Novel betulin derivatives as antileishmanial agents with mode of action targeting type IB DNA topoisomerase

Article abstract of DOI:10.1124/mol.111.072785

Toward developing antileishmanial agents with mode of action targeted to DNA topoisomerases of Leishmania donovani, we have synthesized a large number of derivatives of betulin. The compound, a natural triterpene isolated from the cork layer of Betula spp. plants exhibits several pharmacological properties. Three compounds (disuccinyl betulin, diglutaryl dihydrobetulin, and disuccinyl dihydrobetulin) inhibit growth of the parasite as well as relaxation activity of the enzyme type IB topoisomerase [Leishmania donovani topoisomerase I (LdTOP1LS)] of the parasite. Mechanistic studies suggest that these compounds interact with the enzyme in a reversible manner. The stoichiometry of these compounds binding to LdTOP1LS is 1:1 (mole/mole) with a dissociation constant on the order of ～10^-6 M. Unlike CPT, these compounds do not stabilize the cleavage complex; rather, they abrogate the covalent complex formation. In processive mode of relaxation assay condition, these compounds slow down the strand rotation event, which ultimately affects the relaxation of supercoiled DNA. It is noteworthy that these compounds reduce the intracellular parasite burden in macrophages infected with wild-type L. donovani as well as with sodium antimony gluconate resistant parasite (GE1). Taken together, our data suggest that these betulin derivatives can be exploited as potential drug candidates against threatening drug resistant leishmaniasis. Copyright

Full text of DOI:10.1124/mol.111.072785

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MOLECULAR PHARMACOLOGY
Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
Vol. 80, No. 4
72785/3717289
Mol Pharmacol 80:694–703, 2011
Printed in U.S.A.
Novel Betulin Derivatives as Antileishmanial Agents with Mode
□
S
of Action Targeting Type IB DNA Topoisomerase
Sayan Chowdhury, Tulika Mukherjee, Souvik Sengupta, Somenath Roy Chowdhury,
Sibabrata Mukhopadhyay, and Hemanta K. Majumder
Molecular Parasitology Laboratory (S.C., S.S.G., S.R.C., H.K.M.) and Department of Chemistry (T.M., S.M.), Indian Institute of
Chemical Biology, Kolkata, India
Received April 6, 2011; accepted July 12, 2011
ABSTRACT
Toward developing antileishmanial agents with mode of action sociation constant on the order of ϳ10Ϫ6 M. Unlike CPT, these
targeted to DNA topoisomerases of Leishmania donovani, we compounds do not stabilize the cleavage complex; rather, they
have synthesized a large number of derivatives of betulin. The abrogate the covalent complex formation. In processive mode of
compound, a natural triterpene isolated from the cork layer of relaxation assay condition, these compounds slow down the
Betula spp. plants exhibits several pharmacological properties. strand rotation event, which ultimately affects the relaxation of
Three compounds (disuccinyl betulin, diglutaryl dihydrobetulin, supercoiled DNA. It is noteworthy that these compounds reduce
and disuccinyl dihydrobetulin) inhibit growth of the parasite as well the intracellular parasite burden in macrophages infected with
as relaxation activity of the enzyme type IB topoisomerase [Leish- wild-type L. donovani as well as with sodium antimony gluconate
mania donovani topoisomerase I (LdTOP1LS)] of the parasite. resistant parasite (GE1). Taken together, our data suggest that
Mechanistic studies suggest that these compounds interact with these betulin derivatives can be exploited as potential drug can-
the enzyme in a reversible manner. The stoichiometry of these didates against threatening drug resistant leishmaniasis.
compounds binding to LdTOP1LS is 1:1 (mole/mole) with a dis-
disease. This led to treatment with current drugs, such as
expensive pentamidine (Mishra et al., 1992), amphotericin B
Introduction
Leishmaniasis is one of the major fatal parasitic diseases
(AmBisome; Astellas Pharma, Deerfield, IL) (Wortmann et
affecting millions of people around the world. The disease
al., 2010; Solomon et al., 2011) and the teratogenic drug
presents a variety of symptoms ranging from self-healing
miltefosine (Impavido; Æterna Zentaris, Quebec, QC, Can-
cutaneous lesions through the metastasizing mucocutaneous
ada) (Sindermann and Engel, 2006; Seifert et al., 2007).
form to the often-fatal visceralizing form (Olivier et al.,
2005). Development of parasites resistant to most commonly
Therefore, improvised drug therapy of Leishmania spp. in-
fection is still desirable, and the need for new molecular
used pentavalent antimonials (e.g., glucantime and Pentos-
targets on which to base improved therapies is clear and
tam) (Ashutosh et al., 2007) or arsenite (Haimeur et al.,
1999), together with lack of available vaccine (Kedzierski,
2010; Nyle´n and Gautam 2010), pose a threat to cure the
justified.
DNA topoisomerases are a family of DNA-processing en-
zymes that release torsional stress in the DNA. The DNA-
resolving enzymes catalyze breakage and rejoining of DNA
strands during several vital life processes, such as replica-
tion, repair, recombination, transcription, chromosome seg-
regation, and integration (Liu, 1989; Wang, 1996). These
DNA manipulators modulate the dynamic nature of DNA
secondary and higher order structure by transient nicking/
closing of DNA strands (Champoux, 2001).
This work was supported by the Network Project from Council of Scientific
and Industrial Research (CSIR), Government of India [Grant NWP-0038]; and
by a Senior Research Fellowship from Council of Scientific and Industrial
Research, Government of India (to S.C.).
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.072785.
□S The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
ABBREVIATIONS: LdTOP1L, Leishmania donovani topoisomerase I large subunit; LdTOP1S, Leishmania donovani topoisomerase I small subunit;
CPT, camptothecin; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; pBS, pBluescript; BSA, bovine serum albumin; EtBr, ethidium bromide; FBS,
fetal bovine serum; RPMI-FBS, RPMI 1640 medium (supplemented with 100 IU/ml penicillin and 100 ␮g/ml streptomycin) containing 10% (v/v)
heat-inactivated FBS; DiSB, 3-O,28-O-disuccinyl betulin (4); DiGDHB, 3-O,28-O-diglutaryl dihydrobetulin (8); DiSDHB, 3-O,28-O-disuccinyl
dihydrobetulin (10); LdTOP1LS, Leishmania donovani type IB topoisomerase; SbS, antimony-sensitive; SbR, antimony-resistant; Lk, Linking
number.
694

Betulin Derivatives, Unique LdTOP1LS Inhibitors
695
The topoisomerases can be classified into types I and II,
both of which are of equal importance as chemotherapeutic
targets (Fortune and Osheroff, 2006; Pommier, 2006;
Teicher, 2008; Nitiss, 2009). Unlike eukaryotic type IB topo-
isomerases, kinetoplastid topoisomerases IB possess het-
erodimeric structure consisting of a large subunit of 635
amino acids (LdTOP1L) and a small subunit of 262 amino
acids (LdTOP1S) (Das et al., 2004). Reconstitution of the
large subunit (L) bearing DNA binding “VAILCNH” motif
with the small subunit (S) bearing the active site SKXXY
motif occurs through protein-protein interaction to form an
active heterodimeric enzyme within the parasite (Das et al.,
2004).
