconazole
(Table 3).
molecules targeted human sperm and caused their instantaneous
(within 30 s) and permanent immobilization at concentrations
(MEC = 0.023–0.117%) that was close to that of N-9 (0.015%), the
marketed spermicide (Table 1).
3.5. Flow cytometric evaluation of Trichomonas viability in the
presence of test compounds

3.4. Spermicidal (contraceptive) activity
The selected compounds were also screened against live human
sperm for complementary spermicidal (contraceptive) activity.
Four compounds (15, 16, 18 and 24) among the five promising
Exponentially growing trichomonads incubated in vehicle for
24 h had 0.02% non-viable cells, whilst those incubated separately
with MTZ or compounds 15, 16, 18, 24 or 26 at their MIC had
95.24%, 98.12%, 97.60%, 90.08%, 81.70% and 91.94% non-viable cells,
respectively. At the MIC, all of the compounds behaved almost
 B. Kushwaha et al. / International Journal of Antimicrobial Agents 47 (2016) 36–47 43

Fig. 5. (A) Inhibition of the ␤-haemolytic activity of Trichomonas vaginalis (T.v) on rat red blood cells (RBCs) by metronidazole (MET) (3.12 mg/L) and compounds 15, 16, 18, 24
and 26 (1.56–3.12 mg/L) at their respective trichomonacidal MICs at 24 h. (B) Statistical data of percent inhibition of ␤-haemolytic activity as shown in (A). Mean ± standard
error of three independent experiments. ***Significant difference from the control (P < 0.001). MIC, minimum inhibitory concentration; HBSS, Hank’s Balanced Salt Solution;
MEC, minimum effective concentration.

identically without any significant difference between them and
MTZ (Fig. 1).
3.6. Cytotoxicity of new compounds towards fibroblast (L929)
cells
Since yeasts invade fibroblast cells for germination [18], com-
pounds 16, 24 and 26 were also tested against fibroblast (L929)
cells for general cytotoxicity at their antifungal MIC (6.25 mg/L)
using Giemsa staining. No significant changes in cell number or
morphology were visible. This was further confirmed by the MTT
assay of L929 cell viability after 24 h incubation in the three test
compounds separately at 50–0.04 mg/L serial dilutions. The cells
did not lose more than 20% viability at the highest concentration
tested (50 mg/L) (Fig. 2).
3.7. Cytopathic effects of Trichomonas vaginalis on HeLa cells
in vitro and its inhibition by the test compounds
Human cervical (HeLa) cell monolayers infected with T. vagi-
nalis in vitro exhibited complete loss of cellular morphology with
cell lysis of >70% cells owing to cytopathy, with a large number of
viable, motile trichomonads remaining after 24 h (Fig. 3). However,
HeLa cultures infected with T. vaginalis in the presence of MTZ or
the test compounds at their antitrichomonal MIC (in parallel) were
very effectively rescued from the cytopathic effects of the infection.
Post-incubation images of these cultures indicated healthy HeLa
cells with normal morphology, with few inactivated trichomonads
(Fig. 3B–F). The HeLa cell density was comparable with untreated
controls in the case of MTZ and compounds 15, 16 and 18, whilst it
was somewhat lower in the case of compound 24.
3.8. Efficacy of test compounds against co-infection of L929 cells
with Trichomonas vaginalis and Candida albicans
L929 fibroblast cells were infected with T. vaginalis, or C. albicans,
or both, and after 24 h ca. 80% of cells were lysed by infections with
either T. vaginalis or C. albicans (Fig. 4B and C). However, in the case
of co-infection >90% cells were lost due to very severe cytopathic
effects of the two infections (Fig. 4D). In the presence of MTZ (at its
antitrichomonal MIC), not more than 20–25% of L929 cells could be
rescued from co-infection to survive after 24 h (Fig. 4E). However,
in the presence of the novel compounds 15, 18 and 24 ca. 40–80%
of L929 cells were rescued from co-infections of protozoa and yeast
44 B. Kushwaha et al. / International Journal of Antimicrobial Agents 47 (2016) 36–47

Fig. 6. (I) (A–E) Fluorescent staining of free thiols on Trichomonas (A) and its inhibition by compounds 15, 16, 18 and 26 (1.56 mg/L) (B–E, respectively). Upper panel, phase
contrast; lower panel, fluorescence. (II) Substrate gel electrophoresis showing the effect of compounds 15, 16, 18 and 26 as well as TLCK (tosyl-l-lysyl-chloromethane
hydrochloride), vehicle and control (untreated) on the proteolytic activity of total cysteine proteases of Trichomonas vaginalis. Note the substrate hydrolysis in control and
vehicle lanes by cysteine proteases and its inhibition by TLCK, 15, 16, 18 and 26 (bold arrow). The clear zone created by hydrolysis of substrate by active cysteine proteases
in control and vehicle lanes during electrophoresis (thin arrows).

