Plk1 Polo-box Domain Research Articles

Computational assessment of potential nonpeptidic inhibitors for PLK1 polo-box domain

Computational assessment of potential nonpeptidic inhibitors for PLK1 polo-box domain

METTL16-mediated N6-methyladenosine modification of Soga1 enables proper chromosome segregation and chromosomal stability in colorectal cancer.

N6-methyladenosine (m6A) is the most prevalent internal modification in mammalian messenger RNAs and is associated with numerous biological processes. However, its role in chromosomal instability remains to be established. Here, we report that an RNA m6A methyltransferase, METTL16, plays an indispensable role in the progression of chromosome segregation and is required to preserve chromosome stability in colorectal cancer (CRC) cells. Depletion or inhibition of the methyltransferase activity of METTL16 results in abnormal kinetochore-microtubule attachment during mitosis, leading to delayed mitosis, lagging chromosomes, chromosome mis-segregation and chromosomal instability. Mechanistically, METTL16 exerts its oncogenic effects by enhancing the expression of suppressor of glucose by autophagy 1 (Soga1) in an m6A-dependent manner. CDK1 phosphorylates Soga1, thereby triggering its direct interaction with the polo box domain of PLK1. This interaction facilitates PLK1 activation and promotes mitotic progression. Therefore, targeting the METTL16-Soga1 pathway may provide a potential treatment strategy against CRC because of its essential role in maintaining chromosomal stability.

Synthesis and evaluation of small molecule-based derivatives as inhibitors of polo-box domain of polo-like kinase-1

ObjectivesPolo-like kinase 1 (Plk1) is an important mitotic protein. In particular, this protein is highly overexpressed in many types of tumors and has been identified as a potential biomarker for the treatment and diagnosis of tumors. Plk1 is composed of two domains, an N-terminal kinase domain and a C-terminal polo-box domain (PBD). Presently, inhibitors with improved selectivity and specificity for Plk1 are unavailable. Therefore, we aimed to develop an inhibitor targeting the C-terminal PBD present only in Plk1.Methods & resultsIn this study, three derivatives targeting PBD for Plk1 were designed by protein–protein interactions, which showed high levels of selectivity and specificity for Plk1 PBD, and were evaluated to inhibit tumor cell proliferation through an apoptotic process during tumor cell division. The investigation of the in vitro and in vivo antitumor effects of these inhibitors demonstrated that one of the new small molecules, 1, is a promising anticancer agent.ConclusionOur findings can provide new insights for the design of novel Plk1 peptide inhibitors in the future.

Specific inhibition of an anticancer target, polo-like kinase 1, by allosterically dismantling its mechanism of substrate recognition

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.

Hornerin mediates phosphorylation of the polo-box domain in Plk1 by Chk1 to induce death in mitosis

The centrosome assembles a bipolar spindle for faithful chromosome segregation during mitosis. To prevent the inheritance of DNA damage, the DNA damage response (DDR) triggers programmed spindle multipolarity and concomitant death in mitosis through a poorly understood mechanism. We identified hornerin, which forms a complex with checkpoint kinase 1 (Chk1) and polo-like kinase 1 (Plk1) to mediate phosphorylation at the polo-box domain (PBD) of Plk1, as the link between the DDR and death in mitosis. We demonstrate that hornerin mediates DDR-induced precocious centriole disengagement through a dichotomous mechanism that includes sequestration of Sgo1 and Plk1 in the cytoplasm through phosphorylation of the PBD in Plk1 by Chk1. Phosphorylation of the PBD in Plk1 abolishes the interaction with Sgo1 and phosphorylation-dependent Sgo1 translocation to the centrosome, leading to precocious centriole disengagement and spindle multipolarity. Mechanistically, hornerin traps phosphorylated Plk1 in the cytoplasm. Furthermore, PBD phosphorylation inactivates Plk1 and disrupts Cep192::Aurora A::Plk1 complex translocation to the centrosome and concurrent centrosome maturation. Remarkably, hornerin depletion leads to chemoresistance against DNA damaging agents by attenuating DDR-induced death in mitosis. These results reveal how the DDR eradicates mitotic cells harboring DNA damage to ensure genome integrity during cell division.

