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Aurora A is a serine/threonine kinase with a pivotal role in the cell cycle. By a timely restricted binding to centrosomes it recruits and regulates proteins at the centrosomes, ensures spindle bipolarity and eventually accurate chromosome segregation. Therefore, Aurora A is considered a valuable target for cancer therapy. In the past there has been notable interest in finding inhibitors for Aurora A by screening large compound libraries with biochemical assays. In order to elaborate a structure activity relation (SAR) it is still an open question whether the choice of the construct, modifications of the target protein or the presence of accessory proteins have a significant influence on the outcome of the screening results. Furthermore, it is under debate whether the use of a kinase fragment, e.g. the kinase domain, is a valid surrogate for the full length protein. In order to address these issues we heterologously expressed and purified four variants of Aurora A: 1. Aurora A full length, 2. Aurora A full length pre-activated with ATP after purification, 3. Aurora A full length co-expressed with TPX2 (a microtubule-targeting protein), and 4. Aurora A-ΔN106 (N-terminally truncated and reduced to the kinase domain). All four variants show different levels of activity. Further enzymological studies reveal that also the Km values for ATP differ clearly between the four versions of Aurora A. Since the Km value for ATP approximates the affinity between an enzyme and ATP these varying Km values suggest that the ATP binding site is different between the Aurora A variants. In order to investigate if not only the affinity for ATP but also the affinity for ATP competitive compounds depends on the choice of the variant of Aurora A, IC50s of a variety of inhibitors for Aurora A were determined and compared.

The results clearly show that the choice of construct has a significant impact on the SAR and needs to be carefully considered before starting a screening campaign.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA