Neutral Porphyrin Derivative Exerts Anticancer Activity by Targeting Cellular Topoisomerase i (Top1) and Promotes Apoptotic Cell Death without Stabilizing Top1-DNA Cleavage Complexes

Article abstract of DOI:10.1021/acs.jmedchem.7b01297

Camptothecin (CPT) selectively traps topoisomerase 1-DNA cleavable complexes (Top1cc) to promote anticancer activity. Here, we report the design and synthesis of a new class of neutral porphyrin derivative 5,10-bis(4-carboxyphenyl)-15, 20-bis(4-dimethylaminophenyl)porphyrin (compound 8) as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8 reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP) assays. We established that MCF7 cells treated with compound 8 trigger proteasome-mediated Top1 degradation, accumulate higher levels of reactive oxygen species (ROS), PARP1 cleavage, oxidative DNA fragmentation, and stimulate apoptotic cell death without stabilizing apoptotic Top1-DNA cleavage complexes. Finally, compound 8 shows anticancer activity by targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic process.

Full text of DOI:10.1021/acs.jmedchem.7b01297

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Article
Neutral porphyrin derivative exerts anticancer activity by
targeting cellular topoisomerase I (Top1) and promotes apoptotic
cell death without stabilizing Top1-DNA cleavage complexes
Subhendu Kumar Das, Arijit Ghosh, Srijita Paul Chowdhuri, Nyancy Halder, Ishita Rehman,
Souvik Sengupta, Krushna Chandra Sahoo, Harapriya Rath, and BENU BRATA DAS
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01297 • Publication Date (Web): 30 Dec 2017
Downloaded from http://pubs.acs.org on December 30, 2017
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Neutral porphyrin derivative exerts anticancer activity by targeting cellular
topoisomerase I (Top1) and promotes apoptotic cell death without stabilizing
Top1ꢀDNA cleavage complexes
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Subhendu Kumar Das , Arijit Ghosh , Srijita Paul Chowdhuri , Nyancy Halder , Ishita
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Rehman , Souvik Sengupta , Krushna Chandra Sahoo , Harapriya Rath,
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and Benu Brata Das
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Laboratory of Molecular Biology, Department of Biological Chemistry; Department of
Inorganic Chemistry; Indian Association for the Cultivation of Science, 2A & 2B, Raja S. C.
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Mullick Road, Jadavpur, Kolkataꢀ700032, India. Division of Biological and Life Sciences,
School of Arts and Sciences, Ahmedabad University, Central Campus, Navrangpura,
Ahmedabad, Gujaratꢀ380009, India.
*To whom correspondence should be addressed. Tel: +91 33 2473 4971 (Ext. 2108); Fax: +91
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3 2473 2805; Email: pcbbd@iacs.res.in (BBD) and ichr@iacs.res.in (HR)
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ABSTRACT
Camptothecin (CPT) selectively traps topoisomerase 1ꢀDNA cleavable complexes (Top1cc) to
promote anticancer activity. Here, we report the design, synthesis of a new class of neutral
porphyrin derivative 5,10ꢀbis(4ꢀcarboxyphenyl)ꢀ15, 20ꢀbis(4ꢀdimethylaminophenyl)ꢀporphyrin
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(compound 8), as a potent catalytic inhibitor of human Top1. In contrast to CPT, compound 8
reversibly binds with the free enzyme and inhibits the formation of Top1cc and promotes
reversal of the preformed Top1cc with CPT. Compound 8 induced inhibition of Top1cc
formation in live cells was substantiated by fluorescence recovery after photobleaching (FRAP)
assays. We established that MCF7 cells treated with compound 8 triggers proteasomeꢀmediated
Top1 degradation, accumulates higher levels of reactive oxygen species (ROS), PARP1
cleavage, oxidative DNA fragmentation and stimulates apoptotic cell death without stabilizing
apoptotic Top1ꢀDNA cleavage complexes. Finally, compound 8 shows anticancer activity by
targeting cellular Top1 and preventing the enzyme from directly participating in the apoptotic
process.
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Introduction
DNA topoisomerase I (Top1) are often exploited as imperative anticancer chemotherapeutic
targets due to its critical role in DNA supercoil relaxation which involves three main steps: (a)
DNA strand cleavage by a transesterification reaction initiated by the active site tyrosine
attacking DNA phosphodiester backbone and generating a covalent intermediate of DNA 3'ꢀ
phosphotyrosyl linkage (Top1cc), (b) DNA relaxation by controlled strand rotation and (c) DNA
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religation by a similar transesterification and release of the enzyme from the DNA.
Top1 inhibitors are classified into two groups, class I (poisons) and class II (catalytic
inhibitors). Top1 poisons includes camptothecin (CPT), Topotecan, Irinotecan and other CPT
derivatives as well as few nonꢀCPT Top1 inhibitors like indenoisoquinolines, indolocarbazoles
and thiohydantoin derivatives reveal their anticancer activity by selectively trapping the Top1ꢀ
DNA covalent cleavage complexes (Top1cc) and inhibiting further religation of cleaved DNA
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, 3, 6, 7 8
strands.
with replication or transcription machinery, which activate cellꢀcycle arrest and cell death.
contrast, class II catalytic inhibitors hinder other steps of Top1 catalytic cycle by directly binding
Unrepaired Top1cc generates DNA double strand breaks following collision
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, 9
In
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with enzyme but do not stabilize Top1cc which includes indolizinoquinolinedione.
All types
of topoisomerase inhibitors cause DNA breaks and are accountable for the killing of the
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proliferating cancer cells.
Drug independent trapping of Top1cc's are also evidenced from endogenous DNA
lesions, like UV and IR radiationꢀinduced DNA damage, abasic sites, oxidized bases and
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mismatches.
Production of "apoptotic Top1cc" is independent of Top1 poisons but are
dependent on variety of agents that are inducers of apoptotic cell death, including staurosporine,
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a protein kinase C inhibitor,
Top2 inhibitor like etoposide and tubulin inhibitor like
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vinblastine. All these compounds trigger cellular reactive oxygen species (ROS) that account
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for oxidative DNA damages that promote stabilization of Top1cc.
Despite clinical success of CPT, the major limitations include its unstable chemical
structure, poor aqueous solubility, rapid cellular efflux via membrane pumps and acquisition of
cellular resistance of these drugs impelled the designing and investigation of new nonꢀ
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camptothecin Top1 inhibitors.
Porphyrins are remarkably promising chemotype for
development of anticancer agents and photodynamic therapy, which includes FDAꢀapproved and
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clinically used sensitizer Photofrin.
Porphyrin derivatives have wide pharmaceutical
properties and broad range of biological activities that constitute selective modes of DNA
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9, 21ꢀ24
binding, mimicking photosynthetic centers, vitamin B12 and Pꢀ450,
cellular target of the compounds is still unclear.
nevertheless, the
Here, we discuss our study involving design, synthesis and biological evaluation of a
novel series of neutral porphyrin that inhibits human Top1. Selected neutral porphyrin derivative
5
,10ꢀbis(4ꢀcarboxyphenyl)ꢀ15,20ꢀbis(4ꢀdimethylaminophenyl)ꢀporphyrin
(compound
8),
exhibited highest potency against human Top1 activity both as purified enzyme and as an
endogenous protein in the total cellular extracts of human breast adenocarcinoma (MCF7) cells
from our synthetic library. We have further established that the compound 8 binds with the free
enzyme and targets cellular Top1 for proteasomeꢀmediated degradation and bolster ROSꢀinduced
apoptotic cell death without stabilizing Top1ꢀDNA cleavage complexes. Persistent with
inhibition of human Top1 activity in vitro, compound 8 was effective in killing cancer cells by
targeting cellular Top1.
