CDC25A-inhibitory RE derivatives bind to pocket adjacent to the catalytic site

Mol Biosyst. 2013 May;9(5):1026-34. doi: 10.1039/c3mb00003f.

Abstract

RE derivatives, which are cell-permeable and non-electrophilic dual-specificity protein phosphatase inhibitors developed in our laboratory, inhibit CDC25A/B non-competitively, as determined by means of kinetic experiments. To identify the binding site of RE derivatives, we designed and synthesized the new probe molecule RE142, having a Michael acceptor functionality for covalent bond formation with the enzyme, a biotin tag to enable enrichment of probe-bound peptide(s), and a chemically cleavable linker to facilitate release of probe-bound peptides from avidin beads. LC-MS analysis indicated that RE142 binds to one of the residues Cys384-Tyr386 of CDC25A, within a pocket adjacent to the catalytic site.

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Binding Sites
  • Blotting, Western
  • Catalytic Domain*
  • Crystallography, X-Ray
  • Enzyme Inhibitors / chemistry*
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Kinetics
  • Mass Spectrometry / methods
  • Models, Chemical
  • Models, Molecular
  • Molecular Conformation
  • Molecular Structure
  • Protein Binding
  • Protein Structure, Tertiary*
  • Substrate Specificity
  • cdc25 Phosphatases / antagonists & inhibitors
  • cdc25 Phosphatases / chemistry*
  • cdc25 Phosphatases / metabolism

Substances

  • Enzyme Inhibitors
  • cdc25 Phosphatases