(Fig. 4F–H). Compound 18 appeared to be the most effective against
the co-infection.
3.9. ˇ-Haemolysis of red blood cells with Trichomonas vaginalis
and its inhibition by the test compounds
Incubation of rat RBCs with T. vaginalis for 24 h caused marked
haemolysis, which was inhibited very significantly by ca. 90%
(P < 0.001) in the presence of MTZ or compounds 15, 16, 18 and 24.
Compound 26 inhibited ␤-haemolysis of RBCs by ca. 40% (P < 0.001)
(Fig. 5).
3.10. Inhibition of free thiols on Trichomonas by the test
compounds
Since the compounds were designed to target free thiols on Tri-
chomonas, inhibition of free sulfhydryls of T. vaginalis by the test
compounds was qualitatively evaluated by fluorescent labelling
with a thiol detector. Apparently, significant inhibition of free thiol
fluorescence was evident in the case of compounds 15, 16, 18 and
26 compared with controls (Fig. 6I). Maximum thiol inhibition was
seen with compound 15.
3.11. Effect of test compounds on the proteolytic activity of total
cysteine proteases of Trichomonas vaginalis
Compared with vehicle (0.05% DMSO) and control (untreated),
there was a decrease in the cysteine protease activity of the parasite
in the presence of test compounds incubated at the MIC for 24 h, as
evident by the substrate gel electrophoresis. TLCK (an inhibitor of
cysteine proteases) at 1 mM almost completely abolished cysteine
protease activity (Fig. 6II).
3.12. In vivo efficacy of compounds in the mouse subcutaneous
abscess assay model
Subcutaneous injection of live trichomonads resulted in a small
pustule of ca. 40 mm2 area on Day 8 of injection (Day 1 of
treatment) in experimental and control animals [Fig. 7B(i) and C]
that grew to ca. 90 mm2 in area in controls (Fig. 7C) but was reduced
to ca. 15–25 mm2 after 5 days of treatment with compounds 15
and 18 and MTZ (P < 0.001 vs. parallel controls) (Fig. 7C). There-
after, growth of the abscess was exponential in controls and after
7 days of treatment it was >200 mm2 (Fig. 7B and C), whilst it was
further reduced to ca. 11–15 mm2 in treated animals (Fig. 7B and
C). On the day of autopsy (i.e. the day following 7 days of treat-
ment), the abscess was ca. 225 mm2 in area in untreated controls
and 12.5 mm2 in MTZ-treated, 4.45 mm2 in compound 15-treated
and 8.90 mm2 in compound 18-treated groups (P < 0.001 vs. par-
allel control) (Fig. 7C). The spleen weight at autopsy was ca. 0.2 g
in controls (non-infected), 0.58 g in trichomonas-infected animals,
0.51 g in MTZ-treated, 0.30 g in compound 15-treated and 0.25 g in
compound 18-treated animals (Fig. 7D and E).
