Most bacteria possess a single methionine aminopeptidase (MetAP) that is essential for survival. Therefore, this enzyme is an attractive target for novel broad-spectrum antibacterial agents. This key enzyme catalyzes the removal of N-terminal methionine from 55-70% newly synthesized proteins, which is required for localization, activation and stability. All MetAPs need a divalent metal ion for activation, such as Mn(II), Fe(II), Co(II), Ni(II) or Zn(II), but the question is which of these ions is the most important in bacterial cells. The difficulties for potent MetAP inhibitors to show antibacterial activity indicate the discrepancies between inhibition of a purified enzyme and inhibition of the same enzyme in a cellular environment. Understanding the catalysis and inhibition of MetAP in vitro and in vivo, and elucidating the physiologically relevant metalloform of MetAP in bacterial cells are the keys to realize the therapeutic potential of MetAP inhibitors. In the past a few years, we focused on E. coli MetAP and have discovered unique MetAP inhibitors that can distinguish different metal ions at the enzyme active site, i.e., metalloform-selective inhibitors. With these inhibitors, we characterized the cellular metalloform of MetAP in E. coli and concluded that Fe(II) is the native metal. More importantly, some of the Fe(II)-form selective inhibitors showed antibacterial activity against several E. coli and Bacillus strains. In this proposal, we will expand from E. coli MetAP to MetAPs in other bacteria, especially the drug resistant Staphylococcus aureus and Mycobacterium tuberculosis. We will continue to elucidate the catalysis and inhibition of MetAP as purified proteins, which is import for our understanding and assigning the physiologically relevant metalloform of MetAP enzymes in bacterial cells. Starting with our current metalloform-selective and antibacterial MetAP inhibitors, we will concentrate on improving their potency and selectivity on bacterial MetAP enzymes. The structural modification will be guided by X-ray structures of enzyme-inhibitor complexes, selectivity between human and bacterial MetAPs, toxicity on mammalian cells, and antibacterial activities. Our multidisciplinary approach with biochemistry, medicinal chemistry, structural biology, and microbiology will be synergistic and will accelerate the drug discovery process.