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Chemistry
The macrocycles under biological investigation described in our present manuscript are shown
schematically (Scheme 1). We have taken into consideration, the parent basic porphyrin i.e.
tetraphenyl porphyrin and variation in the periphery of the macrocycles with other mesoꢀ
substituents. After investigating the biological properties of macrocycles compounds 1, 2, 3, 4, 5,
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, 7, 9 and 10, we designed and synthesized 8 following the literature known synthetic
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methodology. Synthesis of porphyrins, compounds 1 , 2 , 3 , 7 , 8, 9 and 10 were carried
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out following Adler Method,
macrocycles bearing four identical mesoꢀsubstituents such as
compound 1, 2 and 10 were obtained by condensation of freshly distilled pyrrole and
corresponding aryl aldehyde under reflux condition using propionic acid as an acidic solvent.
Furthermore, synthesis of compound 3 and 8 (Scheme 1A) was tuned following the mixedꢀ
aldehyde condensation method leading to porphyrins bearing two different types of mesoꢀ
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substituents.
The A B type of porphyrin, 7 and 9 are often obtained in mixedꢀaldehyde
3
condensation methods and their yield was increased by reaction of 3:1 ratio of aldehydes A and
B. It is a wellꢀknown fact that haloporphyrins undergo palladiumꢀcatalyzed crossꢀcoupling
reactions to produce alkylꢀ, vinylꢀ, arylꢀ, pyridylꢀ and alkynylꢀporphyrin monomers, dimers, and
3
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oligomers. Thus, Suzukiꢀcoupling was carried out by using strategically using mixture of 3 and
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ꢀAminophenylboronic acid pinacol ester to afford compound 4 (Scheme 1A). Subsequently,
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the transꢀsubstituted porphyrin, compound 5 and 6 (Scheme 1B), were synthesized based on
the MacDonald 2 + 2 condensation of dipyrromethane (synthesized by acidꢀcatalyzed
condensation of pyrrole with 4ꢀbromobenzaldehyde) with 4ꢀPyridinecarboxaldehyde and 4ꢀ
3
5
(pyridinꢀ4ꢀyl) benzaldehyde respectively.
Detailed spectroscopic characterization data,
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elemental analysis and melting point of all the reported macrocycles in this manuscript have been
documented in the Experimental Section and the Supporting Information (S1 to S40).
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Biological Results and Discussion:
Neutral porphyrin derivative 8 inhibits human Topoisomerase 1 catalytic activity
To test the inhibitory effect of the synthetic library of neutral porphyrin compounds
(Scheme 1) on human Top1 activity, we performed the DNA relaxation assays in a standard
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assay mixture containing the plasmid DNA and recombinant Top1.
Table 1 enlists the
compounds with relative efficiencies of Top1 inhibition as a measure of their effective drug
concentrations (EC50 value), indicating highest activity for the selected neutral porphyrin
derivative compound 8.
To examine the mechanism of Top1 inhibition with compound 8 we used variable
incubation conditions in the relaxation assays (Figure 1A). When Top1 and compound 8 were
mixed together simultaneous in the relaxation assays (Figure 1A, lanes 13ꢀ16), 35ꢀ40 % Top1
inhibition was achieved at 1ꢁµM of compound 8 (Figure 1A, lane 16). Next to test the impact of
compound 8 interaction with Top1 in the relaxation assays, we preꢀincubated Top1 with
compound 8 at indicated concentrations (Figure 1A, lanes 5ꢀ8) separately before the addition of
6
, 13, 36, 38
DNA.
Under these conditions, 1ꢁµM of compound 8 was sufficient to induce 85ꢀ95%
inhibition (Figure 1A, lane 8). Figure 1, panel A and D shows that Top1 inhibition was markedly
increased (~4ꢀ5 fold) when compound 8 was preꢀincubated with the recombinant enzyme
compared to the simultaneous incubation (EC for Top1 inhibition preꢀincubation: 0.381 ± 0.11
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M vs. simultaneous 1.472 ± 0.32 ꢀM), suggesting that compound 8 may bind to the free
enzyme, unlike CPT to inhibit Top1 activity.
To investigate the selectivity of compound 8 towards endogenous Top1 in the whole cell
extracts of human breast adenocarcinoma (MCF7) cells (Figure 1B), we used ex-vivo Top1
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, 39
relaxation inhibition assays.
The advantage of employing whole cell extracts as source of
Top1 is that the enzyme is maintained in its native structure among plethora of other proteins.
Figure 1C shows that compound 8 markedly inhibited Top1 activity when the cellular extracts
were preꢀincubated with compound 8 (Figure 1C, lanes 5ꢀ10) compared to simultaneous
incubation (Figure 1, panel C, lanes 15ꢀ20). Quantification indicates that Top1 inhibition was
significantly increased (~2ꢀ3 fold) when compound 8 was preꢀincubated with whole cell extracts
(
Figure 1E), consistent with the inhibition of recombinant Top1 in preꢀincubation relaxation
assays (Figure 1D).
Here we provide evidence that compound 8 selectively inhibit Top1, both as a purified
enzyme (Figure 1A) and as an endogenous protein in the total cellular lysate (Figure 1C) without
being afflicted by other proteins. Therefore, it is conceivable that compound 8 selectively
interacts with human Top1 to promote the inhibition of DNA relaxation activity.
Compound 8 inhibits Top1ꢀDNA cleavage complexes
Because camptothecin (CPT) stabilizes Top1ꢀDNA cleavable complexes (Top1cc) to
3
, 5, 37
inhibit Top1 activity,
we investigated compound 8 induced Top1cc formation with the
6, 8, 36, 37
plasmid DNA cleavage assays.
Top1 mediated conversion of closed circular DNA (form
I) to nicked circular DNA (form II) in the presence of specific inhibitors are referred as
“cleavable complex”. Figure 2A shows that compound 8 failed to stabilize Top1cc in contrast to
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CPT, suggesting that compound 8 inhibits Top1cc formation. To further investigate the impact of
compound 8 on preformed Top1cc, we preꢀincubated Top1 with plasmid DNA and CPT (Figure
2B) before the addition of compound 8 in the cleavage assays. Figure 2C shows that compound 8
completely abrogated CPTꢀinduced Top1cc in a dose dependent manner (Figure 2, panel C, lanes
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ꢀ9).
In contrast to CPT (Figure 2D, lane 3), we further established that compound 8 failed to
stabilize Top1cc in single turnover equilibrium cleavage assays (Figure 2D, lanes 4ꢀ6) by
reacting recombinant Top1 with 25ꢀmer duplex oligonucleotides harboring preferred Top1
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, 13, 36, 38
cleavage sites.
cleaved oligonucleotides (Figure 2D, lane 7ꢀ9) consistent with plasmid DNA cleavage assays
Figure 2C). Taken together, our data indicate that compound 8 inhibits Top1 without trapping
Top1cc.
