Toxico-pharmacological evaluations of the small-molecule LQFM166: Inducer of apoptosis and MDM2 antagonist

Abstract

Inhibition of p53-MDM2 complex has been emerging as a strategy for antitumoral drug development considering the pro-apoptotic role of functional p53 in tumor cells. In our study, the prototype LQFM166 (2), designed through molecular simplification strategy inspired in the Nutlins compounds, was synthetized, characterized and the mechanisms of cell death were investigated. In addition, we estimated the starting doses for acute oral systemic toxicity tests according to the OECD Guidance Document No.129 - 3T3 NRU. The cytotoxic profile of LQFM166 (2) was determined in K-562 cells, a p53-null cell line, since previous studies also showed activity of LQFM166 (2) on this cells. After 24, 48 or 72 h of compound treatment, using MTT reduction assay, the IC50 values found were 100.1 μM, 56.76 μM and 45.11 μM, respectively. LQFM166 (2) was cytotoxic for leukemia cells in a concentration-time-dependent manner. Cell death mechanisms studies of LQFM166 on K-562 cells, revealed that the compound induced cell cycle arrest, increased the expression of caspase 3/7, 8 and 9, cytochrome c, Bax, p21 and p27. Additionally, a decrease in the expression of the Bcl-2 and cyclin-B1 was observed. The apoptotic inducer profile of the compound was confirmed by phosphatidylserine externalization. Investigation of complexation of p53/MDM2 was carried out by ELISA assay using 3T3 cell, showing a decrease in the p53-MDM2 complex induced by the compound. Furthermore, the cytotoxicity in basal fibroblasts 3T3 was determined to estimate LD50. LQFM166 (2) reduced 3T3 cells viability with the IC50 of 185.3 μM and estimated LD50 of 706.7 mg/kg (category 4 of GHS). The rationally designed of the prototype LQFM166 (2) induced cell death by apoptotic mechanisms in leukemic cells and showed MDM2 complexation antagonism in 3T3 cells.