Scaffold-Hopping of Aurones: 2-Arylideneimidazo[1,2-a]pyridinones as Topoisomerase IIα-Inhibiting Anticancer Agents

Article abstract of DOI:10.1021/acsmedchemlett.6b00242

Scaffold-hopping of bioactive natural product aurones has been studied for the first time. 2-Arylideneimidazo[1,2-a]pyridinones as potential topoisomerase IIα (hTopoIIα)-targeting anticancer compounds were considered. A multifunctional activator, polyphos

Full text of DOI:10.1021/acsmedchemlett.6b00242

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Letter
Scaffold-Hopping of Aurones: 2-Arylideneimidazo[1,2-
a]pyridinones as Topoisomerase II#-inhibiting Anticancer Agents
Garima Priyadarshani, Anmada Nayak, Suyog M. Amrutkar, Sarita Das,
Sankar K. Guchhait, Chanakya Nath Kundu, and Uttam Chand Banerjee
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00242 • Publication Date (Web): 20 Sep 2016
Downloaded from http://pubs.acs.org on September 20, 2016
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ScaffoldꢀHopping of Aurones: 2ꢀArylideneimidazo[1,2ꢀa]pyridinones
as Topoisomerase IIαꢀinhibiting Anticancer Agents
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Garima Priyadarshani,a,‡Anmada Nayak,b,‡ Suyog M. Amrutkar,c Sarita Das,b Sankar K. Guchhait,*,a
Chanakya N. Kundu,*,b and Uttam C. Banerjeec
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.
A. S. Nagar, Mohali, Punjab 160062, India.
bSchool of Biotechnology, KIIT University, Campusꢀ11, Patia, Bhubaneswar, Orissa 751024, India.
cDepartment of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research
(NIPER), Sector 67, S. A. S. Nagar, Mohali, Punjab 160062, India.
KEYWORDS Scaffoldꢀhopping, Human topoisomerase IIα, Imidazo[1,2ꢀa]pyridine, Antiꢀcancer agents, Aurones
ABSTRACT: Scaffoldꢀhopping of bioꢀactive natural product aurones has been studied for the first time. 2ꢀArylideneimidazo[1,2ꢀ
a]pyridinones as potential topoisomerase IIα (hTopoIIα)ꢀtargeting anticancer compounds were considered. A multifunctional actiꢀ
vator, polyphosphoric acid, enabled to realize a cascade reaction of 2ꢀaminopyridine with 2, 3ꢀepoxyesters towards synthesis of 2ꢀ
arylideneimidazo[1,2ꢀa]pyridinones. Most of the compounds exhibited hTopoIIαꢀselective poison activity with efficiency more
than etoposide and DNAꢀbinding property, while not interacting with hTopo I. The compounds showed pronounced antiproliferaꢀ
tive activities in nanomolar range while relatively poor toxicity to normal cells, inhibition of invasiveness, and apoptotic effect. The
activities for inhibition of tubulin assembly, CDK1 and pCDK1 were also observed. Interestingly, the hTopoIIα inhibitory (in vitro
and ex vivo studies) and antiproliferative activities of representative potent compounds were found to be manifold higher compared
to corresponding parent aurones bearing alike substitutions, indicating the importance of such scaffoldꢀhopping strategy in mediciꢀ
nal chemistry research.