Improvised drug therapy of Leishmania spp. infections has
become a need and use of topoisomerase inhibitors for anti-
leishmanial therapy are of immense interest. These inhibi-
tors can be broadly classified into two classes: the class I
inhibitors stabilize the formation of topoisomerase-DNA co-
valent complex (cleavable complex) and are known as “topo-
isomerase poisons.” Inhibitors with property to abrogate only
the catalytic property of the enzyme and thus interfere with
the formation of covalent complex formation are termed class
II inhibitors (Li et al., 1993; Chowdhury et al., 2003). The
most well studied type IB topoisomerase poison camptoth-
ecin (CPT) promotes protein-DNA cleavable complex forma-
tion (Bodley and Shapiro, 1995) leading to apoptosis-like cell
death (Sen et al., 2004) in Leishmania donovani.
We have reported previously that dihydrobetulinic acid,
isolated from the leaves of Bacopa monnieri, has profound
effect on intracellular amastigotes with an IC50 value of 4.1
␮M. Dihydrobetulinic acid inhibits Leishmania spp. topo-
isomerases and induces apoptosis in L. donovani with an
effective clearance of parasites from infected golden ham-
sters (Chowdhury et al., 2003).
Betulin [lup-20(29)-ene-3b,28-diol] is an abundant natu-
rally occurring triterpene and constituent of the cork layer of
the outer bark of Betula alba, Betula pendula, Betula pubes-
cent, and Betula platyphylla. Betulin and the more active
form betulinic acid exhibit antimalarial (Steele et al., 1999),
anti-HIV, and anti-inflammatory (Reutrakul et al., 2010) as
well as cytotoxic activities on cancer cell lines (Laszczyk,
2009).
Synthesis of Several Derivatives of Betulin and Dihydro-
betulin. Hydrogenation of betulin was carried out by dissolving 400
mg of betulin in a minimal volume of dry ethyl acetate (50 ml) and a
little dichloromethane was added to clear the solution. Then catalytic
amount of 10% palladized charcoal was added and exposed to a
hydrogen gas atmosphere, the reaction mixture was stirred over-
night at room temperature and filtered, and the hydrogenated prod-
uct was dried over vacuum.
For general preparations of betulin or dihydrobetulin derivatives,
a mixture of betulin (50 mg, 1 Eq) or dihydrobetulin (50 mg, 1 Eq)
and 4-N,N-dimethylaminopyridine (2 mg) was dissolved in pyridine
(2 ml). Acid anhydride (10 Eq) was added, and the reaction mixture
was placed in 80–90°C pre heated oil bath and stirred for 24 h in
anhydrous condition. After the usual work-up, the organic layer was
collected and dried under reduced pressure. All the reaction products
were purified by chromatography over silica gel (60–120 mesh) and
were monitored through thin layer chromatography (solvent system,
2% methanol in chloroform).
Purification and Reconstitution of Recombinant Proteins
of Topoisomerase I Activity. Escherichia coli BL21 (DE3)pLysS
cells harboring pET16bLdTOP1L and pET16bLdTOP1S, described
previously (Das et al., 2006), were separately induced at OD600 ϭ 0.6
with 0.5 mM isopropyl ␤-D-thiogalactoside at 22°C for 12 h. Cells
harvested from 1 liter of culture were separately lysed by lysozyme/
sonication, and the proteins were purified through Ni2ϩ-nitriloac-
etate-agarose column (QIAGEN, Valencia, CA,) followed by a phos-
phocellulose column (P11 cellulose; Whatman) as described
previously (Das et al., 2004). Finally, the purified proteins LdTOP1L
and LdTOP1S were stored at Ϫ70°C. The concentrations of each
protein were quantified by Bradford reaction using a Protein Esti-
mation Kit (Bio-Rad, Hercules, CA) according to the manufacturer’s
protocol.
Purified LdTOP1L was mixed with purified LdTOP1S at a molar
ratio of 1:1 at a total protein concentration of 0.5 mg/ml in reconsti-
tution buffer [50 mM potassium phosphate, pH 7.5, 0.5 mM DTT, 1
mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 10% (v/v)
glycerol]. The mixture was dialyzed overnight at 4°C, and dialyzed
fractions were used for plasmid relaxation activity (Das et al., 2004,
2005).
Plasmid Relaxation Assay. The type I DNA topoisomerases
were assayed by decreased mobility of the relaxed isomers of super-
coiled pBlueScript (SKϩ) [pBS (SKϩ)] DNA in agarose gel. The re-
laxation assay was carried out as described previously with
LdTOP1LS (Sen et al., 2004; Das et al., 2005), serially diluted in the
relaxation buffer (25 mM Tris-HCl, pH 7.5, 5% glycerol, 0.5 mM DTT,
10 mM MgCl2, 50 mM KCl, 25 mM EDTA, and 150 ␮g/ml BSA) and
supercoiled plasmid pBS (SKϩ) DNA (85–95% was negatively super-
coiled, with remainder being nicked circles). The reconstituted en-
zyme LdTOP1LS was assayed at 50 mM KCl concentration, whereas
human topoisomerase I was assayed at 150 mM KCl concentration
as described previously (Ganguly et al., 2006). For all kinetic studies,
the reaction mixtures containing the buffer and DNA were heated to
37°C before addition of the enzymes. The reactions were rapidly
quenched using stop solution and kept on ice. The gels were stained
with ethidium bromide (EtBr) (0.5 ␮g/ml), and the amount of super-
coiled monomer DNA band fluorescence was quantified by integra-
In the present study, we report some novel derivatives of
betulin, which interfere with the relaxation activity of L.
donovani topoisomerase I. These derivatives act like class II
inhibitors and abrogate topoisomerase I-DNA complex for-
mation. These compounds prevent the strand rotation step
by binding to enzyme in 1:1 ratio through a weak interaction.
It is noteworthy that these betulin derivatives can efficiently
reduce parasite burden from macrophage cultures infected
with antimony resistant and sensitive Leishmania spp. par-
asites having less effect on host cells. Thus, the need for new tion using Gel Doc 2000 under UV illumination (Bio-Rad Quantity
One Software), as described previously (Das et al., 2005). Initial
velocities (nanomoles of DNA base pairs relaxed per min) were
calculated using the following equation: Initial Velocity ϭ ([Super-
coiled DNA]0 – (Intt ꢀ [supercoiled DNA]0/Int0)/t, where ([Supercoiled
DNA]0 is the initial concentration of supercoiled DNA, Int0 is the
area under the supercoiled DNA band at zero time, and Intt is the
therapeutic agents against antimony-resistant strains of
Leishmania spp. can be exploited using modification of plant-
derived betulin.
Materials and Methods
Chemicals. DMSO and camptothecin were purchased from area at the reaction time t (Osheroff et al., 1983). The effect of DNA
Sigma-Aldrich (St. Louis, MO). All drugs were dissolved in 100% concentration on the kinetics of relaxation was examined over a
DMSO at a concentration of 20 mM and stored at Ϫ20°C. Recombi- range of 4 to 60 nM supercoiled pBS (SKϩ) DNA (0.16–2.4 ␮g/25 ␮l
nant human topoisomerase I was purchased from TopoGEN, Inc. of reaction mixture) at constant concentration of 10 mM MgCl2 and
(Port Orange, FL).
0.98 nM enzyme (LdTOP1LS) at 37°C for 1 min. The data were

696
Chowdhury et al.
analyzed by a Lineweaver-Burk plot. Intercept of the y-axis is cillin and 100 ␮g/ml streptomycin at 22°C to obtain promastigotes.
1/Vmax, and catalytic-center activity ϭ Vmax/enzyme concentration Promastigotes were further grown in 10% (v/v) heat-inactivated FBS
(plasmid molecules relaxed per minute per molecule of enzyme).
for 3 to 5 days at 22°C before use.
