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. 2018 Mar 16;430(6):842-852.
doi: 10.1016/j.jmb.2018.01.016. Epub 2018 Feb 2.

Binding and Action of Amino Acid Analogs of Chloramphenicol upon the Bacterial Ribosome

Andrey G Tereshchenkov  1 , Malgorzata Dobosz-Bartoszek  2 , Ilya A Osterman  3 , James Marks  4 , Vasilina A Sergeeva  1 , Pavel Kasatsky  5 , Ekaterina S Komarova  6 , Andrey N Stavrianidi  1 , Igor A Rodin  1 , Andrey L Konevega  7 , Petr V Sergiev  3 , Natalia V Sumbatyan  1 , Alexander S Mankin  4 , Alexey A Bogdanov  8 , Yury S Polikanov  9
Affiliations

Binding and Action of Amino Acid Analogs of Chloramphenicol upon the Bacterial Ribosome

Andrey G Tereshchenkov et al. J Mol Biol. .

Abstract

Antibiotic chloramphenicol (CHL) binds with a moderate affinity at the peptidyl transferase center of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving properties of this inhibitor, we explored ribosome binding and inhibitory activity of a number of amino acid analogs of CHL. The L-histidyl analog binds to the ribosome with the affinity exceeding that of CHL by 10 fold. Several of the newly synthesized analogs were able to inhibit protein synthesis and exhibited the mode of action that was distinct from the action of CHL. However, the inhibitory properties of the semi-synthetic CHL analogs did not correlate with their affinity and in general, the amino acid analogs of CHL were less active inhibitors of translation in comparison with the original antibiotic. The X-ray crystal structures of the Thermus thermophilus 70S ribosome in complex with three semi-synthetic analogs showed that CHL derivatives bind at the peptidyl transferase center, where the aminoacyl moiety of the tested compounds established idiosyncratic interactions with rRNA. Although still fairly inefficient inhibitors of translation, the synthesized compounds represent promising chemical scaffolds that target the peptidyl transferase center of the ribosome and potentially are suitable for further exploration.

Keywords: X-ray structure; antibiotic; peptidyl transferase center; protein synthesis; ribosome.

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Figures

Figure 1
Figure 1
Schematic diagram of chemical synthesis of the histidine analogue of CLM.
Figure 2
Figure 2. Binding and inhibitory properties of AA-CAM-derivatives
(A) Competition binding assay to test the inhibition of BODIPY-ERY binding to the E. coli ribosomes in the presence of increasing concentrations of AA-CAM derivatives measured by fluorescence anisotropy. (B, C) Inhibition of in vitro synthesis of firefly luciferase by AA-CAM derivatives in the E. coli S30 cell extract (B) or in the PURE system (C). All the inhibitors were present in the reaction at 30 μM. All the reactions were performed in triplicates and error bars represent confidence interval (α= 0.05). Inhibitory activity of AA-CAM compounds with free α-amino groups are shown as light grey bars, N-protected AA-CAM compounds – dark grey, positive control CLM – white bars. (D) Primer extension inhibition (toe-printing) analysis of site specificity of action of CLM and AA-CAMs. The synthetic mini-gene was translated in the cell-free translation (‘PURE’) system and sites of antibiotic-induced translation arrest were analyzed by primer extension. The reactions loaded onto lanes 1-6 contained mupirocin, an inhibitor of isoleucyl-tRNA synthetase. The sample in lane 2 (labeled ‘NONE’) contained no other antibiotics besides mupirocin. The control antibiotic retapamulin (RET) inhibits translation initiation and arrests the ribosome at the start codon (black arrowheads). Bands corresponding to the CLM-induced translation arrest at the 5th codon are indicated by the green arrowheads. Stalling of ribosomes at the 7th codon of the ORF due to the presence of mupirocin that causes depletion of isoleucyl-tRNA (lanes 1-6) is indicated by the blue arrowheads. U- and A-specific sequencing lanes are indicated. The nucleotide sequence of the gene and the corresponding encoded amino acids are indicated on the left. Note that the reverse transcriptase stops 15-16 nucleotides downstream from the first nucleotide of the P-site codon as indicated by the dashed arrow.
Figure 3
Figure 3. Chemical structures and electron density maps of CAM-derivatives
Chemical structures and difference Fourier maps of His-CAM (A), D-His-CAM (B), and Lys-CAM (C) in complex with the T. thermophilus 70S ribosome. The refined model of each compound is displayed in its respective electron density before the refinement (green mesh). Carbon atoms are colored yellow for His-CAM, orange for D-His-CAM, magenta for Lys-CAM, nitrogens are blue, oxygens are red.
Figure 4
Figure 4. Structure of His-CAM in complex with the 70S ribosome and A- and P-tRNAs
(A, B) Overview of the His-CAM binding site (yellow) in the T. thermophilus 70S ribosome viewed from the PTC down the tunnel as indicated by the inset (A), or as a cross-cut section through the ribosome (B). The 30S subunit is shown in light yellow, the 50S subunit is in light blue, the mRNA is magenta and the A- and P-site tRNAs are green and dark blue, respectively. The E-site tRNA is omitted for clarity. (C, D) Close-up views of the His-CAM bound in the PTC. The E. coli nucleotide numbering is used throughout. Potential H-bond interactions are indicated with dashed lines. Note that side chain of His-CAM compound forms tilted edge-to-face Π-stacking with the nucleobase of U2506 of the 23S rRNA (shown as spheres).
Figure 5
Figure 5. Side-chain-specific interactions of AA-CAMs with the ribosome
Compound-specific H-bond interactions of His-CAM (A), D-His-CAM (B), or Lys-CAM (C) with the nucleotides of the 23S rRNA are indicated with dashed lines. Stacking interactions of His-CAM are shown with the black arrow.
Figure 6
Figure 6. Structural basis for resistance to CLM (A) and His-CAM (B)
Molecular modeling of the C8-methylation of A2503 (red sphere) catalyzed by the Cfr-methyltransferase reveals a small clash with CLM (A) but not with histidine analogue of CLM (B). Note that the path of the His-CAM in the PTC is located further away from the C8-position of A2503 (compared to CLM) that should allow it to avoid a possible steric clash with this position carrying methyl group in the Cfr-modified ribosome.
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