4. Discussion
Facultative anaerobic cells/organisms such as sperm, Tri-
chomonas, Candida and Staphylococcus are generally very sensitive
to the redox status of the environment and prefer low-redox poten-
tial for active metabolism and motility/vitality. Thiols play a crucial
role in maintaining the optimum redox environment for these
cells, making them very susceptible to thiol-modifying agents. Tri-
chomonas does not synthesize glutathione, the main redox buffer,
and is almost completely dependent on cysteine for redox homeo-
stasis, which forms >70% of the intracellular thiol pool [19]. Cysteine
in association with the thioredoxin reductase/peroxidase system
maintains the major antioxidant defence mechanism of the para-
site [20]. Although the promising new compounds in comparison
with MTZ were much more effective against MTZ-resistant T. vagi-
nalis than the MTZ-susceptible strain, they faced mild resistance
by the MTZ-resistant Trichomonas. Given that the metabolites of
metronidazole also target thiols in Trichomonas [21], this common
mode of action may explain the low degree of cross-resistance. We
have shown previously that thiol-binding agents can serve as top-
ical vaginal microbicides with contraceptive activity [6]. However,
in most of our earlier molecules the spectrum of potent activity was
 B. Kushwaha et al. / International Journal of Antimicrobial Agents 47 (2016) 36–47 45

Fig. 7. (A) Subcutaneous abscess assay in mice using virulent, metronidazole (MTZ)-susceptible Trichomonas vaginalis. (B) Abscess size in untreated mice (I), treated mice (II,
III and IV) at Days 1, 3 and 7 of subcutaneous injections and control non-diseased animal (V). (C) Statistical data of decrease in abscess size at Days 1, 2, 5, 7 and 8 (autopsy
day). (D) Spleen size in control (non-diseased, a), experimental control (diseased, untreated, b), and mice treated with MTZ (c) and compounds 15 (d) or 18 (e) at autopsy. (E)
Variations in spleen size and weight per 100 g of body weight of control, experimental control, and mice treated with MTZ or compounds 15 and 18. Mean ± standard error
of three independent experiments. **, ***Significant difference from the control (**P < 0.01; ***P < 0.001).
46 B. Kushwaha et al. / International Journal of Antimicrobial Agents 47 (2016) 36–47
limited. Nevertheless, our enduring efforts to optimize the activ-
ity resulted in the present series in which potent activity against
Trichomonas could optimally be amalgamated with that against
Candida, Staphylococcus and sperm. Mixed vaginal infections of T.
vaginalis and C. albicans are quite frequent [10], whilst on the other
hand S. aureus often co-exists with these infections in women [22].
Staphylococcus aureus is amongst the prevalent vaginal (bacterial)
pathogen at 11–60 years of age, more in women of reproduc-
tive age [23]. Hence, the new compounds can potentially offer
broad-spectrum prophylaxis in topical vaginal use. Since intrav-
aginal topical agents offering simultaneous protection against
unwanted pregnancy are preferred by women over plain micro-
bicides [6], complementary spermicidal (contraceptive) activity in
four of five promising compounds could help in developing suit-
able alternatives to N-9, the active pharmaceutical ingredient used
in marketed spermicides. N-9 is a non-ionic detergent that kills
sperm, bacteria and protozoa by its non-specific surfactant action,
but it is currently under World Health Organization (WHO) and
FDA caution for general vaginal toxicity, which increases suscep-
tibility to STIs and HIV [24]. Unlike N-9, the selected compounds
were found to be extremely safe towards human cervical (HeLa)
cells and were highly compatible with Lactobacillus, the normal
vaginal flora, hence their N-9-like toxicity is quite unlikely in
vivo.
Trichomonas vaginalis disrupts urogenital epithelial layers facil-
itating HIV-1 passage to underlying layers and activates local
immune cells leading to increased viral replication [25]. Host cer-
vical cells express a multitude of receptors for the binding of
Trichomonas [26], and cytolysis of 80–90% of cervical cells is caused
by Trichomonas contact [27]. Cysteine proteases (thiol proteases)
that play a vital role in cytoadherence of parasites to host cells
[28,29] are readily detected in vaginal washes of infected women
[30], causing apoptosis of vaginal cells [31]. Up to 23 distinct
cysteine proteases have been detected in T. vaginalis [32]. The
ethyl amide group of N-ethylmaleimide alkylates the active-site
thiols making it an irreversible inhibitor of all cysteine peptidases.
Consequently, it exhibits mild spermicidal and trichomonacidal
activities. On the other hand, the unique structural design of our
compounds presents sulphur as a much stronger nucleophile that
readily reacts with cysteine thiols to form disulfides, blocking
the thiol action. Hence our compounds very substantially and
potently oppose the cytolytic/apoptotic action of Trichomonas on
HeLa cells. This was clearly evident in our experiments where
Trichomonas failed to disrupt HeLa monolayers in the presence
of the new (promising) compounds, and their activity was com-
parable with MTZ. The masking of thiols on Trichomonas by the
new compounds is indicative of the compounds targeting thiols on
pathogens. Haemolysis of RBCs is a well known property of vir-
ulent T. vaginalis, which was abolished by the new compounds,
especially compounds 15 and 18 that were apparently more effica-
cious than MTZ. Likewise, in the mouse subcutaneous abscess assay,
the in vivo infection with virulent Trichomonas was seemingly
treated more effectively by the new compounds 15 and 18 than by
MTZ.
