1. An Exploration of in vitro DNA Binding Propensity &
Cytotoxicity of Aromatic Organo Selenium Derivatives
Masood Ahmad Rizvi* and G.M. Peerzada
Department of Chemistry, University of Kashmir, Hazratbal, Srinagar, J&K, India.
INTRODUCTION
Recent investigations ascertain the dual function of selenium compounds as chemo preventives as well as selective anti-tumor therapeutics with the apoptosis and inhibition of cell proliferation as the broader mechanisms in the cancer chemoprevention. Selenium compounds in different chemical architectures target cancer in different ways and with different efficacies. Human exposure to selenium predominantly occurs through selenomethionine in food stuff, Screening organo selenium compounds can be interesting to address challenges of cancer chemotherapy and hence is aimed to potentiate chemotherapy [
Compound 8 Characterization
Methodology
Target synthesis of symmetric aromatic organodiselenides (1-13) using a simple synthetic methodology .
In vitro cytotoxicity against a panel of human cancer cell lines for identification of lead molecule.
The DNA binding ability of lead compound 8 was experimentally explored and theoretically investigated using molecular Docking method
DNA cleavage activity and HSA binding ability was also investigated
Organoselenium Compound Library C H EM I S T R y
Synthesis .
""
K.K Bhasin et al, J. Organomet. Chem , 658, 71
M.A. Rizvi et al, Bioorg. Med. Chem. Lett., 2014, 24, 3440
Mitochondrial membrane potential (mt)
The loss of (ψmt) was observed from 6 to 58% after the treatment (HL-60) with compound 8 in a dose dependant manner. The loss of (ψmt) was measured using Rhodamine123, fluorescent dye.
Lead Identification
The synthesized compounds displayed varied cytotoxicity towards human cancer cell line: human promyelocytic leukemia (HL-60). The lead compound 8 was further explored for its cytotoxicity potential against breast cancer-MCF-7, pancreatic cancer-MIA-PA-Ca-2 and colon cancer- HCT-116, cell lines. B I O A C T V Y .
DNA Cell Cycle Analysis
HL-60 cells when exposed to 8 at concentration of 10, 20 and 30 µM for 24 h exhibited an increase in apoptosis percentage in a dose dependent manner (2, 5 and 24% sequentially). The compound delayed the s-phase of cell cycle with concurrent induction of apoptosis in HL-60 cells.
Apoptotic morphological changes
In Silico DNA binding study
The compound 8 selectively binds to minor groove of DNA. One benzene ring of compound 8 is interacting with G-C rich region including G22B, G4A and C21B bases whereas other benzene ring is stacked with A-T rich region The fluorine atoms of 8 are involved in inter molecular hydrogen bonding interactions with G22B and G4A of opposite chain bases. The binding energy of Kcal mol-1 and hydrogen bonding interactions indicate that compound 8 efficiently binds to the DNA
The apoptotic morphological changes of HL-60 cells assessed by the fluorescent microcopy (Hoechst dye). The results reveal nuclear condensation, membrane blebbing, nuclear fragmentation and apoptotic bodies in cells that had been incubated with lead compound 8.
Absorption and Emission spectra Spectra
Competitive binding with Ethidium Bromide
DNA Clevage Activity D N A B I G S T U Y
The observed decrease in fluorescence intensity at (591nm) accompanied by red shift of 22 nm clearly indicates that the EB molecule was replaced from Its DNA binding sites Ksv= 1.2
Quenching Studies with HSA
The decrease in the fluorescence intensity of HSA was observed at 362 nm with a slight blue shift in the wavelength attributed to the reduction in the polarity of the microenvironment around the Tryptophan (Trp) due to the binding of 8 to the hydrophobic sites of HSA near the subdomain IIA .The calculated KSV and Kq value was found to be M−1 and M−1 s−1,
3D Fluorescence
3D fluorescence spectrum of compound 8 has two peaks, A (λex= 270 nm) and B (λem = 361 nm) . However, upon addition of DNA, the intensity of the peaks A and B decrease indicating either the partial intercalation/electron /energy transfer with double stranded DNA which is in good agreement with the results of absorption and emission measurements.
Summary
A target synthesis of symmetric aromatic diselenides for exploring anticancer lead compounds
Out of thirteen screened molecules (1–13) against a panel of human cancer cell lines, compound 8 exhibited highest cell growth inhibition in Human leukemia HL-60 cells with IC50 value of 8 µM .
Gel electrophoresis in presence of T4 DNA ligase
A good apoptotic potential of 8 is evidenced from several apoptotic protocols like DNA cell cycle analysis, apoptotic bodies formation using phase contrast and nuclear microscopy with Hoechst 33,258.
In vitro DNA binding profile of 8 by various biophysical techniques and molecular docking reveal its DNA binding through external contact and hydrogen bonding, selectively towards the minor groove of DNA
Compound 8 cleaves supercoiled plasmid pBR322 DNA via hydrolytic cleavage mechanism induced by the nucleophilic Se atom further supported by DNA relegation assay
Conclusion
Compound 8 act as a promising candidate to initiate nuclear blebbing in HeLa cancer cell line. Further studies aimed at the detailed molecular mode of action of 8 are underway, efforts are continued for further in vivo investigations.
Docked pose of Compound 8 with GPX
Further Reading
Rizvi, M. A. ; et al; Bioorg. Med. Chem. Lett., 2014, 24, 3440—3446.
Rizvi, M. A. ; et al; Eur. J. Med. Chem., 2015, 90, 876 —888.
Sinha, R.; El-Bayoumy, K. Curr. Cancer Drug Targets, 2004, 4, 13—56.
Naithani, R. Mini-Rev. Med. Chem. 2008, 8, 657—670