New antineoplastic agent based on a dibenzoylmethane derivative: Cytotoxic effect and direct interaction with DNA

Article abstract of DOI:10.1016/j.bpc.2018.04.009

Melanoma accounts for only 4% of all skin cancers but is among the most lethal cutaneous neoplasms. Dacarbazine is the drug of choice for the treatment of melanoma in Brazil through the public health system mainly because of its low cost. However, it is a

Full text of DOI:10.1016/j.bpc.2018.04.009

Accepted Manuscript
New antineoplastic agent based on a dibenzoylmethane
derivative: Cytotoxic effect and direct interaction with DNA
Fernanda R. Nascimento, Tiago A. Moura, Jefferson V.P.B. Baeta,
Bruno C. Publio, Pollyanna M.F. Ferreira, Anésia A. Santos,
Andressa A.P. França, Marcio S. Rocha, Gaspar Diaz-Muñoz,
Marisa A.N. Diaz
PII:
S0301-4622(18)30085-1
doi:10.1016/j.bpc.2018.04.009
BIOCHE 6089
DOI:
Reference:
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Biophysical Chemistry
Received date:
Revised date:
Accepted date:
24 March 2018
28 April 2018
28 April 2018
Please cite this article as: Fernanda R. Nascimento, Tiago A. Moura, Jefferson V.P.B.
Baeta, Bruno C. Publio, Pollyanna M.F. Ferreira, Anésia A. Santos, Andressa A.P. França,
Marcio S. Rocha, Gaspar Diaz-Muñoz, Marisa A.N. Diaz , New antineoplastic agent based
on a dibenzoylmethane derivative: Cytotoxic effect and direct interaction with DNA. The
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ACCEPTED MANUSCRIPT
New Antineoplastic Agent Based on a Dibenzoylmethane
Derivative: Cytotoxic Effect and Direct Interaction with
DNA
a
b
a
Fernanda R. Nascimento , Tiago A. Moura , Jefferson V. P. B. Baeta , Bruno C.
b
a
c
d
Publio , Pollyanna M. F. Ferreira , Anésia A. Santos , Andressa A. P. França ,
Marcio S. Rocha , Gaspar Diaz-Muñoz , Marisa A. N. Diaz
b, *
e
a, **
a
Department of Biochemistry and Molecular Biology, Universidade Federal de
Viçosa, Minas Gerais 36570-900, Brazil
b
c
d
e
Department of Physics, Universidade Federal de Viçosa, Minas Gerais 36570-900,
Brazil
Department of General Biology, Universidade Federal de Viçosa, Minas Gerais
3
6570-900, Brazil
Department of Biology, Universidade Estadual de Minas Gerais, Minas Gerais
3
6500-000, Brazil
Department of Chemistry, Universidade Federal de Minas Gerais, Belo Horizonte,
Minas Gerais 31270-901, Brazil
*
*
Corresponding author. E-mail address: marcios.rocha@ufv.br (Marcio S. Rocha)
* Corresponding author. E-mail address: marisanogueira@ufv.br (Marisa A. N. Diaz)

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ABSTRACT
Melanoma accounts for only 4% of all skin cancers but is among the most lethal
cutaneous neoplasms. Dacarbazine is the drug of choice for the treatment of melanoma
in Brazil through the public health system mainly because of its low cost. However, it
is an alkylating agent of low specificity and elicits a therapeutic response in only 20%
of cases. Other drugs available for the treatment of melanoma are expensive, and
tumor cells commonly develop resistance to these drugs. The fight against melanoma
demands novel, more specific drugs that are effective in killing drug-resistant tumor
cells. Dibenzoylmethane (1,3-diphenylpropane-1,3-dione) derivatives are promising
antitumor agents. In this study, we investigated the cytotoxic effect of 1,3-diphenyl-2-
benzyl-1,3-propanedione (DPBP) on B16F10 melanoma cells as well as its direct
interaction with the DNA molecule using optical tweezers. DPBP showed promising
results against tumor cells and had a selectivity index of 41.94. Also, we demonstrated
the ability of DPBP to interact directly with the DNA molecule. The fact that DPBP
can interact with DNA in vitro allows us to hypothesize that such an interaction may
also occur in vivo and, therefore, that DPBP may be an alternative to treat patients
with drug-resistant melanomas. These findings can guide the development of new and
more effective drugs.
Keywords: dibenzoylmethane; dibenzoylmethane derivative; melanoma; optical
tweezers; DNA.

