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

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

  • J Biol Chem. 2020 Feb 21;295(8):2359-2374. doi: 10.1074/jbc.RA119.011083.
Ian M McDonald 1,  Gavin D Grant 2,  Michael P East 1,  Thomas S K Gilbert 3,  Emily M Wilkerson 3,  Dennis Goldfarb 4,  Joshua Beri 3,  Laura E Herring 3,  Cyrus Vaziri 5,  Jeanette Gowen Cook 2,  Michael J Emanuele 1,  Lee M Graves 6
Affiliations

Affiliations

  • 1 Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599.
  • 2 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599; Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599.
  • 3 Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599; UNC Michael Hooker Proteomics Core Facility, University of North Carolina, Chapel Hill, North Carolina 27599.
  • 4 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110; Institute for Informatics, Washington University School of Medicine, St. Louis, Missouri 63110.
  • 5 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599; Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina 27599.
  • 6 Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599; Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599; UNC Michael Hooker Proteomics Core Facility, University of North Carolina, Chapel Hill, North Carolina 27599. Electronic address: lmg@med.unc.edu.
PMID: 31896573 DOI: 10.1074/jbc.RA119.011083
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.

Keywords

AMPK family; G2/M checkpoint; NVS-MELK8a; cell cycle; cell proliferation; inhibitor; inhibitor selectivity; kinome profiling; mass spectrometry (MS); maternal embryonic leucine zipper kinase (MELK); mitotic delay; multiplexed kinase inhibitor beads/mass spectrometry (MIB/MS); protein kinase.

Figures
Products
嗨！很高兴为您提供帮助！

尊敬的 MCE 客户您好，
请您选择所在区域，我们将转接对应客服为您服务！

热门区域

A

B

C

F

G

H

J

L

N

Q

S

T

X

Y

Z

热 A B C F G H J L N Q S T X Y Z