Peptide Deformylase as an Antibacterial Drug Target: Target Validation and Resistance Development

Abstract

New inhibitors of peptide deformylase (PDF) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. Several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) The inhibition of Escherichia coli growth could be counteracted by overexpression of PDF from different organisms, including E. coli, Streptococcus pneumoniae, and Haemophilus influenzae. Conversely, reduced expression of PDF in S. pneumoniae resulted in an increased susceptibility to the inhibitors. (ii) Proteome analysis on two-dimensional gels revealed a shift for many proteins towards lower pI in the presence of PDF inhibitors, as would be expected if the proteins still carry their N-formyl-Met terminus. (iii) PDF inhibitors show no antimicrobial activity against E. coli under conditions that make growth independent of formylation and deformylation. The antibacterial activity in E. coli was characterized as bacteriostatic. Furthermore, the development of resistance in E. coli was observed to occur with high frequency (10 ؊7 ). Resistant mutants show a reduced growth rate, and DNA sequence analysis revealed mutations in their formyl transferase gene. Taking all these aspects into account, we conclude that PDF may not be an optimal target for broad-spectrum antibacterial agents.Antibiotic resistance is a major health concern, and the existing antibiotics target only a handful of molecules. Therefore, there is an urgent need for antibiotics with novel mechanisms of action. Peptide deformylase (PDF; EC 3.5.1.27) is essential in a variety of pathogenic bacteria but is not required for cytoplasmic protein synthesis in eukaryotes and is therefore an interesting potential target for antibacterial agents. Protein synthesis in eubacteria, under normal conditions, is initiated by formyl-methionyl-tRNA (19). Consequently, all nascent polypeptides are synthesized with N-formyl-methionine at the N terminus. The formyl group is removed by PDF during elongation of the polypeptide chain (1, 7). As methionine aminopeptidase (EC 3.4.11.18) cannot hydrolyze N-blocked polypeptides, deformylation is also a prerequisite for protein maturation (10,22,27). Both PDF and MAP, are essential for growth in Escherichia coli (10,19,21). pdf gene mutants can only be obtained in E. coli strains lacking the gene for formyltransferase, the enzyme that N-formylates the methionyl-tRNA f Met (EC.2.1.2.9) (20). In a recent publication, we described the identification, optimization, and biological characterization of novel PDF inhibitors (3). These compounds were potent inhibitors of the isolated enzyme but only moderately active as antibacterials. In the accompanying paper, we describe transcription-translation assays that allowed us to demonstrate that the inhibitors were active as inhibitors of PDF in cell homogenates as well as in intact cells (4a). The experimental evidence presented here demonstrates that (i) antibacteri...

Reducing the fitness cost of antibiotic resistance by amplification of initiator tRNA genes

“…Actinonin is synthesized by actinomycetes (4), and it inhibits bacterial growth in a bacteriostatic manner (5,6). Unlike protein synthesis in eukaryotes, where translation is initiated with a nonformylated methionyl (Met) initiator tRNA (tRNAi), most eubacteria initiate translation with a formylated Met-tRNAi (5,7).…”

“…The most common, previously identified mutations causing actinonin resistance are loss-of-function mutations in the fmt gene (2,5). This gene encodes methionyl-tRNA formyltransferase (FMT), an enzyme that adds the formyl group to the Met-tRNAi.…”

“…These steps are often required for the nascent polypeptide to mature into a functional protein (1,8). Actinonin inhibits PDF, leading to accumulation of toxic formylated polypeptides in the cell and subsequent growth inhibition (5,6).…”

“…Actinonin is synthesized by actinomycetes (4), and it inhibits bacterial growth in a bacteriostatic manner (5,6). Unlike protein synthesis in eukaryotes, where translation is initiated with a nonformylated methionyl (Met) initiator tRNA (tRNAi), most eubacteria initiate translation with a formylated Met-tRNAi (5,7). When translation is complete, the formyl group is removed by PDF, encoded by the def gene (1,8), and, for many proteins, an amino peptidase subsequently removes the N-terminal methionine.…”

An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

“…Over-expression of FabB protein appeared to lead to increased resistance to thiolactomycin in E. coli [40]. Apfel and colleagues [41] demonstrated that over-expression of peptide deformylase in a cell reduced its susceptibility to inhibitors specific for the essential target, peptide deformylase.…”