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1
. Introduction
Melanoma is a cancer of low incidence but high lethality. It accounts for only 4%
of all cancers in Brazil. About six thousand new cases are expected for the biennium
016–2017, of which 1500 will probably result in death. The southern region of Brazil
2
has the highest incidence rates of the country, 8.02 and 7.06 cases per 100,000 men
and women, respectively. The lowest rates occur in the northern region, 0.20 cases per
1
00,000 men and 0 cases among women [1].
The main causes of melanoma are genetic predisposition and excessive sun
exposure. The use of sunscreen is recommended as a preventive measure. The disease
in its initial stages is treated by surgical removal of the tumor and sentinel lymph
nodes. This procedure aims to eliminate all cancer cells before they reach the
bloodstream and lymphatic vessels and thereby avoid metastasis. For patients with
more advanced stages of the disease, treatment consists in the administration of
antineoplastic drugs, which have low effectiveness and high cost. In addition, the more
advanced the disease, the lower the chances of patient survival, even with treatment.
Drugs that intercalate or interact with DNA have been widely used as
chemotherapeutic agents, such as cisplatin, doxorubicin, and dacarbazine. However,
despite their therapeutic success, these drugs cause serious side effects, and tumor
cells can become resistant to their cytotoxic mechanisms. Dacarbazine (Figure 1) is
the most used drug to treat melanoma. This alkylating agent binds to the DNA,
preventing DNA strands from separating and disrupting protein and DNA synthesis
[
2]. Dacarbazine is a non-specific agent, as it can act at any stage of the cell cycle.
This drug elicits a therapeutic response in only 20% of cases for approximately five
months and a complete response in 5% of cases [3].
O
NH2
N
N
CH3
N
N
N
H
CH3
Figure 1. Chemical structure of dacarbazine, a drug used for the treatment of
melanoma.

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Dibenzoylmethane (DBM, Figure 2) is a -diketone and natural flavonoid found
in families such as Annonaceae, Asteraceae, Menispermaceae, Rosaceae, Salicaceae,
and especially Fabaceae (genera Glycyrrhiza and Lonchocarpus) [4, 5]. DBM is
widely used in sunscreens as an ultraviolet blocking agent [6]. A variety of biological
properties have been attributed to DBM, including anti-inflammatory [7], anti-
mutagenesis [8], and anti-estrogenic activities [9]. In addition, DBM exerts many
anticancer effects. DBM has been shown to induce apoptosis and cell cycle arrest in
human prostate cancer cells [10] and in colon cancer cells [11] and to prevent the
formation of DNA-adducts induced by carcinogens, both in vitro and in vivo, in
mammary glands [12] and in the lungs by activation of the Nrf2 detoxification
pathway [13].
O
O
Figure 2. Chemical structure of dibenzoylmethane (DBM).
As an extension of the pioneering work of Nogueira et al. (2003) [5, 14] reporting
the photoprotective properties and promising results of a series of dibenzoylmethane
derivatives against human melanoma cell lines, in the present study, we evaluate the
anti-melanoma activity of 1,3-diphenyl-2-benzyl-1,3-propanedione (DPBP, Figure 3)
against the B16F10 cell line. DPBP was one of the best exponents of the series; its
complete mechanism of action, however, is still unknown. Here we advance the
understanding of DPBP’s mechanism of action, studying its efficiency at a cellular
level as well as at a single-molecule level by optical tweezers analysis, a technique
used to tether DNA and measure its mechanical properties. This technique allows the
elucidation of the binding modes and the physical chemistry of drug–DNA
interactions [15].
In order to evaluate the performance of DPBP as a potential anticancer agent,
first, we investigated the cytotoxic effect of DPBP on melanoma cells. DPBP gave
promising results against B16F10 cells, showing a high selectivity index (SI) of 41.91.
By comparing the effect of DPBP on normal melanocytes with the effects obtained
using other classical chemotherapeutic agents, we concluded that this molecule is a
potential chemotherapeutic agent for the treatment of melanoma.

