Discovery of Inhibitors of Methionine aminopeptidase from Enterococcus faecalis
- Paper number
IAC-06-A1.P.2.03
- Author
Ms. Omonike Omotoso, The John Hopkins School of Medicine (SOM), United States
- Coauthor
Prof. Jun Liu, The John Hopkins School of Medicine (SOM), United States
- Year
2006
- Abstract
Purpose: The increasing emergence of drug-resistant pathogens has imposed an urgent call for antibiotics with novel mechanisms of action. We have taken a target-based approach to address this issue, by identifying and characterizing inhibitors of methionine aminopeptidase (MetAP) from Enterococcus faecalis. MetAP is a metalloprotease that removes the N-terminal methionine from proteins and peptides. The essential role of MetAP in Escherichia coli and other micro-organisms makes it a promising target for the development of new antibiotics. E. faecalis causes the majority of hospital acquired infections and some strains have gained resistance to vancomycin which is the antibiotic of last resort for some multidrug-resistant pathogens. Thus, the discovery of potent and selective MetAP inhibitors have the potential to contribute to the global basic research efforts by the National Aeronautics and Space Administration (NASA) and other organizations in response to emerging infectious diseases. Moreover, the discovery of antibiotics with new modes of action will be indispensable for astronauts on NASA missions in the case of infection with new or drug-resistance pathogens during space explorations. Methods: E. faecalis MetAP1 (EfMetAP1) was amplified from genomic DNA by PCR and sub-cloned into a pET28a vector. The plasmid encoding the MetAP gene was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography. Using a High-Throughput Screening approach with a coupled methionine-proline aminopeptidase assay, we screened over 175,000 compounds against EfMetAP1. The MetAP inhibitors were tested for antibacterial activity in collaboration with Lance Price at the Johns Hopkins School of Public Health. Results: We have cloned, over-expressed and purified to near homogeneity the only MetAP, EfMetAP1 from E. faecalis. In addition, we have characterized EfMetAP1 using a coupled methionine-proline aminopeptidase assay, by determining its kinetic constants, metal dependence, temperature and pH optima. Furthermore, using a High-throughput screening approach we have identified novel MetAP inhibitors that have activity in both enzymatic assays and bacterial culture. Conclusions: The validation of new antibiotic targets is essential because of the rise in multi-drug resistant pathogens. These results suggest that Enterococcus faecalis MetAP1 can be targeted for antibacterial drug development. We show that inhibitors against EfMetAP1 could be effective against clinically relevant pathogens. In addition, identification of MetAP inhibitors will be a valuable tool to study N-terminal processing of proteins, a universal and essential process in micro-organisms.
- Abstract document