Polo-box Domain Research Articles

Structural Basis for Variations in Polo-like Kinase 1 Conformation and Intracellular Stability Induced by ATP-Competitive and Novel Noncompetitive Abbapolin Inhibitors.

Polo-like kinase 1 (PLK1) is an essential protein kinase with multiple roles in mitotic progression. PLK1 consists of a kinase domain (KD) and a phosphopeptide-binding polobox domain (PBD), which is responsible for substrate recognition and subcellular localization. The regulation of PLK1 involves an autoinhibitory conformation in which KD and PBD interact. Our previous work identified PBD-binding molecules termed abbapolins that inhibit the cellular phosphorylation of a PLK1 substrate and induce the loss of intracellular PLK1. Here, we describe a comparison of the abbapolin activity with that of KD inhibitors to gain insight into conformational features of PLK1. As measured by a cellular thermal shift assay, abbapolins produce ligand-induced thermal stabilization of PLK1. In contrast, KD inhibitors decreased the soluble PLK1, suggesting that catalytic-site binding causes a less thermally stable PLK1 conformation. Binding measurements with full-length PLK1 and a KD inhibitor also demonstrated a conformational change. Interestingly, the cellular consequences of KD versus PBD engagement contrast as KD binding causes the accumulation of intracellular PLK1, whereas PBD binding produces a striking loss of nuclear PLK1. These data are consistent with the relief of autoinhibited PLK1 by KD binders; an explanation for these observations is presented using structures for the catalytic domain and full-length PLK1 predicted by AlphaFold. Collectively, the results highlight an underappreciated aspect of targeting PLK1, namely, conformational perturbations induced by KD versus PBD binding. In addition to their significance for PBD-binding ligands, these observations have implications for the development of ATP-competitive PLK1 inhibitors because catalytic inhibitors may conversely promote PLK1 noncatalytic functions, which may explain their lack of clinical efficacy to date.

The toxicity of dysregulated Plk1 activity revealed by its suppressor mutations.

Polo-like kinase 1 (Plk1) is a mitotic kinase that has multiple functions throughout the cell cycle. Catalytic activation of Plk1 is known to be regulated by phosphorylation of the kinase domain, including Thr210, and by releasing the kinase domain from its inhibitory polo-box domain. However, how Plk1 is activated to fulfill its proper roles, in time and space, is not well understood. In this study, we unintentionally found that the expression of a constitutively active form of human Plk1 is toxic to bacterial cells, such that cells contained point mutations that alleviate the kinase activity. Structural prediction revealed that these mutations are adjacent to the amino acids supporting the kinase activity. When human cells express these mutants, we found decreased levels of Plk1's substrate phosphorylation, resulting in mitotic defects. Moreover, unlike in bacterial cells, the expression of activated Plk1 mutants did not affect cell proliferation in human cells unless localized at the right place in mitosis. Our observations identified new suppressor mutations and underscored the importance of spatiotemporal regulation in Plk1, providing a basis for how we might intervene in this kinase for therapeutic purpose in human cells.

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.

Polo-Like Kinase 2: From Principle to Practice.

Polo-like kinase (PLK) 2 is an evolutionarily conserved serine/threonine kinase that shares the n-terminal kinase catalytic domain and the C-terminal Polo Box Domain (PBD) with other members of the PLKs family. In the last two decades, mounting studies have focused on this and tried to clarify its role in many aspects. PLK2 is essential for mitotic centriole replication and meiotic chromatin pairing, synapsis, and crossing-over in the cell cycle; Loss of PLK2 function results in cell cycle disorders and developmental retardation. PLK2 is also involved in regulating cell differentiation and maintaining neural homeostasis. In the process of various stimuli-induced stress, including oxidative and endoplasmic reticulum, PLK2 may promote survival or apoptosis depending on the intensity of stimulation and the degree of cell damage. However, the role of PLK2 in immunity to viral infection has been studied far less than that of other family members. Because PLK2 is extensively and deeply involved in normal physiological functions and pathophysiological mechanisms of cells, its role in diseases is increasingly being paid attention to. The effect of PLK2 in inhibiting hematological tumors and fibrotic diseases, as well as participating in neurodegenerative diseases, has been gradually recognized. However, the research results in solid organ tumors show contradictory results. In addition, preliminary studies using PLK2 as a disease predictor and therapeutic target have yielded some exciting and promising results. More research will help people better understand PLK2 from principle to practice.

Shedding light on the binding mechanism of kinase inhibitors BI-2536, Volasetib and Ro-3280 with their pharmacological target PLK1.

