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Research Area
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Description
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Non-Hodgkin's lymphoma,Small cell lung cancer |
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Biological Activity
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Description
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BI 2536 is a potent Plk1 inhibitor with IC50 of 0.83 nM. |
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Targets
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Plk1 |
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IC50 |
0.83 nM [1] |
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In Vitro
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BI 2536 blocks the activities of Plk2 and Plk3 to a slightly lesser extent with IC50 of 3.5 nM and 9.0 nM, respectively. BI 2536 displays more than 1000-fold selectivity relative to a panel of 63 other protein kinases with IC50 of >10 μM. In HeLa cells, BI 2536 treatment ranging from 10 - 100 nM leads to the blocking of the recruitment of γ-tubulin and phosphorylation of Apc6 at mitotic centrosomes, inhibition of cohesin release from chromosome arms, induction of monopolar spindles, as well as a range of other mitotic processes that are known to depend on Plk1. Consistent with the effects of Plk1 RNAi, BI 2536 treatment leads to the HeLa cells arrested in G2/M, subsequently a sub-G1 DNA peak indicative of DNA breakdown and apoptosis, and accumulated cleaved PARP p85 fragments in a concentration-dependent manner. BI 2536 inhibits the growth of a panel of 32 human cancer cell lines with EC50 of 2-25 nM, while blocking the proliferation of exponentially growing hTERT-RPE1, human umbilical vein endothelial cells (HUVECs), and normal rat kidney (NRK) cells with EC50 of 12-31 nM. [1] Plk1 inhibition by BI 2536 reduces the growth and viability of anaplastic thyroid carcinoma (ATC) cells such as CAL62, OCUT-1, SW1736, 8505C, and ACT-1 with EC50 values of 1.4-5.6 nM. [2] |
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In Vivo
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BI 2536 given i.v. once or twice per week is highly efficacious in diverse xenograft models with acceptable tolerability by inhibiting cell proliferation through a mitotic arrest, and subsequently induction of tumor-cell death. Administration of BI 2536 at 50 mg/kg once or twice per week significantly inhibits growth of HCT 116 xenografts with T/C of 15% and 0.3%, respectively. BI 2536 treatment twice-weekly also leads to excellent tumor-growth in BxPC-3 and A549 models with T/C of 5% and 14%, respectively. [1] |
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Clinical Trials
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A Phase II study of BI 2536 infusional treatment in patients over 60 years of age with refractory or relapsed acute myeloid leukaemia has been completed. |
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Features
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BI 2536 is the first potent and selective Plk1 inhibitor that induces all hallmarks of Plk1 inhibition. |
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Protocol
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Kinase Assay
[1]
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| Plk1 in vitro kinase assay |
Recombinant human Plk1 (residues 1-603) is expressed as N-terminal, GST-tagged fusion protein with a baculoviral expression system and purified by affinity chromatography with Glutathione-agarose. Enzyme activity assays for Plk1 are performed in the presence of serially diluted BI 2536 with 20 ng of recombinant kinase and 10 μg casein from bovine milk as the substrate. Kinase reactions are performed in a final volume of 60 μL for 45 minutes at 30 °C (15 mM MgCl2, 25 mM MOPS [pH 7.0], 1 mM DTT, 1% DMSO, 7.5 mM ATP, 0.3 μCi γ-33P-ATP). Reactions are terminated by the addition of 125 μL of ice-cold 5% TCA. After transfer of the precipitates to Multi-Screen mixed ester cellulose filter plates, plates are washed with 1% TCA and quantified radiometrically. Dose-response curve is used for calculating IC50 value. |
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Cell Assay
[1]
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| Cell Lines |
HeLa, A43, SKOV-3, HT-29, K562, A549, Saos-2, MCF7, HCT116, COLO 205, Hep G2, Raji and PC-3 cells, etc. |
| Concentrations |
Dissolved in DMSO, final concentrations ~1 μM |
| Incubation Time |
24 and 72 hours |
| Methods |
Cells are exposed to various concentrations of BI 2536 for 24, and 72 hours. Cell growth is assessed by the measurement of Alamar Blue dye conversion in a fluorescence spectrophotometer. For determining the DNA content of the cultures, cell suspensions are fixed in 80% ethanol, treated for 5 minutes with 0.25% Triton X-100 in PBS, and incubated with 0.1% RNase and 10 μg/mL propidium iodide (PI) in PBS for 20 minutes at RT. Cell-cycle profiles are determined by flow cytometric analysis. |
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Animal Study
[1]
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| Animal Models |
Female BomTac:NMRI-Foxn1nu mice injected subcutaneously with HCT 116, NCI-H460, or A549 cells |
| Formulation |
Formulated in hydrochloric acid (0.1 N), and diluted with 0.9% NaCl |
| Doses |
~50 mg/kg |
| Administration |
Injection i.v. once or twice per week |
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