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ORIGINAL PAPER
An orally effective dihydropyrimidone (DHPM) analogue induces
apoptosis-like cell death in clinical isolates of Leishmania
donovani overexpressing pteridine reductase 1
Neeloo Singh & Jaspreet Kaur & Pranav Kumar & Swati Gupta & Nasib Singh &
Angana Ghosal & Avijit Dutta & Ashutosh Kumar & RamaPati Tripathi &
Mohammad Imran Siddiqi & Chitra Mandal & Anuradha Dube
Received: 22 May 2009 /Accepted: 30 June 2009 /Published online: 21 July 2009
# The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract The protozoan parasite Leishmania donovani is
the causative agent of visceral leishmaniasis. The enzyme
pteridine reductase 1 (PTR1) of L. donovani acts as a
metabolic bypass for drugs targeting dihydrofolate reduc-
tase (DHFR); therefore, for successful antifolate chemo-
therapy to be developed against Leishmania, it must target
both enzyme activities. Leishmania cells overexpressing
PTR1 tagged at the N-terminal with green fluorescent
protein were established to screen for proprietary dihydro-
pyrimidone (DHPM) derivatives of DHFR specificity
synthesised in our laboratory. A cell-permeable molecule
with impressive antileishmanial in vitro and in vivo oral
activity was identified. Structure activity relationship based
on homology model drawn on our recombinant enzyme
established the highly selective inhibition of the enzyme by
this analogue. It was seen that the leishmanicidal effect of
this analogue is triggered by programmed cell death
mediated by the loss of plasma membrane integrity as
detected by binding of annexin V and propidium iodide
(PI), loss of mitochondrial membrane potential culminating
in cell cycle arrest at the sub-G0/G1 phase and oligonu-
cleosomal DNA fragmentation. Hence, this DHPM
analogue [(4-fluoro-phenyl)-6-methyl-2-thioxo-1, 2, 3, 4-
tetrahydropyrimidine-5-carboxylic acid ethyl ester] is a
potent antileishmanial agent that merits further pharmaco-
logical investigation.
Introduction
The kinetoplastid parasite Leishmania donovani is the causa-
tive agent of visceral leishmaniasis (VL) known as Kala-azar
in India. Over 90% of VL cases occur in India, Bangladesh,
Sudan, Brazil and Nepal (http://www.oneworldhealth.org/
diseases/leishmaniasis.php). The control of leishmaniasis in
absence of vaccine solely depends on the choice of
chemotherapy. Treatment available for VL is far from ideal
(Berman et al. 2006; Sundar and Chatterjee 2006). The
search for development of a new, safe, effective and
inexpensive drug that can cure VL throughout the world
continues. In the search for better therapeutics against VL,
we focused on a validated drug target viz. folate bio-
synthetic pathway which is unique to the parasite (Bello et
al. 1994). The enzyme pteridine reductase 1 (PTR1;
accession no. AY547305) of L. donovani acts as a
metabolic bypass for drugs targeting dihydrofolate reduc-
tase (DHFR); therefore, for successful antifolate chemo-
N. Singh (*) : J. Kaur : P. Kumar
Drug Target Discovery & Development Division,
Central Drug Research Institute,
Lucknow 226001, India
e-mail: neeloo888@yahoo.com
S. Gupta : N. Singh : A. Dube
Division of Parasitology, Central Drug Research Institute,
Lucknow 226001, India
A. Kumar : M. I. Siddiqi
Division of Molecular and Structural Biology,
Central Drug Research Institute,
Lucknow 226001, India
R. Tripathi
Division of Medicinal and Process Chemistry,
Central Drug Research Institute,
Lucknow 226001, India
A. Ghosal : A. Dutta : C. Mandal
Department of Infectious Disease and Immunology,
Indian Institute of Chemical Biology,
Kolkata 700032, India
Parasitol Res (2009) 105:1317–1325
DOI 10.1007/s00436-009-1557-z
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therapy to be developed against Leishmania, it must target
both enzyme activities. Reduced pterins and folates are
essential for the growth of Leishmania parasites, but anti-
pteridines have not shown much promise clinically against
Leishmania in contrast to other protozoal infections (Hardy
et al. 1997). This establishes the need for continued effort
and research in this direction. Since dihydropyridines and
pyrimidinones (80 thiones) are known inhibitors of DHFR
and exhibit antitubercular activity against Mycobacterium
tuberculosis, a pathogen residing in macrophages like
Leishmania (Küçükgüzel et al. 2007), we were interested
to see whether they exhibit any pteridine reductase
inhibitory activities, and therefore, this study was under-
taken. Dihydropyrimidone (DHPM) analogues have
exhibited important therapeutic and pharmacological prop-
erties as the integral backbone of several calcium channel
blockers (Kappe 2000), antihypertensive agents (Atwal et
al. 1991), α1a-antagonist (Kappe et al. 1997) and neuro-
peptide Y antagonists (Wang et al. 2006). A broad range of
biological effects including antiviral, antitumor, antibacte-
rial and anti-inflammatory activities have been described
for these compounds (Kappe 1993). Further, understanding
the mode of action and binding modes of these DHPM
analogues to specific target sites may be used to design
potent, novel, selective and less toxic antileishmanial
analogues of these compounds on a structural basis. Here,
we report a novel DHPM analogue that is cell permeable and
a potent oral antileishmanial molecule in vivo. We also
sought to determine the mechanism of leishmanicidal
activity of this compound. There are several reports showing
that Leishmania apoptosis occurs in response to antileish-
manial drugs (Wang et al. 2006; Singh et al. 2005). It has
also been demonstrated that the antileishmanial toxicity of
trivalent antimonials is associated with apoptosis (Mann et
al. 2006; Shaha 2006). This analogue induces programmed
cell death (PCD) in Leishmania parasites via externalisa-
tion of phosphatidyl serine involving changes in mitochon-
drial membrane potential leading to DNA fragmentation.