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Natural products are not only used as drugs in the treatment of 28 Towards the aim of topoisomerase IIꢀtargeting anticancer drug
12ꢀ14
various diseases but also have been considered as leading 29 discovery and development,
we considered scaffoldꢀ
templates for the design, which have successfully led to develꢀ 30 hopping of bioꢀactive flavonoids class of compounds. We
opment of a number of approved drugs and clinical trial 31 have already discovered scaffoldꢀhopped analogs of flavones
agents.1,2They introduce molecular diversity and structural 32 and isoflavones, 2/3ꢀarylpyridopyrimidinones as Topo IIꢀ
novelty and have been valuable in recognition of novel targets. 33 targeting anticancer agents.10,15,16 In continuation, we were
In particular, natural products have played critical role in antiꢀ 34 interested in the scaffoldꢀhopping of aurone class of comꢀ
cancer drug discovery and development.3According to the 35 pounds. Aurones have been reported as promising anticancer
latest report by Newman and Cragg5 approximately 83% (113 36 agents which show interference with various targets17 such as
10 of 136) anticancer small molecules approved by FDA were 37 CDK1 inhibition,18 adenosine receptor inhibition,19 DNA scisꢀ
11 either natural products or based thereon or mimicked natural 38 sion and Telomerase inhibition.20 We focussed on amalgamaꢀ
12 products in some form. In order to have desired biological 39 tion of structural features of aurones and the imidazopyridine
13 activity as well as ADMET properties the lead optimizationis 40 class of compounds which we previously discovered as potent
14 required which is usually done by modification/introduction of 41 topoisomerase IIꢀtargeting anticancer agents.11,13 In addition,
21ꢀ23
15 side chains or substituents. In this direction, interestingly, a 42 several other related bicyclic compounds
have been reꢀ
16 complete change in molecular framework can be achieved by 43 ported as topoisomerase II inhibitors. We envisaged scaffoldꢀ
17 scaffoldꢀhopping.
44 hopped analogs of aurones, 2ꢀarylideneimidazo[1,2ꢀ
The term “scaffoldꢀhopping” has been introduced by Schneiꢀ 45 a]pyridinones as potential topoisomerase IIꢀtargeting antiꢀ
18
6
as the identification of isoꢀfunctional molecular 46 cancer agents (Figure 1a).
19 der et al,
20 structures with significantly different molecular backbones. 47 Chemistry: Recently, we have developed a new cascade reacꢀ
21 The new molecule with changed core becomes patentaꢀ 48 tion of 2ꢀaminopyridine with 2, 3ꢀepoxyesters to produce 2ꢀ
22 ble.7There are number of examples of successful drug discovꢀ 49 arylideneimidazo[1,2ꢀa]pyridinones.24The reaction involves a
23 ery and development employing scaffoldꢀhopping.8,9 However, 50 set of sequential transformation: epoxide ring opening, aziridiꢀ
24 a few reports of scaffoldꢀhopping of natural products are 51 nation, nucleophilic opening of aziridine, elimination to form
25 available.10 Recently, scaffold hopping of a natural product 52 enamine, and intramolecular transamidation. These cascade
26 proteasome inhibitor Belactosin A has been reported, which 53 transformations required a distinct catalysis/activation, which
27 led to development of highly potent nonꢀpeptidic inhibitors.11 54 was achieved by polyphosphoric acid as multifunctional actiꢀ
55 vator. Several relevantly substituted 2ꢀarylideneimidazo[1,2ꢀ
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OH
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N
OH
5-/6-
hetAr
HO
O
N
R1
R2
Consideration of
R2 structural features
for Scaffold-
NH
R3
Scaffold-
hopping
O
hopping
hTopo IIα-inhibiting anticancer agents
previously discovered from our lab12, 13
N
N
R1
Ph
N
N
O2N
O
N
HO
O
O
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R
2-Arylideneimidazo[1,2-a]pyridinones as
potential topoisomerase II-targeting
anticancer agents
Cl
O
O
nPr
MeO
O
OH
Anticancer natural and synthetic
aurones
N
R:
COOH
Bicyclic topoisomerase II inhibitors21,22
Figure 1a: Design of the target compounds
Figure 1b: Synthesis of 2-arylideneimidazo[1,2-a]pyridinones
NH2
O
Ar
N
N
EtOOC
PPA, 110 °C
1 - 1.5 h
