Hesperadin(IC50=250 nM) effectively inhibits Aurora B. It potently reduces the activity of AMPK, MAPKAP-K1, MKK1, Lck, CHK1 and PHK, but it could not inhibit MKK1 activity in vivo.

Aurora B:250 nM

在HeLa细胞中，Hesperadin通过抑制Aurora B的功能，干扰染色体连接，造成有丝分裂和细胞质分裂缺陷，导致细胞增殖停止和多倍体化。Hesperadin在20-100 nM浓度作用下，导致有丝分裂活化组蛋白H3在Ser10位点的磷酸化丢失。当Hesperadin浓度达到1 μM时，其他激酶（AMPK, Lck, MKK1, MAPKAP-K1, CHK1, 和 PHK）活性显著降低。在体外激酶测定中，Hesperadin（IC50 = 40 nM）阻断了来自病原性锥虫（Trypanosoma brucei）的TbAUK1（T. brucei Aurora kinase-1）对重组色质组蛋白H3的磷酸化。Hesperadin（IC50 = 48 nM）显著抑制了培养的传染性血液形式（BF）细胞的生长，而以IC50 = 550 nM的浓度弱抑制了昆虫循环阶段（PF）细胞的生长。

For the Aurora B kinase assay, HeLa cells are lysed in a buffer containing 50 mM NaCl, then centrifuging at 13,000 rpm for 20 minutes at 4 °C. Discard supernatant, add 15 mL lysis buffer containing 250 mM NaCl in order to obtain active Aurora B kinase. Centrifuging at low-speed supernatant of the latter extract is used for immunoprecipitation. Monoclonal mouse anti–AIM-1, or mouse anti-HA, is coupled to GammaBind Plus Sepharose, and beads are rotated over-end in the extract for 90 minutes at 4 °C. Beads are washed, aliquoted, and washed in kinase buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM DTT, 10 mM NaF). The kinase assay is performed with 10 μL beads in 20 μL kinase buffer containing 5 μg histone H3, 10 μM ATP, 2.5 μCi [γ-32P]ATP, and different concentrations of Hesperadin for 20 minutes at 37 °C.

HeLa cells and PtK1 cells are added Hesperadin 500 nM for 24 and 48 hours.