Peptide deformylase catalyzes the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria. Its essential character in bacterial cells makes it an attractive target for antibacterial drug design. In this work, we have rationally designed and synthesized a series of peptide thiols that act as potent, reversible inhibitors of purified recombinant peptide deformylase from Escherichia coli and Bacillus subtilis. The most potent inhibitor has a K(I) value of 11 nM toward the B. subtilis enzyme. These inhibitors showed antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations (MIC) as low as 5 microM ( approximately 2 microg/mL). The PDF inhibitors induce bacterial cell lysis and are bactericidal toward all four bacterial strains that have been tested, B. subtilis, Staphylococcus epidermidis, Enterococcus faecalis, and E. coli. Resistance evaluation of one of the inhibitors (1b) against B. subtilis showed that no resistant clone could be found from >1 x 10(9) cells. Quantitative analysis using a set of inhibitors designed to possess varying potencies against the deformylase enzyme revealed a linear correlation between the MIC values and the K(I) values. These results suggest that peptide deformylase is the likely molecular target responsible for the antibacterial activity of these inhibitors and is therefore a viable target for antibacterial drug design.

Peptide-derived thiols of the general structure N-mercaptoacyl-leucyl-p-nitroanilide (1a-c) were synthesized and found to be potent, slow-binding inhibitors of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potencies (K(I)) of these inhibitors against AAP range from 2.5 to 57 nM exceeding that of the natural product bestatin and approaching that of amastatin. The corresponding alcohols (2a-b) are simple competitive inhibitors of much lower potencies (K(I) = 23 and 360 microM). These data suggest that the free thiols are involved in the formation of the E. I and E.I complexes, presumably serving as a metal ligand. To investigate the nature of the interaction of the thiol-based inhibitors with the dinuclear active site of AAP, we have recorded electronic absorption and EPR spectra of Co(II)Co(II)-, Co(II)Zn(II)-, and Zn(II)Co(II)-AAP in the presence of the strongest binding inhibitor, 1c. Both [CoZn(AAP)] and [ZnCo(AAP)], in the presence of 1c, exhibited an absorption band centered at 320 nm characteristic of an S --> Co(II) ligand-metal charge-transfer band. In addition, absorption spectra recorded between 400 and 700 nm showed changes characteristic of 1c interacting with each active-site metal ion. EPR spectra recorded at high temperature (19 K) and low power (2.5 mW) indicated that in a given enzyme molecule, 1c interacts weakly with one of the metal ions in the dinuclear site and that the crystallographically identified micro-OH(H) bridge, which has been shown to mediate electronic interaction of the Co(II) ions, is likely broken upon 1c binding. EPR spectra of [CoCo(AAP)]-1c, [ZnCo(AAP)]-1c, and [CoZn(AAP)]-1c were also recorded at lower temperature (3.5-4.0 K) and high microwave power (50-553 mW). The observed signals were unusual and appeared to contain, in addition to the incompletely saturated contributions from the signals characterized at 19 K, a very sharp feature at g(eff) approximately 6.8 that is characteristic of thiolate-Co(II) interactions. These data suggest that the thiolate moiety can bind to either of the metal ions in the dinuclear active site of AAP but does not bridge the dinuclear cluster. Compounds 1a-c are readily accessible by synthesis and thus provide a novel class of potent aminopeptidase inhibitors.

Peptide deformylase catalyzes the removal of the N-terminal formyl group from nascent polypeptides during prokaryotic protein synthesis and maturation and is essential for bacterial survival. Its apparent absence from mammalian organisms makes it an attractive target for designing novel antibacterial agents. Based on the substrate specificity of peptide deformylase from Escherichia coli, a focused library of peptide thiols was synthesized on TentaGel resin using a disulfide linkage. Screening of the library against the purified deformylase was carried out in solution phase after the inhibitors were released from the resin with a reducing agent. A potent deformylase inhibitor was obtained from a 750-member library and was further optimized through rational modification into a low nanomolar inhibitor (KI = 15 nM against E. coli deformylase).

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