Identification of novel, potent and selective inhibitors of Polo-like kinase 1

A series of pyrimidodiazepines was identified as potent Polo-like kinase 1 (PLK1) inhibitors. The synthesis and SAR are discussed. The lead compound 7 (RO3280) has potent inhibitory activity against PLK1, good selectivity against other kinases, and excellent in vitro cellular potency. It showed strong antitumor activity in xenograft mouse models.     

NMS-P937, a 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivative as potent and selective Polo-like kinase 1 inhibitor

As part of our drug discovery effort, we identified and developed 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as PLK1 inhibitors. We now report the optimization of this class that led to the identification of NMS-P937, a potent, selective and orally available PLK1 inhibitor. Also, in order to understand the source of PLK1 selectivity, we determined the crystal structure of PLK1 with NMS-P937. The compound was active in vivo in HCT116 xenograft model after oral administration and is presently in Phase I clinical trials evaluation.     

Design,Synthesis and Antitumor Activity of FAK/PLK1 Dual Inhibitors with Quinazolinone as the Skeleton

Febrifugine is a kind of quinazolinone compound with high biological activity from a Chinese herb called Chang Shan (Dichroa febrifuga). Febrifugine and its derivatives possess extensive biological activities, some of which exhibited anti-tumor activities as FAK inhibitors. However, they are not very effective at inhibiting tumor metastasis, perhaps because tumors gain energy through compensatory activation of other signaling pathways that promote cell migration and invasion. Therefore, seventeen novel febrifugine derivatives with quinazolinone skeleton were designed, synthesized and acted as potential FAK/PLK1 dual inhibitors. These compounds were determined by ¹H-NMR, ¹³C-NMR and MS. Most of the compounds exhibited good inhibitory activity against cancer cell lines by computer-assisted screening, antitumor activity test and FAK/PLK1 inhibitory activity test, wherein compound 3b was screened as a high-efficiency lead compound.     

PLK1 (polo like kinase 1) inhibits MTOR complex 1 and promotes autophagy

Mechanistic target of rapamycin complex 1 (MTORC1) and polo like kinase 1 (PLK1) are major drivers of cancer cell growth and proliferation, and inhibitors of both protein kinases are currently being investigated in clinical studies. To date, MTORC1′s and PLK1′s functions are mostly studied separately, and reports on their mutual crosstalk are scarce. Here, we identify PLK1 as a physical MTORC1 interactor in human cancer cells. PLK1 inhibition enhances MTORC1 activity under nutrient sufficiency and in starved cells, and PLK1 directly phosphorylates the MTORC1 component RPTOR/RAPTOR in vitro. PLK1 and MTORC1 reside together at lysosomes, the subcellular site where MTORC1 is active. Consistent with an inhibitory role of PLK1 toward MTORC1, PLK1 overexpression inhibits lysosomal association of the PLK1-MTORC1 complex, whereas PLK1 inhibition promotes lysosomal localization of MTOR. PLK1-MTORC1 binding is enhanced by amino acid starvation, a condition known to increase autophagy. MTORC1 inhibition is an important step in autophagy activation. Consistently, PLK1 inhibition mitigates autophagy in cancer cells both under nutrient starvation and sufficiency, and a role of PLK1 in autophagy is also observed in the invertebrate model organism Caenorhabditis elegans. In summary, PLK1 inhibits MTORC1 and thereby positively contributes to autophagy. Since autophagy is increasingly recognized to contribute to tumor cell survival and growth, we propose that cautious monitoring of MTORC1 and autophagy readouts in clinical trials with PLK1 inhibitors is needed to develop strategies for optimized (combinatorial) cancer therapies targeting MTORC1, PLK1, and autophagy.     

5-(2-amino-pyrimidin-4-yl)-1H-pyrrole and 2-(2-amino-pyrimidin-4-yl)-1,5,6,7-tetrahydro-pyrrolo[3,2-c]pyridin-4-one derivatives as new classes of selective and orally available Polo-like kinase 1 inhibitors

The discovery and characterization of two new chemical classes of potent and selective Polo-like kinase 1 (PLK1) inhibitors is reported. For the most interesting compounds, we discuss the biological activities, crystal structures and preliminary pharmacokinetic parameters. The more advanced compounds inhibit PLK1 in the enzymatic assay at the nM level and exhibit good activity in cell proliferation on A2780 cells. Furthermore, these compounds showed high levels of selectivity on a panel of unrelated kinases, as well as against PLK2 and PLK3 isoforms. Additionally, the compounds show acceptable oral bioavailability in mice making these inhibitors suitable candidates for further in vivo activity studies.     

