Mitotic regulators exhibiting gain of function in tumor cells are considered 
useful cancer therapeutic targets for the development of small-molecule 
inhibitors. The human Aurora kinases are a family of such targets. In this 
study, from a panel of 105 potential small-molecule inhibitors, two compounds 
Tripolin A and Tripolin B, inhibited Aurora A kinase activity in vitro. In human 
cells however, only Tripolin A acted as an Aurora A inhibitor. We combined in 
vitro, in vivo single cell and in silico studies to demonstrate the biological 
action of Tripolin A, a non-ATP competitive inhibitor. Tripolin A reduced the 
localization of pAurora A on spindle microtubules (MTs), affected centrosome 
integrity, spindle formation and length, as well as MT dynamics in interphase, 
consistent with Aurora A inhibition by RNAi or other specific inhibitors, such 
as MLN8054 or MLN8237. Interestingly, Tripolin A affected the gradient 
distribution towards the chromosomes, but not the MT binding of HURP (Hepatoma 
Up-Regulated Protein), a MT-associated protein (MAP) and substrate of the Aurora 
A kinase. Therefore Tripolin A reveals a new way of regulating mitotic MT 
stabilizers through Aurora A phosphorylation. Tripolin A is predicted to bind 
Aurora A similarly but not identical to MLN8054, therefore it could be used to 
dissect pathways orchestrated by Aurora kinases as well as a scaffold for 
further inhibitor development.