SummaryCentrosomes associate with spindle poles; thus, the presence of two centrosomes promotes bipolar spindle assembly in normal cells. Cancer cells often contain supernumerary centrosomes, and to avoid multipolar mitosis and cell death, these are clustered into two poles by the microtubule motor protein HSET. We report the discovery of an allosteric inhibitor of HSET, CW069, which we designed using a methodology on an interface of chemistry and biology. Using this approach, we explored millions of compounds in silico and utilized convergent syntheses. Only compound CW069 showed marked activity against HSET in vitro. The inhibitor induced multipolar mitoses only in cells containing supernumerary centrosomes. CW069 therefore constitutes a valuable tool for probing HSET function and, by reducing the growth of cells containing supernumerary centrosomes, paves the way for new cancer therapeutics.

Rhodium-catalyzed carbozincation of ynamides using diorganozinc reagents or functionalized organozinc halides is described. Using a tri(2-furyl)phosphine-modified rhodium catalyst, the reaction course is altered to hydrozincation when diethylzinc is employed as the organozinc reagent. Trapping of the alkenylzinc intermediates produced in these reactions in further functionalization reactions is possible. Collectively, these processes enable access to a range of multisubstituted enamides in stereo- and regiocontrolled fashion.

The combretastatins have been investigated for their antimitotic and antivascular properties, and it is widely postulated that a 3,4,5-trimethoxyaryl A-ring is essential to maintain potent activity. We have synthesized new tetrazole analogues (32-34), demonstrating that 3,5-dihalogenation can consistently increase potency by up to 5-fold when compared to the equivalent trimethoxy compound on human umbilical vein endothelial cells (HUVECs) and a range of cancer cells. Moreover, this increased potency offsets that lost by installing the tetrazole bridge into combretastatin A-4 (1), giving crystalline, soluble compounds that have low nanomolar activity, arrest cells in G2/M phase, and retain microtubule inhibitory activity. Molecular modeling has shown that optimized packing within the binding site resulting in increased Coulombic interaction may be responsible for this improved activity.

Almost a century ago Theodor Boveri suggested that tumor cells differ from normal cells in their high incidence of centrosome amplification. However, only recently have new therapeutic strategies been explored in an attempt to exploit these differences and the role of kinesins in mitosis. Intense interest in the field has led to development of KSP and CENP-E inhibitors that have been tested clinically as treatments for human cancer. Success has been limited because both motor proteins are essential to normal mitosis and inhibition leads to mitotic arrest and associated neutropenia toxicity in normal cells. In contrast HSET is essential for survival of cancer cells with centrosome amplification and has been shown to be dispensable in normal cells. Hence, HSET inhibition offers a unique opportunity to selectively damage malignant cells with supernumerary centrosomes without affecting normal cells. In keeping with these findings we report discovery of a novel allosteric inhibitor of HSET, CW069, that does not disrupt division in normal human fibroblast cells, or in MCF-7 cells with normal centrosome numbers. In fact, CW069 induces multipolar mitosis exclusively in cancer cells with extra centrosomes, causing apoptosis via catastrophic aneuploidy. The increased multipolar mitoses induced in N1E-115 cells by inhibitor CW069 recapitulates the phenotype described here, and by others, for siRNA depletion of HSET. The inhibitor also reduces cell growth and centrosome clustering in cancer cells with a lower incidence of centrosome amplification, including BT549 and MDA-MB-231 breast cancer cells. This is consistent with recent reports that depletion of HSET in DNA damage repair deficient cells may be lethal even to cancer cells with low-level centrosome amplification. Taken together, these data indicate that CW069 inhibition of HSET is not restricted to use in N1E-115 cells with high centrosome amplification, and could be broadly applicable to a range of human cancers. What is more, CW069 does not decrease the clonogenic capacity of primary adult human bone marrow cells, suggesting that it would not cause neutropenia toxicity in normal cells. It is anticipated that HSET inhibition could have a greater therapeutic margin than KSP or CENP-E inhibition, and, to the best of our knowledge we have described the first allosteric inhibitor of HSET that reduces centrosome clustering but does not induce the mitotic phenotypes associated with inhibition of KSP or CENP-E. This selectivity for HSET is consistent with our computational model, which indicates that the HSET loop 5 displays dynamic conformational selection for CW069 that cannot be achieved by closely related KSP. In summary, CW069 not only represents a substantial advance toward new cancer therapeutics, but also offers researchers a unique tool to unveil the full details of HSET function in mitosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B96. Citation Format: Ciorsdaidh A. Watts, Frances M. Richards, Andreas Bender, Peter J. Bond, Oliver Korb, Oliver Kern, Michelle Riddick, Paul Owen, Rebecca M. Myers, Jordan Raff, Fanni Gergely, Duncan I. Jodrell, Steven V. Ley. Design, synthesis and biological evaluation of a novel allosteric inhibitor of HSET that damages cancer cells with supernumerary centrosomes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B96.

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