| The fumagillin analogue TNP-470 was the first anti-angiogenic small molecule to enter clinical trials and was shown to target methionine aminopeptidase-2 (MetAP-2), a metalloprotease that cleaves the N-terminal methionine of proteins. Previously, biochemical binding, in vivo yeast studies, and structural studies of human methionine aminopeptidase-2 bound to fumagillin revealed that these compounds exhibit specificity for MetAP-2 over its family member MetAP-1. In order to elucidate further the nature of this specificity, we developed a yeast-based screen for human MetAP-2 mutations that confer resistance to the fumagillin analogue ovalicin. In three independent experiments, over a million alleles of human MetAP-2 were screened for ovalicin-resistance. Analysis and confirmation of these alleles revealed that the A362T and Y444C alleles of human MetAP-2 confer ovalicin-resistance to the sensitive ΔMAP1 Saccharomyces cerevisiae strain. The majority of clones in three independent screens identified A362T as an ovalicin-resistant mutation, thus suggesting this residue's importance in the interaction of ovalicin and MetAP-2. Alignments of various species isoforms of MetAP-2 and MetAP-1 revealed that the alanine is conserved in MetAP-2 and that the homologous residue in MetAP-1 is a conserved threonine. Mutation of this residue to alanine resulted in an ovalicin-sensitive MetAP-1 allele, demonstrating that the alanine at this position is critical to interaction with ovalicin. These results provide a molecular explanation for the specificity exhibited by this class of anti-angiogenic agents for MetAP-2 over MetAP-1. To confirm whether MetAP-2 mediates the response of endothelial cells to ovalicin, these mutant alleles were expressed in endothelial cells. Unexpectedly, these alleles were not able to confer ovalicin-resistance to endothelial cells, most likely due to interference from endogenous levels of the protein. Further investigation suggested that MetAP-2 regulation in endothelial cells may be more complex than had been presumed and is thus a topic for further studies. |