Candida albicans germinates on HeLa cells within 2 h, destroy-
ing them completely as the host cells fail to phagocytose the yeast
[33]. The immunosuppressive action of mycotoxins produced by
Candida is thiol-dependent and is inhibited by reduced glutathione
[34]. Thiols are also very important for survival of Candida, which is
clearly evident from the fact that the candidacidal activity of human
lactoferrin and diamide is dependent on inactivation of thiols [35].
Whilst C. glabrata and Candida krusei are intrinsically resistant to
fluconazole (preferred for treating AIDS patients with candidiasis),
the emergence of fluconazole-resistant C. albicans strains is also on
the rise [36]. In the present study, compounds 16 and 24 were found
to be more active than fluconazole against C. albicans MTCC-183.8
strain. Since yeast and trichomonal infections often co-exist, the
efficacy of new compounds against mono/co-infections with yeasts
and drug-susceptible/resistant Trichomonas was more promising
than that of MTZ against monoinfections with only susceptible Tri-
chomonas.
As a pathogen, S. aureus must cope with oxidative stress gen-
erated by the human immune system [37], which triggers the
oxidation of glyceraldehydes-3-phosphate dehydrogenase thiols
making the pathogen very susceptible to ROS [38]. Hence, staphylo-
cocci were found to be extremely sensitive to thiol-depleting new
compounds. Targeting both of drug-susceptible and drug-resistant
strains of S. aureus with equal intensity indicates a different mode
of action of compounds than the antibiotics gentamicin and meti-
cillin and signifies their worth in eliminating obstinate strains of S.
aureus in the vagina.
Lactobacilli may prevent vaginal infections with S. aureus by
their potent antibacterial effects [39] and may also help in preven-
ting vaginal yeast infections [40], acting as a commensal microflora
for vaginal health. N-9 shows potent anti-Lactobacilli effects [15].
Nevertheless, the new promising compounds did not prevent Lac-
tobacillus growth and were almost as safe as MTZ towards this
probiotic. Thus, the ability of new structures to counter drug resis-
tance in pathogens, to preserve the commensal lactobacilli and to
inactivate sperm may add considerably to their worth as contra-
ceptive microbicides for vaginal use.
Funding: This work was supported by a grant [no. GAP0155] from
the Indian Council of Medical Research (New Delhi, India) and par-
tially by the CSIR – Network Project BSC0101. Research fellowships
to research scholars were awarded by the Indian Council of Medical
Research (ICMR) (to BK, VB, AP and DP), the Council of Scientific &
Industrial Research (CSIR) (to DM, PS and SKY) and the University
Grants Commission (to LK).
Competing interests: None declared.
Ethical approval: This study was approved by the Institutional
Animal Ethics Committee of CSIR – Central Drug Research Insti-
tute (Lucknow, India) [reference nos. CDRI/IEC/2015/A1 and
IAEC/2014/94]. CDRI communication number is 9119.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ijantimicag.2015.
10.022.
References
[1] World Health Organization. Global prevalence and incidence of selected
curable sexually transmitted infections: overviews and estimates. Geneva,
Switzerland: WHO; 2001. WHO/HIV AIDS/2001.02.
[2] Gottlieb SL, Douglas Jr JM, Foster M, Schmid DS, Newman DR, Baron AE, et al.
Incidence of herpes simplex virus type 2 infection in 5 sexually transmitted dis-
ease (STD) clinics and the effect of HIV/STD risk-reduction counseling. J Infect
Dis 2004;190:1059–67.
[3] Shew ML, Fortenberry JD, Tu W, Juliar BE, Batteiger BE, Qadadri B, et al. Asso-
ciation of condom use, sexual behaviors, and sexually transmitted infections
with the duration of genital human papillomavirus infection among adolescent
women. Arch Pediatr Adolesc Med 2006;160:151–6.
[4] Zuiable AG, Aboud H, Coulson I, Treleaven JG, Powles RL. Razoxane and T-cell
lymphoma. Br J Dermatol 1989;121:149.
[5] Twu O, Dessí D, Vu A, Mercer F, Stevens GC, de Miguel N, et al. Trichomonas
vaginalis homolog of macrophage migration inhibitory factor induces prostate
cell growth, invasiveness, and inflammatory responses. Proc Natl Acad Sci U S
A 2014;111:8179–84.
