Specific, efficient, and selective inhibition of prokaryotic translation initiation by a novel peptide antibiotic

Abstract

Many known antibiotics target the translational apparatus, but none of them can selectively inhibit initiation of protein synthesis and͞or is prokaryotic-specific. This article describes the properties of GE81112, an effective and prokaryotic-specific initiation inhibitor. GE81112 is a natural tetrapeptide produced by a Streptomyces sp. identified by an in vitro high-throughput screening test developed to find inhibitors of the prokaryotic translational apparatus preferentially acting on steps other than elongation. In vivo GE81112 inhibits protein synthesis but not other cell functions such as DNA duplication, transcription, and cell wall synthesis. In vitro GE81112 was found to target the 30S ribosomal subunit and to interfere with both coded and noncoded P-site binding of fMettRNA, thereby selectively inhibiting formation of the 30S initiation complex.30S initiation complex ͉ fMet-tRNA binding ͉ P-site inhibition

Characterization of GE82832, a peptide inhibitor of translocation interacting with bacterial 30S ribosomal subunits

“…2B), indicating that it does not affect the rates of the events responsible for existence of the lag. This behavior is clearly different from that found for GE81112, another antibiotic recently discovered and characterized as a powerful inhibitor of translation initiation, which slows down initiation through a competition with fMet-tRNA for P-site binding, causing lengthening of the lag of luciferase appearance (Brandi et al 2006a). These findings indicate that GE81112 and GE82832 hit different targets within the translational apparatus, both inhibiting early yet different steps of protein synthesis; unlike GE81112, GE82832 is not an inhibitor of translation initiation (see below), in spite of the fact that both antibiotics were identified by the same HTS test.…”

“…In fact, amino acid misincorporation during the synthesis of luciferase (311 amino acids) would be expected to reduce the enzymatic activity, thereby diminishing the level of luminescence without affecting the overall amount of protein synthesized. As seen in Figure 2A, under the conditions of our experiment and in the absence of any inhibitor, the luciferase activity appears after a fairly long (ffi6 min) lag that can be attributed to the low initiation and elongation rates characteristic of translation at low temperature (Farewell and Neidhardt 1998;Brandi et al 2006a) and to the time required for the correct folding of the de novo synthesized enzyme before its activity can be expressed (Fedorov and Baldwin 1995;Kolb et al 2000). As seen from Figure 2A,C, the level of luciferase synthesized and the incorporation of radioactive precursors in the protein diminish in parallel with increasing concentrations of GE82832 (Fig.…”

Antibiotic discovery in the twenty-first century: current trends and future perspectives

“…This program led to the identification of GE81112 (Figure 1), a novel tetrapeptide produced by a Streptomyces sp., which targets specifically the 30S ribosomal subunit by interfering with fMet-tRNA binding to the P-site. 33 The compound was highly effective against a few Grampositive and Gram-negative strains, if grown in minimal or chemically defined medium, suggesting active uptake by the cells. 34 The above examples illustrate how different approaches can lead to novel antibiotic classes.…”

A Bright Future for Antibiotics?

“…Examples are those used recently to target Helix 69 (111), the first 16 residues of the proline-rich antimicrobial peptide mammalian Bac7 (112), the thiazolyl peptide antibiotics (113), and the small peptides that were shown to inhibit translation in prokaryotes (75,114).…”