MEK5 (IC50 = 1.5 nM); ERK5 (IC50 = 59 nM); CSF1R (FMS) (IC50 = 46 nM); LCK (IC50 = 250 nM); JAK3 (IC50 = 440 nM); TGFβR1 (IC50 = 580 nM); RPS6KA6 (RSK4) (IC50 = 990 nM); RPS6KA3 (RSK2) (IC50 = 2.1 μM); FGFR1 (IC50 = 1 μM); KIT (IC50 = 1.1 μM); ABL1 (IC50 = 2.4 μM); MAPK14 (p38 alpha) (IC50 = 3.7 μM); SRC (IC50 = 7.6 μM)
Mitogen-activated protein kinase kinase 5 (MEK5) inhibitor (IC₅₀ = 1.5 nM).
Extracellular signal-regulated kinase 5 (ERK5) inhibitor (IC₅₀ = 59 nM). [1]
MEK5 (IC50 = 1.5 nM)
ERK5 (IC50 = 59 nM)[2]
MEK5/ERK5 pathway (described as a specific MEK5 inhibitor). [3]

BIX02189 blocks phosphorylation of ERK5, without affecting phosphorylation of ERK1/2 in sorbitol-stimulated HeLa cells. A dose-dependent mechanism underlies BIX02189's inhibition of ERK5 phosphorylation[1]. Vascular smooth muscle cells' (VSMCs') proliferation is inhibited by fluvastatin in response to advanced glycation endproducts (AGE). VSMCs are given AGE treatment with or without Fluvastatin to see if this effect is present, and after that, the cells are subjected to an MTT assay. Fluvastatin significantly inhibits the dose-dependent induction of cell proliferation by AGEs that is observed. The same outcomes are obtained with cell counting in addition to the MTT assay. When VSMCs receive BIX02189 as a pretreatment, the suppressive effect of Fluvastatin is avoided. Using Ad-CA-MEK5α, which encodes a mutant form of MEK5 (an upstream kinase of ERK5) that is constitutively active, it is also investigated whether activating ERK5 can inhibit proliferation. In the presence of Ad-CA-MEK5, AGE-induced proliferation as measured by the MTT assay and cell counting is significantly reduced, and Nrf2 depletion using siRNA restored AGE-induced proliferation[2].

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BIX02189 inhibited the catalytic activity of purified MEK5 enzyme in a dose-dependent manner with an IC₅₀ of 1.5 nM. [1]
BIX02189 inhibited the catalytic activity of purified ERK5 enzyme with an IC₅₀ of 59 nM. [1]
BIX02189 showed high selectivity for MEK5 over closely related kinases MEK1 and MEK2 (IC₅₀ > 6200 nM). It also did not significantly inhibit ERK1, ERK2, or JNK2 at concentrations up to 6300 nM. [1]
In a broad kinase selectivity panel (79 kinases), BIX02189 (tested at 3 or 10 µM) showed greater than 100-fold selectivity against 85 out of 87 kinases tested. At the tested concentrations, it inhibited only a limited number of kinases (e.g., CSF1R (FMS), RPS6KA3 (RSK2), RPS6KA6 (RSK4)) by more than 50%. [1]
Dose-response profiling against a selected kinase panel confirmed its primary potency against MEK5, with higher IC₅₀ values for other kinases (e.g., ABL: 2400 nM; CSF1R: 46 nM; FGFR1: 1000 nM; JAK3: 440 nM; KIT: 1100 nM; LCK: 250 nM; SRC: 7600 nM). [1]
In sorbitol-stimulated HeLa cells, BIX02189 (pre-incubated for 90 minutes) inhibited the phosphorylation of ERK5 in a dose-dependent manner, without affecting the phosphorylation of ERK1/2, p38, or JNK1/2. [1]
In a cellular trans-reporter assay (HeLa or HEK293T cells), BIX02189 inhibited MEK5/ERK5/MEF2C-driven luciferase gene expression in a dose-dependent manner after 18-24 hours of treatment, without showing cytotoxicity as assessed by an Alamar Blue assay. [1]
BIX02189 inhibited MEK5 catalytic activity in a dose-dependent manner with an IC50 of 1.5 nM, and ERK5 catalytic activity with an IC50 of 59 nM. It showed high selectivity, with IC50 values >6200 nM for MEK1 and MEK2, >6300 nM for ERK1, and >6200 nM for JNK2. When tested against a panel of 79 kinases at a single concentration (3 µM or 10 µM), BIX02189 exhibited greater than 100-fold selectivity against 85 out of 87 kinases. In sorbitol-stimulated HeLa cells, BIX02189 inhibited ERK5 phosphorylation in a dose-dependent manner but did not inhibit the phosphorylation of ERK1/2, p38, or JNK1/2. Furthermore, in a cellular trans-reporter assay system using HeLa or HEK293T cells co-transfected with constitutively active MEK5, ERK5, and a MEF2C-GAL4 fusion protein, BIX02189 inhibited MEF2C-driven luciferase reporter gene expression in a dose-dependent manner, without showing cytotoxicity as assessed by Alamar Blue assay. [2]
In Human Umbilical Vein Endothelial Cells (HUVECs), pretreatment with 10 µM BIX02189 for 16-24 hours inhibited laminar flow-induced (12 dynes/cm², 16-24 h) phosphorylation of ERK5, as well as the protein expression of downstream Nrf2 target genes HO-1 and NQO1, and the KLF2 target gene eNOS.
In a separate experiment, HUVECs were pretreated with 10 µM BIX02189 for 16-24 hours. Cellular fractionation followed by immunoblotting showed that BIX02189 strongly suppressed laminar flow-induced nuclear translocation of Nrf2. It also inhibited the flow-induced nuclear translocation of ERK5 itself.
Furthermore, in a reporter gene assay where HUVECs were co-transfected with a pGL3-ARE-luciferase construct, pretreatment with BIX02189 (concentration not specified for this assay, but methodology aligns with 10 µM pretreatment) inhibited laminar flow-induced activation of the antioxidant response element (ARE), which is dependent on Nrf2 transcriptional activity. [3]