[6] Jain A, Lal N, Kumar L, Verma V, Kumar R, Kumar L, et al. Novel trichomonacidal
spermicides. Antimicrob Agents Chemother 2011;55:4343–51.
[7] Iizuka N, Onodera S, Kondo N, Kuroda J, Shirakawa H, Hosobe T, et al. Clinical
investigation of 9 cases of urachal carcinoma [in Japanese]. Hinyokika Kiyo
1991;37:17–20.
 B. Kushwaha et al. / International Journal of Antimicrobial Agents 47 (2016) 36–47
[8] Crowell AL, Stephens CE, Kumar A, Boykin DW, Secor WE. Activities of dica-
tionic compounds against Trichomonas vaginalis. Antimicrob Agents Chemother
2004;48:3602–5.
[9] Bauters TG, Dhont MA, Temmerman MI, Nelis HJ. Prevalence of vulvovaginal
candidiasis and susceptibility to fluconazole in women. Am J Obstet Gynecol
2002;187:569–74.
[10] López-Monteon A, Gómez-Figueroa FS, Ramos-Poceros G, Guzmán- Gómez D,
Ramos-Ligonio A. Codetection of Trichomonas vaginalis and Candida albicans by
PCR in urine samples in a low-risk population attended in a clinic first level in
central Veracruz, Mexico. Biomed Res Int 2013;2013:281892.
[11] US Centers for Disease Control and Prevention. CDC fact sheet. 10 ways
STDs impact women differently from men. CDC; 2011. http://www.cdc.gov/
nchhstp/ux-test-2015/newsroom/docs/factsheets/stds-women.pdf [accessed
22.06.15].
[12] Clinical and Laboratory Standards Institute. Reference method for broth dilu-
tion antifungal susceptibility testing of yeasts; approved standard Document
M27-A3. 3rd ed. Wayne, PA: CLSI; 2008.
[13] Liu H, Chen J, Jiang J, Giesy JP, Yu H, Wang X. Cytotoxicity of HC Orange NO. 1
to L929 fibroblast cells. Environ Toxicol Pharmacol 2008;26:309–14.
[14] Humphreys MJ, Allman R, Lloyd D. Determination of the viability of Trichomonas
vaginalis using flow cytometry. Cytometry 1994;15:343–8.
[15] Jain A, Kumar L, Kushwaha B, Sharma M, Pandey A, Verma V, et al. Combining
a synthetic spermicide with a natural trichomonacide for safe, prophylactic
contraception. Hum Reprod 2014;29:242–52.
[16] Tiwari P, Singh D, Singh MM. Anti-Trichomonas activity of Sapindus saponins,
a candidate for development as microbicidal contraceptive. J Antimicrob
Chemother 2008;62:526–34.
[17] Krieger JN, Poisson MA, Rein MF. ␤-Hemolytic activity of Trichomonas vaginalis
correlates with virulence. Infect Immun 1983;41:1291–5.
[18] Merkel GJ, Phelps CL. Factors influencing the interaction of Candida albicans
with fibroblast cell cultures. Infect Immun 1988;56:792–801.
[19] Westrop GD, Georg I, Coombs GH. The mercaptopyruvate sulfurtransferase of
Trichomonas vaginalis links cysteine catabolism to the production of thiore-
doxin persulfide. J Biol Chem 2009;284:33485–94.
[20] Coombs GH, Westrop GD, Suchan P, Puzova G, Hirt RP, Embley TM, et al.
The amitochondriate eukaryote Trichomonas vaginalis contains a diver-
gent thioredoxin-linked peroxiredoxin antioxidant system. J Biol Chem
2004;279:5249–56.
[21] Leitsch D, Kolarich D, Binder M, Stadlmann J, Altmann F, Duchene M. Tri-
chomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced
by the flavin enzyme thioredoxin reductase and disrupt the cellular redox
system. Implications for nitroimidazole toxicity and resistance. Mol Microbiol
2009;72:518–36.
[22] Fan A, Yue Y, Geng N, Zhang H, Wang Y, Xue F. Aerobic vaginitis and mixed
infections: comparison of clinical and laboratory findings. Arch Gynecol Obstet
2013;287:329–35.
