Ulixertinib (BVD-523)

Ulixertinib (VRT752271) is a reversible, ATP-competitive inhibitor of ERK1 and ERK2 kinases, with IC50 of <0.3 nM against ERK2.

Signaling Pathways >> MAPK/ERK Pathway >> ERK

Signaling Pathways >> Stem Cell/Wnt >> ERK

Research Areas >> Cancer

ERK2:0.3 nM (IC50, at KM ATP (60 μM))

ERK1

Ulixertinib (VRT752271) is a reversible, ATP-competitive inhibitor of ERK1 and ERK2 kinases, with IC50 of <0.3 nM against ERK2[2].

In the pharmacokinetic study, the sensitivity and specificity of the assay are found to be sufficient for accurately characterizing the plasma pharmacokinetics of Ulixertinib in Balb/C mice[1].

The MEK autophosphorylation assay is performed using the ADP Glo Kinase Assay kit. Activated MEK protein is expressed and purified in-house. Enzyme and substrate solutions are prepared in assay buffer consisting of 50 mM Tris (pH 7.5), 10 mM MgCl2, 0.1 mM EGTA, 10 mM DTT and 0.01 % Tween 20. 6 nM MEK protein is prepared in assay buffer and 2 µL is dispensed into each well of a 384-well white small volume medium bind plate containing test and reference control compounds. Compound plates are dosed with a 12 point dose response curve from 10 µM down to 0.0625 nM in order to calculate compound IC50s, with a total DMSO concentration in the assay of 1%. Following a 15 minute pre-incubation of enzyme and compound at room temperature, 2 µL of a 20 µM Ultra Pure ATP solution in assay buffer is added to the wells, and the reaction is allowed to progress for 90 minutes at room temperature before the addition of 4 µL ADP Glo reagent R1 to quench the reaction. The plate is incubated at room temperature for 45 minutes before the addition of 8 µL Kinase Detection Reagent, and then the luminescence signal is allowed to equilibrate for 60 minutes before the plates are read on a Pherastar plate reader.

A375 cells are cultured in cell media composed of DMEM, 10% (v/v) Foetal Calf Serum and 1% (v/v) L-Glutamine. After harvesting, cells are dispensed into black, 384-well Costar plates to give 200 cells per well in a total volume of 40 µL cell media, and are incubated overnight at 37°C, 90% relative humidity and 5% CO2 in a rotating incubator. Test compounds and reference controls are dosed directly into the cell plates, into the inner 308 wells. The cells are dosed over a 12 point range from 30 µM down to 0.03 nM in order to calculate compound IC50s, with a total DMSO concentration in the assay of 0.3%. The cell plates are then incubated for 72 hours at 37°C. Cells are fixed and stained by the addition of 20 µL 12% formaldehyde in PBS/A (4% final concentration) and 1:2000 dilution of Hoechst 33342, with a 30 minute room temperature incubation, and then washed with PBS/A. A cell count is performed on the stained cell plates using a Cellomics ArrayScanTM VTI imaging platform. A Day 0 cell plate is also fixed, stained and read to generate a cell count baseline for determining compound cytotoxic effects as well as anti-proliferative effects.

[1]. Kumar R, et al. Determination of ulixertinib in mice plasma by LC-MS/MS and its application to a pharmacokinetic study in mice. J Pharm Biomed Anal. 2016 Jun 5;125:140-4.

[2]. Ward RA, et al. Structure-Guided Design of Highly Selective and Potent Covalent Inhibitors of ERK1/2. J Med Chem. 2015 Jun 11;58(11):4790-801.

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