To obtain direct evidence for compound 8 mediated inhibition of Top1cc formation in
In addition, compound 8 reversed the CPTꢀinduced Top1cc with 12ꢀmer
(
live human carcinoma cells, we used MCF7 cells and transiently expressed EGFPꢀTop1. Live
cells expressing ectopic Top1 were analyzed under laser confocal microscopy equipped with
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0ꢀ42
fluorescence recovery after photobleaching (FRAP) technology
. The FRAP recovery curves
of EGFPꢀTop1 in the untreated samples represents a large (∼80–85%) mobile population (see
Figure 2E, and 2F; Ctr) compared to a small (∼20%) immobile population, suggesting that Top1
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is dynamic, freely exchanged and binds transiently with the DNA (reversible Top1cc).
However CPT covalently trapped Top1 on the DNA in the live cells (Figure 2E; CPT),
which significantly impede FRAP recovery by increasing ∼40–50% of Top1 immobile
population (Figure 2E, and 2F; (+) CPT; 5 ꢂM). Under similar condition fluorescence recovery
of EGFPꢀTop1 was unaffected in the presence of compound 8 (Figure 2E, and the quantification
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in 2F; (+) compound 8; 20 ꢂM), indicating compound 8 failed to trap Top1cc in live cells.
Furthermore, compound 8 can restore ~12ꢀ15% of Top1 mobility in cells pretreated with CPT
(Figure 2EꢀF), suggesting compound 8 promotes reversal of CPTꢀinduced Top1cc in live cells
consistent with the in vitro cleavage assays (Figure 2AꢀD). Taken together our data provide
evidence that compound 8 inhibits cellular Top1cc formation.
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Compound 8 reversibly binds with Top1 at equimolar concentration
Next we investigated the binding nature of compound 8 with the purified Top1 by
measuring the intrinsic tryptophan fluorescence quenching of Top1. Figure 3A shows 1:1
12, 14, 43
binding stoichiometry of compound 8 with Top1 as measured from Job plot,
suggesting
that there is one binding site for the drug in the enzyme. Figure 3B and C shows the quenching
profile of Top1 in the presence of variable doses of compound 8, with a dissociation constant
(K = 0.381 µM) calculated from figure 3B and C.
D
To further investigate the nature of binding of compound 8 with Top1 (reversible vs.
12, 14
irreversible),
we performed dilution assay with recombinant Top1. Top1 was preꢀincubated
with 1 µM of compound 8 at which 90–99% inhibition of enzyme has been achieved (Figure 3D,
lane 4). The subsequent dillution of the reaction mixtures in the relaxation assays showed an
increase in the % relief of inhibition for compound 8. Complete relief of Top1 inhibition was
achieved at 40ꢀfold dilution (Figure 3D, lane 5ꢀ8). This suggests that compound 8 interact in
reversible manner with Top1 consistent with its weak dissociation constant.
To investigate the intercalation capacity of the compound 8 into the plasmid DNA, we
36, 44
performed topoisomerase I unwinding assays
, which depends on the capacity of
intercalating compounds to unwind the duplex DNA and thereby inducing a conformational
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36, 44
change in the DNA.
Figure 3E demonstrates that mꢀAMSA a strong DNA intercalative
agent, incite a net negative supercoiling of the relaxed plasmid DNA at concentration
independent manner (Figure 3E, lanes 3 and 4). Under similar conditions nonꢀintercalative
compounds like etoposide failed to show such effect (Figure 3E, lanes 5 and 6). Figure 3E, lanes
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, 8, 9 shows that compound 8 failed to induce negative supercoiling of the relaxed plasmid
DNA at 50, 100, 300 µM concentrations, suggesting neutral porphyrin derivative 8 is not a DNA
intercalator. Therefore compound 8 interacts in reversible manner with Top1 to inhibit the
plasmid relaxation activity (Figure 1).
Compound 8 triggers proteasomeꢀmediated degradation of cellular Top1 without
stabilizing apoptotic Top1ꢀDNA covalent complexes
Because compound 8 binds with free Top1 (Figure 3) and inhibits Top1cc formation in
live cells (Figure 2E), therefore we examined the impact of compound 8 on endogenous Top1.
Cellular lysates were prepared from MCF7 cells treated with compound 8 for indicated times and
were analyzed by Western blotting. Figure 4A, lanes 2ꢀ4 shows time dependent depletion of
Top1 signal in compound 8 treated cells, suggesting Top1 degradation. Because proteasome has
7
, 9, 45
been implicated for Top1 degradation,
to investigate Top1 proteolysis in compound 8 treated cells. Figure 4B confirmed that compound
ꢀinduced degradation of Top1 (Figure 4B, lane 2) was abrogated in the presence of proteasome
thus we used proteasome specific inhibitor (MG132)
8
inhibitor MG132 (Figure 4B, lane 4), suggesting compound 8 activates proteasome pathway.
Next, to investigate the plausibility between Top1 proteolysis and activation of apoptosis
1
6ꢀ18, 46, 47
we tested PARP cleavage, a "hallmark" event of apoptosis in compound 8 treated cells.
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Figure 4C shows compound 8 treatment for 12 h indeed promote PARP cleavage, suggesting
compound 8 activates apoptosis through degradation of Top1.
Because compound 8 stimulates apoptosis (Figure 4C and Figure 5B), therefore we
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examined accumulation of apoptotic Top1ꢀDNA complexes in compound 8 treated cells (Figure
15, 16
4
D) by ICEꢀbioassays.
Top1cc related to apoptosis are due to secondary DNA modifications
1
5ꢀ17
and are independent from direct Top1ꢀdrug interaction.
Under a condition that triggers
PARP1 cleavage in MCF7 cells treated with compound 8, immunoblotting revealed the absence
of Top1 in the DNAꢀcontaining fractions (Figure 4D; compound 8), in contrast to camptothecin
(Figure 4D; CPT), indicating that compound 8 inhibits Top1cc during apoptosis. Compound 8
abrogates CPTꢀinduced cellular Top1ꢀDNA complexes (Figure 4D; CPT + 8) in keeping with in
vitro cleavage assays (Figure 2B). Therefore proteasome mediated degradation of Top1 triggers
compound 8 induced apoptosis without stabilizing apoptotic Top1ꢀDNA covalent complexes.
Compound 8 accumulates reactive oxygen species induced DNA breaks and promotes
apoptotic cell death
Next to investigate the mechanistic link between compound 8 induced cellular Top1
inhibition (Figure 1), degradation (Figure 4A and B) and activation of apoptosis (Figure 4C), we
tested reactive oxygen species (ROS) formation and accumulation of oxidative DNA
1
5, 17, 46
fragmentation
in compound 8 treated MCF7 cells. To examine ROS accumulation, we
used non fluorescent substrate (2',7'ꢀdichlorodihydrofluorescein diacetate; H2DCFDA) which is
transformed into fluorescent product (2',7'ꢀdichlorofluorescein; DCF) in the presence of ROS
1
3
inside the cells. Figure 5A shows time dependent accumulation of ROS in compound 8 treated
cells. We observed a marked elevation (~3ꢀ5 fold) in ROS accumulation (Figure 5A) under
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similar conditions that activate PARP1 cleavage (Figure 4C). Nonetheless, MCF7 cells preꢀ
treated with Nꢀacetylꢀcysteine (NAC), a specific inhibitor of ROS, reduced compound 8 induced
ROS generation by ~3ꢀfold (Figure 5A).