In the present work, the interactions of the novel kinase inhibitors BI-2536, Volasetib (BI-6727) and Ro-3280 with the pharmacological target PLK1 have been studied by fluorescence spectroscopy and molecular dynamics calculations. High Stern-Volmer constants were found in fluorescence experiments suggesting the formation of stable protein-ligand complexes. In addition, it was observed that the binding constant between BI-2536 and PLK1 increases about 100-fold in presence of the phosphopeptide Cdc25C-p that docks to the polo box domain of the protein and releases the kinase domain. All the determined binding constants are higher for the kinase inhibitors than for their competitor for the active center (ATP) being BI-2536 and Volasertib the inhibitors that showed more affinity for PLK1. Calculated binding free energies confirmed the higher affinity of PLK1 for BI-2536 and Volasertib than for ATP. The higher affinity of the inhibitors to PLK1 compared to ATP was mainly attributed to stronger van der Waals interactions. Results may help with the challenge of designing and developing new kinase inhibitors more effective in clinical cancer therapy.

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.

Tumor suppressor PLK2 may serve as a biomarker in triple-negative breast cancer for improved response to PLK1 therapeutics.

Polo-like kinase (PLK) family members play important roles in cell cycle regulation. The founding member PLK1 is oncogenic and preclinically validated as a cancer therapeutic target. Paradoxically, frequent loss of chromosome 5q11-35 which includes PLK2 is observed in basal-like breast cancer. In this study, we found that PLK2 was tumor suppressive in breast cancer, preferentially in basal-like and triple-negative breast cancer (TNBC) subtypes. Knockdown of PLK1 rescued phenotypes induced by PLK2-loss both in vitro and in vivo. We also demonstrated that PLK2 directly interacted with PLK1 at prometaphase through the kinase but not the polo-box domains of PLK2, suggesting PLK2 functioned at least partially through the interaction with PLK1. Furthermore, an improved treatment response was seen in both Plk2-deleted/low mouse preclinical and PDX TNBC models using the PLK1 inhibitor volasertib alone or in combination with carboplatin. Re-expression of PLK2 in an inducible PLK2-null mouse model reduced the therapeutic efficacy of volasertib. In summary, this study delineates the effects of chromosome 5q loss in TNBC that includes PLK2, the relationship between PLK2 and PLK1, and how this may render PLK2-deleted/low tumors more sensitive to PLK1 inhibition in combination with chemotherapy.

Structure-activity and mechanistic studies of non-peptidic inhibitors of the PLK1 polo box domain identified through REPLACE

Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase involved in cell cycle regulation and mitotic progression. Studies have shown that PLK1 is upregulated in many tumors and high levels are adversely related to a poor prognosis. Knocking down or inhibiting PLK1 results in synthetic lethality in PTEN deficient prostate tumors and Kras mutant colorectal tumors, further validating PLK1 as an oncotarget. Substrate recognition by PLK1 occurs through the Polo-Box Domain (PBD), which is a phospho-peptide binding site also responsible for subcellular localization. Much effort has been directed to target this kinase therapeutically through the ATP-binding site, and a few such inhibitors have advanced to clinical trials however with limited clinical efficacy. Moreover, it has been shown that a point mutation in PLK1 (C67V) confers dramatic cellular resistance to catalytic site inhibitors. An alternative approach to target PLK1 potently and selectively is through the PBD to block its protein-protein interactions. Through the REPLACE strategy, for converting peptide inhibitors into more drug-like non peptidic compounds, a PBD targeting compound series (“ABBAs”), has been identified and the key determinants of potency and selectivity elucidated through structure-activity relationship studies. In cellular experiments, the ABBAs were shown to lead to profound effects on the cell cycle, to inhibit tumor proliferation and overcome resistance of cells expressing the PLK1 C67V mutant to ATP-based inhibitors. These non-ATP competitive inhibitors of PLK1 were also used chemical biology probes to investigate the gene regulatory effects of PLK1, known to act on transcription factors such as p53.

Virtual screening of natural compounds as selective inhibitors of polo-like kinase-1 at C-terminal polo box and N-terminal catalytic domain

The over-expression of Polo-like kinase-1 (PLK1) is associated with cancer prognosis due to its pivotal role in cell proliferation. The N-terminal catalytic domain (NCD) and C-terminal polo box domain (PBD) of PLK1 are critical for the activity of the protein. Drugs that inhibit PLK1 by targeting these domains are on clinical trials, but so far, none has been approved by FDA. Thus, this study targets the two domains of PLK1 to identify compounds with inhibitory potential. Four validated e-pharmacophore models from NCD (PDB ID: 2OU7 and 4J52) and PBD (PDB ID: 5NEI and 5NN2) were used to screen over 26,000 natural compounds from NPASS database. Hits were identified after the well-fitted compounds were subjected to molecular docking study and ADME prediction. The pIC50 and electronic behaviour of the identified hits selectively targeting NCD and PBD of PLK1 were predicted via an externally validated QSAR model and quantum mechanics. The results showed that CAA180504, CAA197326, CAA74619, CAA328856 modulating PLK1 at NCD, and CBB130581, CBB230713, CBB206123, CBB12656 and CBB267117 modulating PLK1 at PBD had better molecular docking scores, pharmacokinetics and drug-like properties than NCD (volasertib) and PBD (purpurogallin) reference inhibitors. The compounds all had satisfactory inhibitory (pIC50) values which range from 6.187 to 7.157. The electronic behaviours of understudied compounds using HOMO/LUMO and global descriptive parameters revealed the atomic portion of the compounds prone to donating and accepting electrons. In conclusion, the hit compounds identified from the library of natural compounds are worthy of further experimental validation. Communicated by Ramaswamy H. Sarma

