In vitro activity: STK16-IN-1 is a novel and highly selective ATP competitive inhibitor of STK16 kinase with an IC50 of 295 nM. STK16 is a serine/threonine protein kinase that is ubiquitously expressed and is conserved among all eukaryotes. STK16 has been implicated to function in a variety of cellular processes such as VEGF and cargo secretion, but the pathways through which these effects are mediated remain to be elucidated. STK16-IN-1 exhibits potent inhibitory activity against STK16 kinase (IC50: 0.295 μM) with excellent selectivity across the kinome as assessed using the KinomeScan profiling assay (S score (1) = 0.0). In MCF-7 cells, treatment with STK16-IN-1 results in a reduction in cell number and accumulation of binucleated cells, which can be recapitulated by RNAi knockdown of STK16. Co-treatment of STK16-IN-1 with chemotherapeutics such as cisplatin, doxorubicin, colchicine, and paclitaxel results in a slight potentiation of the antiproliferative effects of the chemotherapeutics. STK16-IN-1 provides a useful tool compound for further elucidating the biological functions of STK16.

Kinase Assay: STK16-IN-1 is a novel and highly selective ATP competitive inhibitor of STK16 kinase with an IC50 of 295 nM. STK16 is a serine/threonine protein kinase that is ubiquitously expressed and is conserved among all eukaryotes. STK16 has been implicated to function in a variety of cellular processes such as VEGF and cargo secretion, but the pathways through which these effects are mediated remain to be elucidated. STK16-IN-1 exhibits potent inhibitory activity against STK16 kinase (IC50: 0.295 μM) with excellent selectivity across the kinome as assessed using the KinomeScan profiling assay (S score (1) = 0.0). STK16-IN-1 is generally prepared with 1:3 serial dilutions for 4 concentrations (100 nM, 50 nM, 20 nM, and 10 nM); 6 concentrations are used (1 mM to 10 μM) for ATP competition experiments. The kinase reaction is performed with 1×kinase reaction buffer. Reactions in each well are started immediately by adding ATP and kept going for half an hour under 37°C. After the plate cooled for 5 minutes at room temperature, 5 μL of ADP-Glo reagent is added into each well to stop the reaction and consume the remaining ADP within 40 minutes. At the end, 10 μL of kinase detection reagent is added into the well and incubated for 1 hour to produce a luminescence signal.

Cell Assay: In MCF-7 cells, treatment with STK16-IN-1 results in a reduction in cell number and accumulation of binucleated cells, which can be recapitulated by RNAi knockdown of STK16. Co-treatment of STK16-IN-1 with chemotherapeutics such as cisplatin, doxorubicin, colchicine, and paclitaxel results in a slight potentiation of the antiproliferative effects of the chemotherapeutics. STK16-IN-1 provides a useful tool compound for further elucidating the biological functions of STK16. MCF-7, HCT116, HeLa cells are treated with STK16-IN-1 (0, 5, 10 μM) for 72 hours and apoptotic cells are analyzed by flow cytometry using Annexin V/PI apoptosis detection kit.