Evaluation of potential Myt1 kinase inhibitors by TR-FRET based binding assay

The glycoglycerolipid 1,2-dipalmitoyl-3-(N-palmitoyl-6′-amino-6′-deoxy-α-d-glucosyl)-sn-glycerol is no inhibitor of the human Myt1 kinase

“…Again, the concentration of GGL1 used here is about 250 times higher than the reported IC 50 value. For dasatinib, 10 mM equals approximately 80-to 160-fold the reported IC 50 values 7,15 . No relevant functional inhibition occurred in presence of GGL1.…”

“…Recombinant GST-Cdk1 was incubated with full-length Myt1 and ATP in the presence or absence of GGL1 (30 mg/ml). Samples containing 10 mM dasatinib, a confirmed Myt1 kinase inhibitor 7,15,16 , were carried along as inhibitor controls and samples without ATP were used as background controls. Myt1 was present in all samples.…”

“…To be noted, a more general issue is raised by the use of staurosporine for this purpose. Independent reports describe Myt1 to be one of the few examples that are insensitive towards these compounds 7,15 . Other compounds with confirmed inhibitory potency towards Myt1, such as dasatinib or PD166285, should be preferred.…”

“…Full-length Myt1 was expressed in the human cell line HEK293 as described before 7 . Briefly, cells were cultured at 37 C in Dulbecco's modified Eagle medium (DMEM), supplemented with 10% FCS and GlutaMAX in a humidified incubator.…”

“…That report gained a lot of attention and several research groups, including ours, synthesized and derivatized the compound for further biological evaluation [4][5][6][7][8] . However, presumably caused by the restrictive substrate acceptance of this kinase 9 , reliable assay data confirming the original report and elucidating the mechanism of action are still missing.…”