Plasmid Cleavage Assay. Cleavage assay was carried out as
In Vitro Macrophage Infection. BALB/c mice, originally ob-
described previously (Ray et al., 1998). In brief, 50 fmol of pHOT1 tained from The Jackson Laboratory (Bar Harbor, ME) and reared in
supercoiled DNA (containing topoisomerase I cleavage site) and 100 the institute animal facilities, were used for experimental purposes
fmol of reconstituted LdTOP1LS were incubated in standard reac- with prior approval of the animal ethics committee. Macrophages
were isolated from mice 36 to 48 h after injection (intraperitoneal)
with 2% (w/v) hydrolyzed starch by peritoneal lavage with ice-cold
phosphate-buffered saline. Cells were washed and cultured for 18 to
24 h (for adherence) in RPMI 1640 medium (supplemented with 100
IU/ml penicillin and 100 ␮g/ml streptomycin) containing 10% (v/v)
heat-inactivated FBS (RPMI-FBS) at 37°C at 5% CO2 in air on sterile
cover glass (22 ϫ 22 mm) placed in disposable plates 35 mm in
diameter (Tarsons India Ltd., Kolkata, India). The culture medium
was washed off, and fresh RPMI-FBS was added. Approximately 5 ϫ
105 macrophages were maintained for proper distribution on cover
glass. During the course of this study, macrophages were infected
with promastigotes at a macrophage-to-parasite ratio of 1:10 in
RPMI-FBS for 6 h to ensure entry of parasite. After incubation,
unphagocytosed parasites were removed by washing with medium,
and cells were resuspended in RPMI-FBS at 37°C, 5% CO2 and then
incubated for 6 h. Cultures were transferred to a CO2 incubator at
37°C and incubated for another 10 to 12 h. Respective inhibitors
were added at different concentrations (ranging from 1 to 20 ␮M) to
infected macrophages and left for another 24-h period. Cells were
then fixed in methanol and stained with 2% Giemsa. Percentages of
infected cells and total number of intracellular parasites were deter-
mined by manual counting in at least 200 cells using light micro-
scope.
tion mixture (50 ␮l) containing 50 mM Tris-HCl, pH 7.5, 100 mM
KCl, 10 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, and 30 ␮g/ml BSA
in the presence of various concentrations of inhibitors at 37°C for 30
min. The reactions were terminated by adding 1% SDS and 150
␮g/ml proteinase K and further incubated for 1 h at 37°C. DNA
samples were electrophoresed in 1% agarose gel containing 0.5 ␮g/ml
EtBr to resolve more slowly migrating nicked product (form II) from
the supercoiled molecules (form I).
Suicide Cleavage Assay. A 14-mer (5Ј-GAAAAAAGACTT2AG-
3Ј) oligonucleotide (ML14) containing a topoisomerase IB-specific
cleavage site was 5Ј-32P-end-labeled and annealed to 25-mer (3Ј-
CTTTTTTCTGAATCTTTTTAAAAAT-5Ј) oligonucleotides (ML25) as
described previously (Das et al., 2005). The suicidal cleavage reaction
was carried out with 5 nM DNA substrate and 0.2 ␮M enzyme
(LdTOP1LS) in 20-␮l reaction mixtures under standard assay con-
ditions (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 0.5 mM EDTA, and
50 mM KCl) at 23°C for 2 h in the presence or absence of betulin
inhibitors. Finally, all the reactions were stopped by adding SDS,
and DNAs were subsequently precipitated by ethanol. Samples were
digested with 5 ␮l of 1 mg/ml trypsin, electrophoresed in 12% (w/v)
denaturing polyacrylamide gel, and autoradiographed.
Job Plot. The binding stoichiometry for each of the inhibitors
with LdTOP1LS was determined using the method of continuous
variation (Huang, 1982; Ward, 1985). Several mixtures of each of the
three inhibitors and LdTOP1LS were prepared by continuously vary-
ing the concentrations of recombinant LdTOP1LS and one inhibitor
in the mixtures, keeping the total concentration of inhibitor plus
recombinant LdTOP1LS constant at 1.25 ␮M. Reaction mixtures
were incubated for 10 min at 25°C, and the quenching of tryptophan
fluorescence was recorded at 350 nm upon excitation at 295 nm on a
luminescence spectrometer (LS55; PerkinElmer Life and Analytical
Sciences, Waltham, MA).
Spectrofluorimetric Binding Assay. LdTOP1LS (200 nM) was
incubated in fluorescence buffer (20 mM Tris/HCl, 50 mM NaCl, and
10 mM MgCl2) with varying concentrations of each of the inhibitors
(0–22.5 ␮M) at 25°C for 10 min. The fluorescence intensity was
measured at 350 nm upon excitation at 295 nm. Excitation and
emission slit widths were 2.5 and 5 nm, respectively. The measure-
ments in the fluorescent values (performed in duplicate) in the
presence of continuous increasing concentrations of inhibitors were
corrected for the inner filter effect for excitation and emission wave-
length. The fraction of binding sites (B) occupied by inhibitor was
determined by the following equation: B ϭ (F0 Ϫ F)/Fmax, where F0 is
the fluorescence intensity of LdTOP1LS alone in the absence of any
Results
Derivatives of Betulin and Dihydrobetulin Inhibit
the Catalytic Activity of L. donovani Topoisomerase
IB. A series of analogs of betulin (Fig. 1A, 1) [e.g., 3-O,28-O-
diglutaryl betulin (2), 3-O,28-O-diphthaloyl betulin (3),
3-O,28-O-disuccinyl betulin (4), 3-O,28-O-diacetyl betulin (5),
and 3-O,28-O-dicrotonyl betulin (6)] was prepared from bet-
ulin as mentioned under Materials and Methods. Dihydro-
betulin (Fig. 1B, 7) was synthesized by hydrogenation of
betulin, and similar reactions were carried out to generate
3-O,28-O-diglutaryl dihydrobetulin (8), 3-O,28-O-diphthaloyl
dihydrobetulin (9), 3-O,28-O-disuccinyl dihydrobetulin (10),
3-O,28-O-diacetyl dihydrobetulin (11), and 3-O,28-O-dicroto-
nyl dihydrobetulin (12).
The effect of these chemically synthesized derivatives of
betulin and dihydrobetulin on the unusual bisubunit type IB
topoisomerase of L. donovani (LdTOP1LS) was examined by
plasmid relaxation assays as described under Materials and
Methods. The relaxation assays were carried out under stan-
dard assay conditions in which the plasmid DNA and the
enzyme were present at a molar ratio of 3:1. Under
this reaction condition, in the absence of any inhibitor,
inhibitors, and
F is the corrected fluorescence intensity of
LdTOP1LS in the presence of inhibitor. Fmax is obtained from the
plot of 1/(F0 Ϫ F) versus 1/[X] and by extrapolating of 1/[X] to zero as
shown in Fig. 6B, where [X] is the concentration of one of the three
inhibitors. The dissociation constant (Kd) was determined as de-
scribed previously (Acharya et al., 2008) using the following equa- LdTOP1LS relaxes supercoiled plasmid DNA completely af-
tion: Fmax/(F0 Ϫ F) ϭ 1 ϩ Kd/Lf, where Lf denotes the free concen-
tration of inhibitor; Lf ϭ C Ϫ B ϫ [J], where C is the total
concentration of inhibitor and [J] is the molar concentration of li-
gand-binding sites using a stoichiometry from the Job plot.
