Antineoplastic drug development is based on the discovery of efficient molecules with lower cost and specific for the neoplastic cell target. In recent years, hybrid molecules' conceit has gained prominence, as they act synergistically, in reduced doses, and less likely to develop resistance, compared to the individual antineoplastic compounds. The screening of a new molecule can be performed by automated platforms, such as High Content Screening (HCS). This platform uses imaging parameters to determine cytotoxicity, which is then validated analytically and experimentally. The aim is to evaluate the antineoplastic potential of hybrid molecules using an HCS platform, and their ADME parameters in silico. According to our preliminary in silico results (CortellisTM), MDA-MB-231, T-47D and BT-474 breast cell lines will be used to screen for the 15 hybrid molecules. The hits will be obtained using a three-dimensional cell culture model (3D), monitored by fluorescence intensity (Calcein-AM), and subsequent statistical validation. Then, the cytotoxicity of the selected hits will be evaluated by real time (0-72 hours), using fluorescent assay RealTime-Glo" MT, and the evaluation of the parameters of death and cell cycle status will be measured by flow cytometry. Finally, the cells will be treated with the best hybrid molecules (IC50), to assess changes in proliferative signaling and death pathways, using protein arrays phopho-RTK, phopho-MAPK array and apoptosis signaling pathways arrays (R&D Systems).
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