Discovery of a Highly Selective STK16 Kinase Inhibitor

Abstract

STK16, a serine/threonine protein kinase, 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. Through screening of our focused library of kinase inhibitors, we discovered a highly selective ATP competitive inhibitor, STK16-IN-1, which 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.

STK16 regulates actin dynamics to control Golgi organization and cell cycle

“…In our previous study, we discovered a highly selective inhibitor against STK16 kinase, STK16-IN-129. To examine whether the effect of STK16 on the Golgi complex is relevant to its kinase activity, we treated HeLa and HeLa-STK16-GFP-FLAG-WT cells with 10 μM STK16-IN-1 for 6 hours and found that STK16-IN-1 also caused Golgi complex fragmentation (Fig.…”

“…To ensure that the STK16-IN-1 induced Golgi phenotype was specific, we used a HeLa-STK16-GFP-FLAG-F100C cell line (Fig. 1h) which has the hinge binding site of the STK16-IN-1 mutated and was reported to be resistant to STK16-IN-129. Our results show that STK16-IN-1 increased the percentage of cells with fragmented Golgi complex from 9.7% to 33.8% in HeLa cells and from 8.8% to 35.5% in HeLa-STK16-GFP-FLAG-WT cells, but not in HeLa-STK16-GFP-FLAG-F100C cells (Fig.…”

“…STK16 protein was purified as previously described29, and subjected to pyrene-actin polymerization and depolymerization assays. As 1–5 μM actin monomer was used by recent studies on in vitro actin dynamics, we used 3 μM actin in this part of the experiments35363738.…”

“…Previous studies, including ours, found that purified STK16 is able to phosphorylate 4EBP1 and DRG1, as well as autophosphorylation2829. Although it has been discovered for around twenty years, the biological functions of STK16 are still not well understood.…”

“…STK16 is a myristoylated and palmitoylated kinase, localizing to the Golgi and is believed to be involved in the regulation of sorting secretory soluble cargo into the constitutive secretory pathway at the trans-Golgi network2430. Moreover, our previous study showed that STK16 depletion or kinase inhibition induced binucleated cells as well as cell accumulation in the G2/M-phase29. However, the molecular mechanism remains unknown.…”