UAMC-3203 is a potent and selective Ferroptosis inhibitor (IC50: 12 nM).

Ferroptosis:12 nM ( in IMR32 Neuroblastoma cells)

In order to determine IC50-values, human neuroblastoma cells (IMR-32) were seeded in a 96-well plate at a density of 25,000 cells/well. The next day, the cells were pretreated for 1h (in triplicates) with a 1/3 dilution series of ferrostatin-1 analogues ranging from 5μM to 0.68nM and Sytox Green (1.6 μM) and a 1/2 dilution series of ferrostatin-1 analogues ranging from 200 nM to 0.78nM and Sytox Green (1.6 μM) for erastin and ferrous ammonium sulphate respectively. After stimulating the cells with erastin (10 μM) or ferrous ammonium sulphate (600 μM), the plate was transferred to a temperature- and CO2-controled FLUOstar Omega fluorescence plate reader. Sytox Green intensity was measured after 13h using an excitation filter of 485 nm and an emission filter of 520 nm. In each setup, Triton-X100 (0.05%) was used to induce lyses of the cells in 6 wells/plate, and was used as 100% cell death reference. The percentage of the cell death was calculated by the formula ((AVG[erastin] - AVG[background]) /(AVG[Triton-X100] - AVG[background])) × 100. Cell death percentage was plotted in GraphPad Prism 6, and IC50-values were calculated using a sigmoidal dose-response (variable slope) curve.

Vehicle solution (2% DMSO) or compound was administered daily at a concentration of 2 mM (in 0.9% NaCl containing 2% DMSO; 200 μL / 20 g body weight) by intraperitoneal injection. Body temperature and weight were monitored daily. On day 28 the mice were anesthetized with isoflurane and blood was sampled. Hereafter, mice were sacrificed by cervical dislocation. ALT, AST and LDH levels in plasma were measured. CK, creatinine, urea and troponin T were determined.

DMSO: 50 mg/mL (106.01 mM), Sonication is recommended.

10% DMSO+40% PEG300+5% Tween 80+45% Saline: 2 mg/mL (4.24 mM), Sonication is recommended.