A.M. Dar et al.
Computational Toxicology 17 (2021) 100145
study. The crystal structure of the B-DNA dodecamer
d
at 4 ◦C and immunoblotted with antibodies anti-Bcl-XL, anti-Bax and
anti-PARP. Detection of signals was done using ECL Western blotting
reagent and chemiluminescence was exposed on Kodak X-Omat films.
Antibody anti-β-actin was used as loading control. All the antibodies
were procured from cell signalling technology, CA, USA.
(CGCAAATTTCGC)2 (PDB ID : 1BNA) was downloaded from the protein
data bank. All calculations were carried out on an Intel CORE i5, 3.1 GHz
based machine running MS Windows XP as the operating system. First of
all the water molecules were deleted. The DNA was enclosed in a box
with number of grid points in x × y × z directions, 76 × 78 × 120 and a
grid spacing of 0.375 Å. Visualization of the docked pose have been done
using Discovery Studio 3.5 molecular graphics program.
3.8. Computational details
All the DFT calculations were performed using the Gaussian 09 code
[44]. B3LYP functional was employed for the optimization of all the
structures [45]. The B3LYP functional is believed to yield the correct
structures [46]. The geometry optimization was carried out using a
631G** basis set for all the atoms. All structures studied in this work
were fully optimized in gas-phase without any restriction.
3.5. In vitro cytotoxicity
To study the cytotoxic effect of the compounds, in vitro MTT assay
was carried out [36]. Human cancer cell lines SW480 (colon adeno-
carcinoma cells)/ATCC (CCL-228), HeLa (cervical cancer cells)/ATCC
(CCL-2), MCF-7 (breast cancer cells)/ATCC (HTB-22), HepG2 (hepatic
carcinoma cells)/ATCC (CRL-8065) and HL-60 (Leukaemia cells)/ATCC
(CCL-240) were taken for the study. SW480, HL-60 and HepG2 cells
were grown in RPMI 1640 supplemented with 10% foetal bovine serum
4. Conclusion
In conclusion, we have synthesized and characterized pyrimidinone
derivatives. DFT studies revealed a similar reactivity pattern of these
compounds as deduced from the similar HUMO-LUMO energy gap of all
the synthesized pyrimidinones. Spectroscopic titration revealed that
compounds (9–12) are minor groove binders. The absorption and fluo-
rescence studies reveal the stabilization of the energy levels of the
compounds in the presence of DNA. In vitro cytotoxicity screening
revealed that pyrimidinones are potential cytotoxic agents against the
cancer cell lines. Thes relative expressions of relevant apoptotic markers
including the increasing Bax/Bcl-XL ratio, cleaved Caspase-3 and PARP
cleavage clearly indicated that these pyrimidinones have prompt ability
to cause apoptosis. In short, this study reveals that these semi-
synthesized pyrimidinones can be used as template for future drug
development through even different derivatization to prepare more
potent and selective-DNA binding agents.
(FBS), 10U penicillin and 100 μ
g/mL streptomycin at 37 ◦C with 5% CO2
in a humidified atmosphere. HeLa andMCF7 cells were grown in Dul-
becco’s modified Eagle’s medium (DMEM) supplanted with FCS and
antibiotics as described above for RPMI 1640. The cells were incubated
in an incubator at 37 ◦C and 5% CO2. After 24 h, the cells were serum
starved overnight. Compounds 9–12 were then added prepared in
DMSO in a concentration range of 3–100
of 200
μM, ensuring an equal volume
μ
L across the wells of the plate. The plate was further incubated at
37 ◦C and 5% CO2 for 48 h. The cytotoxicity of the compounds was
tested by the addition of the yellow tetrazolium salt MTT (3-(4,5-
dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide) prepared in
culture medium at a working concentration of 0.4 mg/mL across the
plate. The plate was further incubated for 2 h so that MTT is reduced by
the live cells, to produce a purple Formazan product. After this time, the
medium was aspirated and 200 μL of DMSO (Sigma Ltd.) was added to
each well. The experiment was performed in triplicates. The plate was
agitated gently for 5 min. before measuring the optical density at 570
nm in each well using Thermo Scientific multi-plate reader (MultiSkan
EX Elisa reader). Since the absorbance correlated with the number of
viable cells the percentage of viability was calculated from the absor-
bance. The IC50 values of the compounds were determined by plotting
the percentage viability versus concentration on a logarithmic graph and
the reading of the concentration at which 50% of cells are viable relative
to the control.
CRediT authorship contribution statement
Ayaz Mahmood Dar: Conceptualization, Supervision, writing.
Shafia Mir: Data Curation, Investigation. Masrat Jan: Methodology,
Resources, Visualization. Rizwan Nabi: Software, Visualization, Val-
idiation, Formal Analysis, Funding acquisition. Manzoor A Gatoo:
Project adminstration, review and editing.
Declaration of Competing Interest
3.6. Apoptosis studies with (AO)/ (EB) staining method
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Apoptotic studies were performed with a staining method utilising
Acridine Orange (AO) and Ethidium Bromide (EB) [43]. A monolayer of
HepG2 cells was incubated in the absence or presence of compounds 9
Acknowledgments
and 10 at concentration of 10 μ
M at 37 ◦C and 5% CO2 for 24 h. Each cell
culture was then stained with AO/EB solution (100
μ
gmLꢀ 1 AO,
The Author (AMD) also thanks University Grants Commission India
for JRF/SRF and for successful completion of work. The authors (MJ and
SM) thank the Department of Chemistry OPJS University for completion
of this work. The corresponding author also thanks MAG for successful
biological studies. We are greatly thankful to G. Rajaraman IIT Bombay
for providing computational facility.
100
μ
gmLꢀ 1 EB). The cell cultures were observed under a fluorescence
microscope.
3.7. Western blot analysis
MCF-7 cells were grown on 100 mm tissue culture discs at a density
of 1 × 106 cells per plate and incubated overnight. Then the cells were
Appendix A. Supplementary data
exposed to compound 9 (0, 10, 15 and 20 μM) for 48 h prior to harvest.
The cell lysate was prepared by using modified RIPA lysis buffer (50 mM
tris, 150 mM NaCl, 0.5 mM deoxycholate, 1% NP-40, 0.1% SDS, 1 mM
Na3VO4, 5 mM EDTA, 1 mM PMSF, 2 mM DTT, 10 mM β-glycer-
ophosphate, 50 mM NAF, 0.5% triton X-100, protease inhibitor cock-
tail). Protein estimation was done by Bradford’s method. Proteins were
separated in 10% SDSPAGE and transferred to Peripheral vascular dis-
ease membrane. Membranes were blocked overnight in 10% skimmed
milk in 1 × TBS-T (Tris-buffered saline containing 0.05% of Tween-20)
Supplementary data to this article can be found online at https://doi.
References
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