PLK1 inhibition-based combination therapies for cancer management

Polo-like kinase I (PLK1), a cell cycle regulating kinase, has been shown to have oncogenic function in several cancers. Although PLK1 inhibitors, such as BI2536, BI6727 (volasertib) and NMS-1286937 (onvansertib) are generally well-tolerated with a favorable pharmacokinetic profile, clinical successes are limited due to partial responses in cancer patients, especially those in advanced stages. Recently, combination therapies targeting multiple pathways are being tested for cancer management. In this review, we first discuss structure and function of PLK1, role of PLK1 in cancers, PLK1 specific inhibitors, and advantages of using combination therapy versus monotherapy followed by a critical account on PLK1-based combination therapies in cancer treatments, especially highlighting recent advancements and challenges. PLK1 inhibitors in combination with chemotherapy drugs and targeted small molecules have shown superior effects against cancer both in vitro and in vivo. PLK1-based combination therapies have shown increased apoptosis, disrupted cell cycle, and potential to overcome resistance in cancer cells/tissues over monotherapies. Further, with successes in preclinical experiments, researchers are validating such approaches in clinical trials. Although PLK1-based combination therapies have achieved initial success in clinical studies, there are examples where they have failed to improve patient survival. Therefore, further research is needed to identify and validate novel biologically informed co-targets for PLK1-based combinatorial therapies. Employing a network-based analysis, we identified potential PLK1 co-targets that could be examined further. In addition, understanding the mechanisms of synergism between PLK1 inhibitors and other agents may lead to a better approach on which agents to pair with PLK1 inhibition for optimum cancer treatment.     

PLK1, A Potential Target for Cancer Therapy

Polo-like kinase 1 (PLK1) plays an important role in the initiation, maintenance, and completion of mitosis. Dysfunction of PLK1 may promote cancerous transformation and drive its progression. PLK1 overexpression has been found in a variety of human cancers and was associated with poor prognoses in cancers. Many studies have showed that inhibition of PLK1 could lead to death of cancer cells by interfering with multiple stages of mitosis. Thus, PLK1 is expected to be a potential target for cancer therapy. In this article, we examined PLK1’s structural characteristics, its regulatory roles in cell mitosis, PLK1 expression, and its association with survival prognoses of cancer patients in a wide variety of cancer types, PLK1 interaction networks, and PLK1 inhibitors under investigation. Finally, we discussed the key issues in the development of PLK1-targeted cancer therapy.     

Multiple Requirements of PLK1 during Mouse Oocyte Maturation

Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1’s functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.     

LATS1/WARTS phosphorylates MYPT1 to counteract PLK1 and regulate mammalian mitotic progression

In the mitotic exit network of budding yeast, Dbf2 kinase phosphorylates and regulates Cdc14 phosphatase. In contrast, no phosphatase substrates of LATS1/WARTS kinase, the mammalian equivalent of Dbf2, has been reported. To address this discrepancy, we performed phosphoproteomic screening using LATS1 kinase. Screening identified MYPT1 (myosin phosphatase-targeting subunit 1) as a new substrate for LATS1. LATS1 directly and preferentially phosphorylated serine 445 (S445) of MYPT1. An MYPT1 mutant (S445A) failed to dephosphorylate Thr 210 of PLK1 (pololike kinase 1), thereby activating PLK1. This suggests that LATS1 promotes MYPT1 to antagonize PLK1 activity. Consistent with this, LATS1-depleted HeLa cells or fibroblasts from LATS1 knockout mice showed increased PLK1 activity. We also found deoxyribonucleic acid (DNA) damage-induced LATS1 activation caused PLK1 suppression via the phosphorylation of MYPT1 S445. Furthermore, LATS1 knockdown cells showed reduced G2 checkpoint arrest after DNA damage. These results indicate that LATS1 phosphorylates a phosphatase as does the yeast Dbf2 and demonstrate a novel role of LATS1 in controlling PLK1 at the G2 DNA damage checkpoint.     

