Hesperadin

Catalog No.S1529 Batch:S152901

Technical Data

Formula	C₂₉H₃₂N₄O₃S
Molecular Weight	516.65	CAS No.	422513-13-1
Solubility (25°C)*	In vitro	DMSO	103 mg/mL (199.36 mM)
		Ethanol	1 mg/mL (1.93 mM)
		Water	Insoluble
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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Biological Activity

Description

Hesperadin potently inhibits Aurora B with IC50 of 250 nM in a cell-free assay. It markedly reduces the activity of AMPK, Lck, MKK1, MAPKAP-K1, CHK1 and PHK while it does not inhibit MKK1 activity in vivo.

Targets

TbAUK1 ^[2] (Cell-free assay)	Aurora B (human) ^[1] (Cell-free assay)
40 nM	250 nM

In vitro

Hesperadin inhibits the ability of immunoprecipitated Aurora B to phosphorylate histone H3 with IC50 of 250 nM and markedly reduces the activity of other kinases (AMPK, Lck, MKK1, MAPKAP-K1, CHK1, and PHK) at a concentration of 1 μM. In contrast, only 20-100 nM of Hesperadin is sufficient to induce the loss of mitotic histone H3-Ser10 phosphorylation in HeLa cells. Hesperadin treatment causes defects in mitosis and cytokinesis, leading to stoppage of proliferation of HeLa cells and polyploidization, which can be specifically ascribed to the inhibition of Aurora B function during the process of chromosome attachment. Hesperadin (100 nM) quickly overrides the mitotic arrest induced by taxol or monastrol but not by nocodazole. Hesperadin and nocodazole treatment in HeLa cells abolishes kinetochore localization of BubR1 and diminishes the intensity of Bub1 at kinetochores, suggesting that Aurora B function is required for efficient kinetochore recruitment of BubR1 and Bub1, which in turn might be necessary for prolonged checkpoint signaling. ^[1] Hesperadin prevents the phosphorylation of recombinant trypanosome histone H3 by the T. brucei Aurora kinase-1 (TbAUK1) from pathogenic Trypanosoma brucei with IC50 of 40 nM in vitro kinase assays. Hesperadin significantly inhibits cell growth of cultured infectious bloodstream forms (BF) with IC50 of 48 nM, and only weakly inhibits cell growth of insect stage procyclic forms (PF) with IC50 of 550 nM. ^[2]

Protocol (from reference)

Kinase Assay:^{^[1]}	The Aurora B kinase assay For the Aurora B kinase assay, HeLa cells are lysed in a buffer containing 50 mM NaCl. The whole cell extract is spun at 13,000 rpm for 20 minutes at 4 °C using a table top centrifuge. The pellet obtained from 200 mg of whole cell extract is extracted again in 15 mL lysis buffer containing 250 mM NaCl in order to obtain active Aurora B kinase from mitotic chromatin. The low speed supernatant of the latter extract is used for immunoprecipitation. Monoclonal mouse anti–AIM-1, or mouse anti-HA, is coupled to GammaBind Plus Sepharose, and beads are rotated over-end in the extract for 90 minutes at 4 °C. Beads are washed, aliquoted, and washed in kinase buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 10 mM NaF). The kinase assay is performed with 10 μL beads in 20 μL kinase buffer containing 5 μg histone H3, 10 μM ATP, 2.5 μCi [γ-³²P]ATP, and different concentrations of Hesperadin for 20 minutes at 37 °C. SDS sample buffer is added, and samples are boiled and resolved by SDS-PAGE. The gel is dried, and the radioactive signal is detected by PhosphorImager analysis. The data is analyzed using ImageQuant software.
Cell Assay:^{^[1]}	Cell lines HeLa cells and PtK1 cells Concentrations Final concentration ~500 nM Incubation Time 24 and 48 hours Method Cells are exposed to different concentrations of Hesperadin for 24 and 48 hours. At indicated time points, methanol-fixed cell samples are washed with PBS and subsequently stained in PI buffer (50 μg/mL propidium iodide, 10 mM Tris, pH 7.5, 5 mM MgCl₂, 200 μg/mL RNase A) for 20-40 minutes at 37 °C. The DNA content is determined by flow cytometry.