Mice are administered 10 mg/kg of BIX02189 (in 25% DMSO) intraperitoneally, or a vehicle control (the same volume of 25% DMSO). In mice treated with BIX02189, the nuclear localization of Nrf2 is inhibited in the aortic endothelial cells[3].

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In an in vivo mouse model, intraperitoneal injection of BIX02189 (10 mg/kg, dissolved in 25% DMSO) inhibited the nuclear translocation of Nrf2 in endothelial cells of the thoracic aorta under conditions of steady laminar flow (atheroprotective region). This was assessed by en face immunofluorescence staining of the aortic endothelium. [3]

MEK5 protein isolated from the baculovirus expression system is used to measure kinase activity utilizing PKLight ATP Detection Reagent. The assay is conducted with 15 nM GST-MEK5 and 0.75 μM ATP in the presence of various concentrations of BIX02189 in 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. Following a 90-minute incubation period at room temperature, 10 μL of an ATP detection reagent is added to the kinase reaction mixture, which is then incubated for an additional 15 minutes. The relative light unit (RLU) signal is measured, and the RLU signals are converted to percent of control (POC) values to determine the IC50 value.

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MEK5/ERK5 Catalytic Activity Assay: Purified GST-tagged MEK5 (15 nM) or GST-tagged ERK5 (20 nM) enzyme was incubated with 0.75 µM ATP in an assay buffer (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, 1% DMSO) in the presence of varying concentrations of BIX02189. The kinase reaction mixture was incubated for 90 minutes at room temperature. Residual ATP was quantified by adding an ATP detection reagent based on a luciferin-luciferase system, and the luminescence signal was measured. The IC₅₀ values were calculated based on the percent kinase activity relative to a control without inhibitor. [1]
The catalytic activity of MEK5 and ERK5 was measured using proteins isolated from a baculovirus expression system. The assay utilized a homogeneous ATP detection reagent based on luciferin-luciferase to quantify residual ATP. The reaction mixture contained 15 nM GST-MEK5 or 20 nM GST-ERK5 and 0.75 µM ATP in an 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. The kinase reaction was incubated for 90 minutes at room temperature. Subsequently, 10 µL of ATP detection reagent was added and incubated for 15 minutes. The relative light unit (RLU) signal was measured and converted to percent of control values. Inhibitors were tested in a 10-point dose titration starting at 10 µM as the highest concentration, and IC50 values were calculated from the dose-response curves. [2]

The MTT assay is used to measure the proliferation caused by AGE. VSMCs are cultured on 24-well plates, and when they are about 80% confluent, serum-free DMEM is added to the medium. Fluvastatin (5 mM) is then used to stimulate the cells after BIX02189 (2 mM) pretreatment. MTT reagents are added, incubated for 4 hours at 37°C, removed, washed in PBS, and eluted in DMSO. Using a microplate reader at 570 nm, proliferation is measured[2].