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One interpretation of this result is that compound 8 induced ROS may accumulate
3, 13
oxidative DNA strand breaks that activate apoptosis
as revealed by PARP1 cleavage (Figure
4
C). Therefore, we directly measured compound 8 induced DNA strand breaks at single cellular
level by using alkaline comet assays (Figure 5BꢀC), and simultaneously measured the apoptotic
cell death by immunofluorescence staining of the apoptosis marker phosphatidylserine with
Annexin VꢀFITC antibody (Figure 5DꢀE). Compound 8 treated MCF7 cells accumulate ~8ꢀfold
increase in DNA strand breaks (Figure 5B and C) compared to untreated cells resulting ~7ꢀfold
elevation in compound 8 induced Annexin V(+) apoptotic cells (Figure 5D and E). We further
confirmed that ROS inhibitor NAC resulted ~4ꢀfold decrease in compound 8 induced DNA
breaks (Figure 5C) as well as ~4ꢀ5ꢀfold reduction in Annexin V(+) apoptotic cells (Figure 5E).
Therefore, we conclude compound 8 triggers reactive oxygen species induced DNA degradation
and apoptotic cell death without stabilizing Top1ꢀDNA cleavage complexes.
Compound 8 displays potent anticancer activity
Compound 8 was investigated for its cytotoxicity in the cancer cell lines from different
6
, 41
tissue origin
, as well as in the nonꢀcancerous human embryonic kidney cell lines (HEK293)
and mouse embryonic fibroblasts (MEFs). Figure 6 indicates that compound 8 revealed potent
cytotoxicity in cancerous cells including human breast adenocarcinoma cell lines (MCF7: IC :
5
0
2
.17 µM), human cervical cancer cell lines (HeLa: IC : 4.13 µM), human ovarian
50
adenocarcinoma cell lines (NIH:OVCARꢀ3: IC : 5.19 µM) & human colon carcinoma cell lines
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(
HCT116: IC : 4.39 µM) cells, compared to the non cancerous cells like HEK293 or MEFs that
5
0
shows markedly reduced or no toxicity (IC : >10 µM). The MCF7 breast cancer cell lines were
5
0
most susceptible to compound 8.
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TDP1 hydrolyze 3′ phosphotyrosyl linkages that are primarily due Top1cc, therefore
40, 41, 45, 48
TDP1ꢀ/ꢀ cells are hypersensitive to Top1 poisons.
Figure 6F shows that both TDPꢀ/ꢀ
and TDP1+/+ MEFs cells were equally sensitive to compound 8, further providing evidence that
compound 8 exerts cytotoxicity without stabilizing Top1cc (Figure 4D).
To further confirm that Top1 is the cellular target for compound 8 mediated cytotoxicity,
we have performed cytotoxicity assays in siRNA mediated Top1 knockdown cells (Figure 6G,
inset). Figure 6G indicate that compound 8 treated Top1 knockdown cells displayed reduced
cytotoxicity compared to the Top1 proficient cells, establishing Top1 is the cellular target for
compound 8.
Because compound 8 exerts cytotoxicity without stabilizing Top1cc (Figure 4D), next we
tested the impact of compound 8 on CPTꢀinduced preformed Top1cc. Figure 6H shows that
pretreatment of CPT and further addition of compound 8 were not additive in cytotoxicity, rather
showed overlapping cytotoxicity in MCF7 cells treated with compound 8 or CPT independently.
Therefore we conclude compound 8 plausibly suppress the CPT induced cytotoxicity (Figure
6
H) because compound 8 reverse CPTꢀinduced Top1cc formation in cells as well as in cleavage
assays (Figure 2). Taken together these data suggest that compound 8 is a potential Top1
inhibitor and may be an appropriate lead to develop as a potential anticancer agent.
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Conclusion
In summary, we have identified neutral porphyrin based compound 8 as a new potent
anticancer agent which inhibits cellular Top1 without trapping Top1ꢀDNA cleavage complexes,
a mechanism which is unique in comparison to known Top1 poisons like camptothecin (CPT).
We provide evidence that in contrast to CPT, the selected porphyrin derivative 8 binds reversibly
to the free enzyme (Figure 3) and effectively inhibits the formation of Top1ꢀDNA cleavage
complex (Top1cc) both in vitro and in live cell as determined in FRAP assays (Figure 2).
Compound 8 abrogates CPTꢀmediated preformed Top1cc both as recombinant enzyme (Figure
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AꢀD) and as an endogenous Top1 in the human breast adenocarcinoma (MCF7) cells (Figure
4D), suggesting a plausibility to overcome the limitations of CPTꢀresistance.
ROSꢀinduced DNA damage facilitate trapping of Top1cc in cells is an obligatory event in
1
5ꢀ17
the progression of apoptosis
. In contrast, compound 8 activates reactive oxygen species
(ROS), proteolysis of cellular Top1, which may accumulate DNA torsional strain resulting
oxidative DNA damage as revealed by alkaline comet assays and apoptotic cell death (Figure 5)
without stabilization of apoptotic Top1ꢀDNA complexes (Figure 4D). Therefore compound 8
counteracts the cellular Top1 by abrogating its precise engagement in the apoptotic process.
We further show that in contrast to nonꢀcancerous cells, compound 8 is effective against
cancerous cell lines from different tissue origin including MCFꢀ7, HeLa, NIH:OVCARꢀ3 and
HCT116 cells (Figure 6) by targeting cellular Top1 (Figure 6G). Future challenge includes
development of more potent neutral porphyrin based Top1 catalytic inhibitor, which may be
exploited for anticancer therapy.
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Experimental Section
Chemistry.
General Methods: Electronic absorption spectra were measured with a PerkinElmer Lambda
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950 UVꢀvisibleꢀNIR spectrophotometer. H NMR spectra were recorded on a Bruker AVIII 500
MHz spectrometer and chemical shifts were reported as the delta scale in ppm relative to CHCl3
1
(δ = 7.26 ppm) and DMSO (δ = 2.50 ppm ) as internal reference for H. MALDIꢀTOF MS data
were recorded using Bruker Daltonics flex Analyser. All solvents and chemicals were of reagent
grade quality, obtained commercially and used without further purification except as noted. For
spectral measurements, anhydrous dichloromethane was obtained by refluxing and distillation
over CaH . Gravity column chromatography was performed using Merck Silica Gel 230ꢀ400
2
mesh. All reported yields refer to pure isolated compounds. Chemicals and solvents were of
reagent grade and used as obtained from commercial sources without further purification. The
purities of all of the biologically tested compounds were the peak area of the major product was
≥95% as estimated by NMR spectroscopy and C, H, N analysis along with melting point.
