Aza-benzothiophenyl compounds and methods of use
A compound and composition technology, applied in the direction of drug combination, organic chemistry, pharmaceutical formulation, etc., can solve problems such as lack of inactivation
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Embodiment 1a
[0302] Example 1a: MEK Assay (MEK Activity Assay)
[0303] Constitutively activated human mutant MEK1 expressed in insect cells was used as the source of enzymatic activity at a final concentration of 62.5 nM in the kinase assay.
[0304] The assay was performed in the presence of 50 [mu]M ATP for 30 minutes using recombinant GST-ERK1 produced in E. coli (E. Coli) as substrate. Phosphorylation of the substrate 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. 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, energy transfer from the cryptate to allophycocyanin occurs, resulting in emission and resulting phosphorylation The amount of su...
Embodiment 1b
[0307] Example 1b: MEK Assay (MEK Activity Assay)
[0308] 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.
[0309] The assay was performed in the presence of 50 [mu]M ATP for 30 minutes using recombinant GST-ERK1 produced in E. coli (E. Coli) as substrate. Phosphorylation of the substrate 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, energy transfer from the cryptate t...
Embodiment 2
[0313] Example 2: bRaf Assay (MEK Activation Assay)
[0314] A constitutively active bRaf mutant expressed in insect cells was used as a source of enzymatic activity.
[0315] The assay was performed in the presence of 200 [mu]M ATP for 30 minutes using recombinant GST-ERK1 produced in E. coli (E. Coli) as substrate. Phosphorylation of substrates was detected and quantified using HTRF, reagents provided 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, resulting in emission and resulting phosphorylation The amount of substrate is proport...
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