Discovery and Mechanistic Study of a Small Molecule Inhibitor for Motor Protein KIFC1 Jiaquan Wu, †,§,∥ Keith Mikule, ‡,∥ Wenxian Wang, ‡ Nancy Su, † Philip Petteruti, ‡ Farzin Gharahdaghi, ‡ Erin Code, † Xiahui Zhu, † Kelly Jacques, ‡ Zhongwu Lai, ‡ Bin Yang, ‡ Michelle L. Lamb, ‡ Claudio Chuaqui, ‡ Nicholas Keen, ‡ and Huawei Chen* ,‡ † Discovery Sciences and ‡ Oncology Innovative Medicine Unit, AstraZeneca R&D Boston, Waltham, Massachusetts 02451, United States * S Supporting Information ABSTRACT: Centrosome amplification is observed in many human cancers and has been proposed to be a driver of both genetic instability and tumorigenesis. Cancer cells have evolved mechanisms to bundle multiple centrosomes into two spindle poles to avoid multipolar mitosis that can lead to chromosomal segregation defects and eventually cell death. KIFC1, a kinesin-14 family protein, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division, suggesting that KIFC1 is an attractive therapeutic target for human cancers. To this end, we have identified the first reported small molecule inhibitor AZ82 for KIFC1. AZ82 bound specifically to the KIFC1/microtubule (MT) binary complex and inhibited the MT-stimulated KIFC1 enzymatic activity in an ATP-competitive and MT-noncompetitive manner with a K i of 0.043 μM. AZ82 effectively engaged with the minus end-directed KIFC1 motor inside cells to reverse the monopolar spindle phenotype induced by the inhibition of the plus end-directed kinesin Eg5. Treatment with AZ82 caused centrosome declustering in BT-549 breast cancer cells with amplified centrosomes. Consistent with genetic studies, our data confirmed that KIFC1 inhibition by a small molecule holds promise for targeting cancer cells with amplified centrosomes and provided evidence that functional suppression of KIFC1 by inhibiting its enzymatic activity could be an effective means for developing cancer therapeutics. C entrosomes are the main microtubule-organizing centers and play an important role in accurate chromosome segregation during mitosis. 1 Like chromosomes, centrosomes duplicate once per cell cycle, and normal diploid cells have two centrosomes that organize a bipolar mitotic spindle that functions to ensure equal chromosome segregation to daughter cells following mitosis. 2 In contrast, many cancer cells carry amplified (more than two) centrosomes, and accumulating evidence indicates that centrosome amplification has a causal role in tumorigenesis. 3−5 Centrosomes act dominantly to organize spindle poles, and therefore, unless bundled at the poles or inactivated, centrosome amplified cells would be expected to result in multipolar spindle formation. Multipolar metaphase arrangements can result in mitotic catastrophe, 5 multipolar cell divisions, or whole chromosome loss or gains caused by merotelic kinetochore attachments. Each of these outcomes is incompatible with cell viability. To overcome this paradox, cancer cells have evolved mechanisms to cluster amplified centrosomes into two groups for bipolar mitosis. 6−8 Thus inhibition of centrosome clustering may selectively drive cancer cells with amplified centrosomes to undergo multipolar mitosis and subsequent apoptosis without impairing normal cells. 9 Centrosome clustering mechanisms were investigated via an RNAi screen in multicentrosomal cells. Genes linked to centrosome clustering included chromosomal passenger complex (CPC) components, proteins involved in the organization and regulation of the cytoskeleton, and the minus end-directed motor protein KIFC1. 10,11 Among these genes, KIFC1 stands out as an attractive candidate for cancer therapeutic development because it is not required for diploid cell division and small molecule inhibitors for other motor domain proteins have been demonstrated. KIFC1 belongs to the kinesin-14 family of motor proteins. 12 Similar to other motor proteins, KIFC1 contains a MT binding domain, a stalk domain, and a motor domain, 13 but the arrangement of these domains is reversed compared to that of kinesin-5 proteins. The motor domain of kinesin-5 is located at the N-terminus, whereas KIFC1 and other kinesin-14 proteins have their motor domains at the C-terminus. Functionally, during mitosis KIFC1 slides and cross-links MTs from the Received: March 17, 2013 Accepted: July 29, 2013 Published: July 29, 2013 Articles pubs.acs.org/acschemicalbiology © 2013 American Chemical Society 2201 dx.doi.org/10.1021/cb400186w | ACS Chem. Biol. 2013, 8, 2201−2208