Plk1 overexpression is closely associated with tumorigenesis in a wide range of human cancers. It is also associated with aggressive disease stage and poor patient survival in various types of cancers5
, suggesting that Plk1 is an attractive target for therapeutic intervention against human cancers. Over the years, efforts have been made to generate anti-Plk1 inhibitors, resulting in several compounds (BI 2536, GSK Compound 1, Cyclapolin 1, DAP81, and TAL) developed to competitively inhibit the kinase activity of Plk15
. However, largely because of the structural similarities among the catalytic domains of all Plks and other related kinases, it has been difficult to generate Plk1-specific inhibitors. Since the non-catalytic PBD is found only in the members of the Plk subfamily, development of novel inhibitors that target the PBD of Plk1 may prove to be an alternative strategy for selectively targeting Plk1.
While conducting studies on the interaction between Plk1 and its physiological binding target PBIP1, we identified minimal phosphopeptides derived from the T78 region of PBIP1 that exhibit a high level of affinity and specificity for the Plk1 PBD. Similar to the defects associated with the expression of a dominant-negative PBD10
, inhibition of the Plk1 PBD function by non-hydrolyzable p-T78 mimetic peptides induced a chromosome congression defect that leads to mitotic arrest and apoptotic cell death. Since interference with Plk1 function induces apoptosis in most tumor cells but not in normal cells5
, these findings suggest that inhibition of the PBD function is sufficient to interfere with tumor cell proliferation. Furthermore, our results shown here provide the proof-of-principle that specific inhibition of Plk1 PBD is achievable by small mimetic peptides or their relevant compounds.
It has been demonstrated that SpT-dependent electrostatic interactions are critical for the binding of optimal peptides (PMQSpTPL and MQSpTPL) to the Plk1 PBD12
. Comparative in vitro
binding studies and analyses of the phosphopeptide-binding pockets of PBDS+G
, and PBDLH
revealed that, in addition to the SpT motif of the phosphopeptide that acts as a high affinity anchor, the N-terminal residues provide additional binding affinity and specificity to the Plk1 PBD through at least three distinct interactions. First, the polar contact between the carbonyl oxygen N-terminal to the Leu-3 of PLHSpT or LHSpTA and the guanidinium moiety of Arg516 of Plk1 PBD provides a molecular basis for a high affinity and specificity interaction. Unlike Plk1, both Plk2 and Plk3 possess a Lys residue (Lys607 and Lys568, respectively) at the position analogous to the Plk1 Arg516. Second, docking of the N-terminal Pro-4 side chain into the pocket generated by the surrounding Trp414 and Phe535 offers additional affinity and likely another layer of specificity to the interaction. The PBDs from both Plk2 and Plk3 possess Lys and Tyr residues at positions corresponding to the Plk1 Arg516 and Phe535 residues, respectively, and, as a consequence, may fail to generate as favorable an environment to accommodate the N-terminal Pro residue. Third, mutational analyses of PLHSpT suggest that the His-2 residue is also critical in determining Plk1 PBD specificity. Since the NΔ1
of His-2 was involved in a hydrogen bond with the carbonyl oxygen of Ser-1, one possibility is that the hydrogen bond between these two residues is important for conferring Plk1 specificity. Alternatively, the presence of a Gln residue at the − 2 position could be crucial for strong Plk2-mediated interactions.
Besides each amino acid residue of the p-T78 peptide involved in defining the Plk1 PBD binding, the positions of the glycerol in the neighboring pocket and the network of water molecules that mediate contacts between the phosphopeptide and the PBD suggest that both the glycerol and the network of water molecules surrounding the phosphopeptide could be important elements of the PBD recognition by phosphopeptides. Furthermore, the structures of the PBDS+G, PBDS, and PBDPL were remarkably similar, hinting that the glycerol molecule and the sulfate anion that occupy the phosphopeptide-binding cleft may mimic the role of the SpT dipeptide.
In conclusion, our results provided here demonstrate that the Plk1 PBD-binding pocket accommodates (i) the core SpT motif, (ii) the N-terminal hydrophobic residue, (iii) glycerol, and (iv) a network of contacting water molecules. A combination of some or all of these four elements could be potentially used for targeted drug design. Better understanding of the PBD interaction as well as further isolation and development of PBD-binding agents would greatly facilitate the discovery of a new class of Plk1-specific anti-cancer therapeutic agents.