Panc1 and Hella cell lines were obtained from the national
cell bank of Iran and cultured in 75 cm2 cell culture flasks. The
cells were scraped off and centrifuged for 5 minute at RCF1100 g. The harvested
cells were then suspended in fresh medium, counted by Trypan Blue exclusion
(99% viability) and the cell concentration was adjusted to105
cells/ml. 100µl aliquot of this suspension was added to each well of 96-well
cell culture plates (Corning, USA) with RPMI-1640 medium (Cambrex Bioscience)
supplemented with L-glutamine (2mM, Gibco), penicillin–streptomycin (100 IU/ml
penicillin, 100mg/ml streptomycin (Gibco)) and heat inactivated fetal bovine
serum (Biowest) 10% (v/v). The cells were maintained at 37°C in a humidified
incubator with 5% CO2 atmosphere 14.
24 and 48h after
irradiation, (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)MTT
solution (USB Corporation, Cleveland, USA) was added to each well and the
plates were incubated for 4 h at 37°C. The MTT solution was removed and DMSO
was added to dissolve formazan crystals. The absorbance at 540 nm was read on a
680 Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA). The percentage
of viability was calculated as AT/AC × 100; where AT and AC are the absorbance
of treated and control cells, respectively 14.
modeling and docking procedures were based on our previous article 12.The designed
compound 7 and hydroxyurea as reference drug were built and optimized with
standard bond length and angle using HyperChem. Among the all energy minima
conformers, the conformer with the lowest energy was considered in docking
calculations. Docking calculations were executed using AutoDock (version
4.2). Crystal structures of RNR were downloaded from the PDB bank server (PDB
15. A reconstruction of the whole side chain was attempted using Swiss PDB
viewer. The desired compound were docked into the active site as well as hydroxyurea,
which was acting as our reference drug for validation of our procedure. Grid
maps with 60* 60* 60 points were made and the grid point spacing was 0.375 ºA.
The Lamarckian genetic algorithm was used to perform the docking simulation.
After docking, all generated conformers were assigned to clusters based on a
tolerance of 1 ºA all-atom RMS deviation from the lowest energy structure.