Angiogenesis has been demonstrated to be essential for tumor growth and metastasis, and inhibition of angiogenesis is emerging as a promising strategy for treating cancer. Among the most potent inhibitors of angiogenesis is the fumagillin family of natural products. An analog of fumagillin, known as TNP-470 or AGM-1470. has been undergoing clinical trials for treating a variety of cancers. TNP-470 has been shown to block endothelial cell cycle progression in the late G(1) phase. Although the direct molecular target for TNP-470 has been identified as the type 2 methionine aminopeptidase (MetAP2). how inhibition of this enzyme leads to cell cycle arrest has remained unclear. We report that treatment of endothelial and other drug-sensitive cell types leads to the activation of the p53 pathway, causing an accumulation of the G(1) cyclin-dependent kinase inhibitor p21(WAF1/CIP1). The requirement of p53 and p21(WAF1/CIP1) for the cell cycle inhibition by TNP-470 is underscored by the observation that cells deficient in p53 and p21(WAF1/CIP1) are resistant to TNP-470. These results shed significant light on the mechanism of cell cycle inhibition by TNP-470 and suggest an alternative method of activating p53 in endothelial cells to halt angiogenesis and tumor progression.

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.

Angiogenesis inhibitors are a novel class of promising therapeutic agents for treating cancer and other human diseases. Fumagillin and ovalicin compose a class of structurally related natural products that potently inhibit angiogenesis by blocking endothelial cell proliferation. A synthetic analog of fumagillin, TNP-470, is currently undergoing clinical trials for treatment of a variety of cancers. A common target for fumagillin and ovalicin recently was identified as the type 2 methionine aminopeptidase (MetAP2). These natural products bind MetAP2 covalently, inhibiting its enzymatic activity. The specificity of this binding is underscored by the lack of inhibition of the closely related type 1 enzyme, MetAP1. The molecular basis of the high affinity and specificity of these inhibitors for MetAP2 has remained undiscovered. To determine the structural elements of these inhibitors and MetAP2 that are involved in this interaction, we synthesized fumagillin analogs in which each of the potentially reactive epoxide groups was removed either individually or in combination. We found that the ring epoxide in fumagillin is involved in the covalent modification of MetAP2, whereas the side chain epoxide group is dispensable. By using a fumagillin analog tagged with fluorescein, His-231 in MetAP2 was identified as the residue that is covalently modified by fumagillin. Site-directed mutagenesis of His-231 demonstrated its importance for the catalytic activity of MetAP2 and confirmed that the same residue is covalently modified by fumagillin. These results, in agreement with a recent structural study, suggest that fumagillin and ovalicin inhibit MetAP2 by irreversible blockage of the active site.

BACKGROUND:Angiogenesis, the formation of new blood vessels, is essential for tumor growth. The inhibition of angiogenesis is therefore emerging as a promising therapy for cancer. Two natural products, fumagillin and ovalicin, were discovered to be potent inhibitors of angiogenesis due to their inhibition of endothelial cell proliferation. An analog of fumagillin, AGM-1470, is currently undergoing clinical trials for the treatment of a variety of cancers. The underlying molecular mechanism of the inhibition of angiogenesis by these natural drugs has remained unknown. RESULTS:Both AGM-1470 and ovalicin bind to a common bifunctional protein, identified by mass spectrometry as the type 2 methionine aminopeptidase (MetAP2). This protein also acts as an inhibitor of eukaryotic initiation factor 2alpha (elF-2alpha) phosphorylation. Both drugs potently inhibit the methionine aminopeptidase activity of MetAP2 without affecting its ability to block elF-2alpha phosphorylation. There are two types of methionine aminopeptidase found in eukaryotes, but only the type 2 enzyme is inhibited by the drugs. A series of analogs of fumagillin and ovalicin were synthesized and their potency for inhibition of endothelial cell proliferation and inhibition of methionine aminopeptidase activity was determined. A significant correlation was found between the two activities. CONCLUSIONS:The protein MetAP2 is a common molecular target for both AGM-1470 and ovalicin. This finding suggests that MetAP2 may play a critical role in the proliferation of endothelial cells and may serve as a promising target for the development of new anti-angiogenic drugs.

Calcineurin is an important signal-transducing enzyme in many cell types including T lymphocytes and is a common target for the immunosuppressants cyclosporin A and FK506. The crystal structures of both calcineurin [Griffith et al. (1995) Cell 82, 507-522; Kissinger et al. (1995) Nature 378, 641-644] and a related enzyme, protein phosphatase-l [Goldberg et al. (1995) Nature 376, 745-753], revealed that this class of serine/threonine phosphatases contain in their putative active sites a binuclear metal center formed by an Asn, two Asp, and three His residues. In addition, one His and two Arg residues lie in close vicinity of the binuclear metal centers. The importance of the binuclear metal center and its surrounding residues in catalysis by calcineurin has not been investigated experimentally. Herein, we report an efficient bacterial expression and purification system for human calcineurin or. Using this system, a systematic alanine-scan mutagenesis on the residues surrounding the putative active site was performed. It was found that an intact binuclear metal center is essential for the catalytic activity of the enzyme. In addition, His151, Arg122, and Arg254 also exhibited either a loss or a dramatic decrease in catalytic activity upon mutation into alanines. Interestingly, the Arg254Ala mutant retained a small but significant amount of catalytic activity toward the small substrate p-nitrophenyl phosphate, but is completely inactive toward a phosphopeptide substrate, suggesting that this arginine may be involved in the binding of phosphoprotein substrates as well as in catalysis. As all the residues in the putative active site are conserved between different eukaryotic serine/threonine phosphatases, these results should apply to all members of this family of protein phosphatases.