ABSTRACT: The increased emergence of drug-resistant strains of pathogens has heightened the need for novel anti-bacterial, anti-parasitic and anti-viral agents. We used a combination of target and whole cell approaches to address this pressing problem. Our target based methodology is focused on N-terminal methionine excision (NME). NME is a universally conserved process required for the post-translational modifications of some proteins. MetAP is a metalloprotease that removes the N-terminal methionine during protein synthesis. The essential role of MetAP in microbes makes it a promising target for the development of new therapeutics. We have identified MetAP inhibitors that have potent and selevtive activity against clinically relevant pathogens such as Mycobacterium tuberculosis, Enterococcus faecalis, and Leshmainiasis Major. We also assessed the efficacy and toxicity of some of these inhibitors in cellular and animal models of infection. We have identified promising inhibitors of MetAP with potent in vivo activity. We successfully assessed the cytotoxicity of the MetAP inhibitors and observed no significant cytotoxicity with some pharmacophores. In addition, we observed that the in vitro enzymatic inhibitory activity of some of the inhibitors correlates well with in vivo activity. We have validated the therapeutic potential of MetAP for chemotherapy of infectious diseases. Therefore, MetAP is a promising and novel anti-infective drug target.