R
R
N
Ar
2
O
3, 32-76%
1
OMe
N
Ar:
MeO
OMe
Br
Cl
F
Cl
Cl
OMe MeO
N(Me)2
MeO
OMe
Me
Cl
3k
3b
3c
3a
3g
3j
3d
3e
3f
3h
3i
Cl
Cl
Cl
OMe
Me
Br
N
N
N
N
N
N
N
N
Me
N
N
Cl
N
O
O
Br
Br
Cl
O
O
O
O
3m
3n
3l
3o
3p
3
q
Figure 1d: Synthesis of aurones
Figure 1c: Synthesis of 2-(4-hydroxybenzylidene)imidazo[1,2-a]
O
5
pyridinones
O
Ar
O
OH Ar
H
Ar
CuBr2 (20 mol %)
KOH (50 %)
OH
DMF:H2O (8;1),
130 °C, 12 h
OMe
OH
EtOH, 50 °C
O
6
4
O
Br
OMe
N
N
BBr3 in DCM (5 equiv)
N
N
-10 °C→RT, 3 h
O
O
O
O
O
3r, 65%
O
C2, 48%
C1, 60%
Figure 1: Design and synthesis of the target compounds
a]pyridinones were synthesized bearing structural features of
anticancer aurones (Figures 1b and 1c). 2ꢀ(4ꢀ
Hydroxybenzylidene) imidazo [1,2ꢀa] pyridinone was syntheꢀ
sized via BBr3ꢀmediated ether cleavage.25
and C2 bearing the same substitutions as that of compounds 3c
and 3k were prepared following literature reported procedure
(Figure 1d).27,28
Biological Studies:
To evaluate the effectiveness of scaffoldꢀhopping concept on
the topoisomerase II inhibitory activities of aurones, two auꢀ
rones possessing alike substitutions, completely mimicking the
structures of investigated potent topo IIꢀinhibiting 2ꢀ
arylideneimidazo[1,2ꢀa] pyridinones were prepared, such that
a comparison in their activities can be made. Two aurones C1
Inhibition of hTopoIIαꢀmediated kDNAdecatenation:
The synthesized 2ꢀarylideneimidazo[1,2ꢀa]pyridinones (3aꢀr)
and parent aurones (C1ꢀ2) were tested for inhibition
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293T), its corresponding normal cell line (VERO) and breast
cancer cell line (MCFꢀ7) were considered for testing cytotoxic
activities. The IC50 concentration of compound to cause 50%
cell growth inhibition of compounds 3aꢀd, 3i, 3k, 3n, 3o, 3r
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were found remarkably low (10, 150, 60, 80, 30, 45, 45, 80, 40
nM, respectively) in HEK 293T cells compared to IC50 values
obtained in VERO cells (200, 1500, 1500, 1500, 200,
200,1500, 1500 and 1500 nM, respectively) (Table S1a). The
IC50 for Etoposide was found to be 22 ꢁM in HEKꢀ293T cells
and 50 ꢁM in VERO cells. It is interesting to note that parent
compounds C1 and C2 compared to their investigated scafꢀ
foldꢀhopped analogs did not show significant cytotoxicity. The
IC50 values of compounds C1 and C2 were found to be 0.7 and
0.9 ꢁM in HEKꢀ293T cells and 2.5 and 2.4 ꢁM in VERO cells,
respectively (Table S1a). In MCFꢀ7 cells, the cytotoxicities of
the investigated scaffoldꢀhopped analogs were also found to be
multiꢀfold higher compared to Etoposide and parent auꢀ
ronesC1 and C2 (Table S1a). The cell viability was also
checked in other breast cancer cell lines i.e. MDAꢀMBꢀ231
and MDAꢀMBꢀ468. The IC50 for compound 3c were found to
be 64 nM and 60 nM and the IC50 for compound 3k were
found to be 76 nM and 70 nM, for MDAꢀMBꢀ231 and MDAꢀ
MBꢀ468, respectively.
Figure 2: a) Inhibition of hTopoIIαꢀmediated kDNA decatenaꢀ
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tion: kDNA was treated with hTopoIIα in the absence or presence
of 100 ꢁM etoposide (E) or investigated compounds; b) Quantifiꢀ
cation of decatenation products.
of the hTopoIIαꢀmediated decatenation of kinetoplast DNA
(kDNA) (Figure 2).12 Etoposide, a known topoisomerase IIꢀ
inhibiting anticancer drug, was used as a reference. In the
presence of compounds 3bꢀe, 3i, 3k, 3m, 3n, 3p and 3q, relaꢀ
tively higher inhibition of hTopoIIαꢀmediated kDNA decateꢀ
nation compared to Etoposide was observed. Interestingly,
scaffoldꢀhopping based structural modifications were found to
result in increased hTopoIIαꢀinhibitory activities. For examꢀ
ple, compared to parent aurones (C1 and C2), compounds 3c
and 3k which possess alike substitutions were found to be
more potent hTopoIIαꢀinhibitors.
hTopoIIαꢀDNA cleavage complex formation:
A hTopoIIα poison leads to stabilization of a TopoIIꢀcleaved
DNA complex (called as a cleavage complex) which appears
as a linear band (Lin).12 In the presence of the active comꢀ
pounds 3c and 3k (100 µM), characteristic linear band was
observed (Figure S1). It indicates that the compounds 3c and
3k act as hTopoIIα poisons at tested concentration.
