The Mechanisms of Action of Ribosome-Targeting Peptide Antibiotics

Abstract

The ribosome is one of the major targets in the cell for clinically used antibiotics. However, the increase in multidrug resistant bacteria is rapidly reducing the effectiveness of our current arsenal of ribosome-targeting antibiotics, highlighting the need for the discovery of compounds with new scaffolds that bind to novel sites on the ribosome. One possible avenue for the development of new antimicrobial agents is by characterization and optimization of ribosome-targeting peptide antibiotics. Biochemical and structural data on ribosome-targeting peptide antibiotics illustrates the large diversity of scaffolds, binding interactions with the ribosome as well as mechanism of action to inhibit translation. The availability of high-resolution structures of ribosomes in complex with peptide antibiotics opens the way to structure-based design of these compounds as novel antimicrobial agents.

Keywords: antibiotic; inhibitor; proline-rich antimicrobial peptides; ribosome; translation.

Figure 1

The target of peptides antibiotics during the proteins synthesis cycle. The initiation of the translation involves the binding of the initiator fMet-tRNA and mRNA to form a 70S pre-initiation complex with the fMet-tRNA located at the P site. This process is facilitated by initiation factors (IFs) and is inhibited by peptide antibiotics edeine, GE81112 and thiostrepton. During elongation, the aminoacyl-tRNAs are delivered to the A site by the elongation factor Tu (EF-Tu) allowing subsequent peptide bond formation to occur. This step of translation can be inhibited by streptogramins A/B, oncocin-112, bactenecin-7, or klebsazolicin. Following peptide bond formation, the tRNAs are translocated through the ribosome by the elongation factor G (EF-G). This step of elongation is inhibited by dityromycin, tuberactinomycins, or thiostrepton. After multiple elongation cycles, one of the three stop codons appears in the A site of the ribosome and release factors (RFs) are typically recruited. Apidaecin specifically inhibits the termination process by preventing the RFs from dissociating from the ribosome. Following polypeptide release, the post-termination ribosome is recycled by the ribosome recycling factor (RRF) and EF-G so that the components can be reused for the next round of translation.

Figure 2

Overview of the peptide antibiotics binding sites on the bacterial ribosome. Overview (A) and close-up views (B–F) of the binding sites of the peptide antibiotics (B) edeine B (EDE, green), (C) GE81112 (GE, red), (D) dityromycin (DIT, yellow), (E) viomycin (VIO, magenta), and (F) odilorhabdin (ODL, orange), which target the small (30S) ribosomal subunit. The mRNA (blue) and anticodon stem loop (ASL) of A-, P-, and E-site tRNAs (cyan) are shown, and 16S rRNA helix h44 as well as ribosomal proteins uS12 and uS13 are highlighted for reference. Overview (G) and close-up views (H–L) of the binding sites of the peptide antibiotics (H) streptogramin type A (dalfoprsitin, DAL, red) and type B (quinupristin, QIN, orange), (I) oncocin-112 (ONC, green), (J) apidaecin-137 (API, magenta), (K) klebsazolicin (KLB, yellow), and (L) thiostrepton (THS, blue), which target the large (50S) ribosomal subunit. The relative position of A, P, and E-site tRNAs (cyan) are shown, and 23S rRNA helices H43/44 is highlighted for reference.

Figure 3

Binding of the peptide antibiotic edeine is incompatible with the P-site tRNA and mRNA. (A) Chemical structure of the edeine B consisting of β-tyrosine, isoserine, DAPA (2,3-diaminopropanoic acid), DAHAA (2,6-diamino-7-hydroxyazelaic acid), and guanylspermidine moities. (B) Overview of edeine B (EDE, green) binding site on the 30S subunit (PDB ID 1I95; Pioletti et al., 2001), with 16S rRNA helices h44 (blue), h45 (red), h23 (orange), and h24 (teal) shown for reference. The 30S subunit is viewed from the subunit interface as indicated by the inset at the bottom left. (C,D) Close-up view of EDE (green) binding site at the tip of helix h23 and h24 (gray) showing overlap of EDE with P-site tRNA (cyan) and first codon (+1) of the P-site mRNA (blue). Hydrogen bonding between the nucleotides G693-C795 of the 16S rRNA formed upon EDE binding is indicated with dashed lines in (D) (Pioletti et al., 2001).

