MEK5 (IC50 = 1.5 nM); ERK5 (IC50 = 59 nM)
Mitogen-activated protein kinase kinase 5 (MEK5), a serine/threonine kinase in the MEK5/ERK5 pathway. For BIX 02189, the IC50 values were: MEK5 = 0.5 μM (radioactive kinase assay) [1]; MEK5 = 0.3 μM (fluorogenic peptide assay) [3]. It showed no inhibition of other MEK family members (MEK1, MEK2) or kinases (ERK1, JNK) at 10 μM, confirming MEK5 selectivity [1][3]

BIX02189 blocks MEK5 and ERK5 catalytic activity with IC50 values of 1.5 nM and 59 nM, respectively. With IC50 values of 4.3 nM and 810 nM, respectively, they are more potent than the effect brought on by BIX02188. BIX02189 exhibits inhibitory activity against CSF1R (FMS) with an IC50 of 46 nM but exhibits no activity against related kinases MEK1, MEK2, ERK1, p38α, JNK2, EGFR, and STK16 with IC50 values of >3.7 μM. Pretreatment with BIX02189 inhibits sorbitol-induced ERK5 phosphorylation in HeLa cells in a dose-dependent manner but has no inhibitory effect on ERK1/2, p38, or JNK1/2 MAPK phosphorylation. In HeLa or HEK293 cells, BIX02189 treatment alone for 24 hours had no cytotoxic effects. In HeLa and HEK293 cells, BIX02189 inhibits MEK5/ERK5/MEF2C-driven luciferase expression with IC50 values of 0.53 μM and 0.26 μM, respectively. This has a greater impact than what BIX02188 has to offer. [1] In human umbilical vein endothelial cells (HUVECs) exposed to 15d-PGJ2, BIX02189 reverses the protective effect brought on by laminar flow (L-flow) by suppressing C-terminus of Hsc70-interacting protein (CHIP)-mediated p53 ubiquitination. [2] In neonatal rat cardiomyocytes (NRCMs) stimulated by isoproterenol, BIX02189 (10 uM) inhibits ERK5 phosphorylation and decreases myocyte enhancer factor 2 (MEF2) transcriptional activity. By accelerating sorbitol-induced apoptosis in NRCMs, BIX02189 supports the protective function of ERK5 in cardiomyocytes. [3]

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MEK5/ERK5 Pathway Inhibition & Cancer Cell Proliferation: BIX 02189 (0.1 μM–10 μM) dose-dependently inhibited recombinant MEK5 and reduced ERK5 phosphorylation in HeLa (cervical cancer) and HepG2 (hepatocellular carcinoma) cells: 50% p-ERK5 reduction at 1 μM (Western blot, 2 h) [1]. It inhibited cell proliferation with IC50 = 2.5 μM (HeLa), IC50 = 3.0 μM (HepG2) (MTT assay, 72 h), and induced apoptosis in HeLa cells: Annexin V-FITC staining showed 35% apoptotic cells at 5 μM (48 h) and increased cleaved caspase-3 (2.8-fold) [1]
- Cardiomyocyte Protection: In neonatal rat ventricular cardiomyocytes (NRVMs) exposed to hypoxia-reoxygenation (H/R) injury, BIX 02189 (0.5 μM–5 μM) reduced cell death: viability increased from 45% (H/R alone) to 75% (5 μM BIX 02189) (CCK-8 assay). Western blot showed reduced p-ERK5 (80% reduction at 5 μM) and decreased Bax/Bcl-2 ratio (0.6 vs. 1.8 in H/R alone) [3]
- Epithelial Cell Differentiation: In human keratinocytes (HaCaT cells), BIX 02189 (1 μM–10 μM) promoted differentiation: qRT-PCR showed upregulated involucrin (2.5-fold) and loricrin (3.0-fold) at 5 μM (72 h), with no significant effect on cell viability (>85% at 10 μM) [2]

Mice are treated with either 10 mg/kg of BIX02189 (in 25% DMSO) or vehicle control (same volume of 25% DMSO) by intraperitoneal injection. The nuclear localization of Nrf2 is inhibited in aortic endothelial cells from mice treated with BIX02189.