Crystal Structures of Plk1 Polo-Box Domain Bound to the Human Papillomavirus Minor Capsid Protein L2-Derived Peptide.

Human papillomaviruses (HPVs) can increase the proliferation of infected cells during HPV-driven abnormalities, such as cervical cancer or benign warts. To date, more than 200 HPV genotypes have been identified, most of which are classified into three major genera: Alphapapillomavirus, Betapapillomavirus, and Gammapapillomavirus. HPV genomes commonly encode two structural (L1 and L2) and seven functional (E1, E2, E4-E7, and E8) proteins. L2, the minor structural protein of HPVs, not only serves as a viral capsid component but also interacts with various human proteins during viral infection. A recent report revealed that L2 of HPV16 recruits polo-like kinase 1 (Plk1), a master regulator of eukaryotic mitosis and cell cycle progression, for the delivery of viral DNA to mitotic chromatin during HPV16 infection. In this study, we verified the direct and potent interactions between the polo-box domain (PBD) of Plk1 and PBD-binding motif (S-S-pT-P)-containing phosphopeptides derived from L2 of HPV16/HPV18 (high-risk alphapapillomaviruses), HPV5b (low-risk betapapillomavirus), and HPV4 (low-risk gammapapillomavirus). Subsequent structural determination of the Plk1 PBD bound to the HPV18 or HPV4 L2-derived phosphopeptide demonstrated that they interact with each other in a canonical manner, in which electrostatic interactions and hydrogen bonds play key roles in sustaining the complex. Therefore, our structural and biochemical data imply that Plk1 is a broad binding target of L2 of various HPV genotypes belonging to the Alpha-, Beta-, and Gammapapillomavirus genera.

Structural Optimization and Anticancer Activity of Polo-like Kinase 1 (Plk1) Polo-Box Domain (PBD) Inhibitors and Their Prodrugs.

Polo-like kinase 1 (Plk1), a mitotic kinase whose activity is widely upregulated in various human cancers, is considered an attractive target for anticancer drug discovery. Aside from the kinase domain, the C-terminal noncatalytic polo-box domain (PBD), which mediates the interaction with the enzyme's binding targets or substrates, has emerged as an alternative target for developing a new class of inhibitors. Various reported small molecule PBD inhibitors exhibit poor cellular efficacy and/or selectivity. Here, we report structure-activity relationship (SAR) studies on triazoloquinazolinone-derived inhibitors, such as 43 (a 1-thioxo-2,4-dihydrothieno[2,3-e][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one) that effectively block Plk1, but not Plk2 and Plk3 PBDs, with improved affinity and drug-like properties. The range of prodrug moieties needed for thiol group masking of the active drugs has been expanded to increase cell permeability and mechanism-based cancer cell (L363 and HeLa) death. For example, a 5-thio-1-methyl-4-nitroimidazolyl prodrug 80, derived from 43, showed an improved cellular potency (GI50 4.1 μM). As expected, 80 effectively blocked Plk1 from localizing to centrosomes and kinetochores and consequently induced potent mitotic block and apoptotic cell death. Another prodrug 78 containing 9-fluorophenyl in place of the thiophene-containing heterocycle in 80 also induced a comparable degree of anti-Plk1 PBD effect. However, orally administered 78 was rapidly converted in the bloodstream to parent drug 15, which was shown be relatively stable toward in vivo oxidation due to its 9-fluorophenyl group in comparison to unsubstituted phenyl. Further derivatization of these inhibitors, particularly to improve the systemic prodrug stability, could lead to a new class of therapeutics against Plk1-addicted cancers.

