Assessing compound binding to the Eg5 motor domain using a thermal shift assay

Abstract Eg5 is a kinesinwhose inhibition leads to cycle arrest during mitosis, making it a potential therapeutic target in cancers. Circular dichroism and isothermal titration calorimetryof our pyrrolotriazine-4-one series of inhibitors with Eg5 motor domain revealed enhanced binding in the presence of adenosine 5′-diphosphate (ADP). Using this information, we studied the interaction of this series with ADP–Eg5 complexes using a thermal shift assay. We measured up to a 7 °C increase in the thermal melting ( T m ) of Eg5 for an inhibitor that produced IC 50 values of 60 and 130 nM in microtubule-dependent adenosine triphosphatase(ATPase) and cell-based cytotoxicity assays, respectively. In general, the inhibitor potency of the pyrrolotriazine-4-one series in in vitro biological assayscorrelated with the magnitude of the thermal stability enhancement of ADP–Eg5. The thermal shift assayalso confirmed direct binding of Eg5 inhibitors identified in a high-throughput screen and demonstrated that the thermal shift assayis applicable to a range of chemotypesand can be useful in evaluating both potent (nM) and relatively weakly binding (μM) leads. Overall, the thermal shift assaywas found to be an excellent biophysical method for evaluating direct binding of a large number of compounds to Eg5, and it complemented the catalytic assayscreens by providing an alternative determination of inhibitor potency.