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O
O
Figure 3. Chemical structure of 1,3-diphenyl-2-benzyl-1,3-propanedione
(DPBP).
Finally, we investigated the direct interaction of DPBP with DNA using optical
tweezers. DNA is the therapeutic target of many antineoplastic agents, such as
cisplatin and doxorubicin, and it directs the search for new antineoplastics [16]. The
fight against melanoma demands new and more specific drugs that are effective in
cases of drug resistance.
2
. Results and discussion
2
.1. Chemistry
DPBP was synthesized by the reaction of dibenzoylmethane and benzyl bromide
in acetone in the presence of anhydrous potassium carbonate (Scheme 1). DPBP was
1
13
characterized by IR, H and C NMR, and mass spectra. The spectral data of DPBP
were in agreement with the described in the literature [5].
O
K CO , BnBr
2
3
acetone, r.t.
overnight
O
O
O
dibenzoylmethane
1,3-diphenyl-2-benzyl-1,3-propanedione (DPBP)
Scheme 1. Synthesis of 1,3-diphenyl-2-benzyl-1,3-propanedione (DPBP).
2
2
.2. Biological activity
.2.1. Cell cytotoxicity
Data presented in Figure 4 show that DPBP was more effective against B16F10
cells than against melan-A cells. The selectivity indices (SI = IC50 melan-A/IC50

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B16F10) obtained were 41.94 and 1.00 for DPBP and dacarbazine, respectively, which
indicates the high selectivity of DPBP for B16F10 tumor cells (detailed data are
shown in Table 1).
Figure 4. Plots of the percentage of cell death induced by DPBP at different
concentrations on B16F10 and melan-A cells. Results are the mean ± standard
deviation of three independent experiments.
Table 1. IC values and selectivity indices (SI) of dacarbazine, DBM, and DPBP.
5
0
Compound IC B16F10 (μg/mL) IC melan-A (μg/mL) SI (melan-A/B16F10)
5
0
50
1
0
.00
.50
Dacarbazine
DBM
253.97
11.59
6.25
253.91
5.68
4
1.94
DPBP
262.13
The results presented in Figure 4 and Table 1 indicate that DPBP is a promising
agent for the treatment of melanoma, as its SI (41.94) is considerably high and its IC50
is considerably low (6.25 μg/mL = 1.97 μM) when compared with the SI (0.50) and
IC (11.59 µg/mL = 51.7 µM) of DBM. Moreover, cisplatin and doxorubicin, two
5
0
well-known DNA-intercalating drugs, exhibited IC values of approximately 6 µM
5
0
and SI of 1.0 (data not shown). These results demonstrate that DPBP is a molecule of
high efficiency, suggesting that it has a great potential as a chemotherapeutic agent in
the treatment of melanoma.

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2
.2.2. Optical tweezers experiments
In Figure 5, we show the persistence length A of DPBP–DNA complexes as a
function of DPBP concentration in solution (C ). Black dots represent the
T
experimental data, and the red solid line is the fit to the model, discussed in Section
4
.7. Observe that the persistence length presents a non-monotonic behavior: it initially
decreases (for C < 40 M) from the bare DNA value (~47 nm) until reaching ~34 nm
T
and then increases (for C > 40 M). The model returned the characteristic binding
T
4
−1
parameters of the DPBP–DNA interaction: K = (2.7 ± 0.2) × 10 M and n = 5.1 ±
.5. The local persistence lengths were also determined: A = (22 ± 3) nm,
1
1
corresponding to the binding of DPBP to a single binding site; and A = (68 ± 9) nm,
2
corresponding to two bound molecules becoming nearest neighbors.
In Figure 6, we show the contour length (L) of the same DPBP–DNA complexes
as a function of DPBP concentration (C ). This parameter exhibits a monotonic
T
behavior, increasing from the bare DNA value (~16.5 m) until reaching the saturation
value of ~19.1 m.
Figure 5. Persistence length (A) as a function of DPBP concentration in solution (CT)
(black dots). The red solid line is the fit to the model (discussed in Section 4.7).

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Figure 6. Contour length (L) of the same DPBP–DNA complexes as a function of
DPBP concentration (CT).
DNA is one of the main targets in the discovery of new antineoplastic drugs.
Drugs that interact with DNA can prevent transcription and/or replication. A drug can
interact with DNA molecules in many ways, such as by intercalation, groove binding,
covalent binding, and electrostatic binding [15].
Single-molecule stretching experiments such as those performed using optical
and/or magnetic tweezers have become the state of the art in the past years for
characterizing interactions between DNA and small molecules. The basic idea of these
experiments is to monitor changes in the mechanical properties of DNA induced by
the drug upon binding [15, 17]. The binding parameters of the interaction can be
further deduced from the changes in mechanical properties. By using this type of
approach, it is possible to promptly verify if a ligand interacts with DNA and deduce
the possible binding mechanisms [15, 17].
In Figures 5 and 6, we presented the behavior of the two basic mechanical
properties of DNA, persistence and contour lengths, as a function of DPBP
concentration. Both properties changed as DPBP concentration increased, indicating
that DPBP binds to the double helix and alters the mechanical behavior of the formed
complexes.
The behavior of persistence length was non-monotonic, suggesting a complex
binding mechanism between DPBP and DNA [15]. Note that DPBP has three aromatic
rings in its structure (see Figure 3). Antineoplastic drugs are mostly polyaromatic and
interact with DNA mainly by two binding mechanisms: groove binding and
intercalation [18].

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For ligands that interact with the minor groove of the DNA, there is usually a
correlation between the resulting persistence length and changes in the minor groove
width. A narrowing of the minor groove cavity of the double helix is in general
accompanied by a decrease in the bending rigidity of the biopolymer, thus decreasing
the effective persistence length [19]. Olsen et al. (2003) [20], for example, observed
the narrowing of the minor groove cavity after the interaction of DNA with the ligand
distamycin-A, which resulted in a decrease in persistence length. On the other hand,
some ligands, such as netropsin, lengthen the minor groove cavity, increasing the
effective persistence length [21]. The effect of groove binders on the DNA contour
length also depends on the drug. Some molecules, such as diminazene aceturate,
increase contour length by unwinding the DNA double helix [22], whereas other
molecules, such as Hoechst 33258, decrease the apparent value of this parameter by
promoting DNA condensation [23].
Intercalators, on the other hand, are well recognized for increasing DNA contour
and persistence lengths upon binding [24, 25].
Thus, on the basis of data of Figures 5 and 6, we propose the following
mechanism of interaction between DPBP and DNA. For C < 40 M, there was a
T
strong decrease in persistence length, whereas contour length had only a slight
increase. These observations strongly suggest that DPBP binds to the DNA minor
groove at these concentrations. In general, ligands with a molecular weight of less than
1
000 g/mol bind to the DNA minor groove floor instead of the major groove, as is the
case of DPBP (314.38 g/mol) [26]. At higher concentrations, C > 40 M, there was a
T
considerable increase in persistence and contour lengths. This increase in mechanical
parameters may be related to structural changes in the double helix as a result of the
accommodation of DPBP molecules. Alternatively, there is a possibility of partial
intercalation of DPBP molecules into the double helix by insertion of an aromatic ring
between DNA base pairs. In any case, the sigmoidal shape of the contour length curve
strongly suggests that groove binding is the dominant mechanism and that there is a
relatively high cooperativity between DPBP molecules upon binding, a feature
incompatible with simple intercalative binding. Thus, intercalation, if it takes place
here, is only a secondary binding mechanism. In fact, strong intercalators such as
ethidium bromide and anthracycline antibiotics typically increase DNA contour length
by 27–45%. In the present case, DPBP promoted an increase of only 16% in this
mechanical parameter, corroborating the conclusions drawn above.