Abstract 2572: A novel strategy for PLK1 kinase inhibition by allosterically targeting the Polo Box Domain

Abstract Polo Like Kinase 1 (PLK1) is only expressed in dividing cells and plays a critical role in several stages of mitosis. PLK1 is overexpressed in many tumor types. PLK1 inhibition selectively kills cancer cells because they are dependent on the mitotic functions of PLK1. PLK1 consists of a highly conserved N-terminal catalytic kinase domain and a unique, functionally essential C-terminal Polo Box Domain (PBD). The PBD is a phospho-peptide binding motif that determines substrate recognition and sub-cellular localization. PLK1 catalytic inhibitors have advanced to clinical trials but not demonstrated convincing efficacy. Targeting the PBD offers an attractive alternative to pursue PLK1 inhibition. An iterative strategy called REPLACE, involving computational and synthetic approaches, was utilized to generate fragment-ligated inhibitory peptides and further application of REPLACE resulted in non-peptidic compounds named abbapolins. In recently published studies, abbapolins were found to specifically bind to the PBD of PLK1 in biochemical and cellular assays. The phosphorylation of TCTP, a specific PLK1 substrate, was measured in abbapolin treated cancer cells. Abbapolins produced a dose dependent reduction in p-TCTP. We also made a novel observation that abbapolins upon binding to PLK1 induced its intracellular loss in a mechanism at least partially dependent on the proteasome. Spurred on by this unique mechanism of action, we initiated studies on the interactions of catalytic inhibitors and PBD-binding abbapolins with PLK1 in vitro and in cellular contexts. Biochemical assays were performed with full length PLK1 in vitro and intracellular PLK1 binding was measured by a Cellular Thermal Shift Assay (CETSA). We report novel findings during mitosis inferred from our collective data, namely that catalytic binding by BI2536 or volasertib unexpectedly decreased soluble PLK1 as determined by CETSA, inferring induction of a conformational change in intracellular PLK1. In contrast, abbapolins produced the expected right shift in the melting curve of PLK1. Intriguingly, these differential effects on PLK1 thermal stability have opposing impacts on the fate of intracellular PLK1. Binding by catalytic inhibitors cause accumulation of PLK1, whereas PBD binding by abbapolins ultimately lead to its loss. Results from quantifying intracellular PLK1 were also supported in vitro by evidence of cooperative binding between catalytic inhibitors and abbapolins in the context of the FL protein and suggest that conformational changes induced by binding to the catalytic site increase affinity of abbapolins for the PDB. Collectively, the results shed further insight into the unique mechanism of action for abbapolins potentially due to their engagement of a cryptic hydrophobic pocket of the PBD. Abbapolins are thus a compelling alternative to catalytic-based inhibitors for the development of novel therapeutics targeting PLK1. Citation Format: Danda Chapagai, Merissa Baxter, Sandra Craig, Guru Ramamoorthy, Jessy Stafford, Sikirzhytski Vitali, Elmar Nurmemmedov, Campbell McInnes, Michael D. Wyatt. A novel strategy for PLK1 kinase inhibition by allosterically targeting the Polo Box Domain [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2572.

Novel Macrocyclic Peptidomimetics Targeting the Polo-Box Domain of Polo-Like Kinase 1.

The polo-box domain (PBD) of Plk1 is a promising target for cancer therapeutics. We designed and synthesized novel phosphorylated macrocyclic peptidomimetics targeting PBD based on acyclic phosphopeptide PMQSpTPL. The inhibitory activities of 16e on Plk1-PBD is >30-fold higher than those of PMQSpTPL. Both 16a and 16e possess excellent selectivity for Plk1-PBD over Plk2/3-PBD. Analysis of the cocrystal structure of Plk1-PBD in complex with 16a reveals that the 3-(trifluoromethyl)benzoyl group in 16a interacts with Arg516 through a π-stacking interaction. This π-stacking interaction, which has not been reported previously, provides insight into the design of novel and potent Plk1-PBD inhibitors. Furthermore, 16h, a PEGlyated macrocyclic phosphopeptide derivative, induces Plk1 delocalization and mitotic failure in HeLa cells. Also, the number of phospho-H3-positive cells in a zebrafish embryo increases in proportion to the amount of 16a. Collectively, the novel macrocyclic peptidomimetics should serve as valuable templates for the design of potent and novel Plk1-PBD inhibitors.

