It would be difficult to underestimate the importance of polo-like kinase 1 (Plk1) for cancer, given its influence on cell division. As one of five members of the Polo-like family of serine/threonine protein kinases in eukaryotic cells, Plk1 plays a multifaceted role in the cell cycle and thus controls cancer progression fueled by unchecked cell proliferation. It regulates mitotic entry and the G2/M checkpoint, coordinates the assembly of the centrosome and thus dictates timing of cell cycles, regulates the assembly of the mitotic spindle and chromosome segregation, plays key roles at the spindle midzone and during abscission, facilitates DNA replication, and is involved in cytokinesis and meiosis.¹
Cancer requires efficient cell division; thus it is not surprising that Plk1 is up-regulated in many tumor cell types.² Nor is it surprising that Plk1 is viewed as a promising drug target for novel chemotherapeutics.³
What is, perhaps, surprising is a fascinating drug discovery strategy by Chen et al., who engaged in small molecule inhibitor screening using a rather unusual criteria: identifying compounds that both inhibited the kinase activity of Plk1, and blocked nuclear localization during S and G2 phases of mitosis.4
In a very real sense, this might be described (as the saying goes) as a “kill two birds with one stone” approach.
Here’s how they did it: Plk1 protein has an N-terminal kinase domain, and a C-terminal Polo box domain. To be activated, Plk1 has to be phosphorylated by another kinase (such as Aurora A and its partner Bolo) at threonine-210 in its activation loop. This sets up a series of events in which Plk1 binds to different partners that are themselves phosphorylated by either CDK1 or Plk1; these interactions affect the localization patterns of Plk1 to the centrosome, nucleus, and cytoplasm during S and G2 phases of mitosis.4
Confused? Here’s the short version: to fuel mitosis (and therefore to fuel cancer), Plk1 has to retain both 1) kinase ability, and 2) its capacity to be in the right place at the right time during mitosis.
This begged the question: why not try to find a small molecule inhibitor that affects both of these capabilities?
This is precisely the approach of Chen et al., who first conducted in silico (computational) approaches to virtually screen a library of 200,000 compounds in order to identify those that were likely to form a hydrogen bond or electrostatic interaction with key nuclear localization signal (NLS) residues of Plk1, and to form a hydrogen bond with a critical residue for ATP binding, plus have high likelihood of good hydrophobic interaction with the binding sites.4
The result? 200,000 candidates were narrowed down to 95. These were further assessed by immunofluorescence and flow cytometry to determine ability to cause cell cycle arrest.4
By this means, 95 candidates were narrowed to one: inhibitor D110, which was more extensively analyzed for its effect on apoptosis and subcellular localization of Plk1.
It worked. ELISA data confirmed that D110 binds the ATP pocket and the nuclear localization site of Plk1.4 Exposing cultured tumor cells to D110 caused mitotic arrest.4 And treating HeLa cells with D110 induced formation of a monopolar spindle and affects nuclear localization of Plk1 during interphase, indicating that the compound inhibits the catalytic activity of Plk1 in vivo.4
Last but certainly not least, D110 could induce apoptosis of human tumor cells in vivo, suggesting its promise as a primary compound for anticancer drug development. It also showed excellent selectivity for Plk1 versus other Polo-like kinase families, suggesting that it could have lower risk of off-targeted effects4
Will D110 prove to be a compound that is suitable for therapeutic use? Time will tell. But in the meantime, this project is an outstanding example of a creative strategy to discover novel inhibitors for kinases, as well as the relevancy of small molecule kinase inhibitor discovery.
-Robyn M. Perrin, PhD