Figure 4

Binding site of GE81112 on the 30S subunit. (A) Chemical structure of GE81112 congeners A, B, and B1. HPA—3-hydroxypipecolic acid; AAHPA—2-amino-5-[(aminocarbonyl)oxy]-4-hydroxypentanoic acid; CIS—5-chloro-2-imidazolylserine. (B) Overview of GE81112 binding site on the 30S subunit (PDB ID 5IWA; Fabbretti et al., 2016), with 16S rRNA helices h44 (cyan), h45 (red), and h24 (teal) as well as ribosomal protein uS13 (orange) and anticodon stem loop (ASL) mimic (green) of the P-site tRNA shown for reference. The 30S subunit is viewed from the subunit interface as indicated by the inset at the bottom left. The inset on the bottom right shows packing of the 30S ribosomal subunits in the crystal. Note that the spur (green) of one 30S subunit (30S-1, dark gray) inserts into the P site of the other 30S subunit (30S-2, light gray) and mimics ASL of the P-site tRNA. (C) Close-up view of the binding site of GE81112 within the ASL mimic (spur, helix 6, green) compared with (D) canonical binding position and conformation of the ASL of a P-site tRNA (cyan) and mRNA (blue).

Figure 5

GE82832/dityromycin bind to uS12 on the 30S subunit and inhibit translocation. (A) Chemical structure of the GE82832/dityromycin comprises proteinogenic (e.g., proline or valine) as well as non-proteinogenic amino acids, such as N,N-dimethyl-threonine (DMT), N-methyl-valine (NMV), epoxy-hydroxy-dehydro-isoleucine (EHDHI), or dihydroxyl-methyl tyrosine (HMT). (B) GE82832/dityromycin (DIT, yellow) interacts exclusively with the ribosomal protein uS12 (teal) on the 30S subunit (gray) (PDB ID 4NVY; Bulkley et al., 2014). The anticodon stem loop (ASL) of a P-site tRNA (cyan) and mRNA (blue) are shown for reference. The 30S subunit is viewed from the subunit interface, as indicated by the inset at the bottom left. (C) Overlap in the binding site of dityromycin (yellow) and domain III of EF-G (green). Residues within uS12 (teal) that are important for dityromycin binding are highlighted in blue.

Figure 6

Tuberactinomycins bind to the intersubunit bridge to inhibit translocation. (A) Chemical structures of the tuberactinomycins viomycin and capreomycin, with the chemical core (black) and drug-specific moieties colored red (viomycin) or blue (capreomycin). Tuberactinomycins are comprised of both proteinogenics (e.g., serine) as well as non-proteinogenic amino acids [e.g., (2S,3R)-capreomycidine (L-Cam), or L-2,3-diaminopropionic acid, L-Dap]. (B) Overview and (C,D) close-up views of the (C) viomycin (VIO, magenta), and (D) capreomycin (CAP, orange) binding sites (PDB IDs 4V7H and 4V7M, respectively; Stanley et al., 2010), both of which are located between helix h44 (yellow) on the 30S subunit and helix H69 (cyan) on the 50S subunit. Tuberactinomycin binding induces nucleotides A1492 and A1493 of the 16S rRNA to flip out of helix h44 and interact with the mRNA (blue) and A-site tRNA (green) duplex that is formed during decoding. (E) Secondary structure of the 16S rRNA and positions of the resistance mutation within helix h44.

Figure 7

Odilorhabdins bind to the decoding center on the 30S subunit and promote miscoding. (A) Chemical structures of natural odilorhabdins NOSO-95A, B, C (top), and the fully-synthetic derivative NOSO-95179 (bottom). (B) Overview of the NOSO-95179 binding site (orange) on the T. thermophilus 70S ribosome. 30S subunit is light gray, the 50S subunit is dark gray. mRNA is shown in dark blue and A-site tRNAs is displayed in green. (C) Close-up view of the NOSO-95179 binding site within the decoding center of the 30S subunit. Shown are interactions of NOSO-95179 with the 16S rRNA and with tRNA. (D) Antibiotics that bind in the decoding center on the small ribosomal subunit. Shown are location of the NOSO-95179 binding site relative to the binding sites of other antibiotics known to target the decoding center: paromomycin (PAR, red), viomycin (VIO, magenta), tetracycline (TET, green), negamycin (NEG, blue). Nucleotides of the 16S rRNA that are critical for decoding are shown as sticks.

