SP-96

SP-96 shows sub-nanomolar potency in Aurora B enzymatic assays (IC50 = 0.316 ± 0.031 nM). SP-96 shows >2000 fold selectivity against FLT3 and KIT which is important for normal hematopoiesis. Enzyme kinetics of SP-96 shows non-ATP competitive inhibition which makes it a first-in-class inhibitor. SP-96 shows selective growth inhibition in NCI60 screening, including inhibition of MDA-MD-468, a Triple Negative Breast Cancer cell line.^[1]

The growth inhibition of 60 cell lines is obtained by submitting the compound to National Cancer Institute’s NCI60 panel. MCF-7 cells are cultured in RPMI-1640 medium with 5% FBS in an incubator maintained at 37 ℃ and 5% CO2. The media is changed on alternate days. After reaching confluency, the media is aspirated, cells are washed with PBS, detached using 0.25% trypsin and centrifuged. The collected cells are seeded in 96-well microtiter plates at a density of 5000 cells/well. The cells are allowed to adhere to plate overnight. The test compounds, vehicle control and positive controls are added 24 h after the cells are plated and allowed to incubate. Following 24 h of incubation, the media is aspirated, and the cells are washed with PBS. 40 mL of the media and 10 mL of 5 mg/mL of MTT solution prepared in PBS is added to the cells and incubated for 4 h at 37 ℃ and 5% CO2. After incubation, 150 mL of DMSO is added to each well and the cell viability is measured at 570 nM by an ELISA plate reader.