Parasite Maintenance and Cultures. Two strains of L. don-
ovani, one sodium antimony gluconate-sensitive (Sbs) MHOM/IN/
1983/AG83 (AG83) and one sodium antimony gluconate-resistant
(Sbr) GE1 (raised in hamsters) (Basu et al., 2005), were used. Amas-
tigotes obtained from the spleens of infected hamsters were cultured
at 22°C to obtain promastigotes and cultured in M199 containing
ter 30 min of incubation (Roy et al., 2008). Screening of all the
above compounds on relaxation activity of LdTOP1LS was
carried out using 2% (v/v) DMSO. Betulin and dihydrobetulin
have no inhibitory effect on LdTOP1LS but 3-O,28-O-disuc-
cinyl betulin (DiSB) (4) and 3-O,28-O-diglutaryl dihydrobetu-
lin (DiGDHB) (8) completely inhibit the enzyme activity at
200 ␮M, while 3-O,28-O-disuccinyl dihydrobetulin (DiSDHB)
(10) achieves approximately 93% inhibition at the same con-
dition (data not shown). DiSB, DiGDHB, and DiSDHB show
20% (v/v) heat-inactivated FBS supplemented with 100 IU/ml peni- little inhibition at 10 ␮M (Fig. 2A, lanes 4, 9, 14, respectively)

Betulin Derivatives, Unique LdTOP1LS Inhibitors
697
respectively. To rule out the binding of BSA present in the
relaxation assay mixture, the inhibition kinetics was also
studied in the absence of BSA (data not shown). The IC50
values calculated for each of the inhibitors in simultaneous
relaxation assay in such condition were 11.8 ␮M for DiSB,
15.3 ␮M for DiGDHB, and 24.2 ␮M for DiSDHB. All the IC50
values were calculated using the variable slop model for
finding EC50 in Prism (ver. 5.0; GraphPad Software, San
Diego, CA), which fits the nonlinear regression equation.
Betulin and Dihydrobetulin Derivatives Inhibit L.
donovani Topoisomerase IB-Mediated DNA Cleavable
Complex Formation by Abrogating Topoisomerase-
DNA Interaction. Topoisomerase I changes the linking
number (Lk) of DNA by transiently cleaving single strands
and thus producing nick, about which the uncleaved strand
swivels via a “controlled rotation” mechanism, followed by
religation of the nicked phosphodiester bond. The putative
intermediate of this reaction is a covalent enzyme-DNA com-
plex (cleavable complex) that can be irreversibly converted to
topoisomerase I-linked DNA single-strand breaks by addi-
tion of strong protein-denaturing agents such as NaOH or
SDS. Topoisomerase inhibition can be achieved by preven-
tion of enzyme-DNA binary complex formation or by stabiliz-
ing the enzyme-DNA cleavable complex. Camptothecin, the
most established uncompetitive inhibitors of type IB topo-
isomerase, traps the protein-DNA cleavable complex. In the
present study, the ability of these compounds to stabi-
lize cleavable complex formation between LdTOP1LS and
pHOT1 DNA (Fig. 3A), containing a topoisomerase IB-spe-
cific binding site as mentioned under Materials and Methods,
has been investigated. The cleavage assay was performed at
increasing concentrations of these compounds up to 200 ␮M,
with CPT as a positive control under standard assay condi-
tions. As shown in Fig. 3A, both 25 and 50 ␮M camptothecin
convert closed circular DNA (form I) to nicked circular DNA
Fig. 1. Chemical structure of betulin and its derivatives (A) and dihydro-
betulin and its derivatives (B).
under the same conditions. The inhibition increases in a (form II) by stabilization of the “cleavable complex” (lanes 5
dose-dependent fashion for all the three compounds (Fig. 2A, and 6). Lane 3 shows the formation of form II DNA as a result
lanes 5–8 for DiSB, lanes 10–13 for DiGDHB, and lanes of cleavage of pHOT1 DNA with 100 fmol of LdTOP1LS
15–18 for DiSDHB). At 100 ␮M DiSB and DiGDHB, almost alone. Amount of form II DNA increases when the nicked
98% inhibition was achieved (Fig. 2A, lane 7 for DiSB and products are trapped with SDS and proteinase K (lane 4).
lane 12 for DiGDHB), but DiSDHB inhibits only to the extent This serves as the background cleavage of LdTOP1LS. When
of 94% at 200 ␮M (Fig. 2A, lane 18).
the cleavage assay was performed at increasing concentra-
To investigate whether these compounds interact with the tions (50, 100, and 200 ␮M) of these compounds using 100
enzyme, LdTOP1LS was preincubated with these compounds fmol of LdTOP1LS, no remarkable nicked products (Fig. 3A,
at different concentrations for 5 min at 37°C before the ad- lanes 7–9 for DiSB, lanes 10–12 for DiGDHB, and lanes
dition of substrate DNA (Fig. 2B). The inhibitory effect of 13–15 for DiSDHB) were observed, whereas camptothecin at
these compounds in preincubation condition was compared 25 ␮M (lane 5) and 50 ␮M (lane 6) stabilized almost 70 and
with the inhibition by these compounds incubated simulta- 90% of the cleavable complex, respectively. EtBr at a final
neously with the enzyme (LdTOP1LS) and supercoiled DNA concentration of 0.5% ␮g/ml was included in the gel to resolve
in the relaxation reaction (Fig. 2A). Ninety-eight percent the more slowly migrating nicked product (form II) from the
inhibition was achieved at 20 ␮M DiSB (Fig. 2B, lane 7) and relaxed molecules (form IЈ).
DiGDHB (Fig. 2B, lane 13); but for DiSDHB, 98% inhibition
takes place only at 50 ␮M (Fig. 2B, lane 20).
The experiment was further supported by suicidal cleavage
assay by reacting LdTOP1LS with ML14/ML25 duplex oligo-
The percentage of relaxation inhibition was plotted against nucleotides in presence of 200 ␮M concentrations of each
the concentration of the compounds for both simultaneous inhibitor (Supplemental Fig. S1). The result indicates that
and preincubation assay conditions (Fig. 2, C and D). The these inhibitors do not affect the LdTOP1LS-mediated cleav-
IC50 values were determined under various concentrations of age reaction (i.e., second step of the reaction).
these compounds with the same amount of enzyme and sub-
Moreover, when 100 fmol of topoisomerase I was preincu-
strate. The IC50 values of DiSB, DiGDHB, and DiSDHB bated with 25, 50, and 100 ␮M concentrations of these deriv-
under simultaneous relaxation assays were 12.6, 15.9, and atives before addition of 100 ␮M CPT (Fig. 3B, lanes 6–8 for
23.1 ␮M, respectively; under preincubation assay conditions, DiSB, lanes 9–11 for DiGDHB, and lanes 12–14 for
these compounds have IC50 values of 7.35, 8.2, and 10 ␮M, DiSDHB), the CPT-mediated cleavage was inhibited drasti-

698
Chowdhury et al.
Fig. 2. Inhibition of catalytic activity of LdTOP1LS by
derivatives of betulin and dihydrobetulin. A, relaxation of
supercoiled pBS (SKϩ) DNA with reconstituted LdTOP1LS
at a molar ratio of 3:1. Lane 1, 90 fmol of pBS (SKϩ) DNA;
lane 2, same as lane 1, but simultaneously incubated with
30 fmol of LdTOP1LS for 30 min at 37°C; lane 3, same as
lane 2, but in presence of 2% (v/v) DMSO; lanes 4–8, same
as lane 2, but in presence of 10, 20, 50, 100, and 200 ␮M
DiSB, respectively; lanes 9 to 13, same as lane 2, but in
presence of 10, 20, 50, 100, and 200 ␮M DiGDHB, respec-
tively; lanes 14 to 18, same as lane 2, but in presence of 10,
20, 50, 100, and 200 ␮M DiSDHB, respectively. Positions of
supercoiled monomer (SM) and relaxed and nicked mono-
mer (RL/NM) are indicated. B, preincubation of LdTOP1LS
with respective inhibitors followed by addition of DNA.