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ERK5 Phosphorylation Assay (Western Blot): HeLa cells were plated in six-well plates and serum-starved for 20 hours. BIX02189 was added to the cells 90 minutes before stimulation with 0.4 M sorbitol for 20 minutes at 37°C. Cells were then harvested and lysed. Lysates were centrifuged, mixed with sample buffer, boiled, and subjected to SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and immunoblotted with antibodies against phospho-ERK5, total ERK5, phospho-ERK1/2, total ERK1/2, phospho-p38, and phospho-JNK. [1]
MEK5/ERK5/MEF2C Trans-Reporter Assay: HeLa or HEK293T cells were co-transfected using a lipid-based transfection reagent with a mixture of plasmids encoding constitutively active MEK5 (CA-MEK5), ERK5, a MEF2C-GAL4 fusion protein, and a GAL4-Luciferase reporter gene. Approximately 5 hours after transfection, cells were plated into white 96-well plates. BIX02189 was added at various concentrations and incubated for 18-24 hours. Luciferase activity was then measured using a commercial luciferase assay system. Cytotoxicity was assessed in parallel using an Alamar Blue assay. [1]
For Western blot analysis of ERK5 phosphorylation, HeLa cells were plated in six-well plates and serum-starved for 20 hours. Cells were pretreated with BIX02189 for 1.5 hours prior to stimulation with 0.4 M sorbitol for 20 minutes at 37°C. Cells were then harvested and lysed in RIPA buffer containing protease and phosphatase inhibitors. Lysates were centrifuged, mixed with sample buffer, boiled, and subjected to SDS-PAGE on 10% Tris-glycine gels. Proteins were transferred to nitrocellulose membranes and immunoblotted with anti-phospho-ERK5, anti-total ERK5, anti-phospho-ERK1/2, anti-phospho-p38, and anti-phospho-JNK antibodies. [2]
For the trans-reporter assay, HeLa or HEK293T cells were transfected using a lipid-based transfection reagent. The DNA mixture included plasmids for constitutively active MEK5, ERK5, a MEF2C-GAL4 fusion protein, and a GAL4-luciferase reporter vector. Five hours after transfection, cells were plated into white 96-well plates. BIX02189 was added at various concentrations 18-24 hours prior to measuring luciferase activity. Luciferase activity was determined using a commercial luciferase assay reagent. Compound cytotoxicity was assessed in parallel using Alamar Blue reagent. [2]
For Western blot analysis of flow-induced protein expression, HUVECs were grown on gelatin-coated dishes. Confluent cells were exposed to unidirectional laminar flow (12 dynes/cm²) for 16-24 hours at 37°C with 5% CO2 using a cone flow system. When testing BIX02189, the compound (10 µM final concentration, from a DMSO stock) was added to the culture medium 16-24 hours prior to flow exposure. After stimulation, cells were harvested and lysed. Proteins were separated by SDS-PAGE, transferred to membranes, and immunoblotted with specific antibodies against target proteins (e.g., HO-1, NQO1, eNOS, phospho-ERK5, total ERK5, tubulin).
For analysis of protein subcellular localization, HUVECs were pretreated with 10 µM BIX02189 for 16-24 hours and then exposed to laminar flow or kept under static conditions. Cells were then fractionated into cytosolic and nuclear compartments using appropriate buffers. The distribution of proteins like Nrf2 and ERK5 in these fractions was analyzed by immunoblotting, with fraction purity confirmed using antibodies against compartment-specific markers (e.g., tubulin for cytosol, lamin B for nucleus).
For the luciferase reporter assay, HUVECs were co-transfected with a plasmid containing the ARE promoter linked to luciferase (pGL3-ARE-luc) and a control Renilla luciferase plasmid for normalization. After transfection, cells were pretreated with BIX02189 and then exposed to laminar flow for 16-24 hours. Luciferase activity was measured using a dual-luciferase reporter assay kit, and firefly luciferase activity was normalized to Renilla luciferase activity.
For detecting endogenous protein-protein interaction in cells, HUVECs were exposed to laminar flow for various durations. An in situ proximity ligation assay (Duolink) was performed using antibodies against ERK5 and Nrf2 according to the manufacturer's instructions. Interaction was visualized as fluorescent dots under a fluorescence microscope, with nuclei counterstained. [3]

