Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrol

To study the molecular complementarity of the monastrol-Eg5 interaction, we used a combination of synthetic chemistry and targeted mutations in Eg5 to measure the contribution of specific contacts to inhibition of Eg5 in vitro and in cultured cells. Structure-activity data on chemical derivatives, sequence analysis of Eg5 homologs from different species, and the effect of mutations near the drug binding site were consistent with the crystal structure.The mechanism of monastrol revealed by our data rationalizes its specificity for Eg5 over other kinesins and highlights a potential mechanism of drug resistance for anti-cancer therapy targeting this site in Eg5.Kinesins are a diverse family of microtubule-based motor proteins important for intracellular transport and cell division in all eukaryotes [1,2]. Genetic and biochemical dissection of kinesin function implicates specific kinesins in the trafficking of organelles [3], signaling complexes [4], and vesicular cargo [5]. During mitosis and cytokinesis, kinesins are essential for microtubule dynamics regulation, assembly and maintenance of bipolar spindles, and accurate chromosome segregation [6]. Assessing the precise contributions of kinesins to highly dynamic processes during both interphase and mitosis is challenging. Genetic tools such as siRNA are general and specific, but lack temporal resolution and reversibility necessary for detailed analysis of dynamic processes. Reversible, small molecule inhibitors of both microtubule- and actin-based motors are proving to be invaluable tools with which to study their functions during cell division [7,8]. Monastrol, a specific inhibitor of the BimC class kinesin Eg5 (also called kinesin-5 or kinesin spindle protein, KSP) [7], has permitted more critical analyses of Eg5 function during spindle assembly [9,10] and as a reversible agent to synchronize cells in metaphase [8]. Furthermore, inhibitors of Eg5 and other mitotic kinesins are plausible anti-cancer drugs now under