Dual Targeting of EGFR with PLK1 Exerts Therapeutic Synergism in Taxane-Resistant Lung Adenocarcinoma by Suppressing ABC Transporters.

Simple SummaryOur previous studies led us to hypothesize that downregulation of PLK1 expression or its activity can overcome the hurdles of taxane resistance by downregulating ABC transporters. Targeting PLK1 with shRNA or non-functional mutants downregulated ABCB1, ABCC9, and ABCG2 in paclitaxel-resistant lung adenocarcinoma (LUADTXR), similar to the downregulation effects from treatment with PLK1 inhibitors. Since EGFR is highly expressed in LUADTXR cells, gefitinib was combined with PLK1 inhibitors. Under these conditions, LUADTXR cells tend to undergo apoptosis more effectively than parental cells, showing a synergistic effect on downregulation of ABC transporters through c-Myc or AP-1. Clinical data provide evidence for the relationship between survival rates and expressions of PLK1 and EGFR in LUAD patients. Taken together, our data suggest that a combination of gefitinib and PLK1 inhibitors exerts strong synergism in LUADTXR, providing a benefit to overcome the limitations associated with taxanes.To overcome the limitations of chemoresistance, combination therapies using druggable targets have been investigated. Our previous studies led us to hypothesize that the downregulation of PLK1 expression or activity can be one strategy to overcome the hurdles of taxane resistance by the downregulation of ABC transporters. To explore this, various versions of PLK1 including a constitutively active version, kinase-dead form, and polo-box domain mutant were expressed in paclitaxel-resistant lung adenocarcinoma (LUADTXR). Targeting PLK1 using shRNA or non-functional mutants downregulated ABCB1, ABCC9, and ABCG2 in LUADTXR cells, which was similar to the downregulation effects from treatment with PLK1 inhibitors. The high expression of EGFR in LUAD led us to administer gefitinib, showing a markedly reduced EGFR level in LUADTXR cells. When gefitinib and PLK1 inhibitors were combined, LUADTXR cells tended to undergo apoptosis more effectively than parental cells, showing a synergistic effect on the downregulation of ABC transporters through c-Myc and AP-1. Clinical data provide evidence for the relevance between survival rates and expressions of PLK1 and EGFR in LUAD patients. Based on these results, we suggest that a combination of gefitinib and PLK1 inhibitors exerts strong synergism in LUADTXR, which helps to overcome the limitations associated with taxanes.

Ana1 helps recruit Polo to centrioles to promote mitotic PCM assembly and centriole elongation.

ABSTRACTPolo kinase (PLK1 in mammals) is a master cell cycle regulator that is recruited to various subcellular structures, often by its polo-box domain (PBD), which binds to phosphorylated S-pS/pT motifs. Polo/PLK1 kinases have multiple functions at centrioles and centrosomes, and we have previously shown that in Drosophila phosphorylated Sas-4 initiates Polo recruitment to newly formed centrioles, while phosphorylated Spd-2 recruits Polo to the pericentriolar material (PCM) that assembles around mother centrioles in mitosis. Here, we show that Ana1 (Cep295 in humans) also helps to recruit Polo to mother centrioles in Drosophila. If Ana1-dependent Polo recruitment is impaired, mother centrioles can still duplicate, disengage from their daughters and form functional cilia, but they can no longer efficiently assemble mitotic PCM or elongate during G2. We conclude that Ana1 helps recruit Polo to mother centrioles to specifically promote mitotic centrosome assembly and centriole elongation in G2, but not centriole duplication, centriole disengagement or cilia assembly.This article has an associated First Person interview with the first author of the paper.

Nonpeptidic, Polo-Box Domain-Targeted Inhibitors of PLK1 Block Kinase Activity, Induce Its Degradation and Target-Resistant Cells.