Synthesis:
5
, 10, 15, 20ꢀtetraphenylporphyrin (1): By using general method 1 mL (10 mmol) of
benzaldehyde and 50 mL of propionic acid were mixed and the reaction mixture was
magnetically stirred. Freshly distilled pyrrole (0.7 mL; 10 mmol) was then added to the mixture,
the temperature then brought to reflux and allowed to stir for 2 hrs at reflux. After allowing the
reaction mixture to cool to room temperature, the reaction flask was placed in the freezer
overnight to aid precipitation of the porphyrin. The reaction mixture was then vacuum filtered
using a sintered funnel and a dark purple solid was collected, washed with 5 x 50 mL of DCM,
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washed with methanol and dried overnight and purified by silica gel chromatography to give
o
4
.9g, of. 5,10,15,20ꢀtetraphenylporphyrin (79% yields). mp> 300 C
1
H NMR (500 MHz, CDCl 298 K, δ [ppm]): 8.88 (s, 8H, py), 8.25 (m, 8H, PhꢀCH), 7.79 (m,
3
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12H, PhꢀCH), ꢀ2.76(s, 2H,ꢀNH). UVꢀVIS (CH Cl , λ [nm], 298 K): 417nm, 515nm, 548nm,
2
2
5
90nm, 645 nm. MALDIꢀTOF MS (m/z): 615.635 (Calc. for C H N , exact mass: 614.206).
44 30 4
Elemental analysis: Calcd for C H N C, 85.97; H, 4.92; N, 9.11. Found: C, 86; H, 5.00; N,
4
4
30
4
9.08.
5
, 10, 15, 20ꢀtetrakis(4ꢀcarboxyphenyl)porphyrin (2): By using general method 1.5 g (10
mmol) of 4ꢀformylbenzoic acid and 50 mL of propionic acid were added and the reaction
mixture was stirred at ambient temperature. To increase the solubility of 4ꢀformylbenzoic acid,
o
the reaction mixture was heated to 80 C at which point the aldehyde fully dissolved. Freshly
distilled pyrrole (0.7 ml; 10 mmol) was then added to the mixture, and reaction mixture was
allowed to stir for 2 hrs under reflux. The reaction mixture was cooled to room temperature,
placed in the freezer overnight to aid precipitation of the porphyrin. The reaction mixture was
then vacuum filtered using a sintered funnel and a dark purple solid was collected, washed with
5
x50 mL of DCM, washed with methanol and dried overnight to give 1.1g, of. 5,10,15,20ꢀ
o
tetrakis (4ꢀcarboxyphenyl) porphyrin (55% yields). mp> 300 C
1
H NMR (500 MHz, DMSOꢀd 298 K, δ [ppm]): 13.14 (4H, br, ꢀCOOH), 8.86 (8H, s, Py βꢀH),
6
8
.38 (16H, dd, phenyl H), ꢀ2.93 (2H, s, NH). UVꢀVIS (CH OH, λ [nm], 298 K): 413nm,
3
5
13nm, 545nm, 589nm, 646 nm. MALDIꢀTOF MS (m/z): 790.604 (Calc. for C H N O
8
4
8
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exact mass: 790.206). Elemental analysis: Calcd for C H N O : C, 72.90; H, 3.82.; N, 7.09.
4
8
30
4
8
Found: C, 73.00; H, 3.86; N, 7.07.
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5,10-bis(4-bromophenyl)-15,20-di(4-pyridyl)porphyrin (3): By using general method 4ꢀ
Pyridinecarboxaldehyde (0.535g, 5mmol), 4ꢀbromobenzaldehyde (0.925g, 5 mmol) and 50 mL
of propionic acid were added and the reaction mixture was stirred under nitrogen. To increase the
o
solubility of 4ꢀbromobenzaldehyde, the reaction mixture was heated to 80 C at which point the
aldehyde fully dissolved. Freshly distilled pyrrole (0.7 ml; 10 mmol) was then added to the
mixture, and allowed to stir for 4 hrs under reflux. Reaction mixture was brought to room
temperature and the reaction flask was then placed in the freezer overnight to aid precipitation of
the porphyrin. The crude product was repeatedly chromatographed on silica gel. The purest
o
compound was obtained by recrystallization in methanol. Yield: 3.87g (50%). mp> 300 C
1
H NMR (500 MHz, CDCl 298 K, δ [ppm]): ꢀ2.86 (s, 2H, NH), 7.94 (d, J= 7.5Hz, 4H, Phꢀ
3
Br), 8.10 (d, J= 7.5Hz, 4H, PhꢀBr), 8.18 (d, J= 10 Hz , 4H , Pyridylꢀ H), 8.88 (m, 8H, βꢀH Py),
9
.07 (d, J= 10 Hz ,4H, PyridylꢀH). UVꢀVIS (CH Cl , λ [nm], 298 K): 418 nm, 514 nm, 547 nm,
2 2
5
88 nm, 645 nm. MALDIꢀTOF MS (m/z): 775.399 (Calc. for C H Br N exact mass:
42 26 2 6
7
74.520). Elemental analysis: Calcd for C H Br N : C, 65.13; H, 3.38.; N, 10.85. Found: C,
42 26 2 6
65.14; H, 3.30; N, 10.72.
5
,10-bis(4ꢀaminobiphenyl)-15,20-bis(4ꢀpyridyl)- porphyrin (4): A Schlenk tube was charged
with 5,20ꢀbis(4ꢀbromophenyl)ꢀ10,15ꢀdi(pyridinꢀ4ꢀyl)porphyrin (0.054g, 0.07 mmol) , 4ꢀ(4,4,5,5ꢀ
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tetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)aniline (0.092g , 0.42 mmol) , K PO (0.089g , 0.42 mmol)
3
4
and Pd(PPh ) (8mg, 0.007mmol) under nitrogen atmosphere . Dioxane (5mL) and deionized
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water (0.1mL) were added and the resulting mixture was refluxed for 24 hours, brought to room
temperature and filtered through celite in a sintered funnel. Washed several times with DCM,
and the filtrate was evaporated in vacuum. The crude product obtained upon evaporation of
solvent was purified by silica gel column chromatography (100ꢀ200 mesh) using ethyl acetate/
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o
dichloromethane (20:80, v/v) as an eluent. Yield 44.6 mg (80%). mp> 300 C
1
H NMR (500 MHz, CDCl 298 K, δ [ppm]): ꢀ2.76 (s, 2H, NH), 3.75 (s, 4H, NH ), 6.88(d, 2H,
3
2
J = 8.5Hz, βꢀH Py), 7.72 (d, 2H, J =8.5Hz, βꢀH Py), 7.91 (d, 4H, J =7.5Hz, PhꢀH), 8.17 (d, 4H, J
= 5.5Hz, PyridylꢀH), 8.20 (d, 4H, J = 8Hz, PhꢀH), 8.81 (d, 4H, J = 4Hz, PhꢀH), 8.84 (s, 2H, βꢀH
Py), 8.97 (s, 2H, βꢀH Py), 9.01 (d, 4H, J = 4.5Hz, PhꢀH), 9.03 (d, 4H, J = 5Hz, Pyridylꢀ H). UVꢀ
VIS (CH Cl , λ [nm], 298 K): 420nm, 515 nm, 553nm, 592nm, 645 nm.MALDIꢀTOF MS
2
2
(
m/z): 798.820 (Calc. for C H N exact mass: 798.322). Elemental analysis: Calcd for
54 38 8
C H N : C, 81.18; H, 4.79.; N, 14.03. Found: C, 81.14; H, 4.16; N, 14.12.