Lane 1, 90 fmol of pBS (SKϩ) DNA; lane 2, same as lane 1,
but the enzyme was preincubated with 2% (v/v) DMSO;
lanes 3 to 8, same as lane 2, but the enzyme was preincu-
bated with 1, 2, 5, 10, 20, and 50 ␮M DiSB, respectively;
lanes 9 to 14, same as lane 2, but the enzyme was prein-
cubated with 1, 2, 5, 10, 20, and 50 ␮M DiGDHB, respec-
tively; lanes 15 to 20, same as lane 2, but the enzyme was
preincubated with 1, 2, 5, 10, 20, and 50 ␮M DiSDHB,
respectively. Reactions were stopped by the addition of
SDS to a final concentration of 0.5% and electrophoresed in
1% agarose gel. C and D, quantitative representation of
enzyme inhibition in presence of DiSB, DiGDHB, and DiS-
DHB in relaxation experiments. The percentage of relax-
ation inhibition is plotted as a function of inhibitor concen-
trations as indicated. The results depicted are the means
for three independent experiments, and the representative
results from one set of these experiments are expressed as
means Ϯ S.D. The fitted lines (sigmoidal) from these data
points have the R2 values of 0.9828, 0.9793, and 0.9820,
respectively.
cally with increasing concentration of these compounds and condition independent of the association/dissociation rates
was completely inhibited at a 100 ␮M concentration of each (McConaughy et al., 1981). A molar excess of enzyme com-
derivative (Fig. 3B, lane 8 for DiSB, lane 11 for for DiGDHB, pared with substrate also limits the effect of association/
and lane 14 for DiSDHB). These results not only depict the dissociation on relaxation reaction. The relaxation assay was
inability of betulin and dihydrobetulin derivatives to trap the resolved in the presence of 3 ␮g/ml chloroquine to better
topoisomerase I-mediated cleavable complex but also high- resolve the topoisomers and to improve the visualization of
light the antagonistic nature of these derivatives compared the relaxed band.
with camptothecin-mediated cleavage. This is a clear indica-
It was found that in the absence of any inhibitor, relaxed
tion of the fact that these derivatives inhibit the binding of plasmid monomers start appearing from 10 min of incubation
enzyme to substrate DNA and inhibit cleavable complex in low salt buffer (Fig. 4A, lane 2), and complete relaxation
formation.
under processive assay condition was achieved after 60 min
Betulin and Dihydrobetulin Derivatives Interfere of incubation (Fig. 4A, lane 7). On the other hand, in presence
with the Controlled Strand Rotation after the Single of 100 ␮M DiSB, relaxed molecules started to appear from 60
Strand Cleavage of Topoisomerase IB. In the general min of incubation (Fig. 4B, lane 7), whereas after incubation
mechanism of a topoisomerase-mediated DNA relaxation re- with same amount of DiGDHB (Fig. 4C) and DiSDHB (Fig.
action, strand rotation about the nicked topoisomerase I- 4D), the relaxed form appeared from 40 min (Fig. 4C, lane 5)
DNA cleavage complex occurs, leading to change of a linking and 30 min (Fig. 4D, lane 4) of incubation, respectively.
number. The speed of enzyme-associated relaxation may de- These results suggest a slower rate of completion of catalytic
pend on several factors, including DNA association, cleavage, cycle of LdTOP1LS in presence of the respective inhibitors
strand rotation, ligation, and dissociation. The actual aim of with respect to LdTOP1LS alone. Considering the fact that
this experiment was to investigate the speed of the catalytic multiple strand rotations can occur for every cleavage event,
steps occurring in the relaxation reaction (i.e., cleavage, as suggested by the controlled strand rotation model (Stew-
strand rotation, and ligation). Here, we performed the relax- art et al., 1998), the speed of the strand rotation rather than
ation assays under conditions in which the DNA association/ cleavage/ligation rates are likely to be the rate-limiting step
dissociation rates are not rate-limiting. This was achieved by under these conditions. Taken together, the slower rate of
varying the salt concentrations in the reaction mixtures us- LdTOP1LS in presence of each inhibitor compared with
ing a plasmid/enzyme ratio of 1:2, because topoisomerase I LdTOP1LS alone can best be explained by a slower strand
acts in a strictly processive manner at low salt concentrations rotation event that ultimately affects the general relaxation
(15 mM NaCl, coming from the enzyme preparation and 5 reaction.
mM MgCl2). Under these conditions, the enzyme completes
Betulin and Dihydrobetulin Derivatives Act Revers-
the relaxation of bound DNA substrate before it attacks ibly against the LdTOP1LS. The selected derivatives are
another molecule of substrate DNA and thus provides a potent inhibitors of LdTOP1LS. Preincubation experiments

Betulin Derivatives, Unique LdTOP1LS Inhibitors
699
Fig. 4. Relaxation assay to assess the effect on strand rotation event
under processive conditions. The relaxation of negatively supercoiled pBS
(SKϩ) DNA at processive conditions were performed as described under
Materials and Methods. Effect on of relaxation of LdTOP1LS activity
under processive condition in absence of any inhibitor (A), and in pres-
ence of DiSB (100 ␮M) (B), DiGDHB (100 ␮M) (C), and DiSDHB (100 ␮M)
(D) has been assayed by incubating 2 ␮g of negatively supercoiled plas-
mid pBS (SKϩ) (lane 1) with enzyme alone (A) or with respective inhib-
itors at 37°C at molar enzyme: plasmid ratio of 2:1. Reactions were
stopped by addition of 2% (v/v) SDS after indicated times. After protei-
nase K digestion, samples were analyzed in 1.5% agarose gel containing
3 ␮g/ml chloroquine followed by staining with ethidium bromide. Posi-
tions of supercoiled monomer (SM) and relaxed and nicked monomer
(RL/NM) are indicated. The representative results are from one set of
three independent experiments performed with or without any inhibitor.
Fig. 3. Derivatives of betulin and dihydrobetulin do not induce
LdTOP1LS mediated DNA cleavage. A, cleavage reaction and agarose gel
electrophoresis were performed as described under Materials and Meth-
ods. Lane 1, 50 fmol of supercoiled pHOT1 DNA; lane 2, same as lane 1,
but treated with with SDS-proteinase K; lane 3, same as lane 1, but
incubated with 100 fmol LdTOP1LS; lane 4, same as lane 3, but with
SDS-proteinase K treatment; lanes 5 and 6, same as lane 4, but in
presence of 25 and 50 ␮M CPT, respectively, as control; lanes 7 to 9, same
as lane 4, but in the presence of 50, 100, and 200 ␮M DiSB; lanes 10 to 12,
same as lane 4, but in the presence of 50, 100, and 200 ␮M DiGDHB;
lanes 13 to 15, same as lane 4, but in the presence of 50, 100, and 200 ␮M
DiSDHB. Positions of supercoiled monomer (SM; form I) and nicked
monomer (NM: form II) are indicated. Form IЈ, relaxed molecules. The
figure represents set from three independent experiments. B, betulin and
dihydrobetulin derivatives abrogate CPT-mediated cleavage. Reactions
were described under Materials and Methods. Lane 1, 50 fmol of super-
coiled pHOT1 DNA; lane 2, with 100 fmol of LdTOP1LS followed by
treatment with SDS-proteinase K; lanes 3 to 5, same as lane 2, but
incubated with 25, 50, and 100 ␮M CPT, respectively; lanes 6 to 8, same
as lane 2, but the enzyme was preincubated with 25, 50, and 100 ␮M
DiSB before addition of CPT (100 ␮M); lanes 9 to 11, same as lane 2, but
the enzyme was preincubated with 25, 50, and 100 ␮M DiGDHB before
addition of CPT (100 ␮M); lanes 12 to 14, same as lane 2, but the enzyme
was preincubated with 25, 50, and 100 ␮M DiSDHB before addition of
CPT (100 ␮M). The figure shown here is representative of three indepen-
dent experiments.
