Wolfgang Pirson

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 ofEscherichia 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 theirN-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.

Peptide deformylase as an antibacterial drug target: assays for detection of its inhibition in Escherichia coli cell homogenates and intact cells.

ABSTRACT An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.

Substituted spiro[chroman-4,5′-thiazolidine]-2′,4′-diones as aldose reductase inhibitors

Abstract Aldose reductase (EC 1.1.1.21) is believed to be involved in the pathogenesis of diabetic complications. Inhibitors of this enzyme could be useful for the treatment of diabetic cataracts and neuropathies. A series of spiro[chroman-4,5′-thiazolidine]-2′,4′-diones, substituted at the aromatic ring, has been synthesized as potential inhibitors of this enzyme. Their activity was determined with in vitro and in vivo tests. The compounds are very potent inhibitors of aldose reductase. Activity in galactosemic rats was found, especially for derivatives bearing alkyl-carbamate substituents. Drug levels in plasma, lens and nerve were determined with radioactively labelled compounds and by high performance liquid chromatography. The latter method was also used to determine in vivo degradation of the carbamates.