Inhibition of hTopoIꢀmediated DNA relaxation:
In order to evaluate the selectivity of inhibition for hTopoIIα,
selected compounds 3c and 3k were screened for inhibition of
hTopoIꢀmediated relaxation of negatively supercoiled DNA.12
In the presence of tested compounds, relatively less inhibition
of hTopoIꢀmediated DNAꢀrelaxation occurred as compared to
camptothecin (Figure S2). Hence, tested compounds are not
hTopoI inhibitors.
DNAꢀinteraction study:
In order to check the DNA binding efficacy, UVꢀbased study
for in vitro DNAꢀdrug binding was carried out. The exact
spectrum of 260 nm was found for DNA without any agent. In
case of DNA treated with increasing concentrations of comꢀ
pounds 3c and 3k, a hypochromism in absorption was found to
occur, indicating possible binding of the compounds to DNA
(Figure S6). Kd was found to be 2 x106 Mꢀ1 and 2.5x106 Mꢀ1 for
compounds 3c and 3k, respectively.
Figure3: a). Expression pattern of BAX, BCL XL, p21, p53,
CDK1, pCDK1 (Thr 161) and CASPASE 3 after treatment with
compound 3c. GAPDH served as a loading control to check the
equal loading of protein in each lane. b) Immunocytochemistry of
CASPASE 3 in MCFꢀ7 cells treated with compound 3c. The nuꢀ
merical value above each panel shows the relative fold change in
comparison with untreated control measured by densitometric
analysis. All the experiments are carried out at least thrice.
Cytotoxic potential of selected compounds in cancer cells:
Cytotoxic potential of the potent hTopoIIα inhibitors (3aꢀd, 3i,
3k, 3n, 3o, 3r) and control compounds (C1 and C2) was
measured by a wellꢀknown colorimetricꢀbased MTT assay.12
The representative cancer cells, kidney cancer cell line (HEKꢀ
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On the other hand, the IC50 for control compounds C1 and C2 treated control. This indicates that compound 3c inhibited the
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in MDAꢀMBꢀ231 and MDAꢀMBꢀ468 were found to be much
higher, 840ꢀ600 nM (Table S2).To further confirm the cytoꢀ
toxicity a long term cell survival assay (clonogenic assay) was
performed.24 For compounds 3aꢀd, 3i, 3k, 3n, 3o, and 3r the
LC50 values (fifty percent cell death in culture) in HEK 293T
cells were found to be 55, 60, 60, 75, 40, 20, 40, 40 and 50
nM, respectively and in VERO cells 1000, 1500, 1500, 1500,
1500, 1500, 1500, 1500, 1500 nM, respectively (Table S1b).
In MCFꢀ7 cells, these compounds exhibited also pronounced
antiꢀclonogenic properties (LC50 in nM ranges). In both HEKꢀ
293T and MCFꢀ7 cells, the investigated imidazoꢀpyridinones
were found to be prominently more potent (IC50: nanomolar vs
micromolar) than Etoposide and the parent aurones C1 and
C2. In both MTT and clonogenic assays, concentrationꢀ
dependent cytotoxicities of these imidazoꢀpyridinones were
observed (Figure S3).
in vitro cell invasion (Figure S5).
Inhibition of the topoisomerase activity in MCFꢀ7 cells;
Comparison of activities of scaffoldꢀhopped compounds
with parent compounds:
To check the ex vivo topoisomeraseꢀinhibiting property of
compound 3c and compound 3k, a plasmid based topoisomerꢀ
ase inhibition assay using nuclear lysate of MCFꢀ7 and a
plasmid NFkB was carried out according to protocol described
in experimental section. Etoposide was used as a positive conꢀ
trol. According to the principle if any agent inhibits the topoiꢀ
somerase activity then more supercoiled DNA forms and run
faster in the agarose gel compared to relaxed/linear one. Figꢀ
ures 4a and 4b demonstrate the formation of supercoiled DNA
and migration into the gel after treatment with compounds
3cand 3k, respectively. In nuclear lysate without treatment of
compound 3c/3k, the plasmid was relaxed or linear, and thus
did not enter into 0.8% agarose gel. But with increase in the
concentration of the agents (3c or 3k) the formation of lineꢀ
ar/relaxed plasmid decreased, as a result more supercoiled
DNA entered into the gel. The effect of topoisomerase inhibiꢀ
tory activity is directly proportional to the migration of DNA
into the gel. Interestingly, compounds (3c and 3k) in compariꢀ
son with parent compounds (C1/C2) exhibited significantly
higher migration of bands into the gel. This indicates the supeꢀ
rior activities of scaffoldꢀhopped analogs than that of parent
auronesC1 and C2 in exꢀvivo topoisomeraseꢀinhibition.