Quantitative phospho-proteomics to investigate the polo-like kinase 1-dependent phospho-proteome

Polo-like kinase 1 (PLK1) is a key regulator of mitotic progression and cell division, and small molecule inhibitors of PLK1 are undergoing clinical trials to evaluate their utility in cancer therapy. Despite this importance, current knowledge about the identity of PLK1 substrates is limited. Here we present the results of a proteome-wide analysis of PLK1-regulated phosphorylation sites in mitotic human cells. We compared phosphorylation sites in HeLa cells that were or were not treated with the PLK1-inhibitor BI 4834, by labeling peptides via methyl esterification, fractionation of peptides by strong cation exchange chromatography, and phosphopeptide enrichment via immobilized metal affinity chromatography. Analysis by quantitative mass spectrometry identified 4070 unique mitotic phosphorylation sites on 2069 proteins. Of these, 401 proteins contained one or multiple phosphorylation sites whose abundance was decreased by PLK1 inhibition. These include proteins implicated in PLK1-regulated processes such as DNA damage, mitotic spindle formation, spindle assembly checkpoint signaling, and chromosome segregation, but also numerous proteins that were not suspected to be regulated by PLK1. Analysis of amino acid sequence motifs among phosphorylation sites down-regulated under PLK1 inhibition in this data set identified two potential novel variants of the PLK1 consensus motif.     

PLK1 Down-Regulates Parainfluenza Virus 5 Gene Expression

The paramyxoviruses are a family of negative-sense RNA viruses that includes many important human and animal pathogens. Paramyxovirus RNA synthesis requires the viral phosphoprotein (P) and the large (L) protein. Phosphorylation of P is thought to regulate viral gene expression, though direct proof remains elusive. Recently, we reported that phosphorylation of a specific residue (Ser157) of the P protein of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, correlates with decreased viral gene expression and cytokine expression in infected cells. Here, we show that: Polo-like kinase 1 (PLK1), a serine/theronine kinase that plays a critical role in regulating the cell cycle, interacts with PIV5 P through the S157 residue; PLK1 inhibition increases viral gene expression; PLK1 over-expression inhibits viral gene expression; and PLK1 directly phosphorylates P in vitro, indicating that PLK1 down-regulates viral gene expression by phosphorylating P. Furthermore, we have determined the PLK1 phosphorylation site on P and found that mutant recombinant PIV5 whose P proteins cannot either bind to or be phosphorylated by PLK1 have similar phenotypes. Increased viral gene expression in PIV5 with mutations in the PLK1 binding/phosphorylation sites correlates with increased induction of cell death and cytokine expression, suggesting that PIV5 limits its viral gene expression to avoid these host effects. It is possible that targeting PLK1 will enhance host innate immune responses, leading to a novel strategy of clearing paramyxovirus infections quickly.     

Myosin phosphatase-targeting subunit 1 regulates mitosis by antagonizing polo-like kinase 1

Myosin phosphatase-targeting subunit 1 (MYPT1) binds to the catalytic subunit of protein phosphatase 1 (PP1C). This binding is believed to target PP1C to specific substrates including myosin II, thus controlling cellular contractility. Surprisingly, we found that during mitosis, mammalian MYPT1 binds to polo-like kinase 1 (PLK1). MYPT1 is phosphorylated during mitosis by proline-directed kinases including cdc2, which generates the binding motif for the polo box domain of PLK1. Depletion of PLK1 by small interfering RNAs is known to result in loss of gamma-tubulin recruitment to the centrosomes, blocking centrosome maturation and leading to mitotic arrest. We found that codepletion of MYPT1 and PLK1 reinstates gamma-tubulin at the centrosomes, rescuing the mitotic arrest. MYPT1 depletion increases phosphorylation of PLK1 at its activating site (Thr210) in vivo, explaining, at least in part, the rescue phenotype by codepletion. Taken together, our results identify a previously unrecognized role for MYPT1 in regulating mitosis by antagonizing PLK1.     

Imidazopyridine derivatives as potent and selective Polo-like kinase (PLK) inhibitors

A novel class of imidazopyridine derivatives was designed as PLK1 inhibitors. Extensive SAR studies supported by molecular modeling afforded a highly potent and selective compound 36. Compound 36 demonstrated good antitumor efficacy in xenograft nude rat model.     

A bitter PP1 fights the sweet polo

In a recent paper in Developmental Cell, Yamashiro et al. (2008) report that the PP1 regulatory subunit MYPT1 interacts with PLK1 and antagonizes essential mitotic functions of PLK1, at least in part by promoting the dephosphorylation of PLK1 at Thr210.     