[23] Mumtaz S, Ahmad M, Aftab I, Akhtar N, ul Hassan M, Hamid A. Aerobic
vaginal pathogens and their sensitivity pattern. J Ayub Med Coll Abbottabad
2008;20:113–7.
[24] Jain RK, Jain A, Maikhuri JP, Sharma VL, Dwivedi AK, Kumar ST, et al. In vitro test-
ing of rationally designed spermicides for selectively targeting human sperm
in vagina to ensure safe contraception. Hum Reprod 2009;24:590–601.
47
[25] Guenthner PC, Secor WE, Dezzutti CS. Trichomonas vaginalis-induced epithe-
lial monolayer disruption and human immunodeficiency virus type 1 (HIV-1)
replication: implications for the sexual transmission of HIV-1. Infect Immun
2005;73:4155–60.
[26] Okumura CY, Baum LG, Johnson PJ. Galectin-1 on cervical epithelial cells is a
receptor for the sexually transmitted human parasite Trichomonas vaginalis.
Cell Microbiol 2008;10:2078–90.
[27] Lustig G, Ryan CM, Secor WE, Johnson PJ. Trichomonas vaginalis
contact-dependent cytolysis of epithelial cells. Infect Immun 2013;81:
1411–9.
[28] Mendoza-López MR, Becerril-Garcia C, Fattel-Facenda LV, Avila- Gonzalez
L, Ruíz-Tachiquín ME, Ortega-Lopez J, et al. CP30, a cysteine proteinase
involved in Trichomonas vaginalis cytoadherence. Infect Immun 2000;68:
4907–12.
[29] Fichorova RN. Impact of T. vaginalis infection on innate immune responses and
reproductive outcome. J Reprod Immunol 2009;83:185–9.
[30] Alderete JF, Newton E, Dennis C, Neale KA. The vagina of women infected with
Trichomonas vaginalis has numerous proteinases and antibody to trichomonad
proteinases. Genitourin Med 1991;67:469–74.
[31] Sommer U, Costello CE, Hayes GR, Beach DH, Gilbert RO, Lucas JJ,
et al. Identification of Trichomonas vaginalis cysteine proteases that induce
apoptosis in human vaginal epithelial cells. J Biol Chem 2005;280:
23853–60.
[32] Neale KA, Alderete JF. Analysis of the proteinases of representative Trichomonas
vaginalis isolates. Infect Immun 1990;58:157–62.
[33] Farrell SM, Hawkins DF, Ryder TA. Scanning electron microscope study of Can-
dida albicans invasion of cultured human cervical epithelial cells. Sabouraudia
1983;21:251–4.
[34] Bertling A, Niemann S, Uekötter A, Fegeler W, Lass-Flörl C, von Eiff C, et al.
Candida albicans and its metabolite gliotoxin inhibit platelet function via inter-
action with thiols. Thromb Haemost 2010;104:270–8.
[35] Lupetti A, Paulusma-Annema A, Senesi S, Campa M, Van Dissel JT, Nibbering
PH. Internal thiols and reactive oxygen species in candidacidal activity exerted
by an N-terminal peptide of human lactoferrin. Antimicrob Agents Chemother
2002;46:1634–9.
[36] Wong SS, Kao RY, Yuen KY, Wang Y, Yang D, Samaranayake LP, et al. In vitro and
in vivo activity of a novel antifungal small molecule against Candida infections.
PLOS ONE 2014;9:e85836.
[37] Ji Q, Zhang L, Sun F, Deng X, Liang H, Bae T, et al. Staphylococcus aureus CymR is a
new thiol-based oxidation-sensing regulator of stress resistance and oxidative
response. J Biol Chem 2012;287:21102–9.
[38] Weber H, Engelmann S, Becher D, Hecker M. Oxidative stress triggers thiol
oxidation in the glyceraldehyde-3-phosphate dehydrogenase of Staphylococcus
aureus. Mol Microbiol 2004;52:133–40.
[39] Karska-Wysocki B, Bazo M, Smoragiewicz W. Antibacterial activity of
Lactobacillus acidophilus and Lactobacillus casei against methicillin-
resistant Staphylococcus aureus (MRSA). Microbiol Res 2010;165:
674–86.
[40] Strus M, Kucharska A, Kukla G, Brzychczy-Włoch M, Maresz K, Heczko PB.
The in vitro activity of vaginal Lactobacillus with probiotic properties against
Candida. Infect Dis Obstet Gynecol 2005;13:69–75.