PLK1, polo-like kinase 1, is a central player regulating mitosis. Inhibition of the subcellular localization and kinase activity of PLK1 through the PBD, polo-box domain, is a viable alternative to ATP-competitive inhibitors, for which the development of resistance and inhibition of related PLK family members are concerns. We describe novel nonpeptidic PBD-binding inhibitors, termed abbapolins, identified through successful application of the REPLACE strategy and demonstrate their potent antiproliferative activity in prostate tumors and other cell lines. Furthermore, abbapolins show PLK1-specific binding and inhibitory activity, as measured by a cellular thermal shift assay and an ability to block phosphorylation of TCTP, a validated target of PLK1-mediated kinase activity. Additional evidence for engagement of PLK1 was obtained through the unique observation that abbapolins induce PLK1 degradation in a manner that closely matches antiproliferative activity. Moreover, abbapolins demonstrate antiproliferative activity in cells that are dramatically resistant to ATP-competitive PLK1 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.

Recent Progress in Development of Polo-Like Kinase 1 Inhibitors: Efforts So Far

Polo-like kinase 1(Plk1) plays an important role in the inhibition of cell proliferation and which is come under the family of serine/threonine-protein kinase. Which is a highly specific target for cancer therapy. In some clinical studies, Plk1 has been identified as a target for cancer. Currently, so many scientists are working on the development of the Plk1 inhibitors and so many scientists and researchers thinking about working on it. Recent strategy for Plk1 inhibition is the development of small molecule inhibitors which will inhibit the Plk1 through the ATP-binding site of the Plk. Now new generation Plk1 inhibitors being tested clinically which are targeting polo box domain. This review highlights the recent progress made in the development of Plk1 inhibitors as anticancer agents.

A polo-like kinase modulates cytokinesis and flagella biogenesis in Giardia lamblia

BackgroundPolo-like kinases (PLKs) are conserved serine/threonine kinases that regulate the cell cycle. To date, the role of Giardia lamblia PLK (GlPLK) in cells has not been studied. Here, we report our investigation on the function of GlPLK to provide insight into the role of this PKL in Giardia cell division, especially during cytokinesis and flagella formation.MethodsTo assess the function of GIPLK, Giardia trophozoites were treated with the PLK-specific inhibitor GW843286X (GW). Using a putative open reading frame for the PLK identified in the Giardia genomic database, we generated a transgenic Giardia expressing hemagglutinin (HA)-tagged GlPLK and used this transgenic for immunofluorescence assays (IFAs). GlPLK expression was knocked down using an anti-glplk morpholino to observe its effect on the number of nuclei number and length of flagella. Giardia cells ectopically expressing truncated GlPLKs, kinase domain + linker (GlPLK-KDL) or polo-box domains (GlPLK-PBD) were constructed for IFAs. Mutant GlPLKs at Lys51, Thr179 and Thr183 were generated by site-directed mutagenesis and then used for the kinase assay. To elucidate the role of phosphorylated GlPLK, the phosphorylation residues were mutated and expressed in Giardia trophozoitesResultsAfter incubating trophozoites with 5 μM GW, the percentage of cells with > 4 nuclei and longer caudal and anterior flagella increased. IFAs indicated that GlPLK was localized to basal bodies and flagella and was present at mitotic spindles in dividing cells. Morpholino-mediated GlPLK knockdown resulted in the same phenotypes as those observed in GW-treated cells. In contrast to Giardia expressing GlPLK-PBD, Giardia expressing GlPLK-KDL was defective in terms of GIPLK localization to mitotic spindles and had altered localization of the basal bodies in dividing cells. Kinase assays using mutant recombinant GlPLKs indicated that mutation at Lys51 or at both Thr179 and Thr183 resulted in loss of kinase activity. Giardia expressing these mutant GlPLKs also demonstrated defects in cell growth, cytokinesis and flagella formation.ConclusionsThese data indicate that GlPLK plays a role in Giardia cell division, especially during cytokinesis, and that it is also involved in flagella formation.Graphical

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.

Centrosomal Localization of RXRα Promotes PLK1 Activation and Mitotic Progression and Constitutes a Tumor Vulnerability.

Retinoid X receptor alpha (RXRα), a nuclear receptor of transcription factor, controls various physiological and pathological pathways including cellular growth, proliferation, differentiation, and apoptosis. Here, we report that RXRα is phosphorylated at its N-terminal A/B domain by cyclin-dependent kinase 1 (Cdk1) at the onset of mitosis, triggering its translocation to the centrosome, where phosphorylated-RXRα (p-RXRα) interacts with polo-like kinase 1 (PLK1) through its N-terminal A/B domain by a unique mechanism. The interaction promotes PLK1 activation, centrosome maturation, and mitotic progression. Levels of p-RXRα are abnormally elevated in cancer cell lines, during carcinogenesis in animals, and in clinical tumor tissues. An RXRα ligand XS060, which specifically inhibits p-RXRα/PLK1 interaction but not RXRα heterodimerization, promotes mitotic arrest and catastrophe in a tumor-specific manner. These findings unravel a transcription-independent action of RXRα at the centrosome during mitosis and identify p-RXRα as a tumor-specific vulnerability for developing mitotic drugs with improved therapeutic index.

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.