54
38
8
5
,15-bis(4ꢀbromophenyl)-10,20-di(4-pyridyl)porphyrin (5): By using general method 2,2'ꢀ((4ꢀ
bromophenyl)methylene)bis(1Hꢀpyrrole) (0.5g, 1.66 mmol) and 4ꢀpyridinecarboxaldehyde
0.171g, 1.6 mmol) were taken in a round bottom flask and 40 mL propionic acid was added to
(
it. The reaction mixture was then stirred under nitrogen at 298K for 90 mins. Thereafter, the
reaction mixture was refluxed under nitrogen for 90 minutes, brought to room temperature and
left overnight for precipitation at room temperature. The precipitate was poured into sintered
funnel and washed several times with methanol. The crude product thus obtained was vacuum
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dried followed by repeated silica gel column chromatography. The purest compound was
o
obtained by recrystallization from methanol. Yield 620mg (50%). mp> 300 C
1
H NMR (500 MHz, CDCl 298 K, δ [ppm]): ꢀ2.85 (s, 2H, NH), 7.9 (d, J=8Hz, 4H, PhꢀBr),
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
.10 (d, J=7.5Hz, 4H, PhꢀBr), 8.18 (d, J=5.5Hz, 4H, Pyridylꢀ H), 8.85 (br, 4H, βꢀH Py), 8.90 (br,
4
H, βꢀH Py), 9.07 (d, J=5Hz ,4H, PyridylꢀH). UVꢀVIS (CH Cl , λ [nm], 298 K): 417nm, 513
2
2
nm, 548 nm, 590 nm, 644 nm. MALDIꢀTOF MS (m/z): 772.425 (Calc. for C H Br N exact
4
2
26
2
6
mass: 772.059).Elemental analysis: Calcd for for C H Br N : C, 65.13; H, 3.38.; N, 10.85.
4
2
26
2
6
Found: C, 65.24; H, 3.40; N, 10.72.
5
,15-bis(4-bromophenyl)-10,20-bis(4ꢀphenylpyridyl)porphyrin (6): By using general method
2
,2'ꢀ((4 bromophenyl)methylene)bis(1Hꢀpyrrole) (0.5g, 1.66 mmol) and 4ꢀ(pyridinꢀ4ꢀ
yl)benzaldehyde (0.304g, 1.6 mmol) were taken in a round bottom flask and 40 mL propionic
acid was added. The reaction mixture was then stirred under nitrogen at 298K for 90 mins.
Thereafter, the reaction mixture was refluxed under nitrogen for 90 minutes, brought to room
temperature and left overnight for precipitation. The precipitate was poured into sintered funnel
and washed with methanol. The crude product left on the sintered funnel was then dried in
vacuum. The crude product was purified by silica gel column chromatography (100ꢀ200 mesh)
o
using ethyl acetate/dichloromethane (10:90, v/v) as an eluent. Yield: 296 mg (20%). mp> 300 C
1
H NMR (500 MHz, CDCl 298 K, δ [ppm]): ꢀ2.81 (s, 2H, NH), 8.91 (br, 4H, PyridylꢀH),8.87
3
(br, 2H, PyꢀβH), 8.85 (br, 4H, PyridylꢀH ), 8.34 (d, J = 7.5Hz, 2H, PyꢀβH), 8.09 (m,8H, 4ꢀbromo
Ph, Ph ), 8.05 (d, J = 7.5 Hz, 2H, PyꢀβH), 7.92 (m,8H, 4ꢀbromo Ph, Ph ), 7.86 (d, J= 5.5 Hz, 2H,
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2
2
2
2
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2
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4
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5
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5
5
5
5
5
6
PyꢀβH), UVꢀVIS (CH Cl , λ [nm], 298 K): 418nm, 514 nm, 550 nm, 592 nm, 647 nm.
2
2
MALDIꢀTOF MS (m/z): 925.670 (Calc. for C H Br N exact mass: 924.121). Elemental
54 34
2
6
analysis: Calcd for C H Br N : C, 69.99; H, 3.70.; N, 9.07. Found: C, 69.94; H, 3.64; N, 9.02.
54
34
2
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
6
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8
9
0
5
ꢀ(4ꢀbromophenyl)ꢀ10,15,20ꢀtri(4ꢀpyridyl)porphyrin (7): By using general method 4ꢀ
Pyridinecarboxaldehyde (1.605g, 15mmol), 4ꢀbromobenzaldehyde (0.925g, 5 mmol) and 50 mL
of propionic acid were added and the reaction mixture was magnetically stirred. Freshly distilled
pyrrole (1.4 mL; 20 mmol) was then added to the mixture, the temperature then brought to reflux
and allowed to stir for 4 hrs at reflux. After allowing the reaction mixture to cool to room
temperature, the reaction flask was placed in the freezer overnight to aid precipitation of the
porphyrin. The reaction mixture was then vacuum filtered using a sintered funnel and a dark
purple solid was collected, washed with 5 x 50 mL of DCM, washed with methanol and dried
overnight. The solid was purified by silica gel column chromatography. Yield 2.08 g (15%). mp>
o
300 C
1
H NMR (500 MHz, CDCl 298 K, δ [ppm]): 9.06 (d, J= 5 Hz, 6H, PyridylꢀH), 8.90 (d, J= 4.5
3
Hz, 2H, βꢀH Py), 8.86 (s, 4H, βꢀH Py), 8.83 (d, J= 4.5 Hz, 2H, βꢀH Py), 8.17 (d, J= 5 Hz, 6H,
PyridylꢀH ), 8.07 (d, J= 8 Hz, 2H, Phenylꢀ H), 7.92 (d, J= 8 Hz, 2H, Phenylꢀ H). UVꢀVIS
(
CH Cl , λ [nm], 298 K): 416nm, 512nm, 547nm, 588nm, 643nm. MALDIꢀTOF MS (m/z):
2 2
6
96.658 (Calc. for C H BrN exact mass: 696.612). Elemental analysis: Calcd for
41 26 7
C H BrN : C, 70.69; H, 3.76.; N, 14.08. Found: C, 69.70; H, 3.86; N, 14.19.