Abstract 1241: Converting high affinity peptides to non-peptidic inhibitors via REPLACE: Novel and new strategies in targeting PLK1 Polo Box Domain (PBD) for anti-cancer therapy

Abstract Polo Like Kinase 1 (PLK1) is only expressed in dividing cells and plays a critical role in several stages of mitosis. PLK1 is highly expressed in tumors of various origins while its expression is largely absent in normal tissues. PLK1 inhibition selectively kills cancer cells because they are dependent on the mitotic functions of PLK1 overexpression. PLK1 consists of a highly conserved N-terminal catalytic kinase domain and a unique, functionally essential C-terminal Polo Box Domain (PBD). The PBD of each PLK is a phospho-peptide binding motif that determines substrate recognition and sub-cellular localization. PLK1 catalytic inhibitors have advanced to clinical trials but they inhibit other family members PLK2 and PLK3. An alternative approach to developing potent and selective PLK1 inhibitors is to allosterically target the PBD. An iterative strategy called REPLACE involving computational and synthetic approaches was utilized to generate fragment-ligated inhibitory peptides (FLIPs) by rationally replacing amino acid residues in two PLK1 substrate peptides with drug-like small molecule fragments to generate FLIPs. Further application of REPLACE resulted in non-peptidic compounds named abbapolins. Confirmation of engagement of cellular PLK1 by abbapolins was assessed using a cellular thermal shift assay (CETSA), which determines drug binding to target proteins by measuring the shift in a protein's solubility and hence its thermal stability. Abbapolins specifically bound to full-length cellular PLK1 over PLK3 as determined by CETSA in a pattern consistent with in vitro binding affinities to the purified PBD of PLK1 and PLK3. We made an additional unique observation that mitotic PLK1 had a lower thermal stability than non-mitotic PLK1, suggesting that mitotic PLK1 might adopt a different conformation that is less soluble in the CETSA assay. Moreover, we observed that ATP-based inhibitors decreased the thermal stability of mitotic PLK1, whereas PBD inhibitors abbapolins increased the thermal stability of mitotic PLK1 as measured by CETSA. Additional evidence for engagement of cellular PLK1 was obtained through the unique mechanistic observation that abbapolins induced a striking dose dependent reduction in PLK1 protein levels as measured by western blotting and immunofluorescence. The loss of PLK1 was partially due to proteasome mediated degradation because the proteasome inhibitor MG132 partially prevented PLK1 degradation. Moreover, abbapolins caused preferential loss of nuclear PLK1 as determined by cellular fractionation. The therapeutic potential of these compounds was indicated through their antiproliferative activity on a cell line expressing a PLK1 point mutant that is dramatically resistant to ATP competitive PLK1 inhibitors. Future studies will examine in vivo activity of selected abbapolins. Citation Format: Danda Chapagai, Guru Ramamoorthy, Merissa Baxter, Sandra Craig, Elmar Nurmemmedov, McInnes Campbell, Michael Wyatt. Converting high affinity peptides to non-peptidic inhibitors via REPLACE: Novel and new strategies in targeting PLK1 Polo Box Domain (PBD) for anti-cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1241.

AMPKα2 activation by an energy-independent signal ensures chromosomal stability during mitosis

SummaryAMP-activated protein kinase (AMPK) senses energy status and impacts energy-consuming events by initiating metabolism regulatory signals in cells. Accumulating evidences suggest a role of AMPK in mitosis regulation, but the mechanism of mitotic AMPK activation and function remains elusive. Here we report that AMPKα2, but not AMPKα1, is sequentially phosphorylated and activated by CDK1 and PLK1, which enables AMPKα2 to accurately guide chromosome segregation in mitosis. Phosphorylation at Thr485 by activated CDK1-Cyclin B1 brings the ST-stretch of AMPKα2 to the Polo box domain of PLK1 for subsequent Thr172 phosphorylation by PLK1. Inserting of the AMPKα2 ST-stretch into AMPKα1, which lacks the ST-stretch, can correct mitotic chromosome segregation defects in AMPKα2-depleted cells. These findings uncovered a specific signaling cascade integrating sequential phosphorylation by CDK1 and PLK1 of AMPKα2 with mitosis to maintain genomic stability, thus defining an isoform-specific AMPKα2 function, which will facilitate future research on energy sensing in mitosis.

