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]

• Aurora Kinase B enzymatic assay

  Kinase inhibition assay is done by measuring kinase activity in a microfluidics assay. The separation of a phosphorylated product from substrate is monitored. The assay is run using a 12-sipper chip on a Caliper EZ Reader II. The recipe used for separation buffer is 100 mM HEPES, 10 mM EDTA, 0.015% Brij-35, 0.1% CR-3. The compound stocks (20 mM in DMSO) are diluted into kinase buffer. 1 μL of desired stock solution is transferred into a 384-well microtiter assay plate. The Aurora B enzyme is diluted in kinase buffer to a concentration of 2 nM. 5 μL of the enzyme mixture is transferred to the assay plate. The inhibitors/Aurora B enzyme are incubated for 60 min with minor shaking. A substrate mix is prepared containing ATP and 5FAM tagged peptide dissolved in kinase buffer described above. 5 μL of the substrate solution is added to the assay plate. Running concentrations are as follows: peptide (1.5 μM), ATP (190 μM), and compound 12-point ½log dilutions (0.2 mM-0.632 nM). No inhibitor is added for positive control, and no enzymewas added for negative control. For running control, barasertib is used. Percentage inhibition is measured by comparing starting peptide to phosphorylated productpeaks.

• Cell lines

  MCF-7 cells

• Concentrations

  --

• Incubation Time

  24 h

• Method

  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.