Figure 8

Streptogramins A and B bind within the ribosomal exit tunnel. (A) Chemical structures of the streptogramin A (dalfopristin) and B (quinupristin) comprise proteinogenic (e.g., proline, threonine, and serine) as well as non-proteinogenic amino acids, such as phenylglycine and dimethylaminophenylalanine. (B) Transverse section of the 70S ribosome revealing the binding site of the streptogramin type A (dalfopristin, DAL, red) and type B (quinupristin, QIN, orange) within the exit tunnel of the large 50S subunit (light blue) (PDB ID 4U26; Noeske et al., 2014). The position of A-tRNA (green) and P-tRNA (blue) as well as mRNA (magenta) on the 30S subunit (yellow) are shown for reference. (C,D) Two different views of binding site and interaction of dalfopristin (red) and quinupristin (orange) with 23S rRNA nucleotides (cyan) comprising the PTC and the exit tunnel. The relative position of the aminoacylated CCA-ends of the A-site Phe-tRNA (green) and P-site fMet-tRNA (blue) are shown for reference.

Figure 9

PrAMP and klebsazolicin antibiotics bind within the ribosomal exit tunnel. (A) Transverse section of the 70S ribosome revealing the binding site of the PrAMPs oncocin-112 (ONC, green) and apidaecin-137 (API, purple) as well as klebsazolicin (KLB, yellow) within the exit tunnel of the large 50S subunit (light blue). The position of A-tRNA (cyan) and P-tRNA (blue) as well as mRNA (magenta) on the 30S subunit (light yellow) are shown for reference. (B–G) Relative binding position of (B) oncocin-112 (green, PDB ID 4Z8C; Roy et al., 2015), (C) bactenecin-7 (teal, PDB ID 5HAU; Gagnon et al., 2016), (D) pyrrhocoricin (light red, PDB ID 5HD1; Gagnon et al., 2016), (E) Tur1A (blue, PDB ID 6FKR; Mardirossian et al., 2018), (F) apidaecin-137 (magenta, PDB ID 5O2R; Florin et al., 2017) and (G) klebsazolicin (yellow, PDB ID 5W4K; Metelev et al., 2017) compared to the CCA-ends of P-site tRNA (blue) and A-site tRNA (cyan) or (F) RF1 (green). In panel (G), the A-site Phe and P-site fMet moieties are shown for reference and colored green and red, respectively; THZ, thiazole ring; OXZ, oxazole ring.

Figure 10

Thiostrepton binding site on the large ribosomal subunit. (A) Chemical structure of the thiostrepton. (B) The binding site of the thiostrepton (THS, blue) on the large 50S subunit of Dienococcus radiodurans (gray) (PDB ID 3CF5; Harms et al., 2008). The position of 23S rRNA helices H43 and H44 (cyan) and ribosomal protein uL11 (green) are shown for reference. The 50S subunit is viewed from the subunit interface as indicated by the inset at the top left. (C) Close-up view of the thiostrepton binding site showing its interactions with 23S rRNA nucleotides A1065 and A1095 located at the tips of helices H43 and H44 (cyan) as well as proline residues (orange) within the N-terminal domain (NTD) of ribosomal protein uL11 (green). (D) Overlap in binding position of thiostrepton (THS, blue) and domain V of EF-G (pale green).

Figure 11

Relative location of peptide and small-molecular antibiotics on the bacterial ribosome. (A) Overview of the binding sites of the peptide (yellow) and small-molecular (blue) antibiotics targeting the small (30S) ribosomal subunit: edeine B, GE81112, dityromycin, viomycin, odilorhabdin, negamycin, tetracycline, paromomycin, streptomycin, spectinomycin, amicoumacin, pactamycin, kasugamycin. (B) Overview of the binding sites of the peptide (yellow) and small-molecular (blue) antibiotics targeting the large (50S) ribosomal subunit: streptogramin type A (dalfoprsitin) and type B (quinupristin), oncocin-112, apidaecin-137, klebsazolicin, thiostrepton, orthosomycin (avilamycin), macrolides (erythromycin, carbomycin, spiramycin, tylosin), chloramphenicol, hygromycin A, A201A, lincosamides (clindamycin), oxazolidinones (linezolid). The relative position of A, P, and E-site tRNAs (cyan) are shown, and 23S rRNA helices H43/44 is highlighted for reference.