BIX 02188

Catalog No.S1530 Batch:S153001

Technical Data

Formula	C₂₅H₂₄N₄O₂
Molecular Weight	412.48	CAS No.	1094614-84-2
Solubility (25°C)*	In vitro	DMSO	43 mg/mL (104.24 mM)
		Ethanol	3 mg/mL (7.27 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

BIX02188 is a selective inhibitor of MEK5 with IC50 of 4.3 nM, also inhibits ERK5 catalytic activity with IC50 of 810 nM, and does not inhibit closely related kinases MEK1, MEK2, ERK2, and JNK2.

Targets

MEK5 ^[1]

4.3 nM

In vitro

BIX02188 significantly blocks MEK5 catalytic activity with IC50 of 4.3 nM and inhibits ERK5 catalytic activity with IC50 of 0.83 μM. It shows no activity against closely related kinases MEK1, MEK2, ERK1, p38α, JNK2, TGFβR1, EGFR, and STK16 with IC50 values of 1.8 μM for TGFβR1, 3.9 μM for p38α, and >6.3 μM for other kinases. Pretreatment with BIX02188 inhibits sorbitol-induced phosphorylation of ERK5 in HeLa cells in a dose dependent manner and displays no inhibitory activity against the phosphorylation of ERK1/2, p38, and JNK1/2 MAPKs. BIX02188 treatment alone for 24 hours in HeLa or HEK293 cells does not show any cytotoxic effect. BIX02188 inhibits transcriptional activation of MEF2C through the MEK5/ERK5 signaling cascade in active MEK5/ERK5/MEF2C-driven luciferase expression system in HeLa and HEK293 cells with IC50 values of 1.15 μM and 0.82 μM, respectively. ^[1] BIX02188 also inhibits phosphorylation of BMK1 in bovine lung microvascular endothelial cells (BLMECs) which are stimulated with 300 μM H₂O₂, in a dose-dependent manner, with IC50 of 0.8 μM, specifically by blocking the MEK5 signal pathway. BIX02188 completely reverses the inhibitory effect on TNF-mediation; JNK activation had a similar effect on BMK1 inhibition, suggesting that it inhibits TNF signaling through activation of the MEK5-BMK1 signaling pathway. ^[2]

Protocol (from reference)

Kinase Assay:^{^[1]}	Catalytic assay MEK5 protein isolated from a baculovirus expression system is used to measure kinase activity utilizing PKLight ATP Detection Reagent. The assay is performed using 15 nM GST-MEK5 and 0.75 μM ATP in assay buffer consisting of 25 mM Hepes, pH 7.5, 10 mM MgCl₂, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 μM Na₃VO₄, 0.5 mM DTT and 1% DMSO in the presence of varying concentrations of BIX02188. The kinase reaction mixture is incubated for 90 minutes at room temperature followed by addition of 10 μL of an ATP detection reagent for 15 minutes. The relative light unit (RLU) signal is measured and the RLU signals are converted to percent of control (POC) values for the determination of IC50 value.
Cell Assay:^{^[1]}	Cell lines HeLa cells Concentrations Dissolved in DMSO, final concentration ~10 μM Incubation Time Pretreatment for 1.5 hours Method The cells are serum starved for 20 hours prior to stimulation with sorbitol at a final concentration of 0.4 M for 20 minutes at 37 °C. BIX02188 is added 1.5 hours prior to the addition of sorbitol. The cells are harvested and lysed in 50 μL RIPA buffer containing Halt protease and phosphate inhibitors at 4 °C for 5-10 minutes. The lysates are centrifuged for 10 minutes at 14,000 rpm and 50 μL lysate is added to 50 μl 2× sample buffer and boiled for 4 minutes at 95 °C. A 20 µl sample is run on SDS–PAGE 10% Tris-glycine gels and transferred to nitrocellulose. Western blotting is done with appropriate antibodies.