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The binding parameters obtained from the model fitting were K = (2.7 ± 0.2) ×
4
−1
1
0 M and n = 5.1 ± 1.5. The value of the equilibrium constant (K) is compatible
with the results obtained for some antineoplastic drugs, such as cisplatin [27], under
similar experimental conditions (PBS, [Na] = 150 mM). Typical intercalating
antineoplastic drugs, such as doxorubicin, usually present high binding constants, in
5
−1
the order of 10 M [25].
The relatively high value found for the Hill exponent (n = 5.1) confirms the high
positive cooperativity of the present interaction, indicating that DPBP binding
increases the affinity of the DNA molecule for subsequent binding, which can result in
bound clusters along the double helix, as n > 1 [15]. Cisplatin and doxorubicin also
exhibit positive cooperativity upon binding to DNA under similar experimental
conditions, having Hill exponents of approximately 3.5 [25, 27]. This property is
important in chemotherapies because it indicates that DPBP has a narrow
concentration range between the initial effective dose and the saturation dose when
binding to the DNA molecule.
These results suggest that the molecular mechanism of action of DPBP within
cells involves interaction with DNA. Upon binding to the biopolymer, DPBP changes
the double-helix structure locally, which probably hinders fundamental processes such
as transcription and DNA replication.
3
. Conclusion
In the present study, we tested the antineoplastic efficiency of a novel
dibenzoylmethane derivative, DPBP, on B16F10 melanoma cells. The selectivity
results show that DPBP is a promising agent for the treatment of melanoma, as it
presents a considerably high SI and a considerably low IC . In addition, we proposed
5
0
that its molecular mechanism of action within cells involves binding to DNA. In fact, a
direct interaction between DNA and DPBP was verified using optical tweezers. Minor
groove binding was determined as the main binding mode, although the possibility of
a second binding mechanism (partial intercalation) could not be discarded. Important
binding parameters, such as the equilibrium binding constant and the cooperativity
degree, were determined, characterizing the physical chemistry of DPBP–DNA
interaction. Thus, we characterized the efficiency of DPBP at a cellular level as well as
at a molecular level.