特异小干扰RNA敲除PLK1基因的表达

为研究特异小干扰RNA（siRNA）作用于大肠癌细胞株SW480中PLK1 (Polo-like kinase 1)基因表达的mRNA对该细胞分裂生长的影响,设计了对应于PLK1基因表达mRNA不同位点的10种特异siRNA,经化学合成后,用脂质体转染SW480细胞,实时定量PCR检测PLK1基因的表达,观察不同的siRNA作用强度,并计数细胞了解相应细胞的生长情况,western-blot观察PLK1表达蛋白的变化和流式细胞计数分析细胞周期改变。发现10种siRNA均可敲除PLK1基因表达的20 %以上,其中P1、P4和P9 3组敲除mRNA达80 ％以上,这3种siRNA及其混合物对PLK1基因mRNA的作用具有相应浓度效应,在25 nmol/L时达到最佳作用效果,而且相同浓度的混合物作用效果更好（超过95%）,PLK1表达蛋白质明显降低,细胞周期在G2期受到阻碍。72 h后的各种siRNA浓度下细胞生长变化与PLK1基因的mRNA水平变化相一致。结果表明化学合成的特异siRNA对SW480细胞中PLK1基因表达具有消除作用,混合物作用更强,在细胞水平上抑制了SW480细胞的分裂生长。     

Liver kinase B1 regulates the centrosome via PLK1

Liver kinase B1 (LKB1) is a tumor suppressor mutationally inactivated in Peutz–Jeghers syndrome (PJS) and various sporadic cancers. Although LKB1 encodes a kinase that possesses multiple functions, no individual hypothesis posed to date has convincingly explained how loss of LKB1 contributes to carcinogenesis. In this report we demonstrated that LKB1 maintains genomic stability through the regulation of centrosome duplication. We found that LKB1 colocalized with centrosomal proteins and was situated in the mitotic spindle pole. LKB1 deficiency-induced centrosome amplification was independent of AMP-activated protein kinase (AMPK), a well-defined substrate of LKB1. Cells lacking LKB1 exhibited an increase in phosphorylated and total Polo-like kinase 1 (PLK-1), NIMA-related kinase 2 (NEK2), and ninein-like protein (NLP). Overexpression of active PLK1 (T210D) reversed the inhibition of LKB1 on centrosome amplification. In contrast, depletion of PLK1 with siRNA or suppression of PLK1 kinase activity with BTO-1 (5-Cyano-7-nitro-2-benzothiazolecarboxamide-3-oxide) abrogated LKB1 deficiency-induced centrosome amplification. We further characterized that LKB1 phosphorylated and activated AMPK-related kinase 5 (NUAK1 or ARK5) that in turn increased the phosphorylation of MYPT1, enhanced the binding between MYPT1–PP1 and PLK1, and conferred an effective dephosphorylation of PLK1. More importantly, we noted that LKB1-deficient cells exhibited multiple nuclear abnormalities, such as mitotic delay, binuclear, polylobed, grape, large, and micronuclear. Immediate depletion of LKB1 resulted in the accumulation of multiploidy cells. Expression of LKB1 is reversely correlated with the levels of PLK1 in human cancer tissues. Thus, we have uncovered a novel function of LKB1 in the maintenance of genomic stability through the regulation of centrosome mediated by PLK1.     

PTEN regulates PLK1 and controls chromosomal stability during cell division

PTEN functions as a guardian of the genome through multiple mechanisms. We have previously established that PTEN maintains the structural integrity of chromosomes. In this report, we demonstrate a fundamental role of PTEN in controlling chromosome inheritance to prevent gross genomic alterations. Disruption of PTEN or depletion of PTEN protein phosphatase activity causes abnormal chromosome content, manifested by enlarged or polyploid nuclei. We further identify polo-like kinase 1 (PLK1) as a substrate of PTEN phosphatase. PTEN can physically associate with PLK1 and reduce PLK1 phosphorylation in a phosphatase-dependent manner. We show that PTEN deficiency leads to PLK1 phosphorylation and that a phospho-mimicking PLK1 mutant causes polyploidy, imitating functional deficiency of PTEN phosphatase. Inhibition of PLK1 activity or overexpression of a non-phosphorylatable PLK1 mutant reduces the polyploid cell population. These data reveal a new mechanism by which PTEN controls genomic stability during cell division.