41 26
7
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5
,10-bis(4-carboxyphenyl)-15,20-bis(4ꢀdimethylaminophenyl)-porphyrin (8): By using
general method 4(dimethylamino)benzaldehyde (0.745g, 5 mmol) , 4ꢀformylbenzoic acid (0.75g,
5mmol) and 50 mL of propionic acid were added and the reaction mixture was stirred under
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
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2
2
2
2
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4
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4
4
4
4
5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
nitrogen. To increase the solubility of 4ꢀformylbenzoic acid, the reaction mixture was heated to
o
8
0 C at which point the aldehyde fully dissolved. Freshly distilled pyrrole (0.7 ml; 10 mmol)
was then added to the reaction mixture and allowed to stir for 4 hrs under reflux. After allowing
the reaction mixture to cool to room temperature, the reaction flask was placed in the freezer
overnight to aid precipitation of the porphyrin. The precipitate was vacuum filtered through a
sintered funnel and a dark purple solid so collected was washed with methanol and dried. The
solid residue was purified over silica gel chromatography and recrystallized from methanol.
o
Yield: 1.57g (20%). mp> 300 C
1
H NMR (500 MHz, DMSOꢀd6 298 K, δ [ppm]): 12.85 (2H, br, ꢀCOOH), 8.96 (br, 4H,
CarboxyPhꢀCH), 8.82 (br, 6H, CarboxyPhꢀCH and PyꢀβꢀCH), 8.38 (d, J = 7.5 Hz, 4H, N,Nꢀ
Dimethyl Ph), 8.33 (d, J = 7.5 Hz, 4H, N,Nꢀ Dimethyl Ph), 8.03 (d, J = 7.5 Hz, 2H, Pyꢀβꢀ
CH),7.95 (s, 2H, PyꢀβꢀCH), 7.16 (d, J = 8 Hz, 2H, PyꢀβꢀCH),2.89 (s, 12H, Me), ꢀ2.84 (br, 2H,
NH). UVꢀVIS (CH Cl , λ [nm], 298 K): 414 nm, 516 nm, 557 nm, 594 nm, 652 nm. MALDIꢀ
2
2
TOF MS (m/z): 788.681 (Calc. for C H N O exact mass: 788.311).Elemental analysis: Calcd
5
0
40
6
4
for C H N O : C, 76.12; H, 5.11.; N, 10.65. Found: C, 76.14; H, 5.16; N, 10.62.
5
0
40
6
4
5ꢀ(4ꢀcarboxyphenyl)ꢀ10,15,20ꢀtri(4ꢀdimethylaminophenyl)ꢀ porphyrin (9): By using general
method 4(dimethylamino)benzaldehyde (2.23g, 15 mmol), 4ꢀformylbenzoic acid (0.75g,
5
mmol)were added to 50 mL of propionic acid and the reaction mixture was magnetically
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stirred. Freshly distilled pyrrole (1.4 ml; 20 mmol) was then added to the mixture, the
temperature then brought to reflux and allowed to stir for 4 hrs at reflux. After allowing the
reaction mixture to cool to room temperature, the reaction flask was placed in the freezer
overnight to aid precipitation of the porphyrin. The reaction mixture was then vacuum filtered
using a sintered funnel and a dark purple solid was collected, washed with 5x50 mL of DCM,
washed with methanol and dried overnight. The solid residue was purified over silica gel
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
chromatography using 30% EtOAcꢀCH Cl . Yield 3.1g (20%). mp> 300 C
2
2
1
H NMR (500 MHz, CDCl 298 K, δ [ppm]): 12.85 (1H, br, ꢀCOOH), 8.92 (br, 2H,
3
CarboxyPhꢀCH), 8.75 (br, 2H, CarboxyPhꢀCH), 8.57 (s, 4H, PyꢀβꢀCH), 8.42 (br, 6H, N,Nꢀ
Dimethyl Ph), 8.28 (br, 6H, N,Nꢀ Dimethyl Ph), 8.06 (d, J = 7 Hz, 2H, PyꢀβꢀCH), 7.10 (d, J = 7
Hz, 2H, PyꢀβꢀCH), 3.32 (s, 18H, Me), ꢀ2.70 (br, 2H, NH). UVꢀVIS (CH Cl , λ [nm], 298 K):
2
2
417 nm, 519 nm, 561 nm, 598nm, 655 nm. MALDIꢀTOF MS (m/z): 787.918 (Calc. for
C H N O exact mass: 787.363). Elemental analysis: Calcd for C H N O :C, 77.74; H, 5.76.;
51
45
7
2
51 45
7
2
N, 12.44. Found: C, 77.80; H, 5.81; N, 12.41.
5
,10,15,20ꢀtetra(4ꢀpyridyl)porphyrin (10) : By using general method 1.07 g (10 mmol) of 4ꢀ
Pyridinecarboxaldehyde and 50 mL of propionic acid were added and the reaction mixture was
magnetically stirred. Freshly distilled pyrrole (0.7 mL; 10 mmol) was then added to the mixture,
the temperature then brought to reflux and allowed to stir for 2 hrs at reflux. After allowing the
reaction mixture to cool to room temperature, the reaction flask was placed in the freezer
overnight to aid precipitation of the porphyrin. The reaction mixture was then vacuum filtered
using a sintered funnel and a dark purple solid was collected, washed with 5 x 50 mL of DCM,
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washed with methanol and dried overnight to give 5,10,15,20ꢀtetra(4ꢀpyridyl)porphyrin .Yield
o
2
.472 g (20%). mp> 300 C
1
H NMR (500 MHz, CDCl 298 K, δ [ppm]): 8.94 (d, J= 5 Hz, 8H, PyridylꢀH), 8.79(b, 8H,
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Pyrrole βꢀH), 8.16 (d, J= 5 Hz, 8H, Pyridylꢀ H), ꢀ2.99 (b, 2H, pyrrole NH). UVꢀVIS (CH Cl , λ
2
2
[nm], 298 K): 415nm, 511nm, 545nm, 587nm, 642nm. MALDIꢀTOF MS (m/z): 618.629 (Calc.
for C H N exact mass: 618.704). Elemental analysis: Calcd for C H N : C, 77.65; H, 4.24.;
40 26
8
40 26
8
N, 18.11. Found: C, 77.00; H, 4.16; N, 18.07.
Drug and antibodies
Camptothecin, proteasomal inhibitor MG132 and NꢀacetylꢀLꢀCysteine (NAC) were
purchased from Sigma. Mouse monoclonal antiꢀhuman Top1 (C21) antibody, rabbit polyclonal
PARP1 antibody and secondary antibodies: horseradish peroxidase conjugated antiꢀrabbit IgG or
antiꢀmouse IgG were obtained from Santa Cruz Biotechnology (USA). Antiꢀactin (ACTN05)
antibody was from Neo Markers (USA).
Recombinant human topoisomerase 1 and plasmid DNA relaxation assay
The recombinant human Top1 was purified from Sfꢀ9 insect cells, infected with the
6
,
recombinant baculovirus (a kind gift from Prof. James. J. Champoux) as described previously.
13
The types 1 DNA topoisomerase are assayed by decreased mobility of the relaxed
+
isomers of supercoiled pBS (SK ) DNA in 1% agarose gel. The relaxation assay was carried out
with recombinant human Top1 or the whole cell extracts of human breast adenocarcinoma
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8
9
1
1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
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4
4
4
4
4
4
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5
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5
5
5
5
5
5
5
6
(
MCF7) cells as source of endogenous Top1, diluted in the relaxation buffer with supercoiled
6
, 8, 13, 38
plasmid DNA as described previously.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cleavage assay
6, 8
Plasmid DNA cleavage assay was carried out as described previously . Equilibrium Cleavage
assays with 25ꢀmer duplex of oligonucleotide containing a Top1 binding motif was labeled and
1
3, 36, 38
annealed as described previously.
electrophoresis, dried and exposed on PhosphorImager screens and imaged with Typhoon FLA
000 (GE Healthcare, UK).