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4
. Experimental section
1
13
H and C NMR (nuclear magnetic resonance) spectra were recorded on a Bruker
Avance spectrometer at 400 and 100 MHz, respectively. The chemical shifts () are
reported in ppm using TMS ( 0) and residual CHCl ( 77.00) signals as internal
3
1
13
references for H and C NMR spectra, respectively. Mass spectra (HRMS) were
recorded on a VG Auto Spec-10000 instrument using a 70 eV ionizing potential. IR
spectra were recorded on an FTIR spectrometer with a diamond ATR (attenuated total
reflectance) accessory as a thin film. Purification by preparative thin layer
chromatography (TLC) was performed by using 80:20 n-hexane/AcOEt as eluent.
TLC visualization was achieved by spraying with 5% ethanolic phosphomolybdic acid
and heating.
4
.1. Synthesis of 1,3-diphenyl-2-benzyl-1,3-propanedione (DPBP)
Anhydrous potassium carbonate (0.56 g, 4.0 mmol) and DBM (0.3 g, 1.34 mmol)
were suspended in acetone (20 mL) and stirred for 30 min at room temperature.
Benzyl bromide (0.16 mL, 1.34 mmol) was added to this mixture and stirred overnight
at the same temperature. The mixture was filtered, and the solvent was evaporated.
The crude product was purified by TLC by using 80:20 n-hexane/AcOEt as eluent to
afford DPBP as a colorless solid in 85% yield. m.p.: 58.3–58.7 °C; R = 0.74 (80:20 n-
f
−
1
1
hexane/AcOEt); IR (ν/cm ): 3031, 1694, 1664, 999; H NMR: 7.88 (dd, 4H, J = 8.0
and 1.3 Hz, H-2’ and H-6’), 7.53 (tt, 2H, J = 7.5 and 1.2 Hz, H-4’), 7.40 (tt, 4H, J =
7
7
.9 and 1.3 Hz, H-3’ and H-5’), 7.26–7.20 (m, 4H, H-2”, H-3”, H-5”, and H-6”), 7.18–
13
.15 (m, 1H, H-4”), 5.51 (t, 1H, J = 6.7 Hz, H-2), 3.45 (d, 2H, J = 6.7 Hz, CH Ph); C
2
NMR: 195.6 (2 C=O), 139.3, 136.3, 133.7, 129.2, 129.0, 128.8, 126.8, 59.3, 35.4
(
CH Ph); HRMS: calc. for C H O 314.1307, observed 314.1306.
2
22 18
2
4
.2. Cytotoxicity test
The melan-A and B16F10 cell lines have been established from normal murine
melanoblasts and murine melanoma cells, respectively. Melan-A cells were cultured in
Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal
bovine serum and 1% antibiotics (amphotericin B, penicillin, and streptomycin) in 25

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2
cm culture flasks, incubated at 37 °C, 5% CO , and 95% relative humidity. B16F10
2
cells were cultured in Dulbecco’s Modified Eagle’s medium supplemented with 10%
fetal bovine serum and 1% antibiotics (amphotericin B, penicillin, and streptomycin)
and maintained under the same conditions as described above.
4
.3. Cytotoxic activity measured by the MTT assay
4
5
B16F10 cells were plated at 1 × 10 cells/mL; and melan-A cells, at 1 × 10
cells/mL. Each well received 100 μL of culture medium. Plates were incubated for 24
h at 37 °C, 5% CO , and 95% humidity. Then, different concentrations of dacarbazine
2
(positive control, 1–12 µM) and DPBP (0.39–25 µM) were added to the cells, which
were further incubated for 60 h at 37 °C, 5% CO , and 95% humidity. After this
2
period, the MTT assay was performed. Absorbances were read at 570 nm, and the
5
0% inhibitory concentration (IC ) for each cell line was calculated.
50
4
.4. Selectivity index
The SI indicates the potential therapeutic use of compounds in clinical trials. In
this study, SI corresponds to the ratio of the IC for normal cells (melan-A) to the IC
50
5
0
for neoplastic cells (B16F10).
.5. Statistical analysis
The IC values for tumor cells were analyzed using the GraphPad Prism 5.0
4
®
5
0
software, according to the methods indicated in the manual. Statistical differences
between experimental groups were evaluated by one-way ANOVA (p < 0.05).
4
.6. Optical tweezers experiments
The DNA samples consisted of -DNA molecules (48,502 base-pairs, ~16.5 m
contour length) (New England Biolabs) end-labeled with biotin attached by one end to
a streptavidin-coated polystyrene bead of 3 m in diameter (Bangs Labs) and by the
other end to a previously prepared streptavidin-coated coverslip. All measurements
were performed in phosphate buffered saline (PBS) pH 7.4 and 140 mM sodium
chloride. The DPBP concentration in the sample was varied during the experiments by
changing the buffer solution.
The optical tweezers setup was composed of a 1064 nm ytterbium-doped fiber
laser (IPG Photonics) mounted on a Nikon Ti-S inverted microscope with a 100X