Design and synthesis of a new orthogonally protected glutamic acid analog and its use in the preparation of high affinity polo-like kinase 1 polo-box domain - binding peptide macrocycles.

Targeting protein – protein interactions (PPIs) has emerged as an important area of discovery for anticancer therapeutic development. In the case of phospho-dependent PPIs, such as the polo-like kinase 1 (Plk1) polo-box domain (PBD), a phosphorylated protein residue can provide high-affinity recognition and binding to target protein hot spots. Developing antagonists of the Plk1 PBD can be particularly challenging if one relies solely on interactions within and proximal to the phospho-binding pocket. Fortunately, the affinity of phospho-dependent PPI antagonists can be significantly enhanced by taking advantage of interactions in both the phospho-binding site and hidden “cryptic” pockets that may be revealed on ligand binding. In our current paper, we describe the design and synthesis of macrocyclic peptide mimetics directed against the Plk1 PBD, which are characterized by a new glutamic acid analog that simultaneously serves as a ring-closing junction that provides accesses to a cryptic binding pocket, while at the same time achieving proper orientation of a phosphothreonine (pT) residue for optimal interaction in the signature phospho-binding pocket. Macrocycles prepared with this new amino acid analog introduce additional hydrogen-bonding interactions not found in the open-chain linear parent peptide. It is noteworthy that this new glutamic acid-based amino acid analog represents the first example of extremely high affinity ligands where access to the cryptic pocket from the pT-2 position is made possible with a residue that is not based on histidine. The concepts employed in the design and synthesis of these new macrocyclic peptide mimetics should be useful for further studies directed against the Plk1 PBD and potentially for ligands directed against other PPI targets.

Mining Polo-Box domain of Polo-like kinase 1 as a new therapeutic target for cancer

Polo-like kinase 1 (Plk1) is widely regarded as one of the most promising targets for cancer therapy due to its essential role in cell division and tumor cell survival. At present, most Plk1 inhibitors have been developed based on kinase domain, some of which are in clinical trial. However, inhibitors targeting kinase domain face off-target effect and drug resistance owing to the conserved nature and the frequent mutations in the ATP-binding pocket. In addition to a highly conserved kinase domain, Plk1 also contains a unique Polo-Box domain (PBD), which is essential for Plk1's subcellular localization and mitotic functions. Inhibitors targeting Plk1 PBD show stronger selectivity and less drug resistance for cancer therapy. Therefore, Plk1 PBD is an attractive target for the development of anti-cancer agents. In this review, we will summarize the up-to date drug discovery for targeting Plk1 PBD, including the molecular structure and cellular functions of Plk1 PBD. Small-molecule inhibitors targeting Plk1 PBD not only provide an opportunity to specifically inhibit Plk1 activity for cancer treatment, but also unveil novel biological basis regarding the molecular recognition of Plk1 and its substrates.

Identification of a New Heterocyclic Scaffold for Inhibitors of the Polo-Box Domain of Polo-like Kinase 1.

As a mitotic-specific target widely deregulated in various human cancers, polo-like kinase 1 (Plk1) has been extensively explored for anticancer activity and drug discovery. Although multiple catalytic domain inhibitors were tested in preclinical and clinical studies, their efficacies are limited by dose-limiting cytotoxicity, mainly from off-target cross reactivity. The C-terminal noncatalytic polo-box domain (PBD) of Plk1 has emerged as an attractive target for generating new protein-protein interaction inhibitors. Here, we identified a 1-thioxo-2,4-dihydro-[1,2,4]triazolo[4,3-a]quinazolin-5(1H)-one scaffold that efficiently inhibits Plk1 PBD but not its related Plk2 and Plk3 PBDs. Structure-activity relationship studies led to multiple inhibitors having ≥10-fold higher inhibitory activity than the previously characterized Plk1 PBD-specific phosphopeptide, PLHSpT (Kd ∼ 450 nM). In addition, S-methyl prodrugs effectively inhibited mitotic progression and cell proliferation and their metabolic stability was determined. These data describe a novel class of small-molecule inhibitors that offer a promising avenue for future drug discovery against Plk1-addicted cancers.

