Peptide Deformylase: Mechanism and Inhibitor Design

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DESCRIPTION (provided by the applicant): The objective of this project is (1) to characterize the enzymes which deformylate N-formylated peptides (peptide deformylase (PDF), formylmethionine aminopeptidase (fMAP), and formylmethionine deformylase (fMDF)), and (2) to assess PDF as a novel target for antimicrobial drug design. In bacteria and eukaryotic organelles, protein synthesis initiates with N-formylmethionine. Consequently, all newly synthesized polypeptides in bacteria and organelles carry an N-terminal formyl group. Following translational initiation, PDF removes the N-formyl group from the vast majority of bacterial proteins. As an essential activity in bacteria, PDF is being pursued as a new target for antibacterial drug design. Recently, genomic sequencing has revealed PDF-like sequences in certain eukaryotes including man, raising some questions about the validity of PDF as a drug target. A related issue is whether treatment with PDF inhibitors would result in the accumulation of N-formyl peptides and inflammation in a patient, since some of the N-formyl peptides (e.g., f-MLF) are potent chemotactic agents. To address these issues, five specific aims are proposed in this project. Specific Aim 1 is to perform quantitative analyses of the structure-function relationship of E. coil PDF. Conserved residues in the active site will be mutated and kinetic, spectral, and structural characterization will be conducted. Specific Aim 2 is to clone and characterize the PDF-like sequences from human and Plasmodium falciparum, the causative agent of malaria. The goal is to determine whether these sequences actually code for functional PDF's and their physiological functions. Specific Aim 3 is to further improve the potency and specificity of PDF inhibitors against bacterial PDF, to design potent inhibitors against P. falciparum PDF, and to evaluate PDF as a potential target for antimalarial drug design. Specific Aim 4 is to design prodrugs that can be selectively activated by PDF. Such prodrugs are expected to be more specific (thus less toxic) and difficult for bacteria to develop resistance. Specific Aim 5 is to purify, clone, and characterize fMAP and fMDF from rats. These enzymes are thought to be responsible for inactivating the chemotactic peptides released by commensal bacteria and preventing inflammatory responses in mammals.