TCS7010 (Aurora A Inhibitor I) is a novel, potent, and selective inhibitor of Aurora A with IC50 of 3.4 nM in a cell-free assay. It is 1000-fold more selective for Aurora A than Aurora B.

Aurora A:3.4 nM

TCS7010是一种2,4-二苯胺基嘧啶，具有选择性和高效性地抑制Aurora A的活性。该抑制剂能有效抑制HCT116和HT29细胞的增殖，IC50分别为190 nM和2.9 μM。TCS7010对Aurora A而非Aurora B的选择性，依赖于Aurora A的一个氨基酸（Thr217）。[1] 在KCL-22细胞中，TCS7010（1-5 μM）能增加G2/M期细胞比例，诱导组蛋白H3丝氨酸10位点的磷酸化，抑制有丝分裂期Aurora A在Thr288位点的自磷酸化。在0.5-5 μM浓度下，也能抑制KCL-22细胞及BCR-ABL阴性白血病细胞系KG-1和HL-60的细胞增殖。5 μM浓度下，有效诱导KCL-22细胞凋亡。[2] 近期研究发现，TCS7010还能抑制HCT116、HT29和HeLa细胞的生长，IC50分别为377.6 nM、5.6 μM和416 nM。[3]

Auroras A and B Inhibition Assays: Both Auroras A and B are assayed in ELISA format using a GST fusion (pGEX-4T) of the N-terminus of Histone H3 (aa 1?18) as substrate. Plates are coated with 2 μg/mL substrate in PBS then blocked with 1 mg/mL I-block in PBS. Kinase reactions are run for 40 min with 5 ng/mL (0.16 nM) Aurora A or 45 ng/mL (1.1 nM) Aurora B at 30 μM ATP (~ Km) in kinase buffer. Final DMSO concentration is 4%. Product is detected by incubation with antiphosphohistone H3 (Ser10) 6 g3 mouse monoclonal antibody and sheep-anti-mouse HRP conjugate, followed by washing and addition of TMB substrate. After quenching with 1 M phosphoric acid, plates are read at 450 nM.

Cells are seeded in 384-well plates on day 0 in 50 μL of complete medium and incubated overnight in a 5% CO2 atmosphere at 37 °C. On day 1, 10 μL of Aurora A Inhibitor I is added. On day 4, plates are allowed to reach room temperature, and 30 μL Cell Titer-Glo reagent is added to each well to measure total ATP levels. Plates are read after shaking 15 min at room temperature.(Only for Reference)