Kinase Assay:^{^[1]}

Catalytic assay
MEK5 protein isolated from a baculovirus expression system is used to measure kinase activity utilizing PKLight ATP Detection Reagent. The assay is performed using 15 nM GST-MEK5 and 0.75 μM ATP in assay buffer consisting of 25 mM Hepes, pH 7.5, 10 mM MgCl₂, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 μM Na₃VO₄, 0.5 mM DTT and 1% DMSO in the presence of varying concentrations of BIX02188. The kinase reaction mixture is incubated for 90 minutes at room temperature followed by addition of 10 μL of an ATP detection reagent for 15 minutes. The relative light unit (RLU) signal is measured and the RLU signals are converted to percent of control (POC) values for the determination of IC50 value.

Cell Assay:^{^[1]}

Cell lines
HeLa cells
Concentrations
Dissolved in DMSO, final concentration ~10 μM
Incubation Time
Pretreatment for 1.5 hours
Method
The cells are serum starved for 20 hours prior to stimulation with sorbitol at a final concentration of 0.4 M for 20 minutes at 37 °C. BIX02188 is added 1.5 hours prior to the addition of sorbitol. The cells are harvested and lysed in 50 μL RIPA buffer containing Halt protease and phosphate inhibitors at 4 °C for 5-10 minutes. The lysates are centrifuged for 10 minutes at 14,000 rpm and 50 μL lysate is added to 50 μl 2× sample buffer and boiled for 4 minutes at 95 °C. A 20 µl sample is run on SDS–PAGE 10% Tris-glycine gels and transferred to nitrocellulose. Western blotting is done with appropriate antibodies.

References

Customer Product Validation

: Data from [Exp Neurol, 2012, 238(2), 209-17]

: Data from [Data independently produced by , , J Leukoc Biol, 2016, 99(1):143-52]

Selleck's BIX 02188 has been cited by 13 publications

MAP kinase ERK5 modulates cancer cell sensitivity to extrinsic apoptosis induced by death-receptor agonists [ Cell Death Dis, 2023, 14(11):715]	PubMed: 37919293
MAP kinase ERK5 modulates cancer cell sensitivity to extrinsic apoptosis induced by death-receptor agonists [ Cell Death Dis, 2023, 10.1038/s41419-023-06229-6]	PubMed: 37919293
Non-canonical Targets of HIF1a Impair Oligodendrocyte Progenitor Cell Function [ Cell Stem Cell, 2021, 28(2):257-272.e11]	PubMed: 33091368
ERK5 Inhibition Induces Autophagy-Mediated Cancer Cell Death by Activating ER Stress [ Front Cell Dev Biol, 2021, 9:742049]	PubMed: 34805151
Tumor-derived exosomes antagonize innate antiviral immunity. [ Nat Immunol, 2018, 19(3):233-245]	PubMed: 29358709
Dual role of ERK5 in the regulation of T cell receptor expression at the T cell surface [Rovira-Clavé X, et al. J Leukoc Biol, 2016, 99(1):143-52]	PubMed: 26302753
BAFF activation of the ERK5 MAP kinase pathway regulates B cell survival. [ J Exp Med, 2015, 212(6):883-92]	PubMed: 25987726
Erk5 inhibits endothelialmigration via KLF2-dependent down regulation of PAK1 [Komaravolu RK, et al. Cardiovasc Res, 2015, 105(1):86-95]	PubMed: 25388666
Erk5 inhibits endothelial migration via KLF2-dependent down-regulation of PAK1. [Komaravolu RK, et al. Cardiovasc Res, 2015, 105(1):86-95]	PubMed: 25388666
Increased IGF-IEc expression and mechano-growth factor production in intestinal muscle of fibrostenotic Crohn's disease and smooth muscle hypertrophy [ Am J Physiol Gastrointest Liver Physiol, 2015, 309(11):G888-99]	PubMed: 26428636

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SHIPPING AND STORAGE
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