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

Abstract

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.

Proteomics-based Target Identification

“…Cleavage of N-terminal methionine from a protein causes a minimal change in molecular weight and is not expected to lead to large changes of isoelectric points unless accompanied by acetylation or other post-translational processing such as myristoylation of the newly exposed amino group. This possibility may explain why so few changes were observed in the current study, as well as in earlier reports (29).…”

“…Based on the available literature, it remains unclear why the anti-proliferative activity of fumagillin analogues is selective for endothelial cells, because both endothelial and epithelial cells express both known human MetAp isoforms, and because equivalent inhibition has been reported when MetAp2 is immunoprecipitated from endothelial or epithelial cells treated with a fumagillin analogue (28,29). One possibility is that human MetAp isoforms process distinct sets of substrates, as has been suggested for the yeast isoforms (30), and that among the proteins processed by MetAp2, one or more is required for endothelial cell viability.…”

A methionine aminopeptidase-2 inhibitor, PPI-2458, for the treatment of rheumatoid arthritis

“…MetAPs remove the initiator methionine from growing polypeptide chains. They have the same general substrate specificity, but selective differences are determined by the penultimate residue to the initiator methionine (15,16). Removal of the N-terminal methionine is a prerequisite for a variety of biological processes, such as activity, subcellular localization, and protein stability.…”

“…Removal of the N-terminal methionine is a prerequisite for a variety of biological processes, such as activity, subcellular localization, and protein stability. The fungal metabolite fumagillin and several structural analogs such as TNP-470 (AGM-1470) selectively and irreversibly inhibit MetAP-2, via covalent modification of His-231 in the catalytic site of the enzyme (15,(17)(18)(19). This inhibition of the MetAP-2 enzyme activity provides the molecular link that triggers the in vitro growth arrest of human umbilical vein endothelial cells (HUVEC) in the late G 1 phase of the cell cycle (20,21).…”

“…Paw swelling was monitored by using a plethysmometer (Stoelting) according to instrument specifications. The volumes of the two hind paws were measured and averaged on days 1, 4, 5,6,8,11,13,15,17,19,21,23,25,27,29, and 31. Eight animals were assigned to each group, except the vehicle control (n ϭ 4).…”