Protonation States of Methionine Aminopeptidase and Their Relevance for Inhibitor Binding and Catalytic Activity (original) (raw)
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Proceedings of the National Academy of Sciences, 1998
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.
Proceedings of the National Academy of Sciences, 1997
The inhibition of new blood vessel formation (angiogenesis) is an effective means of limiting both the size and metastasis of solid tumors. The leading antiangiogenic compound, TNP-470, has proven to be effective in in vitro and in animal model studies, and is currently being tested in phase III antitumor clinical trials. Despite many detailed pharmacological studies, little is known of the molecular mode of action of TNP-470. Using a derivative of the TNP-470 parent compound, the fungal metabolite, fumagillin, we have purified a mammalian protein that is selectively and covalently bound by this natural product. This fumagillin binding protein was found to be a metalloprotease, methionine aminopeptidase (MetAP-2), that is highly conserved between human and Saccharomyces cerevisiae. In the absence of MetAP-1, a distantly related methionine aminopeptidase, MetAP-2 function is essential for vegetative growth in yeast. We demonstrate that fumagillin selectively inhibits the S. cerevisiae MetAP-2 protein in vivo. The binding is highly specific as judged by the failure of fumagillin to inhibit MetAP-1 in vivo. Hence, these results identify MetAP-2 as an important target of study in the analysis of the potent biological activities of fumagillin.
Antiparasitic activities of novel, orally available fumagillin analogs
Bioorganic & Medicinal Chemistry Letters, 2009
Fumagillin, an irreversible inhibitor of MetAP2, has been shown to potently inhibit growth of malaria parasites in vitro. Here, we demonstrate activity of fumagillin analogs with an improved pharmacokinetic profile against malaria parasites, trypanosomes, and amoebas. A subset of the compounds showed efficacy in a murine malaria model. The observed SAR forms a basis for further optimization of fumagillin based inhibitors against parasitic targets by inhibition of MetAP2. The sesquiterpene fumagillin 1, produced by Aspergillus fumigatus, has been extensively studied for its antimicrobial activity. 1 While toxic, it is employed in veterinary applications and to a limited degree in humans, particularly in immunocompromised individuals. A renewed interest in fumagillin, driven by recognition of its anticancer properties, led to identification of its molecular target, methionine aminopeptidase-2 (MetAP2). 2 MetAP2 is a cytosolic enzyme which removes methionine from the amino terminus of newly synthesized proteins for subsequent post-translational modifications (e.g. myristoylation), which are required for stability, activity, and intracellular localization. 3, 4 A related isoform, MetAP1, can process many of the same substrates, but its relative activity differs from that of MetAP2 based on the nature of the amino acid residues flanking the cleavage site, primarily the residue immediately adjacent to the N-terminal methionine. Fumagillin and structural analogues, such as TNP-470 (AGM-1470), 2a, selectively inhibit MetAP2 enzymatic activity through irreversible, covalent bond formation with histidine-231 in the active site of the enzyme. 5, 6
Aminopeptidases in Biology and Disease, 2004
Angiogenesis, the formation of new blood vessels from existing vasculature, is a key factor in a number of vascular-related pathologies such as the metastasis and growth of solid tumors. Thus, the inhibition of angiogenesis has great potential as a therapeutic modality in the treatment of cancer and other vascular-related diseases. Recent evidence suggests that the inhibition of mammalian methionine aminopeptidase type 2 (MetAP2) catalytic activity in vascular endothelial cells plays an essential role in the pharmacological activity of the most potent small molecule angiogenesis inhibitors discovered to date, the fumagillin class. Methionine aminopeptidase (MetAP, EC 3.4.11.18) catalyzes the non-processive, co-translational hydrolysis of initiator N-terminal methionine when the second residue of the nascent polypeptide is small and uncharged. Initiator Met removal is a ubiquitous and essential modification. Indirect evidence suggests that removal of initiator Met by MetAP is important for the normal function of many proteins involved in DNA repair, signal transduction, cell transformation, secretory vesicle trafficking, and viral capsid assembly and infection. Currently, much effort is focused on understanding the essential nature of methionine aminopeptidase activity and elucidating the role of methionine aminopeptidase type 2 catalytic activity in angiogenesis. In this chapter, we give an overview of the MetAP proteins, outline the importance of initiator Met hydrolysis, and discuss the possible mechanism(s) through which MetAP2 inhibition by the fumagillin class of angiogenesis inhibitors leads to cytostatic growth arrest in vascular endothelial cells.
A Chemical and Genetic Approach to the Mode of Action of Fumagillin
Chemistry & Biology, 2006
Previous mode of action studies identified methionine aminopeptidase 2 (MetAP-2) as the target of the antiangiogenic natural product fumagillin and its drug candidate analog, TNP-470. We report here that TNP-470-mediated MetAP-2 inhibition blocks noncanonical Wnt signaling, which plays a critical role in development, cell differentiation, and tumorigenesis. Consistent with this finding, antisense MetAP-2 morpholino oligonucleotide injection in zebrafish embryos phenocopies gastrulation defects seen in noncanonical Wnt5 loss-of-function zebrafish mutants. MetAP-2 inhibition or depletion blocks signaling downstream of the Wnt receptor Frizzled, but upstream of Calmodulin-dependent Kinase II, RhoA, and c-Jun N-terminal Kinase. Moreover, we demonstrate that TNP-470 does not block the canonical Wnt/b-catenin pathway. Thus, TNP-470 selectively regulates noncanonical over canonical Wnt signaling and provides a unique means to explore and dissect the biological systems mediated by these pathways.
Depletion of Methionine Aminopeptidase 2 Does Not Alter Cell Response to Fumagillin or Bengamides
Cancer Research, 2004
Inhibition of endothelial cell growth by fumagillin has been assumed to be mediated by inhibition of the molecular target methionine aminopeptidase 2 (MetAp2). New data show that depletion of MetAp2 by siRNA does not inhibit endothelial cell growth. Moreover, MetAp2-depleted endothelial cells remain responsive to inhibition by either fumagillin or a newly identified MetAp2 enzyme inhibitor. These data suggest that MetAp2 function is not required for endothelial cell proliferation.
Chapter 2 Methionine Aminopeptidase Emerging role in angiogenesis
2003
Angiogenesis, the formation of new blood vessels from existing vasculature, is a key factor in a number of vascular-related pathologies such as the metastasis and growth of solid tumors. Thus, the inhibition of angiogenesis has great potential as a therapeutic modality in the treatment of cancer and other vascular-related diseases. Recent evidence suggests that the inhibition of mammalian methionine aminopeptidase type 2 (MetAP2) catalytic activity in vascular endothelial cells plays an essential role in the pharmacological activity of the most potent small molecule angiogenesis inhibitors discovered to date, the fumagillin class. Methionine aminopeptidase (MetAP, EC 3.4.11.18) catalyzes the non-processive, co-translational hydrolysis of initiator N-terminal methionine when the second residue of the nascent polypeptide is small and uncharged. Initiator Met removal is a ubiquitous and essential modification. Indirect evidence suggests that removal of initiator Met by MetAP is importa...