Fig. 5. Reversible interaction between betulin and dihydrobetulin deriv-
atives with LdTOP1LS. Lane 1, 50 fmol of pBS (SKϩ) DNA. Lane 2,
LdTOP1LS (100 fmol) incubated at 37°C for 5 min in the reaction mixture
before addition of pBS (SKϩ) DNA. Lane 3, same as lane 2, but in
presence of 2% (v/v) DMSO. Lanes 4 to 6, same as lane 2, but in presence
of 20, 20, and 50 ␮M DiSB, DiGDHB, and DiSDHB, respectively, prein-
cubated with LdTOP1LS for 5 min at 37°C in relaxation buffer followed
by addition of 50 fmol of pBS (SKϩ) DNA and further incubated for 10 min
at 37°C. Lanes 7 to 9, same as lanes 4 to 6, but diluted 5-fold so that the
final inhibitor concentrations became 4, 4 and 10 ␮M DiSB, DiGDHB,
and DiSDHB, respectively. These were followed by addition of pBS (SKϩ)
DNA and further incubated for 10 min at 37°C. Lanes 10 to 12, same as
lanes 4 to 6, but diluted 10-fold so that the final inhibitor concentrations
became 2, 2, and 5 ␮M DiSB, DiGDHB, and DiSDHB, respectively.
Reactions were followed by addition of pBS (SKϩ) DNA and further
incubated for 10 min at 37°C. Lanes 13 to 15, same as lanes 4 to 6, but
diluted 20-fold so that the final inhibitor concentrations became 1, 1, and
2.5 ␮M DiSB, DiGDHB, and DiSDHB, respectively. Reactions were fol-
lowed by addition of pBS (SKϩ) DNA and further incubated for 10 min at
37°C. Lanes 16 to 18, same as lane 2, but in presence of 1, 1, and 2.5 ␮M
DiSB, DiGDHB, and DiSDHB, respectively before addition of DNA. The
experiments were performed three times and representative results are
support that these compounds interact with the enzyme in
almost the same fashion, but it is not clear whether the
interaction is strong enough that they can act on the enzyme
in an irreversible manner. This critical issue has been sorted
out by doing dilution experiments. Reconstituted LdTOP1LS
was preincubated with 20, 20, and 50 ␮M DiSB, DiGDHB,
and DiSDHB, respectively (Fig. 5, lanes 4–6), the concentra-
tion at which 95 to 99% inhibition of enzyme has been
achieved. The reaction mixtures were subsequently diluted
5-fold so that the final concentrations became 4, 4, and 10
␮M, respectively (lanes 7–9). The results show that partial
(approximately 20%) relief of inhibition has been achieved.
Further dilution to 10-fold (lanes 10–12) and 20-fold (lanes
13–15) show that almost 50% and complete relief of inhibi- from one set of experiments.

700
Chowdhury et al.
tion have been achieved, respectively. In the drug control inhibitor interaction were as follows: DiSB, 1.321 Ϯ 0.281
reaction (i.e., inhibition study), 1, 1, and 2.5 ␮M of DiSB, ␮M; DiGDHB, 0.981 Ϯ 0.372 ␮M, and DiSDHB, 0.527 Ϯ
DiGDHB, and DiSDHB, respectively, the results showed the 0.188 ␮M. Detail calculation and formulation from the
expected pattern of inhibition (lanes 16–18). Thus, in this binding data have been summarized under Materials and
dilution experiment, the relief of inhibition upon dilution Methods section, and the data clearly indicate that all the
suggests that the effective derivatives are acting in reversible derivatives possess weak interaction with the LdTOP1LS
fashion against LdTOP1LS.
(Kd ϳ 10Ϫ6 M). Incubation with CPT does not show any
Betulin and Dihydrobetulin Derivatives Bind Weakly significant quenching (Supplemental Fig. S2), suggesting
to L. donovani Topoisomerase IB. To find out the nature of that these inhibitors specifically bind with LdTOP1LS. Dilu-
enzyme-inhibitor interaction, the binding of betulin and dihy- tion experiments with these compounds also suggest that the
drobetulin derivatives to LdTOP1LS was carried out by mea- compounds bind reversibly with the enzyme LdTOP1LS.
suring the quenching of intrinsic tryptophan fluorescence of
Active Derivatives Are Competitive Inhibitors of L.
LdTOP1LS. The stoichiometry of the ligand-protein interaction donovani Topoisomerase IB. Camptothecin, the well es-
was measured using the Job plot (Huang, 1982). In this speci- tablished inhibitor of LdTOP1LS, acts as an uncompetitive
fied plot, concentrations of both LdTOP1LS and one of the inhibitor of topoisomerase IB (Champoux, 2001). It stabilizes
derivatives were continuously varied, keeping the total ligand- the enzyme-DNA covalent complex and slows down the reli-
protein concentration fixed at 1.25 ␮M. The excitation and gation step, which is essential to decrease the Lk of supercoil
emission slit widths were 2.5 and 5 nm, respectively. Appropri- structure. On the other hand, 3,3Ј-di-indolylmethane inter-
ate blanks corresponding to the buffer were subtracted to elim- acts with both the free enzyme as well as DNA-bound enzyme
inate background fluorescence. The results are shown in and acts like a noncompetitive inhibitor to LdTOP1LS (Roy
Fig. 6A. The stoichiometry of binding, calculated using this et al., 2008). Preincubation relaxation assay and cleavage
method of continuous variation, was found to be 1:1.
experiments suggest that betulin and dihydrobetulin deriv-
The dissociation constant has been calculated from Fig. 6, atives do not interact with the DNA-bound enzyme; rather,
B and C. Figure 6B emphasizes on the quenching profile of a they interact with the free enzyme and thus affect their
fixed amount of LdTOP1LS (200 nM) with various concen- binding to the substrate DNA.
trations of one of the derivatives (represented as X) (0–22.5
To investigate the characteristic of LdTOP1LS inhibition
␮M). All the fluorescence readings were corrected for the by these compounds, a time-course relaxation experiment
inner filter effect. The dissociation constants (Kd) of enzyme- was performed under standard relaxation assay conditions at
Fig. 6. Binding of betulin and dihydro-
betulin derivatives with LdTOP1LS.
A, Job’s Plot of DiSB (ꢁ), DiGDHB (F),
and DiSDHB (Œ) (as indicated by X) bind-
ing to LdTOP1LS. The concentrations of
LdTOP1LS and each of the inhibitors
were varied continuously (for each exper-
iment), keeping the total concentration of
LdTOP1LS plus inhibitor constant at
1.25 ␮M. The corrected fluorescence in-
tensities at 350 nm were plotted against
molar fractions of each inhibitor. B, dou-
ble reciprocal plot of inhibitor binding to
LdTOP1LS. Fmax has been determined
from the 1/(F0 Ϫ F) versus 1/[X] plot,
where [X] represents concentration of
each inhibitor in individual experiments.
C, the linear plot of binding of each inhib-
itor to LdTOP1LS.