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Studies of apoptosis by compound 3c and inhibition of
CDK1:
From above cytotoxicity experiments it was observed that
tested compounds exhibited appreciable toxicity in cancer
cells with minimal effect to normal cells. To check whether
these agents caused apoptosis in cancer cells, we considered a
representative compound 3c and measured the apoptosis after
treatment in MCFꢀ7 cells using DAPI nuclear staining.28The
cells were treated with increasing concentrations of compound
3c for 48 h prior to addition of DAPI dye. Significantly higher
chromatin condensation and nuclear fragmentation in treated
cells were noticed compared to untreated cells (Figure S4).
More than 6 fold increase in apoptotic nuclei was observed at
75 nM compared to untreated control.
To confirm that the investigated compounds caused apoptosis
we did western blot analysis29 of various proꢀ and antiꢀ
apoptotic proteins in MCFꢀ7 cellular lysate treated with comꢀ
pound 3c. An increase in the expressions of proꢀapoptotic p
BAX and decrease in the expression of antiꢀapoptotic protein
BCLꢀXL confirmed that compound 3c causes apoptosis in
MCFꢀ7 cells. Next we intended to check the possible involveꢀ
ment of other cell cycle regulatory proteins i.e. CDK1 and
pCDK1 (Thr 161). A significant decreased expression of
CDK1 as well as pCDK1 (Thr 161) with increasing concentraꢀ
tion of compound 3c (Figure 3a) was observed. Inhibition of
these cell cycle regulatory proteins leads to arresting of cells in
G2/M transition phase.
Induction and degradation of mitochondrial enzyme
CASPASE 3 is the hallmark of apoptosis. An increased exꢀ
pression of cleaved CASPASE 3 (17 kDa) was observed in
compound 3c treated MCFꢀ7 cellular lysate (Figure3a). To
further validate this result we have checked the expression of
CASPASE 3 by immunocytochemistry after treating MCFꢀ7
cells with increasing concentrations of compound 3c.Increase
in the expression of CASPASE 3 was noticed which further
validates that compound 3c induced apoptosis in the MCFꢀ7
cells (Figure 3b).
Figure 4: a) Inhibition of Topoisomerase activity by compound
3c in respect to control C1 and positive control Etoposide in
MCFꢀ7 cell nuclear lysate. b) Inhibition of Topoisomerase activity
by compound 3k in respect to control C2 and positive control
Etoposide in MCFꢀ7 cell nuclear lysate. In both the figures(a and
b) Lane 1 is only plasmid, Lane 2 is plasmid with untreated nucleꢀ
ar lysate, Lane 3ꢀ7 are for plasmid and nuclear lysate treated with
various concentrations of compound 3c/3k (10 nMꢀ75 nM). Lane
8, figure (a) was treated with C1 and figure (b) was treated with
C2, respectively. Lane 9 is lysate treated with Etoposide 45 (µM)
for both the figures (a and b).
Compound 3c inhibited the invasiveness of MCF 7 cells:
Invasion into basement membrane is one of important properꢀ
ties of cancer cell. The effect of compound 3c in the invasive
property of MCFꢀ7 cell was measured by a wellꢀestablished
matrigel cell invasion assay.30 It was observed that with inꢀ
crease in concentration of compound 3c the numbers of invadꢀ
ed colonies were significantly decreased with respect to unꢀ
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Determination cell cycle profile in compound 3c and 3k
treated MCFꢀ7 cells:
compounds. These compounds inhibited the invasiveness of
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cancer cells. The effect of compounds on inhibition of tubulin
assembly, CDK1 and pCDK1 were also found to be signifiꢀ
cant.
