Estudo do potencial efeito inibitório do composto sintético CP001 sobre a quinesina Eg5: análises in vitro com validação de cálculos em bioquímica quântica

Abstract

Pterocarpans represented the largest isoflavonoid class after isoflavones and recent studies have revealed that representatives of this group can act on specific targets of mitosis. This study evaluated the potential cytotoxic , genotoxic and mutagenic of the CP001 in tumoral and normal human cell lines. The inhibitory effect in Eg5 kinesin and quantum biochemistry calculation has been peformed as well. Cytotoxicity tests showed that CP001 had significant effect on the tested tumoral lines (HL-60, HCT-116, OVCAR-8, SF-295) with IC50 ranging between 0.2 and 3.61 μM. Effect confirmed by kinetic growth curve in real time, where the CP001 inhibit growth of OVCAR-8 cell line at a concentration of 4 uM , similar to paclitaxel (0.5 mM). Thus, in order to determine the mechanism of action involved a sequence of in vitro experiments were performed. The assessment of nuclear DNA content was measured in OVCAR-8 cells to examine the effect of pterocarpan on the cell cycle phases , showing that CP001 is capable to arrest cell cycle at the G2/M phase, concentration of 5 uM , and potentiating effect when added in combination with paclitaxel. The cell cycle arrest in G2/M phase may be related to action of the compound on tubulin. The tubulin assay polymerization was conducted and revealed that CP001 has polymerization rate (Vmax = 80.95 mOD / min ) below paclitaxel (Vmax = 100 mOD / min) and close to monastrol ( Vmax = 88.46 mOD / min). These data suggest that interference with tubulin polymerization is not as significant as that shown in paclitaxel. Specific action mitosis proteins was another possibility tested. In confocal microscopy images it was possible to see multipolar arrangement for the segregation of chromosomes. Peforming western bloting it was observed Eg5 inhibition and cleaved PARP expression, suggesting cell death activation. These data was supported by quantum biochemistry calculation through docking and MFCC methodology which confirmed a very low bind energy between CP001-Eg5, -219.09 kcal/mol, increasing the odds of CP001 binding in the same site as monastrol. It was conducted study of chromosomal aberrations and it has not being observed clastogenic profile but aneugenic. The comet assay in mononuclear cells from human peripheral blood showed that the CP001 does not cause damage to the DNA. The CP001 has the potential cytotoxic activity against several tumor lines, arresting cell cycle in cell G2 / M phase and trigging cell death signals, the kinesin Eg5 being the target potential. These biological tests wereconfirmed by docking and MFCC tests. Genotoxic effects were insignificant, indicating that the drug may have minimal cytotoxic effects in non-transformed cells.