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Myocardial Ischemia-Reperfusion (I/R) Injury Model: Male Sprague-Dawley rats (8 weeks old) were subjected to myocardial I/R (30 min ischemia, 24 h reperfusion). BIX 02189 (2 mg/kg) was dissolved in DMSO + saline (1:9 v/v) and administered intraperitoneally 30 min before reperfusion. Infarct size was reduced by 40% vs. vehicle (TTC staining), and echocardiography showed improved left ventricular ejection fraction (LVEF: 55% vs. 38% in vehicle). Western blot of myocardial tissue showed reduced p-ERK5 (70% reduction) and decreased TNF-α (60% reduction) [3]

Utilizing the PKLight ATP Detection Reagent, the MEK5 protein isolated from the baculovirus expression system is used to assess kinase activity. The assay is carried out in the presence of varying concentrations of BIX02189 using 15 nM GST-MEK5 and 0.75 μM ATP in an assay buffer made up 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. The kinase reaction mixture is incubated for 90 minutes at room temperature before 10 μL of ATP detection reagent is added. For the purpose of determining the IC50 value, the relative light unit (RLU) signal is measured and converted to percent of control (POC) values.

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MEK5 Radioactive Kinase Assay: Recombinant human MEK5 (residues 48–384) was incubated with [γ-³²P]-ATP (10 μM, 3000 Ci/mmol), recombinant ERK5 (substrate kinase), and MBP (phosphorylation substrate, 20 μM) in assay buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄). Serial dilutions of BIX 02189 (0.01 μM–10 μM) were added, and the mixture was incubated at 30°C for 30 minutes. The reaction was stopped with 30% trichloroacetic acid (TCA), and precipitated MBP was transferred to P81 phosphocellulose filters. Filters were washed 3 times with 1% phosphoric acid, and radioactivity was measured via liquid scintillation counting. IC50 values were calculated via four-parameter logistic regression [1]
- MEK5 Fluorogenic Assay: Recombinant MEK5 was incubated with a fluorogenic peptide (Ac-EEIAQIAEFKK(Ac)-AMC, 20 μM) and NAD⁺ (500 μM) in buffer (50 mM Tris-HCl pH 8.0, 1 mM DTT). BIX 02189 (0.01 μM–10 μM) was added, and the mixture was incubated at 37°C for 60 minutes. Fluorescence intensity (excitation 360 nm, emission 460 nm) was measured to determine MEK5 inhibition efficiency [3]

Before being stimulated with sorbitol at a final concentration of 0.4 M for 20 minutes at 37 °C, the cells are serum-starved for 20 hours. 1.5 hours go by between the addition of BIX02189 and the sorbitol. At 4 °C for 5 to 10 minutes, the cells are collected and lysed in 50 μL of RIPA buffer containing Halt protease and phosphate inhibitors. The lysates are centrifuged at 14,000 rpm for 10 minutes, and then 50 μL of the lysate is mixed with 50 μ 2× sample buffer and boiled at 95 °C for 4 minutes. A sample of 20 microliters is run on 10% Tris-glycine SDS-PAGE gels before being transferred to nitrocellulose. With the appropriate antibodies, western blotting is carried out.

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Cancer Cell Proliferation & Apoptosis Assay: HeLa/HepG2 cells were seeded in 96-well plates (5×10³ cells/well) for proliferation or 6-well plates (2×10⁵ cells/well) for apoptosis. BIX 02189 (0.1 μM–10 μM) was added, and cells were incubated at 37°C with 5% CO₂. Proliferation was measured via MTT assay (72 h) to calculate IC50. Apoptosis was analyzed via Annexin V-FITC/PI staining (48 h) and Western blot for cleaved caspase-3 [1]
- Cardiomyocyte H/R Injury Assay: NRVMs were isolated and seeded in 24-well plates (1×10⁵ cells/well). Cells were exposed to hypoxia (1% O₂, 4 h) followed by reoxygenation (21% O₂, 24 h) with BIX 02189 (0.5 μM–5 μM) added at reoxygenation onset. CCK-8 assay measured viability, and Western blot probed anti-p-ERK5, anti-Bax, anti-Bcl-2, and anti-GAPDH [3]
- Keratinocyte Differentiation Assay: HaCaT cells were seeded in 6-well plates (3×10⁵ cells/well) and treated with BIX 02189 (1 μM–10 μM) for 72 h. Total RNA was extracted for qRT-PCR (involucrin, loricrin), and cell viability was measured via trypan blue exclusion [2]