To investigate the regulation of the cell cycle profile, FACS
analysis was carried out in MCFꢀ7 cells after treatment with
compounds 3c and 3k. The cells were treated with increasing
concentrations of compounds 3c and 3k for 48 h. FACS analyꢀ
sis was performed after staining with PI and the DNA content
of each phase of the cell cycle was measured. It was observed
that both the compounds 3c and 3k arrest the cell at G2/M
phase. In case of compound 3c, significant G2/M arrest was
observed (80.1 %) at IC50 concentration (50 nM) with noticeaꢀ
ble apoptosis at higher concentration i.e. 20.2% apoptosis in
100 nM treatment. Similarly in case of compound 3k the
G2/M population was found to be 33.2% for 70 nM and in 100
nM significant apoptosis (19.9%) occurred. So, this result
suggests that the selective compounds arrest the cell at G2/M
phase. So, to validate this we compared these compounds with
a known compound that arrests the cell at G2/M phase i.e.
combretastatinAꢀ4(CAꢀ4) (Figure S7). Noticeable G2/M arꢀ
rests with apoptosis (G0) were observed in CAꢀ4 treated
MCFꢀ7 cell lysate. No such profile were obtained for control
compounds (C1 and C2), indicating that compounds 3c and 3k
arrest the cell at G2/M phase at IC50 concentration with apopꢀ
tosis at higher concentration which in accordance with the
expression profile of CDK1 and pCDK1.
Interestingly, compared to parent aurones, their scaffoldꢀ
hopped analogs, 2ꢀarylideneimidazo[1,2ꢀa]pyridineꢀ3ꢀones,
were found to be multiꢀfold higher potent (IC50: micromolar vs
nanomolar) antiproliferative agents against various cancer
cells and hTopoIIαꢀinhibitors in in–vitro as well as exꢀvivo
assays. This suggests successful implication of scaffoldꢀ
hopping strategy on natural product aurones for the discovery
of more potent analogs which are outside the patent issues.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Experimental procedure for synthesis and biology, characterizaꢀ
tion data for new compounds (docx)
AUTHOR INFORMATION
Corresponding Author
* S.K. Guchhait: Tel.: +91 (0)172 2214683; fax: +91 (0)172
2214692; Eꢀmail addresses: skguchhait@niper.ac.in; C.N. Kundu,
Telꢀ+916742725466, E.mailꢀcnkundu@gmail.com
Compounds 3c and 3k inhibit tubulin polymerization and
disrupt microtubule dynamics:
Author Contributions
‡These authors contributed equally.
To study possible anti tubulin effect of investigated comꢀ
pounds, an in vitro tubulin assembly assay was carried out
using breast cancer cells (MCF 7). The compounds 3c and 3k
were found to possess good activity in tubulin assembly inhiꢀ
bition (Figure S8a and Table S3). The tubulin assembly inhibiꢀ
tion was found to be optimum with IC50 (concentration reꢀ
quired to inhibit 50% of tubulin assembly) values of 54 nM
and 60 nM for 3c and 3k, respectively comparison to CAꢀ4
which showed IC50 at 2.5 ꢁM. To further validate this result
we performed an immunostaining of α tubulin. The left panel
represents the PI staining of nucleus. The middle panel repreꢀ
sents α tubulin protein assembly in MCFꢀ7 cells conjugated
with FITC and the third panel is the merge image of PI and
FITC conjugated α tubulin. The result indicated that the comꢀ
pound 3c induced significant distortion in the tubulin assembly
along with the formation of abnormal mitotic spindle in the
treated MCFꢀ7 lysate (Figure S8).
ACKNOWLEDGMENT
We gratefully acknowledge financial support from DBT, DST,
and CSIR, Government of India, New Delhi, for this investigaꢀ
tion. GP and SMA are thankful to NIPER for their fellowships.
AN is thankful to ICMR for providing fellowship.
ABBREVIATIONS
hTopoIIα, Human topoisomerase IIα; kDNA, Kinetoplast DNA;
Nck, Nicked; Rel, Relaxed; Lin, Linear; SC, Supercoiled; TAE,
TrisꢀacetateꢀEDTA; MTT, 3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenylteꢀtrazolium bromide.
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