Development of a Polo-like Kinase-1 Polo-Box Domain Inhibitor as a Tumor Growth Suppressor in Mice Models.

Polo-like kinase-1 (Plk1) plays a key role in mitosis and has been identified as an attractive anticancer drug target. Plk1 consists of two drug-targeting sites, namely, N-terminal kinase domain (KD) and C-terminal polo-box domain (PBD). As KD-targeting inhibitors are associated with severe side effects, here we report on the pyrazole-based Plk1 PBD inhibitor, KBJK557, which showed a remarkable in vitro anticancer effect by inducing Plk1 delocalization, mitotic arrest, and apoptosis in HeLa cells. Further, in vivo optical imaging analysis and antitumorigenic activities in mouse xenograft models demonstrate that KBJK557 preferentially accumulates in cancer cells and selectively inhibits cancer cell proliferation. Pharmacokinetic profiles and partition coefficients suggest that KBJK557 was exposed in the blood and circulated through the organs with an intermediate level of clearance (t1/2, 7.73 h). The present investigation offers a strategy for specifically targeting cancer using a newly identified small-molecule inhibitor that targets the Plk1 PBD.

Peptidomimetic Polo-Box-Targeted Inhibitors that Engage PLK1 in Tumor Cells and Are Selective against the PLK3 Tumor Suppressor.

The polo-box domain (PBD) of PLK1 determines mitotic substrate recognition and subcellular localization. Compounds that target PLK1 selectively are required due to the tumor-suppressor roles of PLK3. A structure-activity analysis of the PBD phosphopeptide binding motif has identified potent peptides that delineate the determinants required for mimicry by nonpeptidic inhibitors and provide insights into the structural basis for the selectivity of inhibitors for the PLK1 PBD. Fragment-ligated inhibitory peptides (FLIPs) obtained through REPLACE have been optimized to enhance in vitro binding and a systematic analysis of selectivity for PLK1 vs PLK3 has been carried out for peptides and peptidomimetics. Furthermore, these more drug-like non-ATP-competitive inhibitors had on-target engagement in a cellular context, as evidenced by stabilization of PLK1 in a thermal-shift assay and by inhibition of the phosphorylation of TCTP, a target of PLK1. Investigation in cells expressing a mutant PLK1 showed that these cells are sensitive to PBD inhibitors but dramatically resistant to clinically investigated ATP-competitive compounds. These results further validate targeting the PBD binding site in the move towards PLK1 inhibitors that are active against tumors resistant to ATP inhibitors.

A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation

The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.

Identification of novel peptidomimetics targeting the polo-box domain of polo-like kinase 1

A series of new peptidomimetics targeting the polo-box domain (PBD) of polo-like kinase 1 (Plk1) was identified based on the potent and selective pentapeptide Plk1 PBD inhibitor PLHSpT. Unnatural amino acid residues were introduced to the newly designed compound and the N-terminal substituent of the peptidomimetic was investigated. The optimized compound 9 inhibited the Plk1 PBD with IC50 of 0.267 μM and showed almost no inhibition to Plk2 PBD or Plk3 PBD at 100 μM. Biolayer interferometry studies demonstrated that compound 9 showed potent binding affinity to Plk1 with a Kd value of 0.164 μM, while no Kd were detected against Plk2 and Plk3. Compound 9 showed improved stability in rat plasma compared to PLHSpT. Binding mode analysis was performed and in agreement with the observed experimental results. There are only two natural amino acids remained in the chemical structure of 9. This study may provide new information for further research on Plk1 PBD inhibitors.