Materials and methods
Materials M-199 medium and foetal bovine serum (FBS)
were obtained from Gibco-BRL, dimethyl sulphoxide
(DMSO) from SRL, ethanol from Merck, propidium iodide
(PI), Annexin V-PE and MitoTracker deep red from
Molecular Probes. All other chemicals were from Sigma
unless stated.
Parasite culture Transgenic parasites overexpressing
PTR1–green fluorescent protein (GFP chimaera, PTR1
tagged at the N-terminal with GFP) were cultured at 25°C
in M-199 medium supplemented with 10% heat-inactivated
FBS, 100 U penicillin, 100μg/ml streptomycin and in the
presence of 150μg/ml geneticin sulphate (G418; Kumar et
al. 2007). These GFP-transfected parasites were used to
determine the IC50 of the compound by flow cytometric
analysis as established by us (Singh and Dube 2004). In
vitro antileishmanial activity was expressed as IC50 which
is the concentration that resulted in 50% inhibition of
parasites (Kumar et al. 2008).
Candidate compound Compound [(4-fluoro-phenyl)-6-
methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic
acid ethyl ester] was prepared by our earlier reported
method (Fig. 1; Dwivedi et al. 2005).
Cytotoxicity test upon cells The toxicity of compound was
evaluated on non-activated, freshly isolated normal human
peripheral blood mononuclear cells (PBMC) isolated
according to standard protocol (Fuss et al. 2009). PBMC
concentration was adjusted to 2×106viable cells/ml after
estimation of viability by trypan blue exclusion assay.
Viability was consistently greater than 96%. Cells (1×106)
were incubated with various concentrations of the com-
pound at 37°C in 5% CO2 for 24 h. The mean percentage of
post treatment viable cells relative to control was evaluated
by epifluorescence microscopy (Leica) using Live-Dead
Cell Staining Kit (BioVision).
Antileishmanial activity in vivo Syrian golden hamsters
(Mesocricetus auratus) were infected intracardially with
1×107 amastigotes isolated from the spleen of heavily
infected donor hamsters. Splenic biopsies were carried out
on day25 p.i. for assessing the status of infection by
making spleen dab smears. Animals having ten to 15
amastigotes per 100 spleen cell nuclei were selected and
used for screening purposes. Animals were treated orally
with the compound at 12.5, 25, 50, 75 and 100 mg/kg dose
for five consecutive days. Splenic biopsies were again
performed on day7 post treatment, and the parasite burden
in treated and untreated group was quantitated. Percent
2 5
3
Fig. 1 Chemical structure of
(4-fluoro-phenyl)-6-methyl-2-
thioxo-1,2,3,4
tetrahydropyrimidine-5-
carboxylic acid ethyl ester
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inhibition of parasite multiplication was calculated by the
following formula:
PI ¼
AT� 100
IT� TI
where PI is percent inhibition; AT is actual number of
amastigotes per 100 spleen cell nuclei in treated animals; IT
is initial number of amastigotes per 100 spleen cell nuclei
in treated animals; and TI is times increase in untreated
control animals. The use of animals for all the experiments
was in compliance with the relevant guidelines of the
institutional animal ethics committee.
