Inhibitors of Initiation Phase of Bacterial Protein Synthesis

Abstract

The initiation phase is the rate‐limiting step in protein synthesis where messenger ribonucleic acid (mRNA), initiator‐transfer RNA (tRNA) and ribosomal subunits are assembled together in the presence of specific initiation factors. This process is the target for a diverse subset of antibiotics that inhibit the initiation step in a variety of different ways. Dissecting the mechanism of action of these antibiotics has provided insight not only into their inhibitory action but also into the process of translation initiation itself. With the dramatic increase in resistance of bacterial strains to many clinically relevant antibiotics, the discovery of improved inhibitors is becoming more important. Because the known initiation inhibitors bind to distinct regions of the ribosome compared with clinically used antibiotics, revisiting the initiation inhibitors may open new avenues to the development of novel antimicrobial agents.

Key Concepts:

  • Initiation of protein synthesis in bacteria operates through a 30S pre‐initiation complex.

  • Aurintricarboxylic acid is a nonspecific compound that in vitro at least can prevent translation initiation by perturbing binding of mRNA to the small ribosomal subunit.

  • Kasugamycin binds within the path of the mRNA on the small subunit to prevent correct interaction between initiator tRNA and start codon to prevent translation initiation.

  • Binding of edeine to the small subunit induces base pair formation between G795 and C693 and prevents binding of initiator tRNA to the small subunit.

  • Pactamycin binds in the E‐site in the path of the mRNA and prevents the first translocation reaction in a tRNA‐dependent fashion.

  • Evernimicin binds to the large subunit and prevents IF2‐dependent formation of the 70S initiation complex.

  • The tetrapeptide GE81112 binds to the 30S subunit to inhibit 30S initiation complex formation.

  • Antibiotics that inhibit translation also induce ribosomal assembly defects.

Keywords: antibiotics; initiation; ribosomes; RNA; antibiotic resistance

Figure 1.

Aurintricarboxylic acid inhibits mRNA binding to small subunits. (a) Chemical structure of aurintricarboxylic acid (ATA) and (b) interface view of 30S subunit, with mRNA (green) and anti‐Shine Dalgarno (anti‐SD; orange) as ribbons (Jenner et al., ; Kaminishi et al., ). (c) Top or ‘birds‐eye’ view of the 30S subunit showing mRNA (green) and P‐site tRNA (P‐tRNA, pink). The base pairing between the 3′ end of the 16S rRNA containing the anti‐SD sequence (orange) and the SD of the mRNA is shown as sticks (Jenner et al., ; Kaminishi et al., ).

Figure 2.

Kasugamycin binds in the path of the mRNA. (a) Chemical structure of kasugamycin (Ksg) with the D‐inositol and kasugamine moieties indicated in blue and orange, respectively. Probable hydrogen bonds from ring I of Ksg to G926 and the kasugamine tail to A792/A794 are indicated (Schluenzen et al., ). (b) Overview of the primary kasugamycin (Ksg, orange)‐binding site on the 30S subunit (Schluenzen et al., ; Schuwirth et al., ), compared with the binding position of the aminoglycoside antibiotic paromomycin (Par, red) (Ogle et al., ). Helices 24 (green), h28 (pink), h44 (yellow) and h45 (cyan) are coloured for reference. (c) Position of the primary (Ksg) and secondary (Ksg2) kasugamycin‐binding sites relative to mRNA (green) and A‐ (blue), P‐ (pink) and E‐tRNA (cyan) (Schluenzen et al., ). The first position of the P site codon (+1) and last position of the E site codon (−1) are indicated (Jenner et al., ; Yusupov et al., ). (d) The primary kasugamycin (Ksg; gold)‐binding site, showing hydrogen bonding to G926 in h28 (pink) and A792/A794 in h24 (green). A1418 and A1519 in h45 (cyan) are not directly in the Ksg‐binding site, but influence Ksg binding indirectly through h44 (yellow) and U793 of h24 (green) (Schluenzen et al., ).

Figure 3.