Samples were analyzed by 12 % sequencing gel
7
Cell culture and transfection
Human cancerous cell lines like MCF7, HeLa, HCT116, NIH: OVCARꢀ3 and HEK293
were obtained from the Developmental Therapeutics Program as kind gift from Dr. Yves
+
/+
ꢀ/ꢀ
Pommier (NIH/NCI/USA), TDP1 and TDP1 primary MEF cells were kind gift from Dr
Cornelius F Boerkoel (University of British Columbia, Canada) were cultured as described
6
, 36, 40, 42
previously.
Plasmid DNAs were transfected with Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s protocol.
Photobleaching experiments
4
0ꢀ42
Photobleaching experiments were performed as described formerly
using Andor
Spining disc inverted confocal laserꢀscanning microscope equipped with a 60X/1.42 NA oilꢀ
immersion objective (Olympus) and with a CO ꢀcontrolled onꢀstage heated environmental
2
0
chamber set to 37 C. FRAP analyses were carried out with living MCF7 cells ectopically
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expressing EGFPꢀhuman Top1 grown on chamber cover glass (Genetix, India) and drug treated
as indicated. For FRAP analysis, a subnuclear spot was bleached for 30 ms by solidꢀstate laser
line (488 nm for EGFP) adapted to the fluorescent protein of interest and FRAP curves were
4
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
generated individually normalized to the preꢀbleach signal as described previously.
Job plot and Spectrofluorimetric binding assay
The binding stoichiometry for compound 8 with Top1 was determined using the method
12, 14, 43
of continuous variation
. Briefly, reaction mixtures containing variable Top1 and
compound 8 to a final concentration of 1.25ꢁµM were analyzed for quenching of tryptophan
fluorescence at 350ꢁnm upon excitation at 295ꢁnm on PerkinElmer LS55 luminescence
spectrometer.
1
2, 14, 43
Spectrofluorimetric binding assay was carried out as previously
where Top1 (200
0
nM) was incubated with various concentrations of compound 8 (0 to 11 µM) at 25 C for 10 min.
The equation for determining fraction of binding sites (B) occupied by inhibitor was B = (F ꢀ F)
0
/
Fmax, where F is the fluorescence intensity at 350 nm of Top1 alone in the absence of any
0
inhibitors, F is the fluorescence intensity at 350 nm of Top1 in the presence of inhibitor, and Fmax
is obtained from the plot of 1/(F ꢀF) versus 1/[X] and by extrapolating 1/[X] to zero, where [X] is
0
the concentration of compound 8. The dissociation constant (K ) was determined as described
D
1
2
previously.
Analysis of compound 8ꢀDNA intercalation
The competence of the drug to intercalate into plasmid DNA was determined by Top1
3
6, 44
unwinding assay.
Assays were performed with 50 fmol of pBluscript (SK+) DNA in the
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2
2
2
2
2
2
2
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3
3
3
3
3
4
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5
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5
5
6
presence or absence of compound 8, mꢀAMSA and etoposide at indicated concentrations. Excess
of DNA topoisomerase I was reacted with supercoiled plasmid DNA to generate relaxed DNA
for the unwinding assays. The relaxed DNA was purified by proteolytic digestion with proteinase
K at 37°C, followed by phenol / chloroform extraction and ethanol precipitation. The unwinding
assays were carried out at 37°C for 15 min with independent compounds, and were analyzed by
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
% agarose gel as described above.
Cell extracts and immunoblotting
Preparation of whole cell extracts from MCF7 cells and immunoblotting were carried out
6
, 40, 41
as described.
Immunoreactivity was detected using ECL chemiluminescence reaction
TM
(
Amersham) under ChemiDoc MP System (BioꢀRad, USA).
Immuno Complex of Enzyme (ICE) Bioassay
In vivo Top1 cleavage complexes (Top1cc) were isolated from MCF7 cells using immuno
49
6
complex of enzyme (ICE) bioassay technique. Briefly, 5×10 MCF7 cells were treated with
drugs and were lysed by DNAzol reagent (Invitrogen, USA) in the presence of 0.1% SDS.
Genomic DNA was prepared and was briefly sonicated. Varying concentrations of DNA were
spotted onto nitrocellulose membrane (Millipore, USA) using a slotꢀblot vacuum system (Bioꢀ
4
1
rad, USA). Immunoblotting were carried out with antiꢀhuman Top1 antibodies as described.
Measurement of ROS
Intracellular ROS was detected in drugꢀtreated MCF7 cells with or without pretreatment
1
3, 46
of NꢀacetylꢀLꢀcysteine (NAC) for indicated time as described.
Briefly, cells were washed
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and resuspended in 500 µL of 1× PBS and were loaded with 2 µg/mL of H DCFDA (Molecular
2
Probes) for 30 min, and green fluorescence of 2,7ꢀdichlorofluorescein was measured at 515 nm
by spectrofluorometer.
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1
1
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1
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0
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Alkaline COMET assays
To detect the levels of drugꢀinduced DNA breaks, after treatment MCF7 cells were
subjected to alkaline comet assays according to the manufacturer’s instructions (Trevigen, USA),
comet length was measured and was scored for at least 50 cells. Distributions of comet lengths
1
3, 40, 50
were compared using the Student tꢀtest as described previously.
Immunocytochemistry
Immunofluorescence staining of apoptosis marker phosphatidylserine was performed as
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0, 41
described previously.
After treatment, MCF7 cells were fixed with 2% paraformaldehyde
for 10 min at room temperature and stained with Annexin VꢀFITC antibody (BD, USA),
mounted in antiꢀfade solution with propidium iodide (Vector Laboratories, USA) and examined
under Leica TCS SP8 confocal laserꢀscanning microscope.
Cell survival assay
Cell survival was assessed by 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium
6
bromide (MTT) assay as discussed previously. The percent inhibition of viability for each
concentration of the compounds was calculated with respect to the control and IC values were
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0
estimated.
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Page 28 of 49
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siRNA transfection
Transfections were performed as described previously.
40
5
In brief, cells (1.5 x 10 ) were
transfected with control siRNA or 40 nM Top1 siRNA (Qiagen) using oligofectamine
(Invitrogen) according to the manufacturer’s protocol. Time course experiments revealed a
maximum suppression of Top1 protein at day 3 after transfection, as analyzed by Western
blotting.
0
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Journal of Medicinal Chemistry
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Supporting Information:
The supporting information is available free of charge on the ACS website
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1
1
MALDIꢀTOF MS, H NMR, Hꢀ H 2D COSY Spectra, and UVꢀVIS data of compounds 1 to 10
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are provided in the Supporting information (PDF).
Molecular String Formula and some data (CSV)
Acknowledgment
We wish to thank Dr. Hemanta K Majumder and Dr. Suvendra N Bhattacharyya, CSIRꢀIndian
Institute of Chemical Biology, India for the help during the study. The BBD team is supported by
Wellcome Trust / DBT India Alliance Intermediate Fellowship (Award# IA/I/13/1/500888). AG
and KCS are recipients of CSIRꢀNETꢀSenior Research Fellowship, India. SPC thank to UGCꢀ
CSIRꢀNET for Junior Research Fellowship. SKD, IR and NH thank IACS for SRF and JRF
positions respectively. HR thank DSTꢀSERB (EMR/2016/004705) for the research grant. BBD is
a Wellcome Trust / DBT India Alliance Intermediate fellow.
CONFLICT OF INTEREST
None declared.
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