Kinase Assay:^{^[1]}

The Aurora B kinase assay
For the Aurora B kinase assay, HeLa cells are lysed in a buffer containing 50 mM NaCl. The whole cell extract is spun at 13,000 rpm for 20 minutes at 4 °C using a table top centrifuge. The pellet obtained from 200 mg of whole cell extract is extracted again in 15 mL lysis buffer containing 250 mM NaCl in order to obtain active Aurora B kinase from mitotic chromatin. The low speed supernatant of the latter extract is used for immunoprecipitation. Monoclonal mouse anti–AIM-1, or mouse anti-HA, is coupled to GammaBind Plus Sepharose, and beads are rotated over-end in the extract for 90 minutes at 4 °C. Beads are washed, aliquoted, and washed in kinase buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 10 mM NaF). The kinase assay is performed with 10 μL beads in 20 μL kinase buffer containing 5 μg histone H3, 10 μM ATP, 2.5 μCi [γ-³²P]ATP, and different concentrations of Hesperadin for 20 minutes at 37 °C. SDS sample buffer is added, and samples are boiled and resolved by SDS-PAGE. The gel is dried, and the radioactive signal is detected by PhosphorImager analysis. The data is analyzed using ImageQuant software.

Cell Assay:^{^[1]}

Cell lines
HeLa cells and PtK1 cells
Concentrations
Final concentration ~500 nM
Incubation Time
24 and 48 hours
Method
Cells are exposed to different concentrations of Hesperadin for 24 and 48 hours. At indicated time points, methanol-fixed cell samples are washed with PBS and subsequently stained in PI buffer (50 μg/mL propidium iodide, 10 mM Tris, pH 7.5, 5 mM MgCl₂, 200 μg/mL RNase A) for 20-40 minutes at 37 °C. The DNA content is determined by flow cytometry.

References

Customer Product Validation

: Data from [Nat Commun, 2011, 2, 316]

: Data from [Nat Commun, 2011, 2, 316 ]

: ,

: , , Dr. Zhang of Tianjin Medical University

Selleck's Hesperadin has been cited by 45 publications

Dynamic phosphorylation of FOXA1 by Aurora B guides post-mitotic gene reactivation [ Cell Rep, 2024, 43(9):114739]	PubMed: 39276350
Visualization strategies to aid interpretation of high-dimensional genotoxicity data [ Environ Mol Mutagen, 2024, 10.1002/em.22604]	PubMed: 38757760
Hesperadin suppresses pancreatic cancer through ATF4/GADD45A axis at nanomolar concentrations [ Oncogene, 2022, 10.1038/s41388-022-02328-4]	PubMed: 35551503
CDC20-Mediated hnRNPU Ubiquitination Regulates Chromatin Condensation and Anti-Cancer Drug Response [ Cancers (Basel), 2022, 14(15)3732]	PubMed: 35954396
Role of aurora kinase B in regulating resistance to paclitaxel in breast cancer cells [ Hum Cell, 2022, 10.1007/s13577-022-00675-8]	PubMed: 35088239
TubulinTracker, a Novel In Vitro Reporter Assay to Study Intracellular Microtubule Dynamics, Cell Cycle Progression, and Aneugenicity [ Toxicol Sci, 2022, kfac008]	PubMed: 35094094
In vitro human cell-based aneugen molecular mechanism assay [ Environ Mol Mutagen, 2022, 63(3):151-161]	PubMed: 35426156
Targeting Aurora B kinase prevents and overcomes resistance to EGFR inhibitors in lung cancer by enhancing BIM- and PUMA-mediated apoptosis [ Cancer Cell, 2021, S1535-6108(21)00383-4]	PubMed: 34388376
Bub1 and CENP-U redundantly recruit Plk1 to stabilize kinetochore-microtubule attachments and ensure accurate chromosome segregation [ Cell Rep, 2021, 36(12):109740]	PubMed: 34551298
PRMT6-mediated H3R2me2a guides Aurora B to chromosome arms for proper chromosome segregation. [ Nat Commun, 2020, 11(1):612]	PubMed: 32001712

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