A single amino acid residue defines the difference in ovalicin sensitivity between type I and II methionine aminopeptidases

Abstract

TNP-470, the first anti-angiogenic small molecule to enter clinical trials, targets 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 TNP-470 and its analogs fumagillin and ovalicin revealed that these compounds exhibit specificity for MetAP-2 over its family member MetAP-1. To further elucidate the nature of this specificity, we developed a yeast-based screen for human MetAP-2 mutations that confer ovalicin resistance. Of the three resistant alleles, A362T appeared in the majority of clones and was found to be the most resistant to the ovalicin class of inhibitors. Alignment of human MetAP-2 with human MetAP-1, which is naturally ovalicin-resistant, revealed that the analogous residue in MetAP-1 is also a threonine. Mutation of this residue to alanine resulted in an ovalicin-sensitive MetAP-1 allele, demonstrating that an alanine at this position is critical for inhibition by ovalicin. These results provide a molecular explanation for the specificity exhibited by this class of anti-angiogenic agents for MetAP-2 over MetAP-1 and may prove useful in the development of additional MetAP-2-specific therapeutic agents.

FIG. 1. Human methionine aminopeptidase-2 complements the function of yeast MAP2

ΔMAP2 yeast were co-transformed with the human MetAP-2 pAD4M and YMAP2 pSE319 vectors. Yeast MAP1 was knocked out by transformation of the map1::HIS3 locus. The resulting strain was then grown in medium supplemented with tryptophan to promote segregation of the pSE319 plasmid and development of red sectors. Sectors or colonies that are red are yeast rescued by human MetAP-2.

FIG. 2. Dot titrations of ovalicin-resistant human MetAP-2 alleles

ΔMAP1 yeast were transformed with either wild type (WT) or ovalicin (Ov)-resistant alleles of human MetAP-2 (hMetAP-2). At 20 nm ovalicin the alleles found in the screen confer significant ovalicin resistance to ΔMAP1 yeast. Plates were photographed after a 6-day incubation at 30 °C.

FIG. 3. Further characterization of the A362T allele

Alignment of the amino acid sequences of various MetAP-2 isoforms shows complete conservation of the alanine residue (as denoted by the asterisk). Alignment of MetAP-2 with MetAP-1 reveals that the analogous residue in MetAP-1 is a completely conserved threonine. Residues highlighted in blue are similar; residues highlighted in pink are identical. C. elegans, Caenorhabditis elegans; P. furiosus; Pyrococcus furiosus.

FIG. 4. Discovery of an ovalicin-sensitive human methionine aminopeptidase-1 allele

Wild type (WT) and T334A human MetAP-1 alleles were expressed in ΔMAP1 yeast, and a dot titration was subsequently performed. Shown is substitution of threonine 334 with alanine renders human MetAP-1 (hMetAP-1) sensitive to ovalicin (A), fumagillin (B), and TNP-470 (C). Plates were photographed after a 2-day incubation at 30 °C.