DNA content (percent sub-G1) analysis by flow cyto-
metry L. donovani promastigotes (2×106 log phase) over-
expressing PTR1–GFP chimaera treated with compound
(98μM, 0–48 h) were harvested by centrifugation at
2,000×g, for 5 min at 4°C. Cells were washed once in
1 ml phosphate-buffered saline (PBS) and then fixed by
incubation in 70% ethanol:30% PBS for 1 h at 4°C. Prior to
analysis, fixed cells were harvested by centrifugation at
1,000×g, for 10 min at 4°C, washed in 1 ml PBS and then
resuspended in 1 ml PBS with RNAse A (100μg/ml) and
PI at 10 mg/ml. The cells were incubated at 25°C for
45 min and then analysed by using a Becton Dickinson
FACSCalibur flow cytometer. Ten thousand cells were
analysed for each sample. Cell cycle distribution was
modelled using the ModFit LT software package (Verity
Software House; Hedley et al. 1993).
Flow cytometric analysis of externalised phosphatidyl
serine Externalisation of phosphatidyl serine (PS) on the
outer membrane of promastigotes upon treatment with
compound was determined by using annexin V-PE. Briefly,
2×106 log phase L. donovani promastigotes overexpressing
PTR1–GFP chimaera were incubated with compound
(98μM, 24 h); cells were centrifuged at 2,000×g for
5 min at 4°C, washed twice in phosphate-buffered saline
(0.02 M, pH7.2, PBS) and resuspended in annexin V-PE
binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM
CaCl2; pH7.4). Annexin V-PE was then added according to
the manufacturer’s instructions and incubated for 30 min in
the dark at 20–25°C. Acquisition was done on a FACSCa-
libur flow cytometer (BD) and analysed with CellQuest
software.
Measurement of mitochondrial membrane potential Mito-
chondrial damage upon treatment with compound in L.
donovani promastigotes overexpressing PTR1–GFP chi-
maera was assessed by flow cytometry using a cell-
permeable dye, MitoTracker deep red. MitoTrackers are
aldehyde fixable cationic lipophilic fluorochrome that
passively diffuses through the plasma membrane of viable
cells and is selectively sequestered in mitochondria with an
active membrane potential and permits the examination of
the membrane potential in adherent cells (Haugland 1996).
L. donovani promastigotes were treated with compound
(98μM, 0–5 h), washed in PBS and loaded in dark for
30 min with MitoTracker (10μM) as per the manufacturer’s
instructions (Molecular Probes). Analysis for mean fluores-
cence intensity was done using FACSCalibur and CellQuest
software.
Analysis of nuclear morphology Oligonucleosomal frag-
mentation in L. donovani promastigotes (overexpressing
PTR1–GFP chimaera) treated with the compound was
identified microscopically using the fluorescent nucleic
acid-specific stain PI. Briefly, promastigotes were incubated
with compound (98μM 0–48 h), cells were then centri-
fuged at each time point, and the resultant pellet was loaded
with PI (10μg/ml) for 30 min at 20–25°C, attached on
poly-L-lysine coated glass slides, mounted in glycerol
containing 1,4-diazabicyclo octane and examined under a
confocal microscope (Leica, Germany). Untreated promas-
tigotes at each corresponding time point were also
processed and analysed similarly. At least 20 microscopic
fields were observed for each sample.
Oligonucleosomal DNA fragmentation assay To analyse
the presence of DNA fragments generated as a function of
cell death, total cellular DNA from L. donovani promasti-
gotes exposed to compound (98μM, 0–48 h) were isolated
according to manufacturer’s instructions (Bio-Rad).
Extracted DNA was quantified spectrophotometrically by
the absorbance ratio of 260/280 nm, and DNA (10μg/lane)
was separated by electrophoresis on 1% agarose gel
containing ethidium bromide in Tris base–boric acid–EDTA
buffer (50 mM; pH8.0) for 1.5 h at 75 V, visualised under
UV light and photographed using a gel documentation
system (GelDoc 2000, Bio-Rad).
Statistical analysis
The data are presented as mean±SD. The statistical
significance of differences in percentage between treated
and untreated was analysed by one-way analysis of
variance using GraphPad Prism software.
Results
Determination of cytotoxicity upon cells The toxicity data
against host cell macrophages was gathered before testing
the compound against the intracellular pathogen in vivo.
Cytotoxic potential of compound at 30 and 100μM on
Parasitol Res (2009) 105:1317–1325 1319
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PBMC was determined. The compound showed no cyto-
toxicity against macrophages (Fig. 2). More importantly,
absence of shared toxicity to PBMC has increased its
therapeutic ratio.
Determination of the IC50 of compound-mediated death in
vivo The compound exhibited excellent in vivo antileish-
manial efficacy by oral route against established infection
of L. donovani. It imparted 80.4±2.4% inhibition of
parasite at 50 mg/kg dose. At higher doses of 75 and
100 mg, it exhibited still higher (85.7±5.4% and 93.5±
4.5%, respectively) growth inhibitory effect. An ED50 of
36.3 mg/kg body weight was obtained (Fig. 3). This
compound was devoid of any toxicity towards animals as
no mortality was seen during the course of treatment.
