Potency assay (off target):
Rentosertib was screened at the dose of 10 μM against a panel of 430 enzymes, which consists of both protein and lipid kinases. The results of this screening were expressed as remaining kinase activity after compound treatment normalized to the DMSO control. In total, 43 human kinases with remaining activity under 20% after incubation with Rentosertib were observed. Next, Rentosertib was assayed against each of these kinases in a dose–response manner at the ATP concentration equaled to a kinase’s Km value. TNIK was inhibited by Rentosertib with the highest potency (IC50 of 31 nM). Interestingly, the dose-dependent study did not confirm the results of single-dose screening for Aurora-B and ErbB4. Indeed, Rentosertib demonstrated IC50 > 10 μM against these enzymes. In addition to TNIK, five kinases were inhibited with IC50 < 100 nM, three of which were mutants (PDGFRα, Kit, and EGFR), and two were wild type (YES and ALK4). Notably, nonmutant Kit and PDGFRα were out of the 43 hit kinases list, whereas EGFR was inhibited in a micromolar range.
