poloxin and thymoquinone

For almost a decade, there has been much interest in the development of chemical inhibitors of Polo-like kinase 1 (Plk1) protein interactions. Plk1 is a master regulator of the cell division cycle that controls numerous substrates. It is a promising target for cancer drug development. Inhibitors of the kinase domain of Plk1 had some success in clinical trials. However, they are not perfectly selective. In principle, Plk1 can also be inhibited by interfering with its protein interaction domain, the Polo-Box Domain (PBD). Selective chemical inhibitors of the PBD would constitute tools to probe for PBD-dependent functions of Plk1 and could be advantageous in cancer therapy. The discovery of Poloxin and thymoquinone as PBD inhibitors indicated that small, cell-permeable chemical inhibitors could be identified. Other efforts followed, including ours, reporting additional molecules capable of blocking the PBD. It is now clear that, unfortunately, most of these compounds are non-specific protein alkylators (defined here as groups covalently added via a carbon) that have little or no potential for the development of real Plk1 PBD-specific drugs. This situation should be minded by biologists potentially interested in using these compounds to study Plk1. Further efforts are needed to develop selective, cell-permeable PBD inhibitors.

Topics: Alkylation; Antineoplastic Agents; Benzoates; Benzoquinones; Cell Cycle Proteins; Glycine; Humans; Mitosis; Neoplasms; Polo-Like Kinase 1; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Quinones; Sulfones

Polo-like kinase 1 (Plk1) plays several roles in cell division and it is a recognized cancer drug target. Plk1 levels are elevated in cancer and several types of cancer cells are hypersensitive to Plk1 inhibition. Small molecule inhibitors of the kinase domain (KD) of Plk1 have been developed. Their selectivity is limited, which likely contributes to their toxicity. Polo-like kinases are characterized by a Polo-Box Domain (PBD), which mediates interactions with phosphorylation substrates or regulators. Inhibition of the PBD could allow better selectivity or result in different effects than inhibition of the KD. In vitro screens have been used to identify PBD inhibitors with mixed results. We developed the first cell-based assay to screen for PBD inhibitors, using Bioluminescence Resonance Energy Transfer (BRET). We screened through 112 983 compounds and characterized hits in secondary biochemical and biological assays. Subsequent Structure-Activity Relationship (SAR) analysis on our most promising hit revealed that it requires an alkylating function for its activity. In addition, we show that the previously reported PBD inhibitors thymoquinone and Poloxin are also alkylating agents. Our cell-based assay is a promising tool for the identification of new PBD inhibitors with more drug-like profiles using larger and more diverse chemical libraries.

Topics: Alkylating Agents; Benzoates; Benzoquinones; Bioluminescence Resonance Energy Transfer Techniques; Cell Cycle Proteins; HEK293 Cells; High-Throughput Screening Assays; Humans; Polo-Like Kinase 1; Protein Interaction Domains and Motifs; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Quinones; Small Molecule Libraries; Structure-Activity Relationship

The expression of polo-like kinase 1 (Plk1) correlates with malignancy and is thus recognized as a target for cancer therapy. In addition to the development of ATP-competitive Plk1 inhibitors, the polo-box domain (PBD), a unique functional domain of PLKs, is being targeted to develop Plk1-specific inhibitors. However, the action mechanisms of these two classes of Plk1 inhibitors have not been thoroughly evaluated. Here, we evaluate the differences in cellular effects of ATP-binding domain inhibitors (BI 2536, GSK 461364) and PBD inhibitors (poloxin, thymoquinone) to determine their mechanisms of Plk1 inhibition. Our data show that BI 2536 and GSK461364 increased the population of cells in the G2/M phase compared with controls, while treatment with poloxin and thymoquinone increased cell population in the S phase as well as in G2/M, in a p53-independent manner. The population of cells staining positively for p-Histone H3 and MPM2, mitotic index, was increased by treatment with BI 2536 or GSK461364, but not by treatment with poloxin or thymoquinone. Furthermore, treatment with BI 2536 or GSK461364 resulted in activation of the BubR1 spindle checkpoint kinase, suggesting that treatment with ATP-binding domain inhibitors induces metaphase arrest. However, the administration of poloxin and thymoquinone resulted in an increase in p21(WAF1) and S arrest, indicating that PBD inhibitors also affected interphase before mitotic entry. Taken together, these data suggest that the PDB of Plk1 plays a role in S phase progression through interaction with other proteins, while its ATP-binding domain is important for regulating mitotic progression mediated by its catalytic activity involving consumption of ATP.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Apoptosis; Benzimidazoles; Benzoates; Benzoquinones; Binding Sites; Catalytic Domain; Cell Cycle Proteins; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Drug Design; Female; G2 Phase Cell Cycle Checkpoints; HeLa Cells; Humans; Inhibitory Concentration 50; Mitosis; Molecular Targeted Therapy; Polo-Like Kinase 1; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins; Pteridines; Quinones; S Phase Cell Cycle Checkpoints; Signal Transduction; Thiophenes; Time Factors; Uterine Cervical Neoplasms

Polo-like kinase 1 (Plk1) is widely established as one of the most promising targets in oncology. Although the protein kinase domain of Plk1 is highly conserved, the polo-box domain (PBD) of Plk1 provides a much more compelling site to specifically inhibit the localization and target binding of Plk1. We recently identified, via fluorescence polarization assay, the natural product derivative, Poloxin, as the first small-molecule inhibitor specifically targeting the function of the Plk1 PBD. In this study, we characterized its mitotic phenotype and its function in vitro and in vivo. Poloxin induces centrosome fragmentation and abnormal spindle and chromosome misalignment, which activate the spindle assembly checkpoint and prolong mitosis. Notably, centrosomal fragmentation induced by Poloxin is partially attributable to dysfunctional Kizuna, a key substrate of Plk1 at centrosomes. Moreover, Poloxin strongly inhibits proliferation of a panel of cancer cells by inducing mitotic arrest, followed by a surge of apoptosis. More important, we report, for the first time to our knowledge, that the PBD inhibitor, Poloxin, significantly suppresses tumor growth of cancer cell lines in xenograft mouse models by lowering the proliferation rate and triggering apoptosis in treated tumor tissues. The data highlight that targeting the PBD by Poloxin is a powerful approach for selectively inhibiting Plk1 function in vitro and in vivo.

Topics: Animals; Apoptosis; Benzoates; Benzoquinones; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Centrosome; Chromosomes, Human; Humans; M Phase Cell Cycle Checkpoints; Mice; Neoplasms; Polo-Like Kinase 1; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Proto-Oncogene Proteins; Quinones; Xenograft Model Antitumor Assays