Betulin Derivatives, Unique LdTOP1LS Inhibitors
701
37°C, where the concentration of supercoiled substrate pBS
(SKϩ) DNA was varied over a range of 4 to 60 nM and the
enzyme/DNA ratio was kept within the steady-state assump-
tion. The velocity of the enzyme remains linear for the first 5
min of reaction. All the subsequent velocities for this kinetic
study were measured for the time point up to 1 min, which
falls within the linear range for the velocity examined. The
initial velocities for each substrate concentration were plot-
ted on a Lineweaver-Burk plot (Supplemental Fig. S3). The
maximal velocity (Vmax) for the LdTOP1LS was 6.67 ϫ 10Ϫ8
M base pairs of supercoiled DNA relaxed/min/0.98 nM en-
zyme, which corresponds to a turnover number of approxi-
mately 70 plasmid molecules relaxed/min/molecules of en-
zyme and remains unaffected upon the incubation of each of
the potent inhibitors with LdTOP1LS, whereas increase in
KM suggests that inhibitor-bound LdTOP1LS has low affinity
toward the enzyme, and when DNA binds in this condition,
strand rotation gets affected.
Effect of the Active Derivatives on Human Topo-
isomerase I. As the three inhibitors potentially inhibit bi-
subunit Leishmania spp. topoisomerase I, the effect of these
compounds was next assessed on human topoisomerase I.
The relaxation experiment was performed under standard
assay conditions (Ganguly et al., 2006). It is noteworthy that
all the three compounds showed partial inhibition at 200 ␮M
(Fig. 7A, lane 8 for DiSB, lane 13 for DiGDHB, and lane 18
for DiSDHB) when incubated simultaneously. Also in prein-
cubation relaxation assay, inhibitors at 50 ␮M concentra-
tions are not sufficient to inhibit human topoisomerase I
completely (Fig. 7B, lane 8 for DiSB, lane 13 for DiGDHB,
and lane 18 for DiSDHB).
Discussion
In the present study, we have shown for the first time
that the derivatives of betulin and dihydrobetulin inhibit
LdTOP1LS. The mechanism of inhibition by these com-
pounds differs from that of CPT. These derivatives bind with
enzyme but do stabilize neither the topoisomerase-mediated
cleavable complex, like CPT, nor the cleavage step of topo-
isomerase reaction. On the other hand, these derivatives
prevent the binary complex formed between the drug and the
enzyme to interact with the substrate DNA.
A topoisomerase reaction has three general mechanistic
steps: 1) binding of the enzyme to the substrate DNA,
2) cleavage of one strand by trans-esterification reaction fol-
lowed by strand rotation leading to the change of linking
number by one, and 3) strand religation and turnover of the
enzyme. Suicidal cleavage experimentd prove that these de-
rivatives do not affect the cleavage of DNA (Supplemental
Fig. S1). However, unlike CPT, these derivatives are not only
unable to stabilize the “cleavable complex” (Fig. 3A), but also
Selective Inhibitors Can Reduce Intracellular SbS
and SbR Amastigotes from Cultured Murine Perito-
neal Macrophage Cells. Primary macrophage cells were
obtained from BALB/c mice peritoneal extrudates. After ad-
herence and inactivation, these macrophage cells were in-
fected with early-passaged L. donovani AG83 promastigotes
(SbS) and laboratory developed SbR (GE1) parasites in vitro.
Infected macrophages, after subsequent washing, were incu-
bated with different concentrations (1, 2.5, 5, 7.5, 10, 15, and
20 ␮M) of each of these inhibitors for 24 h (Fig. 8A). Macro-
phages were fixed, and intracellular amastigotes were
counted by Giemsa staining. The EC50 values of each inhib-
itor against SbS and SbR are as follows: DiSB, 6.05 ␮M;
Fig. 7. Inhibition of catalytic activity of human topoisoemearase I by
DiGDHB, 7.94 ␮M; and DiSDHB, 10.54 ␮M. Stability of the betulin and dihydrobetulin derivatives. Relaxation of negatively super-
compounds have been assessed by treating the infected mac-
coiled pBS (SKϩ) DNA with purified hTop I at a molar ratio of 3:1 in both
simultaneous (A) and preincubation (B) conditions. A, lane 1, 90 fmol of
rophages with betulin and dihydrobetulin separately to
check whether active esterases present in FBS or in macro-
phages can cleave the ester bond of the derivatives to pro-
duce parent compounds of active derivatives. Betulin and
dihydrobetulin are inactive and cannot reduce the parasite
burden (data not shown), which emphasizes the availabil-
ity and stability of these derivatives in cell culture me-
dium. In addition, the percentage of infected macrophages
has been reduced after subsequent treatment with these
compounds (Supplemental Fig. S4). The parasite burden is
reduced to almost 98% in case of intracellular SbS AG83
amastigotes, which is very comparable with that of intra-
cellular SbR GE1 parasites when treated with 20 ␮M DiSB
(Fig. 8B). The efficiency of amastigote killing is up to 96%
for DiGDHB and 78% for DiSDHB when infected with SbS
parasites (Fig. 8A).
pBS (SKϩ) DNA; lane 2, same as lane 1, but simultaneously incubated
with 30 fmol of hTop I for 30 min at 37°C; lane 3, same as lane 2, but in
presence of 2% (v/v) DMSO; lanes 4 to 8, same as lane 2, but in presence
of 10, 20, 50, 100, and 200 ␮M DiSB, respectively; lanes 9 to 13, same as
lane 2, but in presence of 10, 20, 50, 100, and 200 ␮M DiGDHB, respec-
tively; lanes 14 to 18, same as lane 2, but in presence of 10, 20, 50, 100,
and 200 ␮M DiSDHB, respectively. Positions of supercoiled monomer
(SM) and relaxed and nicked monomer (RL/NM) are indicated. B, prein-
cubation of hTop I with respective inhibitors followed by addition of DNA.
Lane 1, 90 fmol of pBS (SKϩ) DNA; lane 2, same as lane 1, but the enzyme
was preincubated in relaxation buffer for 5 min followed by addition of
pBS (SKϩ) DNA; lane 3, same as lane 2 but the enzyme was preincubated
with 2% (v/v) DMSO; lanes 4 to 8, same as lane 2, but the enzyme was
preincubated with 2, 5, 10, 20, and 50 ␮M DiSB, respectively; lanes 9 to
13, same as lane 2, but the enzyme was preincubated with 2, 5, 10, 20,
and 50 ␮M DiGDHB, respectively; lanes 14 to 18, same as lane 2, but the
enzyme was preincubated with 2, 5, 10, 20, and 50 ␮M DiSDHB, respec-
tively. Reactions were stopped by addition of SDS to a final concentration
of 0.5% and electrophoresed in 1% agarose gel. The representative results
were from one set of three experiments.

702
Chowdhury et al.
inhibit camptothecin-mediated cleavable complex formation The rotation of the free 5Ј-hydroxyl DNA end around the
(Fig. 3B). Preincubation study with these inhibitors suggests intact strand takes place in a manner that is restricted by
that these derivatives abrogate topoisomerase I-DNA inter- the surrounding protein (Stewart et al., 1998). To assess the
action (i.e., the first step of topoisomerization). Enzyme ki- mode of enzyme inhibition, we analyzed the rate-limiting
netics study reveals that these derivatives act like a compet- relaxation assays for each of the active derivatives under
itive class of inhibitors, unlike CPT. The increase in KM processive condition (molar enzyme:plasmid ratio of ϳ2:1;
suggests that the affinity of the enzyme toward DNA de- low salt buffer). In this condition, the relaxation rate depends
creases when enzyme is bound to inhibitor (Supplemental mainly on the speed of strand rotation; which acts as the rate
Fig. S3).
limiting step of topoisomerization (McConaughy et al., 1981).