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N.A. 1.4 objective. The DNA molecules were stretched by moving the microscope
stage, and consequently the coverslip, under controlled velocity using a piezoelectric
device (PINano P-545, Physik Instrument).
We started the experiments with bare DNA molecules (0.0 M DPBP). First, we
randomly chose a bead and tested if there was a DNA attached to it by moving the
microscope stage. If the measured contour length agreed with the expected value for

-DNA (16–17 m), six more stretching measurements were taken, and the mean
values of persistence and contour lengths were obtained. These two mechanical
parameters were determined by fitting the experimental force–extension curves,
measured in the low-force entropic regime (F < 3 pN), to the Marko–Siggia worm-like
chain (WLC) model [28]. Next, we changed the surrounding buffer solution,
introducing DPBP at the chosen concentration. We waited for about 1 h before
repeating the stretching experiments in order to reach the chemical equilibrium of the
binding reaction. A new set of six measurements were then taken, and the mean values
and standard errors of the mechanical properties at each concentration were calculated.
The complete details of these procedures have been previously described [25].
4
.7. A model to determine the DPBP–DNA binding parameters
In 2012, a quenched-disorder statistical model was formulated to determine the
binding parameters of a ligand–DNA interaction from persistence length [29]. Here,
we will briefly expose this approach. The complete details can be found in another
study [15].
Essentially, it can be rigorously demonstrated [29] that the effective persistence
length (A ) of a ligand–DNA complex can be expressed as follows:
E
n
r (K C )
max
i
f
r 
(1)
n
1
 (K C )
i
f
where A is the persistence length of the bare DNA molecule, A is the local
0
1
persistence length induced by the ligand upon binding to a site, A is the local
2
persistence length when two bound sites become nearest neighbors, and x is the ratio
of the bound site fraction r to its saturation value rmax
.
The effective persistence length (A ) can be connected to the binding parameters
E
of the interaction by choosing an appropriate binding isotherm that captures the
physical chemistry of the system. In the case of the DPBP–DNA interaction, an

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appropriate binding isotherm is the Hill model, as this molecule exhibits positive
cooperativity upon binding to DNA. The Hill model is the simplest binding isotherm
capable of providing an estimation of the cooperativity degree of a binding reaction. It
reads
n
r (K C )
max
i
f
r 
(2)
n
1
 (K C )
i
f
where C is the free-ligand (not bound to DNA) concentration; K is the equilibrium
f
association binding constant; and n is the Hill exponent, a parameter generally used to
measure the degree of cooperativity of binding reactions.
Equation 2 can be plugged into Equation 1 to fit the experimental data of
persistence length. The binding parameters are left as adjustable parameters and can be
determined from the fitting process. Complete details of this fitting procedure can be
found in a previous work [15].
Acknowledgements
We would like to thank FAPEMIG (APQ-00622-14), CNPq (443566/2014-3), and
SUS-PPSUS (APQ-03497-13) for grants awarded to M.A.N.D.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/..............................................

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Graphical Abstract
O
O
1
,3-Diphenyl-2-benzyl-1,3-
propanedione (DPBP)
Figure. Plot of the percentage of cell death obtained for DPBP compound against melan-A and
B16F10 lineages in different concentrations. The selectivity indices (SI = IC50 melan-A/IC50 B16F10)
was 41.94.

ACCEPTED MANUSCRIPT
Highlights

1,3-Diphenyl-2-benzyl-1,3-propanedione (DPBP),
derivative, is a potential chemotherapeutic agent.
a
dibenzoylmethane


DPBP had a high selectivity for B16F10 melanoma cells over melan-A cells.
Its mechanism of interaction with DNA was elucidated using optical tweezers.

Graphics Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6