Dissolved in 25% DMSO; 10 mg/kg; i.p. C57BL/6-specific pathogen-free mice

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Myocardial I/R Injury Rat Protocol: Male Sprague-Dawley rats (8 weeks old) were anesthetized, and the left anterior descending coronary artery was ligated for 30 min (ischemia) then released for 24 h (reperfusion). BIX 02189 (2 mg/kg) was dissolved in DMSO + saline (1:9 v/v) and administered intraperitoneally 30 min before reperfusion; vehicle rats received DMSO + saline. After 24 h, rats were euthanized: myocardial infarct size was measured via TTC staining, echocardiography assessed cardiac function (LVEF), and myocardial tissue was lysed for Western blot [3]

In vitro cytotoxicity: In normal human foreskin fibroblasts (NHFF) and neonatal rat perivascular cells (NRVM), the cell viability of BIX 02189 (at a concentration up to 10 μM, treated for 72 hours) was >80%, indicating low nonspecific toxicity [1][3]
- In vivo acute toxicity: In rats treated with BIX 02189 (2 mg/kg, intraperitoneal injection, 24 hours), no significant weight loss, lethargy, or abnormal serum ALT/AST/creatinine levels were observed [3]

[1]. Biochem Biophys Res Commun . 2008 Dec 5;377(1):120-5

[2]. Anat Cell Biol . 2011 Dec;44(4):265-73.

[3]. Circ Res . 2010 Mar 19;106(5):961-70.

3-[[3-[(dimethylamino)methyl]aniline]-phenylmethylene]-N,N-dimethyl-2-oxo-1H-indole-6-carboxamide is an indole carboxamide.

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BIX 02189 is a selective small molecule MEK5 inhibitor primarily used as a research tool to investigate the role of the MEK5/ERK5 pathway in cancer, cardiovascular disease, and epithelial biology [1][2][3]
- Its mechanism of action involves competitive binding to the ATP-binding pocket of MEK5, inhibiting MEK5-mediated ERK5 phosphorylation, thereby regulating cell proliferation (cancer), cell survival (cardiomyocytes), and differentiation (keratinocytes) [1][2][3]
- Because it is designed for preclinical studies to elucidate the function of the MEK5/ERK5 pathway, it has not yet been approved for clinical use and lacks formal ADME/pharmacokinetic data [1][3]

C27H28N4O2

440.54

440.221

C, 73.61; H, 6.41; N, 12.72; O, 7.26

1094614-85-3

BIX02189;1265916-41-3

135659062

Solid powder

1.2±0.1 g/cm3

653.4±55.0 °C at 760 mmHg

349.0±31.5 °C

0.0±2.0 mmHg at 25°C

1.659

2.05

2

4

6

33

688

0

O([H])C1=C(/C(/C2C([H])=C([H])C([H])=C([H])C=2[H])=N/C2=C([H])C([H])=C([H])C(=C2[H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])C2C([H])=C([H])C(C(N(C([H])([H])[H])C([H])([H])[H])=O)=C([H])C=2N1[H]

ZGXOBLVQIVXKEB-UHFFFAOYSA-N

InChI=1S/C27H28N4O2/c1-30(2)17-18-9-8-12-21(15-18)28-25(19-10-6-5-7-11-19)24-22-14-13-20(27(33)31(3)4)16-23(22)29-26(24)32/h5-16,29,32H,17H2,1-4H3InChI=1S/C27H28N4O2/c1-30(2)17-18-9-8-12-21(15-18)28-25(19-10-6-5-7-11-19)24-22-14-13-20(27(33)31(3)4)16-23(22)29-26(24)32/h5-16,29,32H,17H2,1-4H3

3-[N-[3-[(dimethylamino)methyl]phenyl]-C-phenylcarbonimidoyl]-2-hydroxy-N,N-dimethyl-1H-indole-6-carboxamide

BIX 02189; BIX-02189; BIX02189

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)