Mice: The mice used are C57BL/6-specific pathogen-free mice. Six-week-old male C57BL/6 mice are intraperitoneally treated with BIX02189 (10 mg/kg of body weight in 25% DMSO) or vehicle control to determine the role of ERK5 on laminar flow-dependent Nrf2 nuclear translocation in vivo. Following euthanasia, vascular perfusion with saline is carried out for 5 min, and then the animal is fixed for 5 min in 4% paraformaldehyde. Fat is removed after a 0.1% PBS with Tween incubation on an isolated aorta. Antibody diluents and blocking solutions are made with 5% goat serum. Anti-vascular endothelial-cadherin antibody and Topro3 are used to stain aortic endothelial cells for the endothelial cell junction and the nuclear, respectively. Immunofluorescence staining with anti-Nrf2 antibody and a Confocal microscope are used to identify Nrf2's cellular localization[3].

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Mice were treated with a single intraperitoneal injection of BIX02189 at a dose of 10 mg/kg. The compound was dissolved in 25% DMSO (vehicle control was the same volume of 25% DMSO). After treatment, the thoracic aorta was harvested. The endothelial layer of the aorta was subjected to en face immunofluorescence staining. Tissues were stained with antibodies against Nrf2 (to visualize its localization), vascular endothelial-cadherin (VE-cadherin, to mark endothelial cell junctions), and a nuclear stain. Stained samples were examined under a confocal microscope to assess the nuclear vs. cytosolic localization of Nrf2 in aortic endothelial cells. [3]

[1]. Identification of pharmacological inhibitors of the MEK5/ERK5 pathway. Biochem Biophys Res Commun. 2008 Dec 5;377(1):120-5.

[2]. Fluvastatin inhibits AGE-induced cell proliferation and migration via an ERK5-dependent Nrf2 pathway in vascular smooth muscle cells. PLoS One. 2017 May 22;12(5):e0178278.

[3]. Laminar flow activation of ERK5 protein in vascular endothelium leads to atheroprotective effect via NF-E2-related factor 2 (Nrf2) activation. J Biol Chem. 2012 Nov 23;287(48):40722-31.

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

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BIX02189 is an indole-6-carboxamide compound that has been identified as a novel and highly potent pharmacological inhibitor of the MEK5 kinase. [1]
In cell models, it exhibits high selectivity for the MEK5/ERK5 pathway, superior to other MAPK pathways (ERK1/2, p38, JNK). [1]
This compound is considered a valuable pharmacological tool for studying the role of the MEK5/ERK5 signaling cascade in various biological processes, including cell proliferation, survival, and cardiovascular pathophysiology. [1]
BIX02189 is an indole-6-carboxamide compound that has been identified as a novel and selective pharmacological inhibitor of the MEK5/ERK5 pathway. It is a valuable tool compound that can be used to better characterize the role of this pathway in various biological systems, including cell proliferation, survival, stress response, tumor progression, neuronal survival, and cardiovascular pathophysiology. This study highlights the lack of selective catalytic inhibitors for this pathway prior to this report. [2]
BIX02189 was used as a specific pharmacological inhibitor of MEK5 in this study to investigate the role of the MEK5/ERK5 pathway in laminar flow-mediated signal transduction in vascular endothelial cells. Studies have shown that laminar flow activation of ERK5 can exert anti-atherosclerotic effects by activating the transcription factor Nrf2. BIX02189 confirmed that inhibiting this pathway blocked the nuclear translocation of Nrf2 and the expression of cytoprotective genes (such as HO-1, NQO1), both in cultured endothelial cells and in mouse aortas in vivo. These findings suggest that the ERK5-Nrf2 axis is a key component of blood flow-mediated endothelial protection and may be a potential target for the treatment of atherosclerosis. [3]

C₂₇H₂₈N₄O₂M

440.53682

440.221

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

1265916-41-3

(E/Z)-BIX02189;1094614-85-3

135659062

White to yellow 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=C(N(C)C)C1=CC(NC2=O)=C(/C2=C(C3=CC=CC=C3)/NC4=CC=CC(CN(C)C)=C4)C=C1

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,17H2,1-4H3

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

BIX02189; BIX 02189; BIX-02189

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)

DMSO: ~88 mg/mL (~199.8 mM)
Ethanol: ~88 mg/mL (~199.8 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.67 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.67 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.67 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 10mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)