In this communication, we have identified a lead compound that specifically disrupts the Hec1/Nek2 interaction by directly binding to Hec1. INH1 treatment resulted in defective Hec1 localization on kinetochores and significant Nek2 reduction, correlating with mitotic abnormalities and cell death. The INH1-targeting effects are in part reminiscent of Hec1 or Nek2 depletion mediated by RNAi, suggesting that INH1 treatment impairs the Hec1/Nek2 function in cells. Although the off-target effects of INH1 cannot be excluded, the resulted mitotic phenotypes strongly support that the Hec1/Nek2 pathway is a major cellular target of INH1.
Structural similarities of INH1 and INH2 underlie a common mechanism of action, wherein the INH compounds target a site within the Hec1 C-terminal region critical for the Hec1/Nek2 interaction. The crystal structure of the Hec1 C-terminal region, however, remains to be resolved, precluding lead optimization via structure-based approaches. Instead, we have adopted a ligand-based strategy to test the structure-activity-relationship. Based on the activity of over 20 INH analogues synthesized to date (data not shown), it appears that both the amide and two distant phenyl rings are essential, since modifying the amide or one of the phenyl rings significantly affects the activity. For instance, the amide hydrogen can be replaced by methyl or bulky acyl groups without significant loss of activity (data not shown), suggesting that this domain of the molecule may be accessible to the solvent, and thus can be used for installing a “linker”, as was done for the INH1 affinity matrix. To this end we have not been able to identify an INH1 derivative serviceable as a true “negative” control. Notably, for human cells, the GI50s of INH1 are at a similar micromolar level to other known protein-protein interaction inhibitors such as those for androgen receptor, IL-2 and BCL-2 (43
How is the Hec1/Nek2 pathway impaired? There are several possibilities. First, INH1 binding may trigger Hec1 conformational changes leading to impaired networking with other mitotic regulators. The region of Hec1 responsible for Nek2 interaction also binds to several important mitotic regulators including Nuf2, Zwint-1 and Hice1 (21
). Our preliminary study suggests that the INH1 binding with Hec1 enhances, unlike the case of Nek2, the Hec1/Nuf2 interaction (data not shown). On the other hand, Hec1 is a substrate of Nek2. When the Hec1/Nek2 interaction is abolished, Hec1 may not be properly phosphorylated and hence become defective. Compound targeting effects eventually manifest themselves through defective Hec1 targeting/recruitment on kinetochores, its major functional sites.
Remarkably, INH1 treatment triggered dramatic reduction of Nek2 in cells, but not endogenous Hec1. This seemingly paradoxical observation can be in part explained by indirect drug effects of unknown nature, or that INH1-bound Hec1 may gain a dominant-negative function that somehow triggers Nek2 degradation. Clearly, loss of Nek2 was another important contributing factor to the cell killing effects of INH1. Nek2A, one of the major Nek2 splice variants, was reported to be degraded in an APC/C dependent manner starting at prometaphase (47
). However, whether INH1 treatment may facilitate Nek2 degradation through the APC/C pathway remains to be addressed. Although the precise molecular mechanism remains to be explored, our results support that INH1 treatment may alter the overall Hec1/Nek2 network in ways that promote mitotic errors, ultimately leading to mitotic catastrophe.
INH1 treatment significantly inhibited tumor growth in a xenograft mouse model while no apparent general toxicity was observed. This may be attributed to the expeditious growth of tumor cells compared with normal cells spared in part due to the quiescence status or slower division. In the mean time, extensive toxicology tests remain to be performed to fully address the potential organismal toxicity. Nonetheless, our results clearly indicate that the INH1-mediated Hec1 impairment, but not complete shutdown, may effectively render cancer cells sensitive to mitotic or genotoxic stress, while sparing the surrounding normal cells, consistent with tumor therapy studies involving Hec1 RNAi (16
). In essence, our study provides a proof-of-principle that inhibiting the Hec1/Nek2 pathway by small molecules may serve as an effective approach for cancer intervention.