Phosphorylation reactions were monitored using a coupled-enzyme assay in which ADP production is coupled to NADH oxidation by pyruvate kinase (PK) and lactate dehydrogenase (LDH)
PDB ID for probe-target interaction (3D structure):
2VAG, 2WUZ
Potency (off target):
IC50
Off target protein and potency:
CLK3 530 nMDYRK1A 55 nM
Potency assay (off target):
Binding of KH-CB19 to kinase catalytic domains as assessed in thermal shift assays against 129 human protein kinases revealed limited off-target binding (CLK family members noted above). These Tm data were compared with published AMBIT data sets to predict likely binding constants. These data were then confirmed in kinase enzyme activity assays for 71 protein kinases including DYRK1A.
In cell validation
Potency assay (cells):
KH-CB19 dose-dependently inhibited CLK1 protein substrate phosphorylation (SRp75, SRp55, and SRp20) in HMEC-1 cells. Dose response of KH-CB19 was tested using SRp75 and SRp55, and phosphorylation levels of these two proteins in TNF-α−stimulated cells were significantly reduced at increasing concentration of the inhibitor.
Target engagement assay (cells):
Indirect, inhibition of protein substrate phosphorylation