Dynamic detailing of type IB topoisomerase-catalyzing Strand rotation is affected maximally when the above relax-
DNA relaxation demonstrates that the relaxation proceeds in ation assay is carried out with DiSB in simultaneous assay
a stepwise, torque-dependent manner (Koster et al., 2005). condition, because there is a lesser degree of interaction
between LdTOP1LS and DNA. Consequently, time needed to
relax the total supercoiled plasmid in presence of DiSB is
highest with respect to enzyme alone. Considering the con-
trolled strand rotation model, these results can best be ex-
plained by a drug-induced stalling of the enzyme in a confor-
mation that restricts strand rotation imposed by flexible
LdTOP1LS enzyme.
Direct measurement of enzyme-inhibitor interaction made
by intrinsic tryptophan quenching of LdTOP1LS with each of
these inhibitors reveals Kd values of 1.31 Ϯ 0.27, 0.59 Ϯ 0.20,
and 0.49 Ϯ 0.19 ␮M for DiSB-enzyme, DiGDHB-enzyme, and
DiSDHB-enzyme, respectively. Job Plot analysis also reveals
that there is a 1:1 interaction of each of these inhibitors with
LdTOP1LS.
In the subsequent study of in vitro parasite clearance, we
have shown that all these inhibitors have profound effects on
the clearance of antimony sensitive (SbS) and resistant (SbR)
intracellular parasites in cultured macrophages. Upon infec-
tion with the wild-type L. donovani (SbS) on the isolated
murine peritoneal macrophages followed by treatment with
the inhibitors individually at 20 ␮M for 24 h, there was
significant reduction of the parasite burden within macro-
phages (up to 95% for DiSB, 96% for DiGDHB, and 78% for
DiSDHB). In case of SbR parasites, the extent of clearance of
intracellular amastigotes is similar when treated with DiSB
(Fig. 8B). With the establishment of betulin derivatives as
potent antileishmanial agents targeting LdTOP1LS, it was
important to study the effect of these compounds on host cell
topoisomerase (i.e., human topoisomerase I) or directly on
host cell viability. Our results show that these inhibitors
have less effect on human topoisomerase I compared with
LdTOP1LS (Fig. 7). Cell viability test with isolated murine
macrophages also suggests that these compounds exert no
remarkable cytotoxicity up to 50 ␮M concentrations (data not
Fig. 8. Effectiveness of clearance of internalized SbS L. donovani (AG83)
shown); however, these compounds showed some toxicity
(23% for DiSB, 18% for DiGDHB, and 15% for DiSDHB)
when incubated at 100 ␮M concentration (data not shown).
A close inspection of all the synthesized derivatives (DiSB,
DiGDHB and DiSDHB) that inhibit LdTOP1LS indicates
that variation of carbon chain length at C-3 and C-28 posi-
tions in betulin and dihydrobetulin are important for inhib-
itory effect. The parent compounds betulin and dihydrobetu-
lin do not inhibit enzymatic activity (data not shown),
whereas substitution with succinyl group at C-3 and C-28
position of betulin and glutaryl or succinyl at the same posi-
tions of dihydrobetulin turns out to be very effective inhibitor
of LdTOP1LS. However, substitution with phthaloyl group or
crotonyl or acetyl group fails to convert the compound as
inhibitors. Alakurtti et al. (2010) showed that some chemi-
cally synthesized heterocycloadducts of betulin with acetyl
and SbR L. donovani (GE1) infection from in vitro infected mouse M␾.
Macrophages from peritoneal extrudate of BALB/c mouse were infected
with parasites. Cultures were treated with DiSB (f), DiGDHB (f), and
DiSDHB (f) separately as indicated under Materials and Methods. In-
cubations were carried out for 24 h. Cells were fixed, stained with Gi-
emsa, and counted under bright-field microscope. A, dose-dependent
amastigote clearance by derivatives of betulin and dihydrobetulin from
infected macrophages. The number of internalized AG83 amastigotes
within each infected macrophages was counted under bright-field micro-
scope. The results shown are the means of three independent experi-
ments and plotted as mean Ϯ S.E. ءءء, p Ͻ 0.001 compared with 1 ␮M
inhibitor treatment. B, comparative analysis of effectiveness of DiSB on
AG83 and GE1 amastigotes. The numbers of internalized SbR (GE1)
parasite (F) and SbS (AG83) parasites (Œ) were counted after 24 h treat-
ment with or without any inhibitor. For each DiSB concentration, at least
200 macrophages were counted. The results shown are the means of three
independent experiments with S.E. The fitted lines were generated with
GraphPad Prism using Sigmoidal dose-Response (variable slope model)
equation. The R2 values for fitted nonlinear curves were 0.9915 and
0.9941, respectively.

Betulin Derivatives, Unique LdTOP1LS Inhibitors
703
activity of flavones against camptothecin-resistant topoisomerase I. Nucleic Acids
Res 34:1121–1132.
and methyl substitution displayed approximately 98%
growth inhibition of amastigote with a GI50 of 8.9 ␮M. Sub-
stitution with more bulky groups decreases the antileishma-
nial activity, which is also observed in our derivatives. This
investigation demonstrates in particular that simple aliphatic-
acid group variations of parent compounds such as betulin or
dihydrobetulin can produce potential inhibitors of L. donovani
topoisomerase with less effect on host topoisomerase.
Fortune JM and Osheroff N (2006) Topoisomerase II as a target for anticancer drugs:
when enzymes stop being nice. Prog Nucleic Acid Res Mol Biol 64:221–253.
Ganguly A, Das BB, Sen N, Roy A, Dasgupta SB, and Majumder HK (2006) ‘Leish-
Man’ topoisomerase I: an ideal chimera for unraveling the role of the small subunit
of unusual bi-subunit topoisomerase I from Leishmania donovani. Nucleic Acids
Res 34:6286–6297.
Haimeur A, Guimond C, Pilote S, Mukhopadhyay R, Rosen BP, Poulin R, and
Ouellette M (1999) Elevated levels of polyamines and trypanothione resulting from
overexpression of the ornithine decarboxylase gene in arsenite-resistant Leishma-
nia. Mol Microbiol 34:726–735.
In conclusion, our results indicate that simple substitution
on the parent skeleton of betulin and dihydrobetulin results
in synthesis of potent L. donovani topoisomerase IB catalytic
inhibitors. Structure-function analysis of these inhibitors
and topoisomerase IB interaction along with modeling stud-
ies can be employed for developing more effective and potent
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Acknowledgments
We thank Prof. S. Roy, the Director of our Institute, for interest in
this work. We thank Rupkatha Mukhopadhyay for help in animal
handling.
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Authorship Contributions
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Cancer 6:789–802.
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thoquinone: a novel inhibitor of type I DNA topoisomerase of Leishmania don-
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anti-inflammatory lupanes from the leaves, twigs, and resin of Garcinia hanburyi.
Planta Med 76:368–371.
Participated in research design: Chowdhury, Sengupta, Mukho-
padhyay, and Majumder.
Conducted experiments: Chowdhury, Roy Chowdhury and
Mukherjee.
Contributed new reagents or analytic tools: Mukherjee and
Mukhopadhyay.
Performed data analysis: Chowdhury and Mukherjee.
Wrote or contributed to the writing of the manuscript: Chowdhury
and Majumder.
Roy A, Das BB, Ganguly A, Bose Dasgupta S, Khalkho NV, Pal C, Dey S, Giri VS,
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Address correspondence to: Hemanta K. Majumder, Molecular Parasitol-
ogy Laboratory, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick
Road, Jadavpur, Kolkata-700032, India. E-mail: hkmajumder@iicb.res.in