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

Abstract

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.

Enigmatic MELK: The controversy surrounding its complex role in cancer

“…Further, the binding affinity of OTS for MELK is lower than its binding affinity for 15 other protein kinases (see supporting material of the Klaeger et al study (13) for quantifications). Using a similar MS-based selectivity-profiling method (MIB/MS), our group recently confirmed the low specificity of OTS (14). We showed that 12 protein kinases were inhibited more than MELK by 1 M OTS, and 52 kinases (of 235 that were quantified) were inhibited Ն75% at this concentration.…”

“…Because of its status as the leading MELK inhibitor, OTS has been used in most functional studies of MELK since its development. It has recently been definitively demonstrated that OTS has extremely poor selectivity for MELK (13)(14)(15)(16)), yet unfortunately, it is still regularly used as a tool compound to investigate MELK function.…”

“…Multiple MELK inhibitors have been developed as potential cancer therapeutics (reviewed previously by Pitner et al (20)). The molecules OTS (10), NVS-MELK8a (14,39), HTH-01-091 (16), MELK-T1 (40), IN17 (41), and C1 (23) have all demonstrated preclinical efficacy in slowing cancer cell proliferation, with OTS exhibiting such potent, broad anti-neoplastic effects that it has advanced to phase I/II clinical trials (11,12,25,36,(42)(43)(44). Certainly, all small-molecule inhibitors exhibit some degree of polypharmacology, so the antiproliferative effects of these compounds cannot be solely attributed to MELK inhibition.…”

“…Certainly, all small-molecule inhibitors exhibit some degree of polypharmacology, so the antiproliferative effects of these compounds cannot be solely attributed to MELK inhibition. For the inhibitor NVS-MELK8a, the viability of TNBC cells has been shown to be reduced at concentrations at which NVS-MELK8a is highly selective for MELK, suggesting that inhibition of MELK is a major contributor to its antiproliferative effects (14).…”

“…Accordingly, we recommend that OTS not be used for any future functional studies of MELK. As an alternative, NVS-MELK8a should be used, as it has been shown to exhibit excellent selectivity in both enzyme-and cell-based assays (14,39). Further characterization of other seemingly selective inhibitors, such as MELK-T1 (40,49), would also be beneficial to the field.…”