Compound induces sub-G0/G1 phase cell cycle arrest in L.
donovani promastigotes The least expensive and most
rapid discrimination of apoptotic cells is based on DNA
content analysis. This is an established approach for
screening drug effects in vitro (Darzynkiewicz et al.
1997). The DNA content of cells is measured by the ability
of PI to bind stoichiometrically to DNA under appropriate
staining conditions. The nuclei of these stained cells are
Control
After 100 µM of compound treatment
Fig. 2 Cytotoxicity assay on PBMCs from human peripheral blood;
PBMCs were isolated from human peripheral blood. Cells were
incubated with various concentrations of the compound. The mean
percentage of post treatment viable cells relative to control was
evaluated by epifluorescence microscopy (Leica) using Live-Dead
Cell Staining Kit (BioVision). Magnification ×40. a Differential
interference contrast image of isolated macrophages. b Live cells
with green stain (flourescein isothiocyanate). c Dead cells with red
stain (PI). As very low or no cells were dead, no red spots were found.
Upper panels show the images without any treatment, and lower
panels show the images after 100μM of compound treatment for 24 h
Fig. 3 Analysis of antileishmanial activity of compound in vivo.
Syrian golden hamsters (M. auratus) were infected intracardially with
1×107 amastigotes isolated from the spleen of heavily infected donor
hamsters. Animals were treated orally with the inhibitor at 12.5, 25,
50, 75 and 100 mg/kg dose for five consecutive days. Splenic biopsies
were performed on day7 post treatment, and the parasite burden in
treated and untreated groups were quantitated
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evaluated individually for DNA content by flow cytometry.
The results are displayed graphically as a histogram in
which the fluorescence emitted by each nucleus is directly
proportional to its DNA content. In addition to the
enumeration of apoptotic cells offered by this method, the
cell cycle-specific effects can easily be recognised from
DNA content histograms of the nonapoptotic cell popula-
tions. We performed a cell cycle analysis by flow cytometry
after PI staining of the parasites incubated for 0–48 h at
IC50 (98μM) of the compound. To determine the apoptotic
population, the sub-G0/G1 peak was measured. Figure 4
shows the DNA cell cycle histograms of parasites before
and after treatment of the compound. DNA that is
fragmented is observed in a sub-G1 peak in the DNA
histograms of the treated parasites. The DNA histograms
show that DNA fragments increased significantly in
promastigotes from 24 (32%) to 48 h (48%) compared to
control. This suggests that this analogue could initiate late
events of PCD such as nuclear condensation and DNA
nicking.
L. donovani promastigotes show externalisation of phos-
phatidylserine following treatment with compound In meta-
zoan and unicellular cells, ubiquitous alteration is the
translocation of phosphatidylserine from the inner side to
the outer layer of the plasma membrane during PCD (Mehta
and Shaha 2004). Annexin V, a Ca2+-dependent phospho-
lipid binding protein with affinity for phosphatidylserine, is
routinely used to label externalisation of phosphatidylser-
ine. Hence, for the determination of the mechanism of cell
death triggered by this compound via apoptosis (annexin V
positive), compound-treated L. donovani promastigotes
overexpressing PTR1–GFP chimaera (98μM, 24 h) were
stained with annexin V-PE. A significant percentage (46%)
of promastigotes of treated cells (3 h) stained positive for
both GFP and annexin V as shown in upper right quadrant
as compared to only 2.0% in untreated cells (Fig. 5). The
percentage of double-stained cells (upper right quadrant)
indicated that this inhibitor exerts its leishmanicidal activity
primarily via apoptosis in Leishmania cells overexpressing
PTR1. The increase in annexin V positive cells increased
Control 32%
SubG1
48%
SubG1
Fig. 4 DNA content (percent
sub-G1) analysis of cells upon
treatment with compound. Log
phase L. donovani promasti-
gotes (overexpressing PTR1–
GFP chimaera) were incubated
with inhibitor (98μM) for 24
and 48 h, stained with PI and
analysed by flow cytometry. A
significant sub-G1 was observed
(blue). This is a representative
profile of at least three
experiments
Fig. 5 Externalisation of phos-
phatidylserine in L. donovani
promastigotes overexpressing
PTR1 upon treatment with
compound. Log phase
L. donovani promastigotes
(overexpressing PTR1–GFP
chimaera) were incubated with
compound (98μM) for 24 h,
stained with annexin V-PE and
analysed by flow cytometry.
This is a representative profile
of at least three experiments
Parasitol Res (2009) 105:1317–1325 1321
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