Edeine induces base pair formation between h23 and h24. (a) Chemical structure of edeine (Ede) with the tyrosine (Tyr) and spermidine moieties at each end of the molecule indicated. (b) Overview of the binding site of edeine (Ede) on the 30S subunit (Pioletti et al., ). Helices 23 (green), h24 (magenta) and h44 (yellow) are coloured for reference. (c) Conformation of A795 in h24 and C693 in h23 of the 30S subunit (Brodersen et al., ). (d) Base pair formation between A795 in h24 (magenta) and C693 in h23 (green) on binding of edeine (Ede, purple) to the 30S subunit (Pioletti et al., ).

Figure 4.

Pactamycin binds in the path of the mRNA in the E site. (a) Overview of the binding site of pactamycin (Pct, cyan) (Brodersen et al., ) and edeine (Ede) on the 30S subunit (Pioletti et al., ). Helices 23 (green), h24 (magenta) and h44 (yellow) are coloured for reference. (b) Pactamycin (Pct, cyan) stacks between bases A795 of h24 (magenta) and C693 of h23 (green) (Brodersen et al., ). (c) Pactamycin (Pct, cyan) sits in the path of the mRNA (gold) (Brodersen et al., ). (d) Superimposition of binding sites of pactamycin (Pct, cyan), edeine (Ede, purple) and kasugamycin (Ksg/Ksg2, orange) relative to mRNA (gold) and A‐ (blue), P‐ (pink) and E‐tRNA (cyan).

Figure 5.

Binding site of evernimicin on the large ribosomal subunit. (a) Ribosomal components of the large subunit (grey) involved in evernimicin binding and resistance: Ribosomal protein L16 (green) and 23S rRNA helices 89 (blue) and 91 (pink). Positions of ribosomal proteins L1, L11, the central protuberance (CP) and peptidyltransferase centre (PTC; red sphere) are indicated for reference. (b) Ribosomal components of the large subunit (grey) involved in evernimicin binding and resistance: Resistance mutations at residues (green spheres) in ribosomal protein L16 confer low‐level evernimicin resistance, whereas higher resistance is conferred by mutations at nucleotides (purple) in H89 and H91. Nucleotides that are protected from chemical modification in the presence of evernimicin are shown in light magenta (dark magenta indicates both protection and resistance positions). The noncanonical hydrogen bond between C2475 (H89) and G2529 (H91) is indicated with dashed lines, and the red sphere indicates the location of the PTC.

Figure 6.

GE81112 targets the small subunit to inhibit translation initiation. (a) Chemical structure of the tetrapeptide antibiotic GE81112 isoforms A, B and B1 (Brandi et al., ). (b) Ribosomal components of the small subunit (grey) involved in GE81112 binding, namely 16S rRNA helices 23 (green) and 24 (magenta). (c) GE81112 protects C693 in h23 from Kethoxal modification and enhances the modification of A795 in h24 by DMS (Brandi et al., ). (d) Superimposition of kasugamycin (Ksg/Ksg2, orange), edeine (Ede, purple) and pactamycin (Pct, cyan) relative to h23/h24.

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Further Reading

Brandi L, Dresios J and Gualerzi CO (2008a) Assays for the identification of inhibitors targeting specific translational steps. Methods in Molecular Medicine 142: 87–105.

Brandi L, Fabbretti A, Pon CL, Dahlberg AE and Gualerzi CO (2008b) Initiation of protein synthesis: a target for antimicrobials. Expert Opinion on Therapeutic Targets 12: 519–534.

Cencic R, Robert F and Pelletier J (2007) Identifying small molecule inhibitors of eukaryotic translation initiation. Methods in Enzymology 431: 269–302.

Maguire BA (2009) Inhibition of bacterial ribosome assembly: a suitable drug target? Microbiology and Molecular Biology Reviews 73: 22–35.

Nierhaus KH and Wilson DN (eds) (2004) Protein Synthesis and Ribosome Structure: Translating the Genome. Weinheim: WILEY‐VCH Verlag GmbH & Co.

Tenson T and Mankin A (2006) Antibiotics and the ribosome. Molecular Microbiology 59: 1664–1677.

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Marquez, Viter, Sohmen, Daniel, and Wilson, Daniel N(Dec 2009) Inhibitors of Initiation Phase of Bacterial Protein Synthesis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021835]