Development of drug resistance during cancer chemotherapy is one of the major causes of chemotherapeutic failure for the majority of clinical agents. The aim of this study was to investigate the underlying molecular mechanism of resistance developed by the mitotic kinesin Eg5 against the potent second-generation ispinesib analogue SB743921 (1), a phase I/II clinical candidate. Biochemical and biophysical data demonstrate that point mutations in the inhibitor-binding pocket decrease the efficacy of 1 by several 1000-fold. Surprisingly, the structures of wild-type and mutant Eg5 in complex with 1 display no apparent structural changes in the binding configuration of the drug candidate. Furthermore, ITC and modeling approaches reveal that resistance to 1 is not through conventional steric effects at the binding site but through reduced flexibility and changes in energy fluctuation pathways through the protein that influence its function. This is a phenomenon we have called "resistance by allostery".

The complex between the motor protein Eg5 and the phase II clinical candidate ispinesib provides insights into the mechanism of action of this important class of inhibitors.

Human kinesin Eg5 is a target for drug development in cancer chemotherapy with compounds in phase II clinical trials. These agents bind to a well-characterized allosteric pocket involving the loop L5 region, a structural element in kinesin-5 family members thought to provide inhibitor specificity. Using X-ray crystallography, kinetic, and biophysical methods, we have identified and characterized a distinct allosteric pocket in Eg5 able to bind inhibitors with nanomolar K(d). This pocket is formed by key structural elements thought to be pivotal for force generation in kinesins and may represent a novel site for therapeutic intervention in this increasingly well-validated drug target.

The precise regulation of microtubule dynamics is essential during cell division. The kinesin-13 motor protein MCAK is a potent microtubule depolymerase. The divergent non-motor regions flanking the ATPase domain are critical in regulating its targeting and activity. However, the molecular basis for the function of the non-motor regions within the context of full-length MCAK is unknown. Here, we determine the structure of MCAK motor domain bound to its regulatory C-terminus. Our analysis reveals that the MCAK C-terminus binds to two motor domains in solution and is displaced allosterically upon microtubule binding, which allows its robust accumulation at microtubule ends. These results demonstrate that MCAK undergoes long-range conformational changes involving its C-terminus during the soluble to microtubule-bound transition and that the C-terminus-motor interaction represents a structural intermediate in the MCAK catalytic cycle. Together, our work reveals intrinsic molecular mechanisms underlying the regulation of kinesin-13 activity.DOI: http://dx.doi.org/10.7554/eLife.06421.001