Mass spectrometry-based selectivity profiling identifies a highly selective inhibitor of the kinase MELK that delays mitotic entry in cancer cells.

The maternal embryonic leucine zipper kinase (MELK) has been implicated in the regulation of cancer cell proliferation. RNAi-mediated MELK depletion impairs growth and causes G2/M arrest in numerous cancers, but the mechanisms underlying these effects are poorly understood. Furthermore, the MELK inhibitor OTSSP167 has recently been shown to have poor selectivity for MELK, complicating the use of this inhibitor as a tool compound to investigate MELK function. Here, using a cell-based proteomics technique called multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS), we profiled the selectivity of two additional MELK inhibitors, NVS-MELK8a (8a) and HTH-01-091. Our results revealed that 8a is a highly selective MELK inhibitor, which we further used for functional studies. Resazurin and crystal violet assays indicated that 8a decreases triple-negative breast cancer cell viability, and immunoblotting revealed that impaired growth is due to perturbation of cell cycle progression rather than induction of apoptosis. Using double-thymidine synchronization and immunoblotting, we observed that MELK inhibition delays mitotic entry, which was associated with delayed activation of Aurora A, Aurora B, and cyclin-dependent kinase 1 (CDK1). Following this delay, cells entered and completed mitosis. Using live-cell microscopy of cells harboring fluorescent proliferating cell nuclear antigen, we confirmed that 8a significantly and dose-dependently lengthens G2 phase. Collectively, our results provide a rationale for using 8a as a tool compound for functional studies of MELK and indicate that MELK inhibition delays mitotic entry, likely via transient G2/M checkpoint activation.

Crystal structure of the kinase and UBA domains of SNRK reveals a distinct UBA binding mode in the AMPK family.

Sucrose non-fermenting (Snf1)-related kinase (SNRK) is a novel member of the AMP-activated protein kinase (AMPK) family and is involved in many metabolic processes. Here we report the crystal structure of an N-terminal SNRK fragment containing kinase and adjacent ubiquitin-associated (UBA) domains. This structure shows that the UBA domain binds between the N- and C-lobes of the kinase domain. The mode of UBA binding in SNRK largely resembles that in AMPK and brain specific kinase (BRSK), however, unique interactions play vital roles in stabilizing the KD-UBA interface of SNRK. We further propose a potential role of the UBA domain in the regulation of SNRK kinase activity. This study provides new insights into the structural diversities of the AMPK kinase family.