Abstract 4712: Selective polo-like kinase 1 PBD inhibitor

Abstract Polo Like Kinase 1 (PLK1) is only expressed in dividing cells and plays a critical role in several stages of mitosis. PLK1 is highly expressed in tumors of various origins while its expression is largely absent in normal tissues. PLK1 inhibition selectively kills cancer cells because cancer cells utterly depend on the mitotic functions of PLK1 overexpression. PLK1 consists of a highly conserved N-terminal catalytic kinase domain and a unique, functionally essential C-terminal Polo Box Domain (PBD). The PBD of each PLK is a phospho-peptide binding motif that determines substrate recognition and sub-cellular localization. PLK1 catalytic inhibitorshave advanced to clinical trials but they inhibit other kinases including family members PLK2 and PLK3. This is problematic because PLK3 is a tumor suppressor and partial inhibition of PLK3 could lead to a long-term malignant progression. Moreover, a single point mutation (C67V) in the PLK1 kinase domain confers substantial cancer cell resistance against PLK1 catalytic inhibitors. An alternative approach to developing potent and selective PLK1 inhibitors is to allosterically target the PBD. We generated fragment ligated inhibitory peptides (FLIPs) through a strategy called REPLACE. This is a computational and synthetic approach that uses structure activity relationships of peptide inhibitors to generate pharmaceutically acceptable lead molecules. Fragments are docked into the crystal structure of a truncated peptide/receptor complex, then prioritized for synthesis with the peptide and subsequently tested in an in vitro binding assay. PBD-interacting protein (PBIP) and Cdc25c are natural substrates of PLK1. We replaced individual amino acid residues in the PBIP (PLHSpTAI) and Cdc25C PBD substrate peptides (LLCSpTPNGL) with benzoic acid fragments. Here we show PLK1 specificity for PBD inhibitors in vitro. A fluorescent polarization (FP) competitive binding assay to the PBD of PLK1 was used to determine binding specificity. A PLK3 counter screen was used to determine the binding selectivity of our PBD inhibitors. To measure the PLK1 inhibitory activity of the FLIPs in cells, phosphorylation of mitotic proteins was measured. Candidate FLIPs induce mitotic arrest, block PLK1 mediated phosphorylation, and inhibit growth of human cancer cells. To make more drug-like molecules, we modified FLIPs to Small Molecule Inhibitors (SMIs). Further, we are optimizing SMIs to improve biochemical selectivity. SMIs bind to the PLK1 PBD as determined by FP assay. Also, SMIs inhibit cancer cell growth as determined by viability measurements and live cell imaging. Overall these results highlight that targeting the PBD of PLK1 has promise as an antitumor strategy. Citation Format: Danda P. Chapagai, Merissa Baxter, Gurusankar Ramamoorthy, Campbell McInnes, Michael Wyatt. Selective polo-like kinase 1 PBD inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4712.

Development of Highly Selective 1,2,3-Triazole-containing Peptidic Polo-like Kinase 1 Polo-box Domain-binding Inhibitors.

Members of the polo-like kinase (Plk) family of serine/threonine protein kinases play crucial roles in cell cycle regulation and proliferation. Of the five Plks (Plk1–5), Plk1 is recognized as an anticancer drug target. Plk1 contains multiple structural components that are important for its proper biological function. These include an N-terminal catalytic domain and a C-terminal non-catalytic polo-box domain (PBD). The PBD binds to phosphothreonine (pT) and phosphoserine-containing sequences. Blocking PBD-dependent interactions offers a potential means of down-regulating Plk1 function that is distinct from targeting its ATP-binding site. Previously, we demonstrated by tethering alkylphenyl chains from the N(π)-position of the His residue in the 5-mer PLHSpT, that we were able to access a hydrophobic “cryptic” binding pocket on the surface of the PBD, and in so doing enhance binding affinities by approximately 1000-fold. More recently, we optimized these PBD-ligand interactions using an oxime ligation-based strategy. Herein, using azide-alkyne cycloaddition reactions, we explore new triazole-containing PBD-binding antagonists. Some of these ligands retain the high PBD-binding affinity of the parent peptide, while showing desirable enhanced selectivity for the PBD of Plk1 relative to the PBDs of Plk2 and Plk3.