Bicyclic heterocycles as MEK kinase inhibitors
A cyclopropyl, CR14R15 technology, applied in anti-inflammatory agents, non-central analgesics, medical preparations containing active ingredients, etc., can solve problems such as lack of inactivation
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Embodiment 1
[0292] Example 1: MEK assay (MEK activity assay)
[0293] Constitutively activated human mutant MEK1 expressed in insect cells was used as the source of enzymatic activity at a final concentration of 15 nM in the kinase assay.
[0294] The assay was performed for 30 minutes in the presence of 50 μM ATP using recombinant GST-ERK1 produced in E. coli (E. Coli) as substrate. Phosphorylation of substrates was detected and quantified using HTRF reagent supplied by Cisbio. The HTRF reagent described above consists of an anti-GST antibody conjugated to allophycocyanin (XL665) and an anti-phospho(Thr202 / Tyr204) ERK antibody conjugated to europium cryptate. The above antibodies were used at final concentrations of 4 μg / mL and 0.84 μg / mL, respectively. Anti-phospho antibody recognizes ERK1 doubly phosphorylated on Thr202 and Tyr204. When both antibodies are bound to ERK1 (i.e. when the substrate is phosphorylated), upon excitation at 340 nm, a transfer of energy from the cryptate to ...
Embodiment 2
[0298] Example 2: bRaf Assay (MEK Activation Assay)
[0299] A constitutively active bRaf mutant expressed in insect cells was used as a source of enzymatic activity.
[0300] The assay was performed for 30 minutes in the presence of 200 μM ATP using recombinant GST-MEK1 produced in E. coli (E. Coli) as substrate. Phosphorylation of substrates was detected and quantified using HTRF and these reagents were supplied by Cisbio. The HTRF reagent described above consists of an anti-GST antibody conjugated to allophycocyanin (XL665) and an anti-phospho(Ser217 / Ser221) MEK antibody conjugated to a europium cryptate. Anti-phospho antibodies recognize MEK that is dually phosphorylated on Ser217 and Ser221 or solely phosphorylated on Ser217. When both antibodies are bound to MEK (i.e., when the substrate is phosphorylated), upon excitation at 340 nm, energy transfer from the cryptate to allophycocyanin occurs, which results in emission and resulting phosphorylation The amount of subst...
Embodiment 3
[0303] Example 3: Cell Proliferation Assay
[0304] Compounds were tested in cell proliferation assays using the following cell lines:
[0305] HCT116 Human Colorectal Cancer (ATCC)
[0306] A375 Human malignant melanoma (ATCC)
[0307] at 37°C in 5% CO 2 Both cell lines were maintained in DMEM / F12 (1:1) medium (Gibco) supplemented with 10% FCS in a humidified incubator.
[0308] Cells were seeded in 96-well plates at 2,000 cells / well, and after 24 hours, were exposed to different concentrations of compounds in 0.83% DMSO. Cells were grown for an additional 72 hours before an equal volume of CellTiter-Glo reagent (Promega) was added to each well. This lyses the cells and produces a luminescent signal proportional to the amount of ATP released (and thus the number of cells in the well), which can be detected using a multiwell luminometer.
[0309] will EC 50 Defined as the concentration of a given compound that achieves 50% inhibition relative to the control. IC 50 Valu...
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