By phosphorylating Thr3 of histone H3, Haspin promotes centromeric recruitment 
of the chromosome passenger complex (CPC) during mitosis. Aurora B kinase, a CPC 
subunit, sustains chromosome bi-orientation and the spindle assembly checkpoint 
(SAC). Here, we characterize the small molecule 5-iodotubercidin (5-ITu) as a 
potent Haspin inhibitor. In vitro, 5-ITu potently inhibited Haspin but not 
Aurora B. Consistently, 5-ITu counteracted the centromeric localization of the 
CPC without affecting the bulk of Aurora B activity in HeLa cells. 
Mislocalization of Aurora B correlated with dephosphorylation of CENP-A and Hec1 
and SAC override at high nocodazole concentrations. 5-ITu also impaired 
kinetochore recruitment of Bub1 and BubR1 kinases, and this effect was reversed 
by concomitant inhibition of phosphatase activity. Forcing localization of 
Aurora B to centromeres in 5-ITu also restored Bub1 and BubR1 localization but 
failed to rescue the SAC override. This result suggests that a target of 5-ITu, 
possibly Haspin itself, may further contribute to SAC signaling downstream of 
Aurora B.