Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells

Chem Biol. 1999 Nov;6(11):823-33. doi: 10.1016/s1074-5521(99)80129-x.


Background: The angiogenesis inhibitors TNP-470 and ovalicin potently suppress endothelial cell growth. Both drugs also specifically inhibit methionine aminopeptidase 2 (MetAP2) in vitro. Inhibition of MetAP2 and changes in initiator methionine removal in drug-treated endothelial cells have not been demonstrated, however.

Results: Concentrations of TNP-470 sufficient to inactivate MetAP2 in intact endothelial cells were comparable to those that inhibited cell proliferation, suggesting that MetAP2 inhibition by TNP-470 underlies the ability of the drug to inhibit cell growth. Both drug-sensitive and drug-insensitive cell lines express MetAP1 and MetAP2, indicating that drug sensitivity in mammalian cells is not simply due to the absence of compensating MetAP activity. With a single exception, detectable protein N-myristoylation is unaffected in sensitive endothelial cells treated with TNP-470, so MetAP1 activity can generally compensate when MetAP2 is inactive. Analysis of total protein extracts from cells pulse-labeled with [(35)S]-methionine following TNP-470 treatment revealed changes in the migration of several newly synthesized proteins. Two of these proteins were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin A. Purification and amino-terminal sequencing of GAPDH from TNP-470-treated cells revealed partial retention of its initiator methionine, indicating that methionine removal from some, but not all, proteins is affected by MetAP2 inactivation.

Conclusions: Amino-terminal processing defects occur in cells treated with TNP-470, indicating that inhibition of MetAP2 by the drug occurs in intact cells. This work renders plausible a mechanism for growth inhibition by TNP-470 as a consequence of initiator methionine retention, leading to the inactivation of as yet unidentified proteins essential for endothelial cell growth.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Aminopeptidases / antagonists & inhibitors*
  • Angiogenesis Inhibitors / pharmacology*
  • Animals
  • Aorta
  • Cattle
  • Cell Division / drug effects
  • Cells, Cultured
  • Cyclohexanes
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / drug effects*
  • Endothelium, Vascular / metabolism
  • Glyceraldehyde-3-Phosphate Dehydrogenases / chemistry
  • Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism*
  • Humans
  • Metalloendopeptidases / antagonists & inhibitors*
  • Methionine / metabolism*
  • Molecular Sequence Data
  • Myristic Acid / metabolism
  • O-(Chloroacetylcarbamoyl)fumagillol
  • Peptide Fragments / chemistry
  • Protein Processing, Post-Translational / drug effects*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sesquiterpenes / pharmacology*
  • Umbilical Veins


  • Angiogenesis Inhibitors
  • Cyclohexanes
  • Peptide Fragments
  • Recombinant Proteins
  • Sesquiterpenes
  • Myristic Acid
  • ovalicin
  • Methionine
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • Aminopeptidases
  • methionine aminopeptidase 2
  • Metalloendopeptidases
  • O-(Chloroacetylcarbamoyl)fumagillol