A new class of cytotoxic agents targets tubulin and disrupts microtubule dynamics.

Despite the advances in treatment strategies, cancer is still the second leading cause of death in the USA. A majority of the currently used cancer drugs have limitations in their clinical use due to poor selectivity, toxic side effects and multiple drug resistance, warranting the development of new anticancer drugs of different mechanisms of action. Here we describe the design, synthesis and initial biological evaluation of a new class of antimitotic agents that modulate tubulin polymerization. Structurally, these compounds are chalcone mimics containing a 1-(1H-imidazol-2-yl)ethan-1-one moiety, which was initially introduced to act as a metal-binding group and inhibit histone deacetylase enzymes. Although several analogues selectively inhibited purified HDAC8 with IC50 values in low micromolar range, tissue culture studies suggest that HDAC inhibition is not a major mechanism responsible for cytotoxicity. The compounds demonstrated cell growth inhibition with GI50 values of upper nanomolar to low micromolar potency with significant selectively for cancer over normal cells. Interestingly, several compounds arrested HeLaM cells in mitosis and seem to target tubulin to cause mitotic arrest. For example, when combined with inhibitors of Aurora B kinase, they led to dramatic disassembly of the mitotic spindle. In-vitro tubulin polymerization studies showed that the compounds reduced the rate of polymerization of microtubules during the elongation phase and lowered the amount of polymerized tubulin during the plateau phase. Finally, in silico docking studies identified binding of IPE-7 to the colchicine site with similar affinity as the test compound D64131. These compounds represent a new antimitotic pharmacophore with limited HDAC inhibitory activity.

Small-Molecule Ferroptotic Agents with Potential to Selectively Target Cancer Stem Cells.

Effective management of advanced cancer requires systemic treatment including small molecules that target unique features of aggressive tumor cells. At the same time, tumors are heterogeneous and current evidence suggests that a subpopulation of tumor cells, called tumor initiating or cancer stem cells, are responsible for metastatic dissemination, tumor relapse and possibly drug resistance. Classical apoptotic drugs are less effective against this critical subpopulation. In the course of generating a library of open-chain epothilones, we discovered a new class of small molecule anticancer agents that has no effect on tubulin but instead kills selected cancer cell lines by harnessing reactive oxygen species to induce ferroptosis. Interestingly, we find that drug sensitivity is highest in tumor cells with a mesenchymal phenotype. Furthermore, these compounds showed enhanced toxicity towards mesenchymal breast cancer populations with cancer stem cell properties in vitro. In summary, we have identified a new class of small molecule ferroptotic agents that warrant further investigation.

Multiple Levels of Regulation of Sororin by Cdk1 and Aurora B.

The cohesin complex holds sister chromatids together until all chromosomes are properly attached to the mitotic spindle. Cleavage of the cohesin subunit Rad21 at the metaphase to anaphase transition allows separation of sister chromatids and is fundamental for the creation of identical daughter cells. Sororin blocks removal of cohesin from chromosomes from S phase until mitosis. In mitosis, Sororin is phosphorylated by Cdk1 releasing it from the cohesin complex. Aurora B phosphorylation of Sororin may play a similar role as Cdk1. Using PhosTag electrophoresis, we detect multiple Sororin species suggesting that phosphorylation of Sororin in mitosis is heterogeneous. Mutating the Cdk1 consensus site S21 to alanine eliminates many of the phosphorylated species suggesting that S21 is a key site of phosphorylation in vivo. Inhibiting Aurora B reduces phosphorylation of Sororin in cells, but only if Cdk1 sites are intact suggesting that some phosphorylation events on Sororin may be sequential. Surprisingly, mutating Aurora B consensus sites in Sororin causes it to relocalize to the nucleolus during interphase and blocks its interaction with chromosomes in Aurora B-inhibited cells. These observations indicate that phosphorylation plays unexpected roles in regulating the subcellular localization of Sororin.

Borealin dimerization mediates optimal CPC checkpoint function by enhancing localization to centromeres and kinetochores.

The chromosomal passenger complex (CPC) localizes to centromeres where it activates the mitotic checkpoint in response to inappropriate inter-kinetochore tension. This error correction function is essential for proper chromosome segregation. Here we define several critical features of CPC localization and function. First, the Borealin dimerization domain suppresses dynamic exchange at the centromere to allow optimal CPC function. Second, Borealin dimerization is essential to target a subpopulation of CPC proximal to the kinetochore when the mitotic spindle is disrupted. This subpopulation is also needed for full CPC checkpoint function. The existence of a pool of CPC at the kinetochore suggests that error correction is more complicated than predicted from the Aurora B phosphorylation gradient model. Finally, Haspin kinase plays a key role in maintaining the slowly exchanging centromere Borealin pool, while Aurora B and Mps1 play minimal roles in maintaining CPC localization once cells are in mitosis.